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Engineering the Metabolism of Escherichia coli for the Synthesis of Oxidized Products: Acetate and Pyruvate Production


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ENGINEERING THE METABOLISM OF Escherichia coli FOR THE SYNTHESIS OF OXIDIZED PRODUCTS: ACETATE AND PYRUVATE PRODUCTION By THOMAS B. CAUSEY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Thomas B. Causey

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This dissertation is dedicated to my wife Celeste and my family for their support through the years.

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ACKNOWLEDGMENTS I would like to acknowledge my Ph.D. committee (Dr. Ingram, Dr. Shanmugam, Dr. Preston, Dr. Luli and Dr. Stewart) for their guidance. I would also like to acknowledge Lorraine Yomano and Shengde Zhou for providing some of the strains used in this study. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.......................................................................................................................xi CHAPTER 1 INTRODUCTION........................................................................................................1 Renewable Fuels and Chemicals..................................................................................1 Why Use Escherichia coli as a Biocatalyst?................................................................2 Overview of Central Carbon Metabolism in Escherichia coli.....................................2 Aerobic versus Anaerobic Metabolism .................................................................2 ATP and Glycolytic Flux.....................................................................................10 Combine Beneficial Effects of Aerobic and Anaerobic Metabolism..................14 2 ENGINEERING THE METABOLISM OF Escherichia coli W3110 FOR THE CONVERSION OF SUGAR TO ACETATE............................................................16 Introduction.................................................................................................................16 Materials and Methods...............................................................................................17 Microorganisms and Media.................................................................................17 Genetic Methods..................................................................................................20 Fermentation........................................................................................................20 ATPase Assay......................................................................................................22 Analyses..............................................................................................................22 Results.........................................................................................................................24 Elimination of Fermentation Products (Strain SZ47)..........................................24 Disruption of Oxidative Phosphorylation (Strains TC24 and TC57)..................24 Inactivation of Alcohol Dehydrogenase E (Strain TC31) ...................................27 Interruption of the Tricarboxylic Acid Cycle (Strain TC32). .............................27 Elimination of Succinate Requirement (Strain TC36)........................................30 Disruption of Pyruvate Oxidase (Strain TC42)...................................................31 Restoration of Pyruvate Formate-lyase Activity (Strain TC51)..........................31 Effects of Gene Disruptions on Growth and Glycolytic Flux.............................32 v

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Production of Acetate and Other Organic Acids.................................................37 Pyruvate Oxidase Contributes to Acetate Production during Stationary Phase..40 Restoration of focA pflB Had No Effect on Acetate Production from Glucose. .41 Elimination of ATPase Activity..........................................................................42 Improving Acetate Yields....................................................................................42 Discussion...................................................................................................................44 3 CONVERSION OF ARABINOSE, MANNOSE, SORBITOL AND XYLOSE TO ACETATE............................................................................................................50 Introduction.................................................................................................................50 Materials and Methods...............................................................................................51 Microorganisms and Media.................................................................................51 Fermentations......................................................................................................51 Results.........................................................................................................................52 Fermentation of Sugars Derived from Hemicellulose (Arabinose, Mannose and Xylose)......................................................................................................52 Oxidation of Sorbitol to Acetate..........................................................................54 Discussion...................................................................................................................54 4 ENGINEERING Escherichia coli FOR THE EFFICIENT CONVERSION OF GLUCOSE TO PYRUVATE.....................................................................................57 Introduction.................................................................................................................57 Materials and Methods...............................................................................................59 Microorganisms and Media.................................................................................59 Genetic Methods..................................................................................................59 Deletion of Acetate Kinase (Strains TC38 and TC44)........................................60 Fermentation........................................................................................................61 Analyses..............................................................................................................62 Results.........................................................................................................................62 Pyruvate as a Co-Product during Acetate Fermentations....................................62 Acetate Kinase (ackA) Deletion Enhances Pyruvate Production and Inhibits Glycolysis (Strain TC38).................................................................................65 Pyruvate Oxidase (poxB) Mutation Causes a Modest Reduction in Acetate Accumulation...................................................................................................65 Combining Mutations for Pyruvate Oxidase (poxB) and Acetate Kinase (ackA) Improved Pyruvate Production (Strain TC44).....................................69 The Beneficial Role of a poxB Mutation for Pyruvate Production.....................70 Improving Pyruvate Yields and Titers of TC44 by Altering Fermentation Conditions........................................................................................................71 Discussion...................................................................................................................71 5 EFFECT OF BETAINE ON THE FERMENTATION PERFORMANCE OF TC44 (PYRUVATE PRODUCTION) AND TC36 (ACETATE PRODUCTION)...78 Introduction.................................................................................................................78 vi

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Materials and Methods...............................................................................................80 Microorganisms and Media.................................................................................80 Acetate and Pyruvate Tolerance..........................................................................80 Results.........................................................................................................................81 Effect of Betaine on Growth and Acetate Production for E. coli TC36..............81 Effect of Betaine on Growth and Pyruvate Production for E. coli TC44............82 Determination of the Minimal Inhibitory Concentration of Acetate and Pyruvate on E. coli W3110 in NBS Medium...................................................84 Discussion...................................................................................................................87 6 CONCLUSIONS........................................................................................................89 LIST OF REFERENCES...................................................................................................93 BIOGRAPHICAL SKETCH...........................................................................................103 vii

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LIST OF TABLES Table page 2-1 Sources and characteristics of strains.......................................................................18 2-2 Sources and characteristics of plasmids...................................................................19 2-3 Comparison of metabolic rates.................................................................................34 2-4 Summary of fermentation products..........................................................................35 2-5 Effect of atpA or atpFH deletions on metabolic rates..............................................42 3-1 Products formed by TC36 during aerobic catabolism of various sugars.................53 4-1 Sources and characteristics of strains and plasmids.................................................60 4-2 Products formed from glucose catabolism by E. coli strains used in our study...................................................................................................................66 4-3 Comparison of metabolic rates.................................................................................69 4-4 Comparison of biocatalysts for pyruvate production...............................................75 5-1 Comparison of metabolic rates for TC36 in the presence or absence of 1 mM betaine..........................................................................................................82 5-2 Comparison of metabolic rates for TC44 in the presence or absence of 1 mM betaine..........................................................................................................82 viii

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LIST OF FIGURES Figure page 1-1 Reaction summary for glucose transport....................................................................3 1-2 Reaction summary for the energy consuming reactions of glycolysis.......................3 1-3 Central carbon metabolism in Escherichia coli.........................................................4 1-4 Reaction Summary for glycolysis..............................................................................5 1-5 Reaction summary of TCA cycle reactions................................................................6 1-6 Electron flow from NADH in Escherichia coli ........................................................7 1-7 Summary of anerobic and aerobic metabolism in E. coli.........................................11 1-8 Futile cycles constructed in E. coli...........................................................................13 2-1 Reaction summary for the conversion of hexose to acetate.....................................17 2-2 Diagram summarizing genetic modifications used to redirect glucose metabolism to acetate...............................................................................................23 2-3 Diagram summarizing plasmid construction............................................................28 2-4 Effect of selected mutations on E. coli metabolism.................................................33 2-5 Effect of selected mutations on the production of acetate........................................38 2-6 Comparison of acetate production between TC36 (poxB + ) and TC42 (poxB )........40 2-7 Effect of restoring PflB activity on growth and acetate production.........................41 2-8 Metabolite production in TC36 and TC57...............................................................43 2-9 Fermentation of 6% glucose to acetate by TC36 in mineral salts medium..............45 3-1 Growth comparison of TC36 with xylose or glucose as the sole carbon source ......................................................................................................................52 ix

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4-1 Genetic modifications used to redirect glucose metabolism for pyruvate production.................................................................................................................63 4-2 Effect of oxygen level on pyruvate production by TC36.........................................64 4-3 Batch fermentation of glucose by mutant strains of E. coli.....................................68 5-1 Effect of 1 mM Betaine on growth and acetate production for TC36......................83 5-2 Effect of 1 mM Betaine on growth and pyruvate production for TC44...................78 5-3 Effect of 1 mM betaine on the minimum inhibitory concentration of acetate and pyruvate for E. coli W3110...................................................................86 6-1 Fermentation profiles for selected strains during the construction of E. coli TC36.............................................................................................................91 x

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENGINEERING THE METABOLISM OF Escherichia coli FOR THE SYNTHESIS OF OXIDIZED PRODUCTS: ACETATE AND PYRUVATE PRODUCTION By Thomas B. Causey August 2004 Chair: Lonnie O. Ingram Major Department: Microbiology and Cell Science To demonstrate a novel approach that combines attributes of fermentative metabolism and oxidative metabolism into a single biocatalyst for production of oxidized chemicals, Escherichia coli was genetically engineered for acetate and pyruvate production. Acetate producing E. coli TC36 was constructed by sequentially assembling chromosomal deletions to inactivate oxidative phosphorylation (atpFH), disrupt the tricarboxylic acid cycle (sucA), and eliminate fermentation pathways (focA-pflB frdBC ldhA adhE ). StrainTC36 produced 572 mM acetate from 6% glucose (333 mM) and a maximum of 878 mM acetate with glucose excess. Although strain TC36 produced a maximum of 4 ATP (net) per molecule of glucose as compared to 33 ATP (net) for wild type strains, it grew in glucose minimal medium ( = 0.49, 82% of W3110) without supplements. Glycolytic flux in strain TC36 was estimated to be 0.3 mol min -1 (mg protein) -1 twice that of the wild type parent (W3110). Pyruvate producing strain TC44 (focA-pflB frdBC ldhA atpFH adhE sucA poxB::FRT xi

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ackA) was constructed from strain TC36 by inserting additional chromosomal deletions in the acetate kinase (ackA) and pyruvate oxidase (poxB) genes. In mineral salts medium containing glucose as the sole carbon source, strain TC44 converted glucose to pyruvate with a yield of 0.75 g pyruvate per g glucose (77.9% of theoretical yield; 1.2 g pyruvate L -1 h -1 ). The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl~CoA. The basic principles demonstrated using strains TC36 and TC44 can be applied for the production of a variety of other chemicals, irrespective of their relative oxidation states. xii

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CHAPTER 1 INTRODUCTION Renewable Fuels and Chemicals The current trend toward increasing demand for foreign petroleum in the United States is forecast to continue, reaching 70% imported petroleum in 2025 as opposed to 37% in 1980 (USDOE/EIA 2004). Subordination to foreign countries, some of which demonstrate mercenary ideals toward US interests, for such a critical component of the day-to-day functioning of society is a national security problem. The use of microbial biocatalysts for the conversion of renewable plant biomass to fuels and chemicals can significantly reduce the United States' dependence on imported petroleum. The commercial production of commodity chemicals by microbial fermentation has been limited to reduced and redox neutral compounds such as ethanol and lactic acid, respectively, under anaerobic conditions where substrate loss to cell mass or CO 2 are minimal and product yields are high. In aerobic processes, Escherichia coli is widely used as a biocatalyst for production of higher value products such as recombinant proteins (Akesson et al. 2001; Aristidou et al. 1995; Contiero et al. 2000; Luli and Strohl 1990) and amino acids (Chotani et al. 2000; Eggeling et al. 2001). Traditional chemical companies have begun to develop infrastructure for the production of compounds using biocatalytic processes (reviewed in Schmid et al. 2001; Stringer 1996; Strohl, 2001). Considerable progress has been reported toward new processes for commodity chemicals (Danner and Braun 1999) such as ethanol (Ingram et al. 1999; Underwood et al. 2002), lactic acid (Chang et al. 1999; Dien et al. 2001; Zhou et al. 2003), 1,3-propanediol 1

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2 (Nakamura et al. US Patent 6,013,494; Tong et al. 1991), and adipic acid (Niu et al. 2002). In addition, advances have been made in the genetic engineering of microbes for high-value specialty compounds such as polyketides (Beck et al. 2003; Dayem et al. 2002) and carotenoids (Lee and Shmidt-Dannert 2002; Wang et al. 2000). Why Use Escherichia coli as a Biocatalyst? Escherichia coli is a facultative anaerobe, possessing a robust metabolism, capable of growing on mineral salts media with only the addition of a carbon source. The capability E. coli to respire, exploiting a variety inorganic electron acceptors, and ferment, using internal metabolites as an electron sink, provide the metabolic engineer with flexibility to explore the production of many compounds regardless of redox state. Escherichia coli is capable of catabolizing hexose and pentose sugars which constitute the majority of carbohydrates in plant biomass commonly used as feedstock for fermentation processes. The efficient conversion of substrate into product is essential if a process is to be economically viable. The physiology and biochemistry of E. coli has been well documented, affording numerous tools for genetic manipulation. Many years of laboratory and industrial experience has demonstrated that E. coli is safe and reliable. Therefore, the broad substrate range and robust metabolism combined with years of technical experience makes E. coli an attractive biocatalyst for the production of fuels and chemicals. Overview of Central Carbon Metabolism in Escherichia coli Aerobic Versus Anaerobic Metabolism Escherichia coli is a heterotrophic, facultative anaerobe. Hexose and pentose sugars are transported into the cell using either phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), ATP-binding cassette (ABC) transporter (the energy

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3 for transport provided by ATP) or cation (H + and Na + ) symporters (Linton and Higgins. 1998; Neidhardt et al. 1996). The PTS system transports sugars, such as glucose, using phosphoenolpyruvate (PEP) as the phosphoryl donor and as the energy source needed for translocation (Fig. 1-1). PEPinGlucoseout+PyruvateinGlucose-6-Pin+PTS PEPinGlucoseout+PEPinGlucoseout+PyruvateinGlucose-6-Pin+PyruvateinGlucose-6-Pin+PTS PTS Figure 1-1. Reaction summary for glucose transport Glucose-6-P can then be used as a carbon and energy source (glucose is also a source of oxygen for biosynthesis). Glucose catabolism can be directed through various pathways dependent on the biosynthetic needs of the cell. The research described in this document is primarily related to central carbon metabolism, therefore only these pathways are discussed in detail. The first stage in glucose-6-P metabolism is the transformation of glucose-6-P to fructose-6-P followed by phosphorylation. (Fig. 1-3A). The additional phosphate is supplied by ATP, therefore the first stage of glycolysis results in an investment of two ATP equivalents (one from PEP and the other from ATP). The net reaction for stage 1 is summarized in Figure 1-2. PEPinGlucoseout+PyruvateinGlucose-6-Pin+PTS Glucose-6-P+ATPADPFructose-1,6-diP+ PEPinGlucoseout+PyruvateinGlucose-6-Pin+PTS PEPinGlucoseout+PyruvateinGlucose-6-Pin+PTS Glucose-6-P+ATPADPFructose-1,6-diP+ Glucose-6-P+ATPGlucose-6-P+ATPADPFructose-1,6-diP+ADPFructose-1,6-diP+ Figure 1-2. Reaction summary for the energy consuming reactions of glycolysis

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4 As As H2ONADH Electron TransportSystem PiH+O2 + einout F0atpIBEFHAGDC +ADP ATPF1 H+H+BNAD+ GlucoseNADHptaTriose-PPEP~P~P pykA ~P ackA~P poxBCO2CO2 + NAD+ HCOOHaceEFlpdApflB NADHNAD+LactateldhA NAD+NADHAcetyl-CoAEthanoladhE 2 NAD+2 NADHA NADPHCytb1(red)Cytb1(ox) FADH2frdABCDFAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc PyruvatepykF Glutamate2-KetoglutarateaspCpartate NH3aspAAcetate H2ONADH Electron TransportSystem PiH+O2 + einout F0atpIBEFHAGDC +ADP ATPF1 H+H+BNAD+ GlucoseNADHptaTriose-PPEP~P~P pykA ~P ackA~P poxBCO2CO2 + NAD+ HCOOHaceEFlpdApflB NADHNAD+LactateldhA NAD+NADHAcetyl-CoAEthanoladhE 2 NAD+2 NADHA NADPHCytb1(red)Cytb1(ox) FADH2frdABCDFAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc PyruvatepykF Glutamate2-KetoglutarateaspCpartate NH3aspAAcetate Figure 1-3. Central carbon metabolism in Escherichia coli. Dashed arrows represent fermentative pathways. A) Central carbon metabolism B) Oxidative phosphorylation. Genes: aceA, isocitrate lyase; aceB, malate synthase B; aceEF, E1p and lipoate acetyltransferase/dihydrolipoamide acetyltransferase component of the PDHc; ackA, acetate kinase; acnB, aconitase; adhE, alcohol/aldehyde dehydrogenase pyruvate formate-lyase deactivase; atpIBEFHAGDC, (F 1 F 0 )H + -ATP synthase; frdABCD, fumarate reductase; glcB, malate synthase G; gltA, citrate synthase; icdA, isocitrate dehydrogenase; ldhA, lactate dehydrogenase; lpdA, dihydrolipoate dehydrogenase/dihydrolipoamide dehydrogenase component of the PDHc and 2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase; poxB, pyruvate oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II; pykF, pyruvate kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-oxoglutarate dehydrogenase; sucDC, succinyl-CoA synthetase.

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5 Glyceraldehyde-3-P oxidation to pyruvate is the energy yielding stage (stage 2) of glycolysis. Glyceraldehyde-3-P dehydrogenase incorporates an inorganic phosphate with the concomitant reduction of NAD + and the formation of 1,3-diphosphoglycerate. The acyl phosphate is then used to phosphorylate ADP to ATP. The incorporation of the inorganic phosphate results in the net gain in ATP from glycolysis. Another ATP is formed from PEP during the production of pyruvate. A reducing equivalent is produced during the oxidation of glyceraldehyde-3-P (Fig. 1-3A and1-4). Glucose+++2ADP 2 Pi2 NAD+++2 ATP2 Pyruvate2 NADH2 H++ Glucose+++2ADP 2 Pi2 NAD+++2 ATP2 Pyruvate2 NADH2 H++ Figure 1-4. Reaction Summary for glycolysis Glycolysis is not only an energy generating pathway but also provides precursors for the biosynthesis of several amino acids, lipids, polysaccharide, and components of peptidoglycan. The fate of pyruvate produced during glycolysis is dependent upon oxygen available in the environment. When E. coli is growing in an aerobic environment, pyruvate is oxidatively decarboxylated to acetyl-CoA by the pyruvate dehydrogenase complex (PDHc) with the concomitant production of NADH. Acetyl-CoA can then enter the tricarboxylic acid cycle (TCA cycle), serve as the substrate for acetate production (phosphotransacetylase and acetate kinase reactions) yielding an ATP by substrate level phosphorylation, or serve as the basic building block for fatty acid biosynthesis. Entry into the TCA cycle is the major route for acetyl-CoA during aerobic metabolism which, like glycolysis, serves energetic and biosynthetic functions. The cyclic functioning of the TCA cycle results in the production of four reducing equivalents, two carbon dioxides,

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6 and one GTP (Fig 1-3A and 1-5). Therefore, four ATP equivalents are produced by substrate-level phosphorylation from catabolism of one glucose molecule by glycolysis and the TCA cycle. Acetyl-CoA++++++2 H2OGDPPiFADNADP+2 NAD+++++++2 CO2GTPFADH2NADPH2 NADH3 H+CoASH Acetyl-CoA++++++2 H2OGDPPiFADNADP+2 NAD+++++++2 CO2GTPFADH2NADPH2 NADH3 H+CoASH Figure 1-5. Reaction summary of TCA cycle reactions The reducing equivalents produced during aerobic growth are re-oxidized via the electron transport chain with molecular oxygen as the final electron acceptor (Fig.1-3B and 1-6). Electrons from NADH enter the electron transport chain by reduction of ubiquinone (UQ) to ubiquinol (UQH 2 ). The NADH dehydrogenases, NDH I and NDH II, catalyze the reduction of UQ to UQH 2 (Campbell and Young 1983; Ingledew and Poole 1984; Jaworowski et al. 1981; Meinhardt et al. 1989; Weidner et al. 1992; Young et al. 1981). There are two terminal cytochrome oxidases, cytochrome bo oxidase and cytochrome bd oxidase, in E. coli that transfer the electrons from UQH 2 to oxygen (Ingledew and Poole 1984; Koland et al. 1984; Miller and Gennis 1985; Neidhardt et al.1990; Puustinen et al. 1991; Rice and Hempfing 1978). Cytochrome o oxidase is predominant under aerobic conditions and has a low affinity, with a K m for oxygen of 0.20 M (Rice and Hempfing 1978). Cytochrome bo oxidase functions as a scalar transporter of protons as well as a proton pump (Neidhardt et al. 1996; Puustinen et al. 1989; Puustinen et al. 1991). Therefore, the H + /e for cytochrome bo oxidase is 2. The high affinity cytochrome bd complex, which is predominant under micro-aerobic conditions (Tseng et al. 1996), has a K m for oxygen of 0.024 M (Rice and Hempfling 1978). The respiratory efficiency of E. coli depends on the stoichiometry of the NADH dehydrogenases and cytochrome oxidases (Fig. 1-6). Cytochrome bd oxidase can only

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7 NDH I (nuo)? 2 H+/eNDH II (ndh)0 H+/eCytochrome bo oxidase (cyoABCDE)2 H+/e-O22H2O Cytochrome bd oxidase (cydAB)1 H+/eQuinonePool NDH I (nuo)? 2 H+/eNDH II (ndh)0 H+/eCytochrome bo oxidase (cyoABCDE)2 H+/e-O22H2O Cytochrome bd oxidase (cydAB)1 H+/eQuinonePool QuinonePool Figure 1-6. Electron flow from NADH in Escherichia coli (figure adapted from Neidhardt, F. C. et al. 1996). NADH is oxidized by the NADH deydrogenases, nuo and ndh. Quinones serve as intermediate electron carriers which are oxidized by the terminal cytochrome oxidases, cyoABCDE and cydAB. transport protons by scalar chemistry resulting in a H + /e of 1 (Neidhardt et al. 1990; Puustinen et al. 1991). Figure 1-6 is a simplified diagram of the process and summarizes the H + /e for each redox reaction. The NADH dehydrogenases, like the cytochrome oxidases, differ in the number of protons that are extruded (H + /e ) on reduction of the substrate (UQ or O 2 respectively). NDH I has a calculated H + /e of 1.5-2.0 (Bogachev et al. 1996; Calhoun et al. 1993) and NDH II does not contribute to the membrane potential gradient (Young et al. 1981). The number of protons extruded by NDH I is yet to be established. Difficulty in obtaining evidence for the actual H + /e arises from the many respiratory chain linked dehydrogenases present in E. coli (Ingledew and Poole 1984). Oxygen pulse experiments performed under anaerobic conditions conducted by Calhoun et al. (1993) and Bogachev et al. (1996) have indicated a H + /e = 2 and H + /e = 1.5, respectively. Since, a H + /e = 1.5 is unlikely, two protons transported per electron is a reasonable assumption. Another line

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8 of evidence which suggests the H + /e = 2 is that NDH I is the homolog of the mitochondrial complex I (Meinhardt et al. 1989; Weidner et al. 1992) which has been demonstrated to extrude two protons per electron (Pozzan et al. 1979; Wikstrm and Penttil 1982). The stoichiometry of the NADH dehydrogenases and cytochrome oxidases, which depend on the growth conditions (Spiro and Guest 1991), result in 1 H + /e to as m any as 4 H + /e delivered to the periplasm under aerobic conditions (Fig. 1-6). The greater the H + /e the higher the respiratory efficiency i.e., increasing the membrane potential difference results in more ATP produced per mole of oxygen consumed (P/O). If all of the electrons originating from glucose catabolism are transported through NDH I and cytochrome bo oxidase then a maximum of 33 ATP can be produced. However, growth yield experiments conducted by Stouthamer (1979) have demonstrated that a maximum of 26 ATP are produced per molecule of glucose when E. coli is grown under optimal conditions. This lower value is the result of 1) differential electron flux through the NADH dehydrogenases and terminal oxidases, 2) the maintenance energy requirement (Pirt 1982; Pirt 1987) and 3) proton leakage reducing the energized state of the cytoplasmic membrane (Stouthamer and Bettenhaussen 1977). The large amount of ATP produced and the high respiratory efficiency of E. coli, grown under aerobic conditions and limiting glucose, result in the production of carbon dioxide and cell mass in a 50:50 ratio (Calhoun et al. 1993; Neidhardt et al. 1990). When E. coli is grown anaerobically on glucose with out an exogenous terminal electron acceptor, NAD is regenerated by reduction of internal organic compounds (Fig.1-3A). The major end products of glucose fermentation are acetate, ethanol, lactate,

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9 and formate in a 1:1:2:2 ratio (Neidhardt et al. 1996). Ten percent of the glucose consumed is converted to succinate and cell mass (Neidhardt et al. 1996). In contrast to the large amount of ATP produced during aerobic metabolism, only two net ATP are produced (by substrate-level phosphorylation) during fermentation of glucose to pyruvic acid (Fig.1-3A). An additional ATP is produced with the formation of acetate through acetate kinase. In addition to the low ATP yield and low cell mass, fermentative metabolism also differs from aerobic metabolism in that the TCA cycle functions in a non-cyclic manner. The transcription of 2-oxoglutarate dehydrogenase (sucAB gene product) is repressed under anaerobic conditions (Amarasingham and Davis 1965; Cunningham and Guest. 1998; Park et al. 1997) which seperates the TCA cycle into reductive and oxidative branches. The anaerobic TCA cycle functions only in a biosynthetic capacity. During fermentative growth, many of the genes involved in aerobic metabolism are repressed and expression of genes involved in anaerobic metabolism are activated. Two global regulatory systems play a major role in the transition to fermentative metabolism. The ArcAB (aerobic respiratory control) two-component signal transduction pathway (ArcA is the response-regulator and ArcB is the sensor kinase) and the redox-sensitive protein, FNR (fumarate and nitrate reduction) function to regulate gene expression in response to oxygen availability (Iuchi and Weiner 1996; Kiley and Beinert 1999). The ArcAB system functions mainly to repress expression of aerobic genes under anaerobic conditions (Spiro and Guest 1991; Iuchi and Weiner 1996). Under micro-aerobic conditions the expression of the cyoABCDE (cytochrome bd, the high affinity terminal oxidase) operon is enhanced by ArcAB (Iuchi and Weiner. 1996). The

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10 expression of both oxidases is repressed by FNR under anaerobic conditions (Kiley and Beinert 1999; Spiro and Guest 1991). FNR is known to activate 22 operons and repress eight operons under anaerobic conditions (Spiro and Guest 1991). When E. coli is growing fermentatively, FNR activates the transcription of pflB, pyruvate formate-lyase. The products of pyruvate formate-lyase are acetyl-CoA and formate (Fig 1-3A). There are no reducing equivalents produced during the decarboxylation of pyruvate under fermentative conditions. The lipoamide dehydrogenase (lpdA) protein, shared by PDHc and 2-oxoglutarate dehydrogenase, is repressed by ArcAB under anaerobic conditions (Cunningham and Guest 1998). Transcription of sucAB, which codes for the E1 and E2 subunits of 2-oxoglutarate dehydrogenase, is repressed by FNR from the sdhC promoter (Park et al. 1997). The repression of sucAB under anaerobic conditions results in the non-cyclic functioning of the TCA cycle mentioned above. In summary, high growth rate and conversion of glucose into high levels of cell mass and large amounts of carbon dioxide, are characteristic of aerobic metabolism in E. coli. During anaerobic metabolism, approximately 95% of the glucose is converted to organic acids and ethanol, while less than 5% is converted to cell mass. Figure 1-7 is a simplified diagram of carbon flow in E. coli during aerobic and anaerobic metabolism. Typically, the growth rate is also decreased during anaerobic conditions. ATP and Glycolytic Flux Studies conducted by Patnaik et al. (1992) and Chao and Liao (1994) have demonstrated that creation of futile cycles in E. coli, which consume ATP, cause a two-fold increase in glycolytic flux. Over-expression of phosphoenolpyruvate synthase (pps) (Fig. 1-8A) by 30-fold resulted in a two-fold increase in glycolytic flux (Patnaik et al. 1992). Low induction levels of Pps resulted in increased respiration to compensate for

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11 Up to 95 % of carbon converted to products (low CO2production)2 ATP expectedCell Mass5% of CarbonA Anaerobic (-oxygen)Glucose, C6H12O6 50% of carbon converted to CO233 ATP (max ) expectedB Aerobic (+ oxygen)Cell Mass 50% of CarbonGlucose, C6H12O6 Up to 95 % of carbon converted to products (low CO2production)2 ATP expectedCell Mass5% of CarbonA Anaerobic (-oxygen)Glucose, C6H12O6 50% of carbon converted to CO233 ATP (max.) expectedB Aerobic (+ oxygen)Cell Mass 50% of CarbonGlucose, C6H12O6 Figure 1-7. Summary of anerobic and aerobic metabolism in E. coli. A) Anaerobic metabolism. B) Aerobic metabolism the loss of ATP. High levels of Pps induction resulted in both increased respiration and increased glycolytic flux. The authors explained the observation at high Pps induction levels by 1) saturation of the respiratory chain and 2) increased glucose uptake due to a high phosphoenolpyruvate/pyruvate ratio resulting in an increase in the phosphorylated forms of enzyme I, Hpr, and enzyme III glc of the PTS (Patnaik et al. 1992). Although the increase in glucose transport may contribute to the increased glycolytic flux in the mutant, it is unlikely the only determinant. Phosphoenolpyruvate is an allosteric inhibitor of phosphofructokinase which may become a bottleneck at high phosphoenolpyruvate levels. The Pyk-Pps futile cycle resulted in a net loss of ATP concomitant with elevated respiration (Patnaik et al. 1992), indicative of a reduction in respiratory efficiency. Therefore, the increased glycolytic flux may be due to decreased ATP production coupled with increased availability of ADP, an allosteric activator of phosphofructokinase (Deville-Bonne et al. 1991), under aerobic conditions.

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12 Introduction of a futile cycle between phosphoenolpyruvate and oxaloacetate by over-expression of phosphoenolpyruvate carboxykinase (pck) and phosphoenolpyruvate carboxylase (ppc) also resulted in an increase in glycolytic flux (Fig. 1-8B) (Chao and Liao 1994). Increasing the magnitude of the futile cycle for ATP consumption was followed by (in order) an increase in respiration rate, increase in glucose consumption and excretion of acetate and pyruvate (Chao and Liao 1994). These results suggest that E. coli compensates for decrease in ATP by first, increasing respiration to form ATP at a higher rate, secondly, by increasing glycolytic flux and the formation of ATP by substrate-level phosphorylation. Another interesting observation was that inactivation of 2-oxoglutarate dehydrogenase in addition to the futile cycle resulted in even greater increase in glycolytic flux than the futile cycle alone (Chao and Liao 1994). This is a result of the decreased amount of electrons available for electron transport and oxidative phosphorylation. The authors comment that, "cells with an incomplete TCA cycle cannot effectively compensate for the energy stress caused by the induced futile cycle" (Chao and Liao 1994).Our observations with the atpFH mutant are contrary to those of Chao and Liao and will be discussed in Chapter 2. Deletion of the (F 1 F 0 )H + -ATP synthase has also been found to increase glycolytic flux as well as acetate production (Jensen and Michelsen 1992). A two-fold increase in glycolytic flux was observed in an E. coli strain carrying a deletion in the atpIBEFHAGDC operon, consistent with the experiments using artificially induced futile cycles. By inactivating the (F 1 F 0 )H + -ATP synthase E. coli can no longer derive ATP from the proton motive force generated during oxidative respiration. The growth rate for the (F 1 F 0 )H + -ATP synthase mutants was decreased to ~74% of the wild type level and the

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13 CitrateATPGlucosePEPPyrOAAAcetyl-CoAppcCO2ADP, CO2pck Pi Pyr ATPppsADP + Pi pykA, pykFGlucosePEPPyrOAAAcetyl-CoACitrate Pyr AMP + PPiATP2Pi CO2ppcPi A Pyk-Pps futile cycleB Pck-Ppc futile cycle CitrateATPGlucosePEPPyrOAAAcetyl-CoAppcCO2ADP, CO2pck Pi Pyr ATPppsADP + Pi pykA, pykFGlucosePEPPyrOAAAcetyl-CoACitrate Pyr AMP + PPiATP2Pi CO2ppcPi A Pyk-Pps futile cycleB Pck-Ppc futile cycle Figure 1-8. Futile cycles constructed in E. coli. A) The pps gene was cloned into a plasmid behind an IPTG-inducible bacteriophage T7 promoter modified to overlap two lac operators. Over-expression of pps during growth on glucose results in net loss of ATP due to formation of a futile cycle involving Pps and Pyk. Abbreviations: ppc, phosphoenolpyruvate carboxylase; pps, phosphoenolpyruvate synthase; pykA and pykF, major and minor pyruvate kinases, respectively (adapted from Patnaik, R. et al. 1992). B) The structural genes of ppc and pck onto a plasmid behind the IPTG inducible tac promoter. Simultaneous expression of ppc and pck result in a net loss of ATP forming a futile cycle. Abbreviations: pck, phosphoenolpyruvate carboxykinase; ppc, phosphoenolpyruvate carboxylase (Chao, Y., and J. C. Liao. 1994). ATP/ADP ratio was decreased from 19 in the wild type to 7 in the mutant strain (Jensen and Michelsen 1992). Deletion of the atp operon also resulted in increased respiration proceeded by accumulation of acetate. The following observations comparing the growth characteristics between wild type E. coli and the atpIBEFHAGDC mutant: 1. increased respiration 2. increased glycolytic flux 3. accumulation of acetate

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14 led the investigators to conclude that E. coli will oxidize NADH by respiration even if it is not coupled to ATP synthesis leading to enhanced substrate level phosphorylation (Jensen and Michelsen 1992). The approaches described above demonstrate that limiting ATP by enhancing futile cycles or suppression of the oxidative phosphorylation results in increased glycolytic flux. Recently, Koebmann et al. (2002) describe a genetic approach that induces ATP hydrolysis to elucidate the nature of control on glycolytic flux. The genes atpAGD, encoding the F 1 subunit of the (F 1 F 0 )H + -ATP synthase, were cloned behind synthetic constituative promoters of different strengths. These investigators discovered that control of glycolytic flux varies with ATP/ADP ratio (Koebmann et al. 2002). During growth of wild type E. coli on glucose minimal medium the anabolic (ATP consuming) reactions exert a majority of the control on glycolytic flux. When the ATP/ADP increases flux control shifts to the catabolic (ATP producing) reactions. These findings are consistent with mathematical models that indicate greater than 75% of glycolytic flux is controlled by ATP/ADP in E. coli (Hofmeyer and Cornish-Bowden. 2000). Combine Beneficial Effects of Aerobic and Anaerobic Metabolism. It is possible to combine the beneficial effects of aerobic and anaerobic metabolism into a single biocatalyst for the production of oxidized and redox neutral compounds. This can be accomplished by combining the high rate of glycolysis and low cell yield of fermentative metabolism with the high growth rate and availability of an external electron acceptor of aerobic metabolism. The advantages for production of oxidized compounds are expected to be increased product yield and rate of product formation combined with decreased production of carbon dioxide and cell mass. The major advantage for production of reduced compounds would be the increase in process stability due to the

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15 elimination of the energy advantage of NADH oxidation by the electron transport chain. To demonstrate this concept, the metabolism of Escherichia coli was engineered for the production of acetate and pyruvate.

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CHAPTER 2 ENGINEERING THE METABOLISM OF Escherichia coli W3110 FOR THE CONVERSION OF SUGAR TO ACETATE Introduction In all aerobic and anaerobic processes, acetate production by the native E. coli pathway (phosphotransacetylase and acetate kinase) has been generally regarded as an undesirable consequence of excessive glycolytic flux (Akesson et al. 2001; Aristidou et al. 1995; Contiero et al. 2000; Farmer and Liao 1997; Underwood et al. 2002; Zhou et al. 2003). Although acetate is equivalent to glucose in redox state, pathways in E. coli that produce acetate also produce CO 2 resulting in a combined redox state of carbon more oxidized than in glucose with the production of NADH. To facilitate expansion into more oxidized commodity chemicals, such as acetate, we developed a novel genetic approach that combines the attributes of fermentative metabolism and oxidative metabolism (rapid growth, external electron acceptor for NADH oxidation) into a single biocatalyst. The biological production of acetic acid is currently limited to food uses. Petrochemical routes have largely displaced biological processes as uses expanded to plastics, solvents, and road de-icers (Berraud 2000; Cheryan et al. 1997; Freer 2002). There are two major petrochemical routes for commercial acetic acid production; methanol carbonylation and liquid-phase oxidation of n-butane. Approximately 35% of commercial acetic acid is recovered from other processes, most significantly, from the production of cellulose acetates and polyvinyl alcohol (Kirschner 2003). Synthesis of acetate in the United States is estimated at 7.8 billion pounds per year, with a projected 16

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17 growth of 2% per year through 2008 (Kirschner 2003). In the current biological process, sugars are fermented to ethanol by Saccharomyces. Ethanol in the resulting beer is subsequently oxidized to acetic acid by Acetobacter sp. under aerobic conditions (Berraud 2000; Cheryan et al. 1997; Freer 2002). This process can summarized as follows: C6H12O6+2 H2O4 H2O 8 [H]2 O2+2 C2H4O22 CO28 [H]++ C6H12O6+2 H2OC6H12O6+2 H2O4 H2O 8 [H]2 O2+8 [H]2 O2+2 C2H4O22 CO28 [H]++2 C2H4O22 CO28 [H]++ Figure 2-1.Reaction summary for the conversion of hexose to acetate In our study, we have genetically modified E. coli W3110 to produce acetic acid as the primary product from glycolysis. Although ethanol was eliminated as an intermediate, the stoichiometry of the reaction remained as shown above. The resulting biocatalyst (TC36) contains multiple chromosomal alterations (Fig. 2-2A and B) that direct carbon flow to acetate and minimize carbon loss to cell mass, CO 2 and alternative products. This strain is devoid of plasmids, foreign genes, and antibiotic resistance markers and grows well in mineral salts medium without complex nutrients. Materials and Methods Microorganisms and Media Strains and plasmids used in our study are listed in Table 1. Working cultures of E. coli W3110 (ATCC 27325) derivatives were maintained on minimal medium containing mineral salts (per liter: 3.5 g KH 2 PO 4 ; 5.0 g K 2 HPO 4 ; 3.5 g (NH 4 ) 2 HPO 4 0.25 g MgSO 4 7 H 2 O 15 mg CaCl 2 2 H 2 O, 0.5 mg thiamine, and 1 mL of trace metal stock), glucose (2% in plates; 3% in broth), and 1.5% agar. The trace metal stock was prepared in 0.1 M HCl (per liter: 1.6 g FeCl 3 0.2 g CoCl 2 6 H 2 O, 0.1 g CuCl 2 0.2 g

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18 Table 2-1. Sources and characteristics of strains Strains Relevant Characteristics Reference W3110 K12 wild type ATCC 27325 TOP10F lacI q (episome) Invitrogen LY01 E. coli B, frd pfl::pdo ZM adhE ZM cat Yomano et al. 1998 LY74 W3110, poxB::FRT-tet-FRT Causey et al. 2004 LY117 W3110, ycaC::FRT-aap-FRT::ycaD This study SE2279 MG1655, pflB ldhA::Tn10 Causey et al. 2003 SZ33 W3110, ldhA::Tn10 Causey et al. 2003 SZ40 W3110, (focA-pflB)::FRT frdBC Zhou et al. 2003 SZ46 W3110, (focA-pflB)::FRT frdBC ldhA::Tn10 Causey et al. 2003 SZ47 W3110, (focA-pflB)::FRT frdBC ldhA Causey et al. 2003 TC20 W3110, adhE::FRT-tet-FRT Underwood et al. 2002 TC21 W3110, atpFH::FRT-tet-FRT Causey et al. 2003 TC23 W3110, (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT-tet-FRT Causey et al. 2003 TC24 W3110, (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT Causey et al. 2003 TC25 W3110, sucA::FRT-tet-FRT Causey et al. 2003 TC30 W3110, (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT-tet-FRT Causey et al. 2003 TC31 W3110, (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT Causey et al. 2003 TC32 W3110, (Succ ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT-tet-FRT Causey et al. 2003 TC35 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT-tet-FRT Causey et al. 2003 TC36 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT Causey et al. 2003 TC41 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT poxB::FRT-tet-FRT Causey et al. 2004 TC42 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT poxB::FRT Causey et al. 2004 TC45 W3110, atpA::FRT-tet-FRT This study TC49 W3110, (Succ + ), ycaC::FRT-aap-FRT::ycaD focA + pflB + frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT This study TC51 W3110, (Succ + ), ycaC::FRT::ycaD focA + pflB + frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT This study TC55 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT atpA::FRT-tet-FRT This study TC56 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT atpA::FRT This study

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19 Table 2-2. Sources and characteristics of plasmids Plasmids Relevant Characteristics Reference pCR2.1-TOPO bla kan, TOPO TM TA cloning vector Invitrogen pFT-A bla flp low-copy vector containing recombinase and temperature-conditional pSC101 replicon Posfai et al. 1997 pKD46 bla exo low-copy vector containing red recombinase and temperature-conditional pSC101 replicon Datsenko and Wanner 2000 pLOI2065 bla, SmaI fragment with FRT flanked tet gene Underwood et al. 2002 pLOI2075 bla kan poxB Causey et al. 2004 pLOI2078 bla poxB Causey et al. 2004 pLOI2080 bla poxB::FRT-tet-FRT Causey et al. 2004 pLOI2403 bla Martinez-Morales et al. 1999 pLOI2745 kan temperature conditional replicon This study pLOI2800 bla kan sucA Causey et al. 2003 pLOI2801 bla kan sucA::FRT-tet-FRT Causey et al. 2003 pLOI2802 bla kan adhE Underwood et al. 2002 pLOI2803 bla kan adhE::FRT-tet-FRT Causey et al. 2003 pLOI2805 bla kan atpEFH Causey et al. 2003 pLOI2820 bla kan atpA This study pLOI2821 bla kan atpA::FRT-tet-FRT This study pLOI3421 EcoRI fragment containing FRT flanked aap gene This study pLOI3430 bla kan ycaC ycaD This study pLOI3439 kan ycaC ycaD temperature conditional replicon This study pLOI3447 bla kan ycaC FRT-aac-FRT ycaD used for repairing focA pflB deletion This study ZnCl 2 4 H 2 O, 0.2 g NaMoO 4 and 0.05 g H 3 BO 3 ). MOPS (0.1 M, pH 7.1) was added to both liquid and solid media (filter-sterilized) when needed for pH control, but was not included in medium used for 10 L fermentations. Minimal medium was also prepared using succinate (1 g L -1 ) as a sole source of carbon (nonfermentable substrate). Succinate (1 g L -1 ) was also added as a supplement to glucose-minimal medium when needed. During plasmid and strain construction, cultures were grown in Luria-Bertani (LB) broth or on LB agar plates (1.5% agar) (Miller 1992). Glucose (2%) was added to LB medium for all strains containing mutations in (F1F0)H+-ATP synthase. Antibiotics were included as appropriate (kanamycin, 50 mg L -1 ; ampicillin, 50 mg L -1 ; and tetracycline,

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20 12.5 or 6.25 mg L -1 ). Fusaric acid plates were used to select for loss of Tn10-encoded tetracycline resistance (Zhou et al. 2003). Genetic Methods Standard methods were used for plasmid construction, phage P1 transduction, electroporation, and polymerase chain reaction (PCR) (Miller 1992; Sambrook and Russell 2001). Chromosomal DNA from E. coli W3110 (and derivatives) served as a template to amplify genes using primers complementary to coding regions (ORFmers) purchased from the Sigma-Genosys, Inc. (The Woodlands, TX). PCR products were initially cloned into plasmid vector pCR2.1-TOPO. During plasmid constructions, restriction products were converted to blunt ends using either the Klenow fragment of DNA polymerase I (5' overhang) or T4 DNA polymerase (3' overhang) as needed. Integration of linear DNA was facilitated by using pKD46 (temperature conditional) containing an arabinose-inducible Red recombinase (Datsenko and Wanner 2000). Integrants were selected for tetracycline resistance (6.25 mg L -1 ) and screened for appropriate antibiotic resistance markers and phenotypic traits. At each step, mutations were verified by analyses of PCR products and fermentation products. FRT-flanked antibiotic resistance genes used for selection were deleted using a temperature-conditional plasmid (pFT-A) expressing FLP recombinase from a chlortetracycline-inducible promoter (Martinez-Morales et al. 1999; Posfai et al. 1997). Fermentation Acetate production was examined in glucose-minimal medium containing 167 mM glucose using a New Brunswick Bioflow 3000 fermentor (New Brunswick Scientific, Edison, NJ) with a 10 L working volume (37C, dual Rushton impellers, 450 rpm). Dissolved oxygen was maintained at 5% of air saturation (unless otherwise stated) by

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21 altering the proportion of N 2 and O 2 Broth was maintained at pH 7.0 by the automatic addition of 11.4 M KOH. During fed batch experiments, additional glucose was added from a sterile 60% stock. Three fed batch regimes were investigated: A. 3% glucose initially with the addition of 3% after 12 h (6% total); B. 6% glucose initially with the addition of 4% glucose after 16 h (10% total); C. 3% glucose initially with multiple additions to maintain glucose levels above 100 mM. Five liter fermentations (7 L vessel, 350 rpm using dual Rushton impellers) were used to examine the effects of deletion of atpFH and atpA or the effect(s) of restoring pyruvate formate-lyase activity. For these fermentations the dissolved oxygen was allowed to fall from 100% to 5% of air saturation at which time O 2 was mixed with air to maintain a dissolved oxygen concentration higher than 5%. Seed cultures were prepared by inoculating colonies from a fresh plate (48 h) into 3 mL of glucose-minimal medium (13 x 100 mm tube) containing 0.1 M MOPS. After incubation for 14 h (120 rpm rotator), cultures were diluted 400-fold into 1 L baffled flasks containing 200 mL of mineral salts medium (37C, 280 rpm). When cells reached 1.5-2.2 OD 550nm sufficient culture volume was harvested (5000 x g, 25C) to provide an inoculum of 33 mg dry cell weight L -1 in the 10 L working volume. Broth samples were removed to measure organic acids, residual glucose, and cell mass. Volumetric and specific rates were determined from measured values for glucose and acetate using GraphPad Prism (GraphPad Software, San Diego, CA). A smooth curve was generated with 10 points per min (Lowess method) to fit measured results. The first derivative (acetate or glucose versus time) of each curve served as an estimate of

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22 volumetric rate. Specific rates (mmoles L -1 h -1 [mg dry cell weight] -1 ) were calculated by dividing volumetric rates by respective values for cell mass. ATPase Assay Cells were grown for enzyme assays as described above for seed cultures. Upon reaching 0.75-1.0 OD 550nm cultures were chilled on ice and harvested by centrifugation (8000 x g, 5 min at 4 o C). Cell pellets were washed 5 times with 0.1 M Tris-HCl (pH 7.55), resuspended in 1 mL of this buffer, and broken using a model W220F ultrasonic cell disruptor (Heat Systems Ultrasonics, Plainview, NY). Total ATPase activity in disrupted cell preparations was assayed at pH 7.55 essentially as described by Evans (Evans 1969). Inorganic phosphate was measured by the method of Rathbun and Betlach (Rathbun and Betlach 1969). Results represent an average of three cultures for each strain. Specific activity is expressed as mol P i released min -1 (mg protein) -1 Analyses Organic acids and glucose concentrations were determined using a Hewlett Packard HPLC (HP 1090 series II) equipped with a UV monitor (210 nm) and refractive index detector. Products were separated using a Bio-Rad HPX-87H column (10 l injection) with 4 mM H 2 SO 4 as the mobile phase (0.4 mL min -1 45C). Cell mass was estimated by measuring OD 550nm (1.0 OD 550nm is equivalent to 0.33 g dry cell weight L -1 ) using a Bausch & Lomb Spectronic 70 spectrophotometer with 10 x 75 mm culture tubes as cuvettes. Protein concentration was determined using the BCA Protein Assay Kit from Pierce (Rockford, IL).

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23 GlucoseNADH AcetateptaTriose-PPEP~P~P pykA ~P ackA~P poxBCO2CO2 + NAD+ HCOOHaceEFlpdApflB NADHNAD+LactateldhA NAD+NADHAcetyl-CoA EthanoladhE 2 NAD+2 NADHA NADPHCytb1(red)Cytb1(ox) FADH2FAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc PyruvatepykF frdABCD H2OH+ O2 + einout Electron TransportSystem F0atpIBEFHAGDC Pi+ADP ATPF1 H+H+B F1 C NADHNAD+ADP + PiATP GlucoseNADH AcetateptaTriose-PPEP~P~P pykA ~P ackA~P poxBCO2CO2 + NAD+ HCOOHaceEFlpdApflB NADHNAD+LactateldhA NAD+NADHAcetyl-CoA EthanoladhE 2 NAD+2 NADHA NADPHCytb1(red)Cytb1(ox) FADH2FAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc PyruvatepykF frdABCD H2OH+ O2 + einout Electron TransportSystem F0atpIBEFHAGDC Pi+ADP ATPF1 H+H+B F1 C NADHNAD+ADP + PiATP Figure 2-2. Diagram summarizing genetic modifications used to redirect glucose metabolism to acetate. Bold arrows mark principal metabolic routes in TC36. Dashed arrows represent fermentative pathways. Reactions that have been blocked are highlighted. A) Central carbon metabolism. Acetate is the principal product from sugar metabolism with the net production of four ATP equivalents per glucose molecule. B) Oxidative phosphorylation. The (F 1 F 0 )H + -ATP synthase is inactive in TC36, although the electron transport system remains functional as the primary route for NADH oxidation. C) F 1 -ATPase. TC36 lacks the essential subunits coupling the ATP synthase to the membrane and thus defective in ATP synthesis but retains a functional cytoplasmic F 1 -ATPase. Genes: aceA, isocitrate lyase; aceB, malate synthase B; aceEF, E1p and lipoate acetyltransferase/dihydrolipoamide acetyltransferase component of the PDHc; ackA, acetate kinase; acnB, aconitase; adhE, alcohol/aldehydrogenase pyruvate formate lyase deactivase; atpIBEFHAGDC, (F 1 F 0 )H + -ATP synthase; frdABCD, fumarate reductase; glcB, malate synthase G; gltA, citrate synthase; icdA, isocitrate dehydrogenase; ldhA, lactate dehydrogenase; lpdA, dihydrolipoate dehydrogenase/dihydrolipoamide dehydrogenase component of the PDHc and 2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase; poxB, pyruvate oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II; pykF, pyruvate kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-oxoglutarate dehydrogenase; sucDC, succinyl-CoA synthetase.

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24 Results Elimination of Fermentation Products (Strain SZ47) Inspection of native pathways in E. coli (Fig. 2-2A) indicated that the production of acetate and CO 2 as sole metabolic products from glucose would require an external electron acceptor such as oxygen. Due to low oxygen solubility, however, it is difficult to satisfy the oxygen demand from active E. coli metabolism. Typically a portion of substrate is converted into fermentation products (Tsai et al. 2002; Varma et al. 1993). This problem was eliminated by combining deletions in genes encoding lactate dehydrogenase, pyruvate formatelyase, and fumarate reductase as follows. The ldhA::Tn10 mutation in E. coli SE2279 was transduced into E. coli W3110 using phage P1 to produce strain SZ33. P1 phage grown on SZ33 was used to transfer this mutation into SZ40((focA-pflB)::FRT frdBC) to produce SZ46. Tetracycline-sensitive derivatives of SZ46 were selected using fusaric acid medium (Zhou et al. 2003). One clone was designated SZ47 ((focA-pflB)::FRT frdBC ldhA). The ldhA mutation in SZ47 was confirmed by the absence of lactate in fermentation broth, inability to grow anaerobically in glucose-minimal medium, and by PCR analysis using ldhA ORFmers (1.0 kbp for the wild type ldhA as compared to 1.1 kbp for SZ47). The slightly larger size of the amplified product from SZ47 is attributed to remnants of Tn10. Disruption of Oxidative Phosphorylation (Strains TC24 and TC57). Growth under oxidative conditions is characterized by conversion of up to 50% of substrate carbon to cell mass (Neidhardt et al. 1990). To reduce the potential drain of substrate into cell mass, a mutation was introduced into SZ47 that deleted portions of two subunits in (F 1 F 0 )H + -ATP synthase required for assembly to the plasma membrane (Sorgen et al. 1998), disrupting oxidative phosphorylation while preserving the hydrolytic

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25 activity of F 1 -ATPase in the cytoplasm. To construct this deletion, the atpEFH coding region of the atpIBEFHAGDC operon was amplified by PCR using primers (ORFmers) complementary to the 5'end of the atpE gene and the 3'end of the atpH The amplified fragment (1.3 kbp) was cloned into pCR2.1-TOPO and one clone was selected in which the atpEFH genes were oriented to permit expression from the lac promoter (pLOI2805; Fig. 2-3). The atpF gene and 117 nucleotides at the 5' end of atpH gene were removed from pLOI2805 by digestion with HpaI and BstEII (Klenow-treated). This region was replaced with a 1.7 kbp SmaI fragment from pLOI2065 containing the FRT-tet-FRT cassette to produce pLOI2807 (Fig.2-3). After digestion with ScaI, pLOI2807 served as a template for amplification of the atpE(FH)::FRT-tet-FRT region (2.4 kbp) using the 5' atpE and 3' atpH primers. Amplified DNA was precipitated, digested again with ScaI to disrupt any residual plasmid, and purified by phenol extraction. This DNA was introduced into E. coli W3110(pKD46) by electroporation while expressing Red recombinase. Plasmid pKD46 was eliminated by growth at 42 o C. Recombinants (double cross-over) were identified using antibiotic markers (tetracycline resistant; sensitive to ampicillin and kanamycin) and by the inability to grow on succinate-minimal plates in the absence of glucose (fermentable carbon source). Integration was further confirmed by PCR analysis using the 5' atpE primer and the 3' atpH primer (1.3 kbp fragment for W3110; 2.3 kbp fragment for mutants). One clone was selected and designated TC21(atp(FH)::FRT-tet-FRT). Phage P1 was used to transduce the atp(FH)::FRT-tet-FRT mutation in TC21 to SZ47 and produce TC23. The tet gene was removed from TC23 by the FLP recombinase (pFT-A). After elimination of pFT-A by growth at 42 o C, the atp(FH)::FRT mutation

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26 was further confirmed by PCR analysis using the 5' atpE primer and the 3' atpH primer (0.8 kbp for deletion and 1.3 kbp for SZ47). The resulting strain was designated TC24 ((focA-pflB)::FRT frdBC ldhA atp(FH)::FRT). TC57 was unable to grow in minimal medium without a fermentable carbon source (substrate level phosphorylation) but retained the ability to oxidize NADH by the electron transport system. In order to demonstrate the effectiveness of preserving the hydrolytic activity of the F 1 subunit, a deletion was inserted into the atpA gene which codes for the alpha subunit of the (F 1 F 0 )H + -ATP synthase. To construct this deletion, the atpA coding region of the atpIBEFHAGDC operon was amplified by PCR using primers (ORFmers) complementary to atpA The amplified fragment (1.5 kbp) was cloned into pCR2.1-TOPO and one clone was selected in which the atpA gene was oriented to permit expression from the lac promoter (pLOI2820). A 249 bp section was removed from the central region of atpA by digestion with SmaI (blunt). This region was replaced with a 1.7 kbp SmaI fragment from pLOI2065 containing the FRT-tet-FRT cassette to produce pLOI2821. After digestion with HindIII and PvuII, pLOI2821 served as a template for amplification of the atpA::FRT-tet-FRT region (2.9 kbp) using the atpA primers. Amplified DNA was gel-purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). This DNA was introduced into E. coli W3110(pKD46) by electroporation while expressing Red recombinase. Plasmid pKD46 was eliminated by growth at 42 o C. Recombinants (double cross-over) were identified using antibiotic markers (tetracycline resistant; sensitive to ampicillin and kanamycin) and by the inability to grow on succinate-minimal plates in the absence of glucose (fermentable carbon source). Integration was further confirmed by PCR analysis using atpA primers (1.5 kbp fragment

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27 for W3110; 2.9 kbp fragment for mutants). One clone was selected and designated TC45 (atpA::FRT-tet-FRT). Phage P1 was used to transduce the atpA::FRT-tet-FRT mutation in TC45 to TC36 and produce TC55. The tet gene was removed from TC45 by the FLP recombinase (pFT-A). After elimination of pFT-A by growth at 42 o C, the atpA::FRT mutation was further confirmed by PCR analysis using the atpA primers (1.2 kbp for deletion and 1.5 kbp for TC36). The resulting strain was designated TC57 ((focA-pflB)::FRT frdBC ldhA adhE::FRT sucA::FRT atpFH::FRT atpA::FRT). Inactivation of Alcohol Dehydrogenase E (Strain TC31) The native bifunctional alcohol/aldehyde dehydrogenase (adhE) in E. coli catalyzes the reduction of acetyl~CoA to ethanol, a reaction that directly competes with the conversion of acetyl~CoA to acetate by phosphotransacetylase and acetate kinase (Fig. 2-2A). To eliminate this activity, phage P1 was used to transduce the adhE::FRT-tet-FRT mutation in TC20 to TC24 and produce TC30. Chromosomal integration was confirmed by PCR analysis using adhE primers (2.7 kbp for TC24 and 3.2 kbp for the adhE::FRT-tet-FRT mutant). The tet gene was deleted from TC30 by FLP recombinase using pFT-A. After elimination of pFT-A by growth at 42 o C, a clone was selected and designated TC31((focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT). Interruption of the Tricarboxylic Acid Cycle (strain TC32). During oxidative growth, up to 50% of substrate carbon can be released as CO 2 (Neidhardt et al. 1990). This loss of carbon can be attributed in large measure to the high efficiency of the tricarboxylic acid cycle and the electron transport system (NADH oxidation). During fermentative metabolism, the production of CO 2 and NADH are

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6730 bps 5247 bps 4356 bps 7259 bps 6254 bps PvuIPvuIPvuIPvuIPvuIScaIScaIAccISnaBISmaISmaIHpaIBstEIIBstXIBstXIpLOI2800pLOI2805pLOI2065pLOI2807pLOI2801Col E1sucAblakanblablablablakankankantettettetCol E1Col E1Col E1Col E1atpEatpF`atpHatpE`sucAsucAFRTFRTFRTFRTFRTFRT SnaBI AccI(Klenow)SmaISmaIHpaIBstEII(Klenow)atpHScaI6730 bps 5247 bps 4356 bps 7259 bps 6254 bps PvuIPvuIPvuIPvuIPvuIScaIScaIAccISnaBISmaISmaIHpaIBstEIIBstXIBstXIpLOI2800pLOI2805pLOI2065pLOI2807pLOI2801Col E1sucAblakanblablablablakankankantettettetCol E1Col E1Col E1Col E1atpEatpF`atpHatpE`sucAsucAFRTFRTFRTFRTFRTFRT SnaBI AccI(Klenow)SmaISmaIHpaIBstEII(Klenow)atpHScaI 28 Figure 2-3. Diagram summarizing plasmid construction

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29 reduced primarily by repression of sucAB encoding 2-oxoglutarate dehydrogenase (Cunnungham and Guest 1998; Park and Gunsalus 1997), disrupting the cyclic function of the tricarboxylic acid cycle. We have genetically imposed a similar restriction in carbon flow during oxidative metabolism by deleting part of the sucA gene. The sucA coding region was amplified using ORFmers. The resulting 2.8 kbp PCR product was cloned into pCR2.1-TOPO to produce pLOI2800 (Fig. 2-3) in which the sucA coding region was oriented to permit expression from the lac promoter. A 1.1 kbp fragment was removed from the central region of sucA by digestion of pLOI2800 with SnaBI and AccI (Klenow-treated). This region was replaced with a 1.7 kbp SmaI fragment containing the FRT-tet-FRT cassette from pLOI2065 to produce pLOI2801 (Fig. 2-3). Plasmid pLOI2801 was digested with PvuI and ScaI and used as a template to amplify the 3.3 kbp region containing sucA::FRT-tet-FRT using sucA ORFmers. Amplified DNA was precipitated, digested with PvuI and ScaI to disrupt any residual circular plasmid, and extracted with phenol. Purified DNA was electroporated into E. coli W3110(pKD46) while expressing Red recombinase. Plasmid pKD46 was eliminated by growth at 42C. Disruption of sucA was confirmed by PCR analysis using sucA ORFmers (2.8 kbp fragment for wild type and 3.3 kbp for sucA::FRT-tet-FRT mutants) and designated TC25. Phage P1 was used to transduce the sucA::FRT-tet-FRT mutation from TC25 into TC31. Transfer of this mutation was verified by PCR analysis (2.8 kbp for wild type sucA and 3.3 kbp for sucA::FRT-tet-FRT mutants) and phenotype (Succ ). Inactivation of 2-oxoglutarate dehydrogenase (sucA) in this frdBC background resulted in an undesirable auxotrophic requirement for succinate (Succ ) during growth on glucose

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30 minimal medium. The resulting strain was designated TC32(Succ , (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT-tet-FRT). Elimination of Succinate Requirement (Strain TC36) Spontaneous mutants of TC32, that did not require succinate in the growth medium (Succ + ), were readily obtained after serial transfers in glucose-minimal broth containing decreasing amounts of succinate (4 mM to 0.4 mM) followed by selection on glucose minimal plates without succinate. Over 170 clones were recovered per mL of culture after enrichment (approximately 3% of viable cells). Ten clones were tested and all grew in glucose minimal broth without succinate and produced acetate as the dominant product. One was selected (TC35) for deletion of the tet gene using the FLP recombinase. This deletion was confirmed by analysis of PCR products using sucA primers (3.3 kbp for TC35 and 1.8 kbp after tet deletion). The resulting strain was designated TC36 (Succ + ; (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT). Although the genetic change that eliminated the succinate requirement was not identified, likely candidates include mutations which provide a low constitutive expression of aceAB (glyoxylate shunt) or citC (citrate lyase). Total ATPase activity was examined in disrupted cell extracts of TC36 and W3110 (wild type). The activity in TC36 (0.355 U [mg protein] -1 ) was equivalent to 71% of the unmodified parent (0.502 U [mg protein] -1 ), confirming that F 1 -ATPase was not inactivated by the atpFH::FRT mutation. This is similar to the levels of ATPase reported for an atpH mutant of E. coli which blocked membrane assembly and coupling to oxidative phosphorylation (Sorgen et al. 1998).

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31 Disruption of Pyruvate Oxidase (Strain TC42) A single derivative of pCR2.1-TOPO was selected in which the amplified poxB gene was oriented in the same direction as the lac promoter (pLOI2075). To eliminate extraneous BsaBI sites in the vector, the small EcoRI fragment containing poxB was ligated into the corresponding site of pLOI2403 to produce pLOI2078. The SmaI fragment from pLOI2065 containing a tet gene flanked by FRT sites was ligated into the unique BsaBI site in poxB to produce pLOI2080. Plasmid pLOI2080 served as a template for the amplification of poxB::FRT-tet-FRT (3.4 kbp) using poxB primers. Amplified DNA was integrated into E. coli W3110(pKD46) as previously described (Causey et al. 2003) to produce LY74. The poxB::FRT-tet-FRT mutation in LY74 was transduced into TC36 to produce TC41. The tet gene was removed using FLP recombinase and the resulting strain designated TC42 (focA-pflB::FRT frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT poxB::FRT). Restoration of Pyruvate Formate-lyase Activity (Strain TC51) Strain LY117 was constructed for restoring pyruvate formate-lyase activity by a colleague, Lorraine Yomano, in which an apramycin marker flanked by FRT sites was inserted downstream of the focA-pflB operon as follows. The ycaC and ycaD region (2.1 kbp), located 2.6 kbp downstream from pflA, was PCR amplified and cloned into TOPO-pCR2.1 using the 3' ycaC primer (ORFmer) and the 3' ycaD primer (ORFmer) to generate plasmid pLOI3430. In order to limit the background after electroporation the EcoRI fragment from pLOI3430 containing ycaC ycaD was ligated into the EcoRI site of pLOI2745 generating a new plasmid, pLOI3439, which contains a temperature conditional replicon. The EcoRI fragment, containing an apramycin resistance marker flanked by FRT sites (1.9kbp), was treated with the Klenow fragment of DNA

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32 polymerase I to generate blunt ends and ligated into the XmnI site of pLOI3439. The new plasmid contained a ycaC::FRT-aap-FRT::ycaD EcoRI fragment (3.5 kbp) and a temperature conditional replicon. Plasmid pLOI3447 was digested with EcoRI and the 3.5 kbp ycaC::FRT-aap-FRT::ycaD fragment was purified by gel extraction (Qiagen, Valencia, CA). The DNA was integrated into E. coli W3110(pKD46) as previously described (Causey et al. 2003) to produce LY117 (ycaC::FRT-aap-FRT::ycaD). The ycaC::FRT-aap-FRT::ycaD mutation in LY117 was transduced into TC36 to produce TC49. The aap gene was was removed using FLP recombinase and the resulting strain designated TC51 (focA + pflB + frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT poxB::FRT). The focA + pflB + genotype was verified by PCR and pyruvate formate-lyase activity was verified by observing formate in fermentation broths. Effects of Gene Disruptions on Growth and Glycolytic Flux Strain TC36 was genetically engineered for the production of acetate from carbohydrates such as glucose (Fig. 2-2A, B and C). Batch fermentations with pH control were used to compare the performance of this strain with W3110 (wild type) and two intermediate strains used in the construction, SZ47((focA-pflB) frdBC ldhA) and TC24((focA-pflB) frdBC ldhA atpFH). Based on preliminary experiments, 5% oxygen saturation and 3% glucose (37C) were selected as test conditions. Broth pH was maintained at 7.0 to minimize toxicity from undissociated acids. Disruption of oxidative phosphorylation and the cyclic function of the tricarboxylic acid cycle, elimination of the primary fermentation pathways, and the production of acetate as the primary endproduct from glycolysis had relatively little effect on the growth of E. coli. The maximum growth rates for strains W3110 (wild type) and SZ47

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33 (lacking the three native fermentation pathways) were similar although the cell yield for SZ47 was higher (Fig. 2-4A; Table 2 and Table 3). Inactivation of oxidative phosphorylation (atpFH) in SZ47 resulted in a modest reduction (12%) in growth rate 0 3 6 9 12 15 18 21 24 27 30 0 1 2 3 4 5 6Time (h)Cell Mass (g/liter) 0 3 6 9 12 15 18 21 24 27 30 0 25 50 75 100 125 150 175Time (h)Glucose (mM)AB 0 3 6 9 12 15 18 21 24 27 30 0 50 100 150 200 250 300 350Time (h)Base Consumed(mmoles/liter)C Figure 2-4. Effect of selected mutations on E. coli metabolism. A) growth B) glucose utilization C) KOH consumption. Solid squares, W3110 (wild type); Open squares, SZ47 ([focA-pflB]::FRT frdBC ldhA); Open circles, TC24 ([focA-pflB]::FRT frdBC ldhA atp[FH]::FRT); Solid circles, TC36 ([focA-pflB]::FRT frdBC ldhA atp[FH]::FRT adhE::FRT sucA::FRT).

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34 (TC24). Cell yield for strain TC24 was equivalent to that of W3110. Strain TC36, containing additional mutations in 2-oxoglutarate dehydrogenase (sucA) and alcohol dehydrogenase (adhE), had a lower cell yield and growth rate; approximately 80% of the unmodified parent W3110. Rates for glucose utilization (maximum specific and average volumetric) were higher for TC36 and TC24 than for W3110 and SZ47 (Table 2). This increase in metabolic activity can be primarily attributed to the atpFH mutation. ATP levels serve as an allosteric regulator of several key glycolytic enzymes (Neidhardt et al. 1990), and acetate kinase (Suzuki 1969). In strains containing the atpFH mutation, increased glycolytic flux and substrate level phosphorylation could partially compensate for the loss of ATP production from respiration-coupled phosphorylation. Differences between strains were particularly evident when comparing incubation times required to complete sugar metabolism (Fig. 2-4B). With TC36 and TC24, glucose was exhausted in 16-18 h as compared to 26 h for SZ47 and 30 h for W3110. The maximum specific rate of glucose utilization (glycolytic flux) was 9 mmole h -1 (g dry cell weight) -1 in the unmodified parent (W3110), 20 mmole h -1 (g dry cell weight) -1 in TC24, and 18 mmole h -1 (g dry cell weight) -1 in TC36. Table 2-3. Comparison of metabolic rates Glucose consumption rate a Pyruvate production rate a Strain (h -1 ) Average Vol. b (mmol L-1 h-1) Max. Spec. b (mmol L-1 h-1 g-1) Average Vol. b (mmol L-1 h-1) Max. Spec. b (mmol L-1 h-1 g-1) W3110 0.60 7 9 4 10 SZ47 0.55 9 11 4 10 TC24 0.53 10 20 10 16 TC36 0.49 11 18 12 16 a Data are derived from averages of two fermentations. b Average volumetric rates for glucose consumption and acetate production or maximum specific rates (dry cell weight basis) for glucose consumption and acetate production.

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Table 2-4. Summary of fermentation products 35 DO, diss olved oxygen. Fermentation Products a (mmoles L -1 ) Strain Conditions Cell yield (g L -1 ) Acetate 2-Oxoglutarate Fumarate Lactate Pyruvate Succinate Yield (% b ) Carbon recovery (% substrate C c ) W3110 3% glucose 5% DO 4.5 30 39 0.8 33 <1 5 9 60 SZ47 3% glucose 5% DO 5.3 6 11 0.9 <1 1 3 2 20 TC24 3% glucose 5% DO 4.4 156 1 1.0 < 1 < 1 2 47 66 TC36 3% glucose 5% DO 3.50.2 22414 166 0.40.1 <1 00.5 41 68 89 TC36 3% glucose 15% DO 3.2 190 24 < 1 < 1 < 1 3 57 88 TC36 3% glucose 5% DO N-limited 2.5 220 31 < 1 < 1 < 1 10 66 95 a Concentrations in broth after all glucose had been depleted, except as noted. b Yield expressed as a percentage of the maximal theoretical yield (0.67 g acetate per g glucose). c Carbon recovery represents the percentage of substrate carbon recovered. Recovered carbon was calculated as the sum of carbon in cell mass, fermentation products, and CO 2 (CO 2 calculated as the difference of [acetate + 2-oxoglutarate]-[succinate + fumarate]). d In the final sample, 44 mM glucose was present. e Excess glucose (9.5%) was added to maintain levels above 100 mM; 107 mM glucose was present in the final sample.

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Table 2-4. Continued Fermentation Products a (mmoles L -1 ) Strain Conditions Cell yield (g L -1 ) Acetate 2-Oxoglutarate Fumarate Lactate Pyruvate Succinate Yield (% b ) Carbon recovery (% substrate C c ) TC36 3+3% glucose 5% DO 3.8 523 21 <1 3 14 2 78 95 TC36 3+3% glucose 5% DO N-limited 3.0 572 33 < 1 < 1 < 1 6 86 102 TC36 6% glucose 5% DO 4.2 415 47 0.3 <1 46 7 62 92 TC36 6+4% glucose 5% DO d 4.5 767 37 0.5 <1 72 5 72 97 TC36 Fed batch 5% DO e 4.1 878 33 3.4 < 1 < 1 25 75 88 DO, dissolved oxygen. a Concentrations in broth after all glucose had been depleted, except as noted. 36 b Yield expressed as a percentage of the maximal theoretical yield (0.67 g acetate per g glucose). c Carbon recovery represents the percentage of substrate carbon recovered. Recovered carbon was calculated as the sum of carbon in cell mass, fermentation products, and CO 2 (CO 2 calculated as the difference of [acetate + 2-oxoglutarate]-[succinate + fumarate]). d In the final sample, 44 mM glucose was present. e Excess glucose (9.5%) was added to maintain levels above 100 mM; 107 mM glucose was present in the final sample.

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37 The slightly lower glycolytic flux in TC36 as compared to TC24 may be related to the increase in ATP yield resulting from improvements in acetate yield (1 ATP per acetate). Assuming protein represents 55% of dry cell weight, maximal glycolytic flux in TC36 is approximately 0.55 moles glucose min -1 (mg protein) -1 Production of Acetate and Other Organic Acids A substantial portion of glucose carbon was not recovered in the carbon balance (Table 3) for W3110 (40%) and SZ47 (80%). This loss is attributed to production of volatile products by high flux through the tricarboxylic acid cycle (CO 2 ) but may also include the reduction of acetyl~CoA to acetaldehyde and ethanol (Fig. 2-2A). Although ethanol was absent in broth samples from all pH-controlled fermentations (sparged at 1 L min -1 ), a small amount of ethanol (6 mM) was found in seed cultures of W3110 (shaken flasks). No ethanol was present in seed cultures of TC36, which contains a mutation in alcohol dehydrogenase E. The native electron transport system (5% dissolved oxygen) and fermentation pathways in W3110 (Table 3) serve as complementary routes for NADH oxidation. Eliminating the fermentation pathways (SZ47) doubled the loss of carbon as volatile products (Table 3) and increased cell yield but decreased the rate of acetate production in comparison to W3110 (Table 2 and Table 3). Inactivation of oxidative phosphorylation (atpFH ) in SZ47 to produce TC24 resulted in a 26-fold increase in acetate yield and a 3-fold improvement in carbon recovery (Table 3, Fig. 2-5A). Acetate yield and carbon recovery increased by another 30% with the introduction of the sucA and adhE mutations (TC36). TC36 produced an average of 224 mM acetate in 16 h with small amounts of other products. This represents 68% of the maximum theoretical yield using native pathways (2 acetates per glucose), remaining carbon being divided between cell mass, dicarboxylic acids, and CO 2 The

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38 0 3 6 9 12 15 18 21 24 27 30 0 50 100 150 200 250Time (h)Acetate (mM) 0 3 6 9 12 15 18 21 24 27 30 0 10 20 30 40 50Time (h)Total DicarboxylicAcids (mM) 0 3 6 9 12 15 18 21 24 27 30 0 4 8 12 16 Time (h)Pyruvate (mM)ABC Figure 2-5. Effect of selected mutations on the production of acetate. A) Acetate B) dicarboxylic acids (2-oxoglutarate, succinate and fumarate) C) pyruvate. Symbols: Solid sqaures, W3110 (wild type); Open squares, SZ47 ((focA--plfB)::FRT frdBC ldhA); Open circles, TC24 ((focA--plfB)::FRT frdBC ldhA atp(FH)::FRT) Solid circles, TC36 (((focA--plfB)::FRT frdBC ldhA atp(FH)::FRT sucA::FRT adhE::FRT).

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39 maximum specific and average volumetric rates of acetate production were approximately 1.6-fold and 2.5-fold higher, respectively, for TC24 and TC36 than for SZ47 and W3110 (Table 2), a difference which can be attributed solely to the mutation in the (F 1 F 0 )H + -ATP synthase. This mutation eliminated ATP production by oxidative phosphorylation while retaining cytoplasmic F 1 -ATPase for the gratuitous consumption of ATP.Strain W3110 accumulated the highest levels of dicarboxylic acids (primarily succinate and 2-oxoglutarate) during glucose metabolism, approximately 3-fold that of the engineered strains (Fig. 2-5B). The order of appearance of dicarboxylic acids in the broth correlated with growth rate and the order of entry into stationary phase for the four strains examined. Dicarboxylic acids were partially consumed as glucose levels declined, and may represent spillover products from excessive glycolysis during the transition from exponential to stationary phase. It is interesting to note that pyruvate levels in the broth of TC36 also increased (16 mM at 12 h) during this transition (Fig. 2-5C). Although this pyruvate was subsequently metabolized, the excretion of pyruvate indicates that glucose uptake and glycolysis per se may not be limiting for acetate production. No significant accumulation of pyruvate was observed for the three other strains (W3110, SZ47 or TC24). The consumption of base to maintain pH 7.0 provides an overall measure of total organic acid production (Fig. 2-4C). Higher rates and maxima for TC24 and TC36 are consistent with more rapid glucose metabolism. In general, variations in glucose utilization were accompanied by corresponding changes in base utilization. The exponential nature of the early time points reflects growth of the biocatalysts.

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40 Pyruvate Oxidase Contributes to Acetate Production during Stationary Phase Pyruvate can be metabolized to acetate by the membrane-bound protein pyruvate oxidase using the electron transport system to couple oxygen as a terminal electron acceptor. The poxB gene is typically repressed during exponential growth but is induced by stress or entry into stationary phase (Chang and Cronan 1983; Chang et al. 1994). An isogenic Pyruvate oxidase mutant of TC36 was constructed by introducing stop codons in the central region of the gene to produce strain TC42. Introducing the poxB mutation had no effect on glucose utilization (Fig. 2-6). No difference was observed in the rate of acetate production for the poxB mutant during logarithmic growth (Fig. 2-6). Upon entry into late logarithmic/stationary phase the rate of acetate production by the poxB mutant declined resulting in a 20% decrease in final acetate concentration (Fig. 2-6). 0 4 8 12 16 20 0.01 0.1 1 10 Time (h)Cell Mass (g L-1) 0 4 8 12 16 20 0 50 100 150 200 250Time (h)Glucose/Acetate (mmoles L-1)AB 0 4 8 12 16 20 0.01 0.1 1 10 Time (h)Cell Mass (g L-1) 0 4 8 12 16 20 0 50 100 150 200 250Time (h)Glucose/Acetate (mmoles L-1)AB Figure 2-6. Comparison of acetate production between TC36 (poxB + ) and TC42 (poxB ). Data from triplicate fermentations. A) Growth. Symbols: Solid squares, TC36; Open circles, TC42. B) Glucose consumption and acetate production. Symbols: Solid squares, TC36 glucose; Solid circles, TC42 glucose; Open squares, TC36 acetate; Open circles, TC42 acetate.

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41 Restoration of focA pflB Had No Effect On Acetate Production from Glucose Pyruvate formate-lyase catalyzes the non-oxidative decarboxylatation of pyruvate to formate and acetyl-CoA under fermentative conditions. Under the 5% dissolved oxygen E. coli W3110 produced 33 mM lactate (Table 2-4) suggesting the cells experience fermentative conditions once the cell density is greater than 2 g L -1 dry weight (data not shown). Therefore PFL activity was restored to provide more acetyl-CoA for the phosphotransacetylase reaction in an attempt to increase acetate production. Pyruvate formate-lyase had no effect on growth rate, final cell mass, glucose consumption, or acetate production (Fig. 2-7A and B). 0 3 6 9 12 15 0.1 1 10 Time (h)Cell Mass (g L-1) 0 3 6 9 12 15 0 20 40 60 80 100 120 140 160 180 200 220Time (h)Glucose/Acetate (mmoles L-1)AB Figure 2-7. Effect of restoring PFL activity on growth and acetate production. A) Growth. B) Glucose consumption and acetate production. Symbols: Circles, TC51; Squares, TC36; Solid symbols represent glucose and open symbols represent acetate in part B.

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42 Elimination of ATPase Activity The efficacy of the atpFH deletion by constructing an isogenic strain containing an additional deletion in the atpA gene (TC57). The atpA gene codes for the alpha subunit of the (F 1 F 0 )H + -ATP synthase, an essential protein for catalysis (Boyer 1997). Growth rate and volumetric glucose consumption were reduced by 20% and 25% respectively, for the atpA mutant compared to the atpFH mutant (Table 2-5). The most striking difference between the two strains was the 62% reduction in the specific glucose consumption rate for TC57. Unfortunately, the elevated glucose consumption rate did not result in an increased rate of acetate production, demonstrated by the modest increase in acetate production rate for TC36 (Table2-5). Where did the rest of the carbon go? Inspection of Figure 2-8 A and B reveals that the increased glucose consumption rateresults in excretion of pyruvate by TC36. The pyruvate is converted to acetate once glucose levels diminish. The increase in pyruvate excretion in TC36 may be due to elevated ADP levels which would allow for increased flow though glycolysis. Improving Acetate Yields Dicarboxylic acids and cell mass were the dominant competing co-products from glucose. A limited set of experiments was conducted to evaluate the potential for process Table 2-5. Effect of atpA or atpFH deletions on metabolic rates Glucose consumption rate a Pyruvate production rate a Strain (h -1 ) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) TC36 0.510.01 10.70.9 29.73.5 10.71.6 31.94.7 TC57 0.41 8.0 11.3 9.2 22.1 a Five liter fermentations were conducted in 7 L vessels. Dissolved oxygen was allowed to fall from 100% to 5% of air saturation. The data for TC36 represent an average of three fermentations and the data for TC57 represent and average of two fermentations. b Average volumetric rates. c Maximum specific rates per gram of dry cell weight (dcw).

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43 0 3 6 9 12 15 18 21 24 0 25 50 75 100 125 150 175 200 225 250A AcetateTime (h)Acetate (mmoles L-1) 0 3 6 9 12 15 18 21 24 0 10 20 30 40 50 60 70 80 90 100B PyruvateTime (h)Pyruvate (mmoles L-1) Figure 2-8. Metabolite production in TC36 and TC57. A) Comparison of acetate production between strains. B) Comparison of pyruvate production between strains. Symbols: Solid squares, TC36. Open squares, TC57.

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44 changes to improve acetate yield (Table 2-4). Acetate yield was not improved by increasing the dissolved oxygen level from 5% to 15% of air saturation, reducing ammonia nitrogen (2 g L -1 ammonium phosphate) by 40% to limit growth, or increasing the initial concentration of glucose from 3% to 6% (Table 2-4). However, a simple two-step batch feeding strategy was beneficial. A second addition of 3 % glucose at the end of the growth phase (12 h) was metabolized to completion and produced 523 mM acetate with minimal loss to cell mass (Fig. 2-9). Acetate yield for this one-step addition (6% total glucose) was 78 % of the theoretical maximum as compared to 68% for 3% glucose. The highest acetate yield, 86% of the theoretical maximum, was obtained by combining the one-step addition of 3% glucose with the nitrogen limitation to further limit loss of carbon into cell mass (Table 2-4). Additional fed-batch experiments were conducted in which multiple additions were made to glucose levels above 100 mM. With this approach, 878 mM acetate was produced representing 75% of the maximum theoretical yield (Table 2-4). Discussion E. coli was genetically engineered to combine the attributes of fermentative metabolism (low cell mass, high product yields) and oxidative metabolism (external electron acceptor) into a single biocatalyst (strain TC36) for the production of oxidized chemicals such as acetate (Fig. 2-2). Chromosomal deletions were used instead of point mutations to maximize stability. All antibiotic resistance genes and auxotrophic requirements were eliminated to permit growth in simple mineral salts medium. During

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45 0 6 12 18 24 30 36 0 100 200 300 400 500 0 1 2 3 4 5Glucose Added Time (h)Glucose & Acetate (mM)Cell Mass (g/liter) Figure 2-9. Fermentation of 6% gluocse to acetate by TC36 in mineral salts medium. Fermentation was initiated at 3% glucose followed by a second addition of 3% glucose after 12 h. Symbols: Open squares, cell mass; Solid circles, acetate. oxidative metabolism, up to half of the substrate carbon can be converted to roughly equal amounts of cell mass and CO 2 (Contiero et al. 2000; Neidhardt et al. 1990; Varma et al. 1993) with minimal carbon flow into alternative products. To reduce the opportunity for excessive growth during oxidative metabolism, ATP production from NADH oxidation (oxidative phosphorylation) was eliminated by deleting the portion of (F 1 F 0 )H + -ATP synthase involved in membrane assembly while preserving a functional cytoplasmic F 1 -ATPase to provide gratuitous hydrolysis of ATP. With this mutation, a maximum of 4 ATP molecules (net) can be produced per glucose (assumes all pyruvate is metablized to acetyl~CoA and acetate) as compared to a theoretical maximum of 33 ATP molecules for wild-type strains of E. coli. Excessive oxidation of substrate to CO 2 and production of NADH were eliminated by disrupting the cyclic function of the tricarboxylic acid cycle (sucA) with the added benefit of reducing oxygen demand for

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46 NADH oxidation. Additional mutations were introduced to eliminate all major fermentation pathways as alternative routes for NADH oxidation, minimizing the formation of alternative products. The resulting strain, TC36, has absolute requirements for a fermentable carbon source (substrate level phosphorylation) and for an external electron acceptor that can couple to the electron transport system during growth in mineral salts medium to maintain redox balance. With genetic blocks in all major fermentation pathways and oxidative phosphorylation, this strain is relatively insensitive to variations in dissolved oxygen. The (F 1 F 0 )H + -ATP synthase and 2-oxoglutarate dehydrogenase mutations introduced into TC36 to miminize the levels of ATP and NAD(P)H produced from glucose under oxidative conditions would also be expected to promote glycolysis through native allosteric controls (Neidhardt et al. 1990; Underwood et al. 2002a; Underwood et al. 2002b), providing a mechanism for the observed 2-fold increase in glycolytic flux as compared to W3110 (wild type). With additional mutations in fermentation pathways, further metabolism of pyruvate was limited primarily to small biosynthetic needs and conversion to acetyl~CoA by the pyruvate dehydrogenase complex. Although pyruvate dehydrogenase is activated by low NADH, acetyl~CoA production may be limited by the availability of free CoA (note pyruvate accumulation in TC36 broth between 9 h and 15 h; Fig. 5C). Resulting increase in pyruvate pools would serve as an allosteric activator of phosphotransferase (Suzuki 1969), the first committed step for acetate production from acetyl~CoA. Gratuitous ATP hydrolysis by F 1 -ATPase should ensure the availability of ADP for the final step in acetate production catalyzed by acetate kinase (Fig. 2-2A). Excess pyruvate can also be directly oxidized to acetate by pyruvate oxidase (poxB), an

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47 enzyme that is induced during the later stages of growth and by environmental stress (Chang et al. 1994). This enzyme may also contribute to acetate production by TC36. Eliminating oxidative phosphorylation while preserving (F 1 ) H + ATPase resulted in a 2-fold increase in glycolytic flux (TC24 and TC36). Previously, Chao and Liao (1994) and Patnaik et al. (1992) demonstrated a similar 2-fold stimulation of glycolytic flux in E. coli using plasmids to express genes that created futile cycles to consume ATP. Recently, Koebmann et al. (2002) independently concluded that glycolytic flux is limited by ATP utilization during the oxidative metabolism of glucose. In their studies, flux increased in a dose-dependent manner with controlled expression of F 1 -ATPase genes from a plasmid. Our results with TC24 and TC36 containing a chromosomal deletion (atpFH ) provide further support for this hypothesis. Thus, glycolytic flux appears to be regulated by the economy of supply and demand as proposed by Hofmeyer and Cornish-Bowden (2000). During the oxidative metabolism of glucose, glycolytic flux is limited by the metabolic ability to utilize ATP (availability of ADP) rather than by glucose transport or the catalytic capacities of central glycolytic enzymes. With this in mind, similar strategies that delete subunits essential for membrane assembly of the (F 1 F 0 )H + -ATP synthase, create futile cycles for ATP consumption, or increase cytoplasmic levels of the ATPase activities may prove useful to decrease cell yield, increase metabolic flux, and increase product yield in many other bioconversion processes. Strain TC36 can be used as a biocatalyst platform for the efficient production of oxidized products. Under conditions of excess glucose, strain TC36 produced a maximum of 878 mM acetate, 75% of the maximum theoretical yield (Table 2-4) or 0.50 g acetate per g glucose. Only cell mass and small amounts of organic acids were

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48 produced as co-products with acetate. It is likely that 878 mM acetate approaches the upper limit of tolerance for metabolism in TC36. Concentrations as low as 50 mM acetate have been shown to induce a stress response in E. coli (Kirkpatrick et al. 2001). The minimal inhibitory concentration for growth has been previously reported as 300 400 mM acetate at neutral pH (Lasko et al. 2000; Zaldivar and Ingram 1999). Oxygen transfer often becomes limiting during aerobic bioconversion processes, promoting the accumulation of reduced products (Tsai et al. 2002; Varma et al. 1993). Synthesis of reduced products was eliminated by mutations in genes (focA-pflB frdBC ldhA adhE) encoding the four major fermentation pathways. Excessive oxygen demand and NADH production were also reduced by a deletion in 2-oxoglutarate dehydrogenase (sucA). The resulting strain, TC36 (focA-pflBfrdBC ldhA atpFH sucA adhE) metabolizes sugars to acetate with the efficiency of fermentative metabolism, diverting a minimum of carbon to cell mass (biocatalyst) and CO 2 By replacing the acetate pathway, a variety of alternative oxidized products can be produced using the mutational strategies employed for the construction of TC36. Commercial production of acetate with biocatalysts involves a 2-step process in which sugar is first fermented to ethanol by yeasts followed by aerobic oxidation to acetate by Acetobacter (Berraud 2000; Cheryan et al. 1997). Although titers of around 650 mM are typically produced, higher titers can be readily achieved by the addition of complex nutrients in fed-batch processes requiring 60-120 h. Overall, yields for commercial processes have been estimated to be 76% of the theoretical maximum (2 acetate per glucose; 0.67 g acetic acid per g glucose). Genetically engineered E. coli TC36 can produce acetate in a simpler, single step process using glucose with titers and

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49 yields equivalent or higher than current batch processes. Although maximum titers with TC36 were lower than can be achieved by ethanol oxidation using Acetobacter (Berraud 2000), acetate production rates from glucose by TC36 were almost two-fold higher than ethanol oxidation and required only mineral salts as nutrients. E. coli TC36 offers a unique set of advantages over currently employed biocatalysts for the commercial production of acetate: 1. The fermentation is a single step process using sugars as substrates. 2. Strain TC36 has high rates of acetate production, high acetate yields and a simple nutritional requirement (mineral salts plus carbon source). 3. Robust metabolism permitting the bioconversion of hexoses, pentoses, and many disaccharides and sugar alcohols. Derivatives of this strain may prove useful as biocatalysts for the production of other compounds that are equivalent to, or more oxidized than the substrate.

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CHAPTER 3 CONVERSION OF ARABINOSE, MANNOSE, SORBITOL AND XYLOSE TO ACETATE Introduction One advantage to employing E. coli as a biocatalyst is its ability to consume hexose and pentose sugars, the carbohydrate constituents of lignocellulosic plant biomass. The plant cell wall is primarily composed of two major carbohydrate polymers: cellulose and hemicellulose (Ingram et al. 1999; McKendry 2002). The carbohydrate composition of the cell wall differs with respect to the type of plant (hardwood, softwood, grass, etc.). Generally, cellulose constitutes 20-55% of agricultural residues and hemicellulose constitutes 20-40% (Sun and Cheng 2002). Pectin, a methylated polymer of galacturonic acid, typically constitutes a small percentage (2-4%) of agricultural residues (Ingram et al 1999). Cellulose is a polymer of glucose (cellobiose) therefore once treated with fungal cellulase the monomers become available for degredation. Hemicellulose is a complex carbohydrate polymer composed of xylose and arabinose in hardwoods and crop residues (Asghari et al. 1996; Ingram et al 1999). Xylose, galactose and mannose are the major sugars in softwoods (Asghari et al. 1996; Ingram et al 1999). The stoichiometry of hemicellulose sugars vary depending on the type of plant. Our laboratory is interested in the conversion of biomass, such as sugarcane bagasse, into fuels and chemicals. The hemicellulose fraction of sugarcane is composed mostly of xylose (89%) with minor amounts of arabinose and glucose (Asghari et al. 1996). In this chapter the engineered E. coli strain TC36 is shown to efficiently convert 50

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51 all major sugar constituents of plant biomass into acetate. In addition, strain TC36 also metabolized sorbitol, a sugar alcohol, into acetate. Materials and Methods Microorganisms and Media Escherichia coli TC36 and TC51 (described in Chapter 1) were used for our study. The NBS salts described in Chapter 1 were used in minimal salts agar and for fermentations as described in Chapter 1. Arabinose, mannose, sorbitol, or xylose was added (2% in plates and 3% in broth) as the sole carbon and energy source, when appropriate, from a 50% stock solution. When needed for pH control, 3-[N-morpholino]propanesulfonic acid (0.1 M, pH 7.4) was added, but was not included in pH-controlled fermentations. Fermentations Fermentations (5 L) were conducted at 37 o C (dual Rushton impellers, 350 rpm; pH 7.0) in Bioflow 3000 fermentors (New Brunswick Scientific, Edison, NJ). Dissolved oxygen levels were 100% of air saturation at the time of inoculation and allowed to fall to 5% of air saturation during cell growth with continuous air sparging (0.2 vvm). This level of oxygen was maintained by mixing O 2 with air at a constant flow rate of 1.0 L min -1 Broth pH was controlled with 11.4 M KOH. Seed cultures were started as described in Chapter 1. Fermentors were inoculated at an initial OD 550 of 0.05 except for xylose fermentations. For xylose fermentations an initial OD 550 was not sufficient for logarithmic growth therefore these fermentations were inoculated at an initial OD 550 of 0.5.

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52 Results Fermentation of Sugars Derived from Hemicellulose (Arabinose, Mannose and Xylose) Pentose sugars constitute the majority of sugars found in hemicellulose. Xylose is the most abundant sugar found in sugarcane derived hemicellulose. Escherichia coli TC36 converted 180 mM xylose into 205 mM acetate, 69% of the maximum theoretical yield (Table 3-1). All of the xylose carbon was accounted for by HPLC analysis of the fermentation broth (Table 3-1). Minor quantities of glycolytic and TCA cycle intermediates were observed in the fermentation broth, 2-oxoglutarate being the most abundant (Table 3-1). Acetate accounted for 88% of the organic acids in the broth. The maximum specific growth rate of TC36 grown on xylose is 75% of the maximum specific growth rate when grown on glucose under the same conditions (Fig. 3-1). The growth rate reduction may be attributed to decreased ATP production on xylose compared to glucose (see discussion). 0 2 4 6 8 10 12 14 16 0.1 1 10 Time (h)Cell Mass (g L-1)Glucose = 0.44Xylose = 0.33 0 2 4 6 8 10 12 14 16 0.1 1 10 Time (h)Cell Mass (g L-1)Glucose = 0.44Xylose = 0.33 Figure 3-1. Growth comparison of TC36 with xylose or glucose as the sole carbon source. Fermentations were begun at an initial OD 550 of 0.5. Symbols: Solid rectangles, xylose; Open rectangles, glucose.

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Table 3-1. Products formed by TC36 during aerobic catabolism of various sugars 53 Concentration mmolesL -1a Condition b Cell mass (g L -1 ) max (h -1 ) c Carbon Balance (%) Acetate Yield (%) d Acetate Pyruvate 2-Oxoglutarate Succinate Malate Arabinose e 4.01 0.46 94 63 220 <1 21 3 4 Glucose f 3.47 0.51 87 62 250 <1 19 14 <1 Mannose e 3.26 0.23 83 59 187 <1 13 2 2 Sorbitol f 3.44 0.30 87 61 195 <1 15 2 <1 Xylosef 3.85 0.33 102 69 205 7 19 2 <1 a Final concentrations at the end of fermentation. Fumarate was not included in Table 3-1 because the concentrations was observed to be less than 1 mM for all fermentations. b All fermentations were conducted with 3% initial sugar concentration. Arabinose, mannose, and sorbitol fermentations were inoculated at an initial OD 550 of 0.05 and the xylose fermentations were inoculated at an intial OD 550 of 0.5. c Maximum specific growth rate. d Acetate yield as a percentage of the maximum theoretical yield (0.67 g of acetate per g of glucose). e Results from one fermentation. f Averages of two fermnetations.

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54 Arabinose constitutes the second most abundant sugar in hemicellulose derived from sugarcane. E. coli TC36 converted 207 mM arabinose into 220 mM acetate, 63% of the maximum theoretical yield (Table 3-1). Again, acetate was the dominant (89%) organic acid recovered from the fermentation broth. The final cell mass and maximum specific growth rate were equivalent to those of TC36 grown on glucose under the same conditions (Tables 2-3 and 3-1). Mannose is an optical isomer of glucose and constitutes a small fraction of the sugars found in hemicellulose. E. coli TC36 converted mannose to acetate with a maximum theoretical yield of 59% (Table 3-1). The specific growth rate (0.23 h -1 ) and final cell density on mannose was the lowest of all sugars tested (Table 3-1). Acetate accounted for 92% of all organic acids recoved, although 17% of the substrate carbon was not recovered (Table 3-1). Oxidation of Sorbitol to Acetate Sorbitol is a six carbon sugar alcohol which is more reduced than glucose. One more NADH is generated from the oxidation of sorbitol than from glucose. The specific growth rate on sorbitol was 59% of the growth rate on glucose and the final cell density was 99% of glucose grown cultures (Table 3-1). The maximum theoretical acetate yield attained was 61%, which is comprable to that acheived on glucose under the same conditions. Discussion Arabinose and xylose are pentose sugar constituents of hemicellulose. Both are transported into the cell by high affinity ABC (ATP binding cassette) transporters specific for the respective sugar (Neidhardt et al. 1996). There are also low affinity H + -linked symporters involved in transport of the pentoses (Henderson 1990). Once in the

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55 cell, arabinose and xylose are catabolized through the pentose phosphate pathway (PPP). Arabinose enters the PPP at the level of ribulose-5-phosphate and xylose enters at the level of xylulose-5-phosphate. Net ATP yield is much lower for the catabolism of the pentoses (0.67 per xylose or arabinose) than for glucose (2 per glucose) with lactate as the end-product (Tao et al. 2001). The NADH produced during xylose and arabinose catabolism is the same as that produced from glucose catabolism. Acetate yield and final cell density were equivalent for the pentose fermentations and glucose fermentations. If ATP was to become limiting during pentose fermentations one would expect greater flux to acetate to generate more ATP or a reduction in the final cell mass. These results imply that net ATP is not limiting the growth of TC36 on arabinose and xylose during aerobic growth. One may expect reduced maximum specific growth rate on xylose due to a slower rate of ATP production, although the maximum specific growth rate for arabinose grown cells is 1.4-fold greater than that of xylose grown cells. If the rate of ATP production was limiting growth rate, one would expect arabinose and xylose cultures to exhibit similar rates. Arabinose was demonstrated to be used preferentially by E. coli K12 when xylose or ribose is present in minimal medium (Kang et al. 1998). The regulatory mechanism was determined to be at the transcriptional level (Kang et al. 1998). Preferential use of arabinose suggests that E. coli derives some thermodynamic or catalytic advantage from catabolism of arabinose over the other pentoses. Unfortunately, the mechanism has not been documented. Mannose was the poorest carbon source examined for growth and acetate yield. Growth on mannose was characterised by a 53% reduction in specific growth rate, as compared to glucose grown cells, and an acetate yield of 59%. Mannose is transported

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56 into the cell by a PTS-linked mannose permease (Erni et al. 1987) and isomerized to fructose-6-phosphate. The growth rate reduction observed for mannose may reflect a slower rate of transport into the cell or isomerization (decreased V max ) to fructose-6-phosphate for mannose than for glucose. Sorbitol is a polyalcohol commonly found in ripe berries. Commercial production of sorbitol uses high-pressure hydrogenation or electrolytic reduction of glucose. Sorbitol was used as a substrate for TC36 in order to demonstrate the ability of the organism to convert a reduced substrate into an oxidized product, acetate. Strain TC36 was able to converted sorbitol efficiently to acetate and maintain a growth rate 61% that of glucose-grown cultures. Alteration of the process conditions, such as increasing the dissolved oxygen above 5%, providing more electron acceptor for respiration, may improve growth rate and/or acetate yield. Conversion of sorbitol to acetate demonstrates the utility of the mutations constructed in TC36. This biocatylist is not limited to the production of compounds reduced or neutral relative to the substrate. In this chapter, the ability of E. coli TC36 to efficiently convert the major sugars found in hemicellulose into acetate is demonstrated. In addition, strain TC36 is capable of converting of substrate carbon into an oxidized product, such as acetate and CO 2

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Chapter 4 ENGINEERING Escherichia coli FOR THE EFFICIENT CONVERSION OF GLUCOSE TO PYRUVATE Introduction There is a growing trend in the commodity and specialty chemicals industries towards the use of microorganisms as biocatalysts. Many commodity chemicals (ethanol, lactate, 1,3-propanediol, and adipic acid) and specialty chemicals (polyketides and carotenoids) can be made directly or indirectly from the same central metabolite, pyruvic acid. The metabolic fate of pyruvate, a key branch point in central carbon metabolism, is regulated in large part by the redox state of the cell. During the fermentative metabolism of glucose, NADH levels are high, and > 95% of pyruvate is used as terminal electron acceptor for NADH oxidation; < 5% is partitioned into biosynthesis (see Figure 1-3 for an overview). During oxidative metabolism, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHc) to produce CO 2 and acetyl-CoA. Oxygen or other external compounds serves as the terminal electron acceptor, and NADH levels remain relatively low. Up to half of the glucose carbon is used for biosynthesis, the remainder being fully oxidized by the tricarboxylic acid cycle to CO 2 Construction of an E. coli strain that excretes pyruvate as the major product of glucose catabolism requires that pyruvate utilizing pathways be eliminated (Fig. 4-1A) and carbon accumulation in the form of cell mass be minimized. 57

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58 Pyruvic acid is currently manufactured for use as a food additive, nutriceutical, and weight control supplement (Li et al. 2001a; Anonymous 2002). Pyruvic acid can also be used as a starting material for the synthesis of amino acids such as alanine, tyrosine, phenylalanine, and tryptophan (Li et al. 2001a) and for acetaldehyde production (Ingram and Conway 1987). Pyruvate is produced commercially by both chemical and microbial processes (Li et al. 2001a). Chemical synthesis from tartrate involves toxic solvents and is energy-intensive (Ondrey 2001; Blair 1999) with an estimated production cost of $8,650 per ton of pyruvate (Li et al. 2001a). Microbial pyruvate production is based primarily on two microorganisms, a multi-vitamin auxotroph of the yeast Torulopsis glabrata (Li et al. 2001b) and a lipoic acid auxotroph of Escherichia coli containing a mutation in the F 1 subunit of the (F 1 F 0 )H + -ATP synthase (Yokota et al. 1994). Both strains require precise regulation of media composition during fermentation and complex supplements. Estimated production costs for pyruvate production with these strains is 14.5% ($1,255 per ton pyruvate) that for chemical synthesis (Li et al. 2001a). Recently, Tomar et al. (2003) have described a new E. coli mutant for pyruvate production from glucose and acetate in complex medium. This strain contains mutations in phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceF), and alcohol dehydrogenase (adhE). Nutrients in culture medium often represent a major cost associated with commercial fermentations. The use of a mineral salts medium and an inexpensive carbon source offer the potential to improve the economics of many biological processes by reducing the costs of materials, product purification, and waste disposal (Zhang and Greasham 1999). A microbial platform that combines attributes of aerobic and

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59 fermentative metabolism for the production of compounds that are redox neutral or oxidized relative to glucose, described in Chapter 2, was developed by our laboratory (Causey, PNAS. 2003). In order to demonstrate further the flexibility of the biocatalyst with regard to the variety of compounds that can be produced, we have engineered Escherichia coli W3110 for the efficient production of pyruvic acid as the major end product of glucose catabolism. Materials and Methods Microorganisms and Media E. coli W3110 (ATCC 27325) and derivatives (Table 4-1) were grown on mineral salts medium (Causey et al. 2003) containing, glucose (2% in plates; 3% in broth). When needed for pH control, 3-[N-morpholino]propanesulfonic acid (0.1 M, pH 7.4) was added, but was not included in pH-controlled fermentations. During plasmid and strain construction, cultures were grown in Luria-Bertani (LB) broth or on LB plates (1.5% agar) (Miller 1992). Glucose (2%) was added to LB medium for all strains containing mutations in (F 1 F 0 )H + -ATP synthase. Antibiotics were included as appropriate (kanamycin, 50 mg L -1 ; ampicillin, 50 mg L -1 ; apramycin, 50 mg L -1 ; and tetracycline, 12.5 or 6.25 mg L -1 ). Genetic Methods Standard methods were used for plasmid construction, phage P1 transduction, electroporation, and polymerase chain reaction (PCR) (Miller 1992; Sambrook and Russell 2001). Coding regions for ackA and poxB were amplified using ORFmer primers (Sigma-Genosys, Inc., The Woodlands, TX) and initially cloned into pCR2.1-TOPO. Chromosomal integration of mutated genes was facilitated by pKD46 containing an arabinose-inducible Red recombinase (Datsenko and Wanner 2000).

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60 Table 4-1. Sources and characteristics of strains and plasmid Strains/Plasmids Relevant Characteristics Reference Strains W3110 K12 wild type ATCC 27325 TOP10F lacI q (episome) Invitrogen SZ61 W3110, ackA::FRT-tet-FRT Zhou et al. 2003 TC36 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT Causey et al. 2003 TC37 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT ackA::FRT-tet-FRT Causey et al. 2004 TC38 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT ackA::FRT Causey et al. 2004 TC42 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT poxB::FRT Causey et al. 2004 TC43 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT ackA::FRT poxB::FRT-tet-FRT Causey et al. 2004 TC44 W3110, (Succ + ), (focA-pflB)::FRT frdBC ldhA atp(FH)::FRT adhE::FRT sucA::FRT ackA::FRT poxB::FRT Causey et al. 2004 Plasmids pCR2.1-TOPO bla kan, TOPO TM TA cloning vector Invitrogen pFT-A bla flp low-copy vector containing recombinase and temperature-conditional pSC101 replicon Posfai et al. 1997 pKD46 bla exo low-copy vector containing red recombinase and temperature-conditional pSC101 replicon Datsenko and Wanner 2000 Integrants were selected using antibiotic resistance, screened for appropriate antibiotic resistance markers and phenotypic traits, and verified by analyses of PCR products and fermentation profiles. FRT-flanked antibiotic resistance genes were deleted using FLP recombinase (Martinez-Morales et al. 1999; Posfai et al. 1997). Deletion of Acetate Kinase (Strains TC38 and TC44) The ackA::FRT-tet-FRT mutation was transduced from SZ61 into TC36 and TC42 to produce strains TC37 (focA-pflB::FRT frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT ackA::FRT-tet-FRT) and TC43(focA-pflB::FRT frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT poxB::FRT ackA::FRT

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61 tet-FRT), respectively. After removal of the tet gene using FLP recombinase, resulting strains were designated TC38 ((focA-pflB::FRT frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT ackA::FRT) and TC44 (focA-pflB::FRT frdBC::FRT ldhA atpFH::FRT adhE::FRT sucA::FRT poxB::FRT ackA::FRT), respectively. Fermentation Fermentations (5 L and 10L) were conducted at 37 o C (dual Rushton impellers, 350 450 rpm; pH 7.0) in Bioflow 3000 fermentors (New Brunswick Scientific, Edison, NJ). Unless stated otherwise, dissolved oxygen levels were 100% of air saturation at the time of inoculation and allowed to fall to 5% of air saturation during cell growth with continuous air sparging (0.2 vvm). This level of oxygen was maintained by mixing O 2 with air at a constant flow rate of 1.0 L min -1 Broth pH was controlled with 11.4 M KOH. For fed-batch studies, glucose was added from a sterile 4 M stock. Two fed batch regimes were investigated: 1) 3% initial glucose followed the addition of 3% glucose after 15 h (6% total); 2) 3% initial glucose with the addition of 590 mL of 4 M glucose at a constant rate over a 20-h period (9.8% total glucose). Seed cultures prepared as previously described (Causey et al. 2003) were used to provide an inoculum of 16.5 mg dry cell weight L -1 Broth samples were removed to measure organic acids, residual glucose, and cell mass. Volumetric and specific rates were estimated from measured values for glucose and acetate using GraphPad Prism (GraphPad Software, San Diego, CA). A smooth curve was generated with 10 points per min (Lowess method) to fit measured results. The first derivative (acetate or glucose versus time) of each curve served as an estimate of volumetric rate. Specific rates (mmoles L -1 h -1 [mg dry cell weight] -1 ) were calculated by dividing volumetric rates by respective values for cell mass.

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62 Analyses Organic acids and glucose were measured using a Hewlett Packard HPLC (HP 1090 series II) equipped with a UV monitor (210 nm) and refractive index detector (Causey et al. 2003). Cell mass was estimated using a Bausch & Lomb Spectronic 70 spectrophotometer (1.0 OD 550nm is equivalent to 0.33 g dry cell weight L -1 ). Results Pyruvate as a Co-Product during Acetate Fermentations Escherichia coli TC36 (focA-pflB frdBC ldhA atpFH adhE sucA) was previously engineered from W3110 (wild type) for the production of acetate (Fig. 3-1A) by combining chromosomal deletions that minimize cell yield, oxygen consumption, CO 2 evolution, and reduced fermentation products (Causey et al. 2003). In this strain, glycolytic flux was higher than the parent due to a deletion of genes (atpFH) encoding two membrane proteins that couple the F 1 and F 0 components of the F 1 F 0 (H + )ATP synthase complex. This mutation eliminated ATP production by oxidative phosphorylation and created an active, cytoplasmic F 1 ATPase (Fig. 3-1B and 3-1C). Glycolytic flux in TC36 exceeded the capacity for acetate production under our initial test conditions (5% air saturation at inoculation and during fermentation), resulting in transient accumulation of approximately 16 mM pyruvate near the end of exponential growth (Fig. 3-2). The peak level of pyruvate was increased to 81 mM (Fig. 4-2) by inoculating the fermentor at an initial dissolved oxygen level of 100% air saturation (rather than 5% of saturation) with continuous air sparging until the oxygen level declined to 5% air saturation, then adding oxygen to maintain 5% of air saturation. Under this condition, pyruvate yield at the peak was 25% of the maximum theoretical yield and 11% of the maximum theoretical yield at the end of fermentation (Fig. 4-2; Table 4-2).

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63 GlucoseNADHAcetateptaTriose-PPEP~P~P pykA ~P ~PCO2CO2 NAD+LactateldhA NAD+NADHA NADPHUQH2UQ EthanoladhE HCOOHaceEF lpdpflB NADHNAD+Acetyl-CoA2 NAD+2 NADHFADH2frdABCDFAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc pykFpoxBackA PyruvateH2OH+ O2 + einout Electron TransportSystem F0atpIBEFHAGDC Pi+ADP ATPF1 H+H+B F1 C NADHNAD+ATPPi+ADP GlucoseNADHAcetateptaTriose-PPEP~P~P pykA ~P ~PCO2CO2 NAD+LactateldhA NAD+NADHA NADPHUQH2UQ EthanoladhE HCOOHaceEF lpdpflB NADHNAD+Acetyl-CoA2 NAD+2 NADHFADH2frdABCDFAD+ FumarateNADHMalateNAD+gltA~P~P CitrateOxaloacetate IsocitrateSuccinyl-CoAlpdAglcB aceBGlyoxylateAcetyl-CoAmdhacnBicdAsucDCfumABCaceACO2 NADP+NAD+ NADH sucAB2-OxoglutarateCO2 sdhABCDUQH2UQ Succinate CO2ppc pykFpoxBackA PyruvateH2OH+ O2 + einout Electron TransportSystem F0atpIBEFHAGDC Pi+ADP ATPF1 H+H+B F1 C NADHNAD+ATPPi+ADP H2OH+ O2 + einout Electron TransportSystem F0atpIBEFHAGDC Pi+ADP ATPF1 H+H+B F1 C NADHNAD+ATPPi+ADP Figure 4-1. Genetic modifications used to redirect glucose metabolism for pyruvate production. Metabolic steps that have been blocked by mutation or deletion of genes are highlighted. A) Central carbon metabolism. B) Oxidative phosphorylation. C) Cytoplasmic F 1 ATPase subunit (active). Genes: aceA, isocitrate lyase; aceB, malate synthase B; aceEF, E1p and lipoate acetyltransferase/dihydrolipoamide acetyltransferase component of the PDHc; ackA, acetate kinase; acnB, aconitase; adhE, alcohol/aldehydrogenase pyruvate formate lyase deactivase; atpIBEFHAGDC, (F 1 F 0 )H + -ATP synthase; frdABCD, fumarate reductase; glcB, malate synthase G; gltA, citrate synthase; icdA, isocitrate dehydrogenase; ldhA, lactate dehydrogenase; lpdA, dihydrolipoate dehydrogenase/dihydrolipoamide dehydrogenase component of the PDHc and 2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase; poxB, pyruvate oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II; pykF, pyruvate kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-oxoglutarate dehydrogenase; sucDC, succinyl-CoA synthetase.

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64 We have explored additional genetic modifications of TC36 to increase the efficiency of pyruvate production from glucose. Although many metabolic routes lead to acetate, two primary routes are present in E. coli (Fig. 4-1A): 1) conversion of acetyl~CoA to acetate by phosphotransacetylase (pta) and acetate kinase (ackA); and 2) direct oxidation of pyruvate to acetate by pyruvate oxidase (poxB) (Fig. 4-1A). Derivatives of TC36 were constructed with mutations in both pathways. 0 2 4 6 8 10 12 14 16 18 20 0 20 40 60 80 100 120 140 160 180 200Glucose (5% DO) Glucose (100 5% DO) Pyruvate (5% DO) Pyruvate (100 5% DO) 0 10 20 30 40 50 60 70 80 90 100Time (h)Glucose (mmoles L-1)Pyruvate (mmoles L-1) Figure 4-2. Effect of oxygen level on pyruvate production by TC36. Cells were inoculated into fermentation broth at 100% air saturation and continuously sparged with air until the oxygen levels declined to 5% saturation. At this time, oxygen was blended to maintain 5% O 2 saturation during the remaining period of incubation (open symbols). Alternatively, media was sparged with a mixture of air and nitrogen to provide 5% air saturation prior to inoculation. Sparging was switched to air and oxygen as needed to maintain 5% air saturation (closed symbols).

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65 Acetate Kinase (ackA) Deletion Enhances Pyruvate Production and Inhibits Glycolysis (Strain TC38) To block acetate production by the acetate kinase pathway, the central region of the ackA gene was deleted in TC36 to produce TC38. Acetate production was reduced by 85% (Fig. 4-3C; Table 4-2), consistent with the acetate kinase pathway being the dominant route for acetate production in TC36. However, this deletion also reduced net ATP production (estimated) by 30% (Fig. 4-1A), cell yield by 36% (Fig. 4-3A; Table 4-2), and maximum specific growth rate by 45% (Table 4-3). Glycolytic flux was reduced by 45% (Table 4-3) increasing the time required to complete fermentations from 18 h for TC36 to 24 h for TC38 (Fig. 4-3B). Although both volumetric and specific rates of glucose metabolism were lower for TC38 (Table 4-3), pyruvate yield was 5.5-fold higher (Table 4-2; Fig. 4-3D) and the specific rate of pyruvate production was 4-fold higher (Table 4-3) than for TC36. Small amounts of 2-oxoglutarate, succinate, and fumarate were produced by both strains. From 10% to 15% of the carbon was not recovered as cell mass or acidic fermentation products and is presumed to be lost as CO 2 due to metabolite cycling. With strain TC38, the pyruvate yield (195 mM) was 58% of the theoretical maximum. Acetate (28.9 mM) remained as the second most abundant product. Pyruvate Oxidase (poxB) Mutation Causes a Modest Reduction in Acetate Accumulation Pyruvate can be metabolized to acetate by the membrane-bound protein pyruvate oxidase using the electron transport system to couple oxygen as a terminal electron acceptor. The poxB gene is typically repressed during exponential growth but is induced by stress or entry into stationary phase (Chang and Cronan 1983; Chang et al. 1994). Pyruvate oxidase was inactivated in TC36 by constructing three stop codons, one in each reading frame, in the central region to produce TC42.

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Table 4-2. Products formed from glucose catabolism by E. coli strains used in our study Product Concentrations (mmoles L -1 ) a Strain Condition Replicates Cell mass (g L -1 ) Carbon Balance (%) Pyruvate Yield (%) b Pyruvate Acetate 2-Oxoglutarate Succinate Fumarate W3110 3% glucose 2 4.13 80.6 5.9 20.8 180.0 8.3 13.7 0.9 TC36 3% glucose 3 3.470.23 89.02.7 10.57.9 38.127.2 c 197.721.1 16.616.2 13.713.2 1.40.2 TC38 3% glucose 3 2.210.09 84.35.2 57.52.6 194.59.1 28.916.7 10.51.9 8.19.1 0.80.7 TC38 3% glucose d 2 2.40 84.7 68.8 241.9 7.0 7.9 <2.0 <0.2 TC42 3% glucose 2 3.40 86.8 29.1 79.0 178.4 76.2 24.3 1.7 a Unless stated otherwise the concentrations represent measurements at the time of glucose exhaustion. b Maximum theoretical yield is 2 moles pyruvate per mol glucose (0.978 g pyruvate per g glucose) 66 c The pyruvate concentrations during glucose fermentations ranged from 14.9 to 111.9 mM. Pyruvate production was very sensitive to dissolved oxygen flucuations. d Dissolved oxygen was allowed to fall from 100% to 50% of air saturaiton. e Three percent initial glucose followed by the addition of 208 mL of a 4 M glucose solution. f Three percent initial glucose followed by the addition of 590 mL of a 4 M glucose solution over a period of 20 h.

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Table 4-2. Continued Product Concentrations (mmoles L -1 ) a Strain Condition Replicates Cell mass (g L -1 ) Carbon Balance (%) Pyruvate Yield (%) b Pyruvate Acetate 2-Oxoglutarate Succinate Fumarate TC44 3% glucose 3 2.360.10 88.50.6 69.31.5 252.56.2 11.61.2 3.61.2 16.80.7 1.10.2 TC44 3% glucose, Nitrogen 2 2.02 73.6 38.8 125.2 50.3 30.0 7.7 2.9 TC44 3%+3% glucose e 2 2.63 86.7 72.3 479.8 39.8 31.7 10.9 0.7 TC44 6% glucose 2 1.95 94.8 588.9 46.0 26.1 <2.0 0.7 TC44 excess glucose f 2 2.51 --749.0 62.4 45.3 14.7 3.3 a Unless stated otherwise the concentrations represent measurements at the time of glucose exhaustion. b Maximum theoretical yield is 2 moles pyruvate per mol glucose (0.978 g pyruvate per g glucose) c The pyruvate concentrations during glucose fermentations ranged from 14.9 to 111.9 mM. Pyruvate production was very sensitive to dissolved oxygen flucuations. d Dissolved oxygen was allowed to fall from 100% to 50% of air saturaiton. 67 e Three percent initial glucose followed by the addition of 208 mL of a 4 M glucose solution. f Three percent initial glucose followed by the addition of 590 mL of a 4 M glucose solution over a period of 20 h.

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0 4 8 12 16 20 24 28 0.01 0.1 1 10 TC36 TC38 (ackA-) TC42 (poxB-) TC44 (ackA-poxB-) ATime (h)Cell Mass (g L-1) 0 4 8 12 16 20 24 28 0 50 100 150 200 250 300TC36 TC38 (ackA-) TC42 (poxB-) TC44 (ackApoxB-) BTime (h)Glucose (mmole L-1) 0 4 8 12 16 20 24 28 0 50 100 150 200 250 300TC36 TC38 (ackA-) TC42 (poxB-) TC44 (ackApoxB-) CTime (h)Acetate (mmole L-1) 0 4 8 12 16 20 24 28 0 50 100 150 200 250 300TC36 TC38 (TC36 ackA-) TC42 (TC36 poxB-) TC44 (TC36 poxB-ackA-) DTime (h)Pyruvate (mmoles L-1) 68 Figure 4-3. Batch fermentation of glucose by mutant strains of E. coli. A) Cell Growth B) Glucose utilization C) Acetate production D) Pyruvate Production.

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69 In contrast to the ackA deletion (TC38), the poxB mutation (TC42) caused a relatively small decease in acetate and pyruvate (Table 4-2; Fig. 4-3C and 4-3D) consistent with a minor role for the POX pathway. Pyruvate yield with TC42 was less than 30% of the theoretical maximum. Unlike the mutation in ackA (TC38), inactivation of poxB (TC42) did not reduce the rate of growth or glucose metabolism (Table 4-3). Combining Mutations for Pyruvate Oxidase (poxB) and Acetate Kinase (ackA) Improved Pyruvate Production (Strain TC44) Strain TC44 (focA-pflB frdBC ldhA atpFH adhE sucA poxB::FRT ackA) was constructed in which both the acetate kinase and pyruvate oxidase mutations were combined in the TC36 background. Inactivation of poxB was beneficial for growth and pyruvate production (Fig. 4-3A; Table 4-2 and Table 4-3) in comparison to TC38 (isogenic strain containing functional poxB). Both volumetric and specific rates of glucose metabolism were higher for TC44 than for TC36 or TC38 (Table 4-3). Acetate production by TC44 was half that of TC38 and pyruvate yield was 17% higher. Table 4-3. Comparison of metabolic rates Glucose consumption rate a Pyruvate production rate a Strain (h -1 ) Average Vol b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) W3110 0.69 11.9 17.4 nd nd TC36 d 0.490.03 10.12.6 17.61.5 nd nd TC36 e 0.510.01 10.70.9 29.73.5 3.83.0 5.33.1 TC38 e 0.280.01 6.70.6 16.32.2 8.30.7 21.13.7 TC38 f 0.21 6 28 8 28 TC42 e 0.55 10 17 6 10 TC44 e 0.340.02 9.70.7 27.24.1 13.10.3 40.47.4 a Standard deviations are included for data from three or more fermentations; others represent an average of two fermentations. b Average volumetric rates. c Maximum specific rates. dcw, dry cell weight. d Dissolved oxygen controlled at 5% of air saturation by adjusting the ratio of O 2 to N 2 e Dissolved oxygen allowed to fall from 100% to 5% of air saturation during growth with air sparging; O 2 added to maintain 5% air saturation. f Dissolved oxygen allowed to fall from 100% to 50% of air saturation during growth with air sparging; O 2 added to maintain 50% air saturation.

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70 The specific rate of pyruvate production by TC44 was 8-fold that of TC36 and twice that of TC38 (Table 4-3). The time required to complete fermentation with TC44 was 30% shorter than with TC38 (Fig. 4-3B). Broth containing 3% glucose (167 mM) was metabolized to 2.2% pyruvate (252 mM) after 18 h in mineral salts medium (Fig. 4-3D). Although acetate levels were substantially reduced by combining the poxB and ackA mutations (Fig. 4-3C), acetate (12 mM) and dicarboxylic acids (20 mM) remained as minor products. The Beneficial Role of a poxB Mutation for Pyruvate Production Although eliminating the primary route for acetyl~CoA dissimilation (ackA) in TC38 increased pyruvate production, this was accompanied by detrimental effects on growth and fermentation rates (Fig. 4-2; Table 4-2 and Table 4-3) that were substantially reduced by inactivation of PoxB (TC44). Pyruvate oxidase competes with NADH oxidation for oxygen as a terminal electron acceptor and represents a potential mechanism for this action. Oxygen transfer rates are frequently limiting during aerobic fermentations at relatively high levels of saturation (Varma et al. 1993), and may be even more problematic under our fermentation conditions (5% of air saturation). Increased PoxB activity resulting from larger pyruvate pools in the acetate kinase mutant (TC38) would be expected to decrease the availability of NAD + for glycolysis and to increase the levels of NADH, an allosteric inhibitor of key biosynthetic enzymes such as citrate synthase (Weitzman 1966) and phosphoenolpyruvate carboxylase (Krebs and Bridger 1976). This hypothesis was tested in part by examining the effect of increasing the oxygen level during fermentation with TC38. Increasing oxygen saturation from 5% to 50% during TC38 fermentations (Table 4-2 and Table 4-3) was beneficial, consistent with our hypothesis. Cell yield, pyruvate

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71 yield, and the specific rate of glucose metabolism were 8% to 41% higher for TC38 at 50% air saturation than at 5% air saturation. These results were very similar to those observed for the isogenic poxB mutant, TC44, during fermentation at 5% air saturation. Increasing the oxygen saturation during TC38 fermentations also decreased the final concentrations of acetate to a level equivalent to TC44 at 5% air saturation and decreased the production of dicarboxylic acids (Table 4-2). Improving Pyruvate Yields and Titers of TC44 by Altering Fermentation Conditions Prior experience with the acetate-producing strain, TC36, demonstrated that yields could be improved by simple changes in fermentation conditions (Causey et al. 2003). Unlike TC36, decreasing the ammonia level by half did not increase product yields for TC44 (Table 4-2). However, doubling the initial concentration of glucose or providing a second addition of glucose (3% plus 3%) increased pyruvate yields by 11% and doubled the final pyruvate titer. With excess glucose, 749 mM pyruvate was produced. This may represent the limit for pyruvate tolerance. Addition of 600 mM pyruvate to mineral salts medium inhibited the growth of wild type W3110 (data not included). Discussion Pyruvate occupies a key branch point in central carbon metabolism, its fate dependent on the redox state of the cell. During respiration, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex (PDHc) to form NADH, CO 2 and acetyl-CoA. During fermentative metabolism, pyruvate acts as a final electron acceptor for NADH oxidation and is converted to reduced fermentation products such as lactic acid. During fermentation, approximately 5% of the metabolized pyruvate is converted to acetyl-CoA and formate (pyruvate formate-lyase; pflB gene) for

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72 biosynthesis. Construction of an E. coli strain that excretes pyruvate at high yield during glucose catabolism required the elimination of non-essential pathways for pyruvate utilization and mutations that reduce the utilization of pyruvate for cell growth. Previously, we described a derivative of Escherichia coli W3110 (strain TC36) that combines many of the attributes of aerobic and fermentative metabolism and efficiently converts glucose to acetate, a redox neutral product (Causey et al. 2003). This strain was used as a microbial platform to engineer TC44 for the conversion of glucose to pyruvate, a more oxidized product, by mutating genes encoding acetate kinase (ackA) and pyruvate oxidase (poxB). Strain TC44 converted glucose to pyruvate with a yield of 0.75 g pyruvate g -1 glucose (77.9% of theoretical yield) under optimal conditions in minimal medium. Under conditions of glucose excess, a maximum of 749 mM pyruvate was produced. Strain TC44 contains multiple mutations that ensure the rapid and efficient conversion of sugars into pyruvate. The minimal effect of these multiple mutations on growth rate was surprising. Indeed, glycolytic flux and maximal growth rate for TC44 were only 20% lower than the unmodified W3110 under similar growth conditions (unpublished results). The pyruvate oxidase mutation was surprisingly beneficial for growth and pyruvate production. Introduction of this mutation into the acetate-producing strain (TC36) resulted in a small improvement in growth rate in contrast to the mutation of acetate kinase that decreased growth rate by half (Table 4-3). The decreased growth rate of the acetate kinase mutant, TC36, cannot be explained by the predicted 30% reduction in ATP yield since growth rate was substantially restored by adding a subsequent mutation in pyruvate oxidase. These results suggested that the reduction in

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73 growth rate by the acetate kinase mutation may be due to increased PoxB activity associated with a larger pyruvate pool. Pyruvate oxidase transfers electrons from pyruvate to ubiquinone upon the decarboxylation of pyruvate (Cunningham and Hager 1975 ). An increase in pyruvate metabolized by pyruvate oxidase would increase oxygen demand and reduce the oxygen available for the oxidation of NADH. Since the total nicotinamide adenine dinucleotide pool is relatively constant (Wimpenny and Firth 1972) and the NAD + /NADH ratio is responsive to changes in oxygen availability (Wimpenny and Firth 1972), increased pyruvate oxidase activity would also reduce the level of NAD + available for glycolysis. High NADH levels also serve as an important allosteric regulator of key enzymes such as citrate synthase (Weitzman 1966) and phosphoenolpyruvate carboxykinase (Krebs and Bridger 1976). Allosteric inhibition of these enzymes in minimal medium would limit both glycolytic flux and the partitioning of carbon into biosynthesis. Oxidation of NADH is also essential to provide the stochiometric levels of NAD + required for glycolysis. Thus, inactivating pyruvate oxidase may be expected to improve growth by eliminating the increase in oxygen demand, restoring the NAD + pools for glycolysis, and decreasing the level of NADH. This hypothesis was supported by further experiments in which the availability of oxygen was increased from 5% of air saturation to 50% of air saturation during fermentation with the acetate kinase mutant, TC38. The increase in oxygen (50% air saturation) substantially reversed the negative impact of the acetate kinase mutation on growth rate and metabolism, analogous to the effect of adding a pyruvate oxidase mutation. A similar increase in oxygen was of no benefit for TC44 in which both acetate pathways had been mutated (data not included).

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74 During the construction of TC44 from the acetate-producing strain TC36, mutations in acetate kinase and pyruvate oxidase were tested individually prior to constructing the combination. These intermediate strains allowed an assessment of the relative roles of each pathway for acetate production in TC36. The acetate kinase pathway represents the dominant route for acetate production since deletion of the ackA gene decreased acetate production by 85% (Table 4-2). The pyruvate oxidase mutation decreased acetate production by 10% while the combination of both mutations decreased acetate production by 94%. The low level of acetate produced by TC44 containing both mutations is presumed to reflect a combination of multiple minor pathways and remains as a potential target for future improvement in pyruvate yield. Pyruvate can be produced by a variety of microorganisms including mutants of yeasts and bacteria (Table 4-4). Typical production rates for these biocatalysts are around 1 g L -1 h -1 with yields exceeding half the weight of substrate. Torulopsis glabrata, a yeast strain currently used for the commercial production, can achieve pyruvate titers of 69 g L -1 although yields are somewhat lower than for TC44 and more elaborate process controls are required. T. glabrata strains used in the commercial process are multivitamin auxotrophs requiring tight regulation of vitamin concentrations which result in complex vitamin feeding strategies during fermentation (Li et al. 2001a). Previous E. coli strains constructed for pyruvate production were cultured in complex media and have been plagued by low titers and yields (Tomar et al. 2003; Yokota et al. 1994). In contrast to previous biocatalysts where vitamins and other complex nutrients are required for effective production of pyruvate by fermentation, E. coli TC44 requires only mineral salts, glucose and very simple process controls. The lack of a requirement for vitamin

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Table 4-4. Comparison of biocatalysts for pyruvate production 75 Strain Relevant genotype/ phenotype Carbon Source Nitrogen Source Ferm-entation Time (h) [Pyruvate] (g L -1 ) Vol. Prod. (g L -1 h -1 ) Pyruvate yield (g g -1 ) Ref. Candida lipolytica AJ 14353 B 1 Met glucose NH 4 NO 3 72 44 0.61 0.44 Li et al. 2001a Debaryomyces hansenii Y-256 B 1 Bio glucose Peptone 96 42 0.44 0.42 Li et al. 2001a Torulopsis glabrata ACII-3 B 1 Bio B 6 NA acetate leaky glucose Soy hydrolysate (NH 4 ) 2 SO 4 47 60 1.28 0.68 Li et al. 2001b Torulopsis glabrata WSH-IP 303 B 1 Bio B 6 NA glucose NH 4 Cl 56 69 1.23 0.62 Li et al. 2001b Escherichia coli TBLA-1 lipA2 bgl + atpA401 glucose Poly-peptone 24 30 1.25 0.60 Yokota et al. 1994 Escherichia coli CGSC7916 aceF fadR adhE ppc glucose acetate Tryptone (NH 4 ) 2 HPO 4 36 35 0.97 0.65 Tomar et al. 2003 Escherichia coli TC44 pflB frdBC ldhA atpFH adhE sucA ackA poxB glucose (NH 4 ) 2 HPO 4 43 52 1.21 0.76 Causey et al. 2004

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76 supplements, complex nutrients or complicated process controls for TC44 should provide a substantial savings in production costs. In addition, the lack of complex nutrients in the fermentation broth should also reduce costs associated with product purification and waste disposal. Based on this comparison, E. coli TC44 appears to represent a competitive alternative to the current pyruvate-producing biocatalysts due to high yields, high product titers, simple fermentation conditions, and the ability to grow well in mineral salts medium with glucose as the sole carbon source. The remarkable tolerance of E. coli to drastic changes in metabolic flow that allows the production of acetate or pyruvate as alternative products of metabolism implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl~CoA. It is interesting to note that pyruvate transiently accumulated in broth during fermentations with TC36, but was rapidly co-metabolized with glucose during the latter stages of fermentation. Similar transient accumulation and re-utilization was previously observed in minimal medium for other central metabolites (carboxylic acids and acetate) in other derivatives of W3110 (Causey et al. 2003). Exogenously added pyruvate was shown to be rapidly mixed with intracellular pyruvate followed by dissimilation (Yang et al. 2001). Carboxylic acids (Lutgens and Gottschalk 1980; Underwood et al. 2004) and acetate (Causey et al. 2003; Underwood et al. 2002) are also co-metabolized with sugars. Active transport systems have been identified in E. coli for pyruvate (Lang et al. 1987), acetate (Gimenez et al. 2003), and dicarboxylic acids (Lo et al. 1972; Pos et al. 1998; Seol and Shatkin 1991). Thus for pyruvate and perhaps any other compound, the extracellular milieu may be reasonably regarded as a reservoir for the temporary expansion of metabolic pools. A similar argument can be made for

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77 CO 2 (Merlin et al. 2003) and for acetaldehyde during ethanol production (Dombek and Ingram 1984; Stanley and Pamment 1993; Stanley et al.1993). The transient accumulation of metabolites in the aqueous milieu may be of evolutionary advantage. This accumulation could arguably regarded as a form of metabolic conditioning of the environment, as a storage reservoir, or as an expanded metabolic pool to relieve a temporary imbalance in metabolic flux. Enzymes are relatively stable in E. coli. Substantial remodeling of metabolism is primarily regulated at the level of transcription with dilution during subsequent growth. Transient storage of products from imbalanced metabolism in the extracellular milieu could increase the metabolic flexibility of E. coli during adaptation to a changing environment

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CHAPTER 5 EFFECT OF BETAINE ON THE FERMENTATION PERFORMANCE OF TC44 (PYRUVATE PRODUCTION) AND TC36 (ACETATE PRODUCTION) Intoduction The membranes that encompass bacterial cells are permeable to water but provide a barrier against most solutes. High osmolarity of the extracellular environment would result in dehydration unless the cell is able to increase the intracellular solute concentration (compatable solutes). Compatable solutes may be taken up from the environment (K + proline, betaine, etc.) or synthesized by the organism (glutamate and trehalose). Escherichia coli compensates for hyperosmotic stress, first, by increasing intracellular K + and glutamate concentrations and, second, synthesis of trehalose (Csonka and Hanson 1991). In minimal medium, trehalose is synthesized and replaces K + and glutamate as the major compatable solute in the cytoplasm (Csonka and Hanson 1991). Other osmoprotectant compounds, such as betaine, are also used for osmoprotection when they are available (Perroud and Le Rudulier 1985). Betaine (and glycine betaine) is an effective osmoprotectant not synthesized by E. coli. Betaine has been demonstrated to protect bacteria against hyperosmotic stress as well as heat (reviewed in Csonka and Hanson 1991; Holtmann and Bremer 2004). In addition, the growth of ethanologenic E. coli KO11 was shown to be improved by the presence of betaine or choline in the growth medium (Underwood et al. 2004). Transport of betaine into the cytoplasm is through an energy dependent proton symport system (Perroud and Le Rudulier 1985; Styrvold et al. 1986). The transport protein, ProU, is 78

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79 membrane bound and is transcribed from the proU promoter (Csonka and Hanson 1991). The proV and proW gene products are the membrane-associated proteins and the proX gene product is the periplasmic binding protein (Csonka and Hanson 1991). Transport does not depend on the solute causing osmotic stress but on the magnitude of the osmotic shock (Perroud and Le Rudulier 1985). E. coli can also transport choline into the cell and oxidize the choline to glycine betaine (Landfald and Strm 1986). Betaine cannot be used as a carbon or nitrogen source (Perroud and Le Rudulier 1985). Betaine is effective as an osmoprotectant under both aerobic and anaerobic conditions but choline is only effective under aerobic conditions (Landfald and Strm 1986). Cayley et al. (1991) measured the cytoplasmic osmolytes of E. coli grown in MOPS-buffered glucose minimal medium, similar to the NBS medium used in our studies, with or without betaine. In the absense of betaine, K + accounted for 53% of the total osmolytes measured in the cytoplasm (Cayley et al. 1991; Cayley et al. 1992). Glutamate, trehalose and MOPS were present in minor quantities. Addition of betaine resulted in a modest increase in total cytoplasmic osmolyte pool (~ 10%), betaine accounted for 58% of the measured osmolytes (Cayley et al. 1991; Cayley et al. 1992). Potassium and glutamate were the only other osmolytes in the cytoplasm at concentrations above the limits of detection. In this chapter the effects of betaine on the growth and product formation of TC36 and TC44 are described. Growth of E. coli W3110 in glucose minimal medium supplemented with increasing concentrations of acetate or pyruvate in the presence or absence of betaine will also be discussed.

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80 Materials and Methods Microorganisms and Media Escherichia coli W3110 (wild type) was used to determine the minimum inhibitory concentration of acetate and pyruvate in the presence and absence of 1 mM betaine. The acetate producing strain TC36 (Table 2-1) and the pyruvate producing strain TC44 (Table 4-1) were used in 5 L fermentations. Strains were carried on NBS 2% glucose minimal plates as described in Chapter 2. Acid tolerance experiments were conducted with NBS 2% glucose minimal broth containing 0-1 M acetate or pyruvate. Seed culture and Batch fermentations were conducted as described in Chapter 4 (5 L working volume, 37 o C, 5% dissolved oxygen, 0.5 vvm gas flow and 350 rpm agitation) except that 10 % final glucose concentration was used. Duplicate fermentations were conducted. Acetate and Pyruvate Tolerance Acetate and pyruvate tolerance studies were conducted using NBS 2% glucose medium at pH 7 or pH 8 containing 0-1 M acetate or pyruvate. Betaine HCl was added to a final concentration of 1 mM or excluded from the broth. One millimolar betaine was previously determined to be the optimum concentration for protection against osmotic shock (Perroud and Le Rudulier 1985). Ten microliters of a logarimically growing culture of E. coli W3110 (~ 1 OD 550 ) was inoculated into 3 mL of the appropriate medium in a 13 x 100 mm test tube (triplicate cultures). The cultures were grown in a 37 o C water bath with shaking at 120 rpm. After incubation for 24 h the OD 550 was read using a Bausch & Lomb Spectronic 70 spectrophotometer.

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81 Results Effect of Betaine on Growth and Acetate Production for E. coli TC36 Betaine has previously been shown to protect E. coli from osmotic shock (reviewed in Csonka 1991; Underwood et al. 2004). Acetate fermentations with TC36, conducted using excess glucose, have acetate concentrations greater than 800 mM resulting in a 1.6 osmolar increase of the broth after neutralization with KOH. It was expected that addition of 1 mM betaine to the growth medium would improve growth rate by protecting TC36 from the osmotic stress imposed on the organism by 10% glucose. In addition, betaine was expected to increase acetate productivity by reducing the effect of the rising potassium acetate concentration throughout the fermentation. E. coli TC36 fermentations were conducted in minimal medium containing 10% glucose with or without 1 mM betaine. The maximum specific growth rate was increased 2.8-fold by the addition of 1 mM betaine to the culture medium (Table 5-1) although both cultures entered stationary phase at approximately 24 h (Fig. 5-1A). Betaine had no effect on volumetric consumption of glucose or acetate production rates (Table 5-1; Fig. 5-1B,C). The maximum specific glucose consumption rate was reduced by 19% when betaine was incorporated into the medium (Table 5-1). Initially, the specific rate of acetate production was higher in the betaine-grown cultures (Fig. 5-1C) resulting in an increase in the maximum specific acetate production rate of 17% (Table 5-1). The rate of acetate production was lower during stationary phase with betaine in the medium (Fig. 5-1C). Overall, betaine did not improve the performance of TC36.

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82 Table 5-1. Comparison of metabolic rates for TC36 in the presence or absence of 1 mM betaine Glucose consumption rate a Acetate production rate a E. coli TC36 (h -1 ) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Betaine 0.15 10 16 10 20 +Betaine 0.42 11 13 11 24 a The data represent an average of two fermentations. b Average volumetric rates. c Maximum specific rates per gram of dry cell weight. Effect of Betaine on Growth and Pyruvate Production for E. coli TC44 Similar to acetate fermentations with TC36, strain TC44 experiences hyperosmotic environments when glucose and pyruvate concentrations are high. It was expected that addition of 1 mM betaine would at least partially alleviate the osmotic stress imposed on TC44. The maximum specific growth rate was increased 3.2-fold by the addition of betaine (Table 5-2). Cell mass reached 2 g L -1 by 24 h, more than double the cell mass at 60 h when betaine was not present (Fig. 5-2A). Betaine had a dramatic positive effect on both glucose consumption and pyruvate production (Fig. 5-2B,C). All of the glucose was consumed in 60 h when betaine was present, yet in the absence of betaine 90% of the glucose remained after 60 h (Fig. 5-2B). The average volumetric rate of glucose consumption was increased 10-fold and the specific rate of glucose consumption was increased 5.8-fold by the incorporation of betaine in the medium (Table 5-2). Table 5-2. Comparison of metabolic rates for TC44 in the presence or absence of 1 mM betaine Glucose consumption rate a Pyruvate production rate a E. coli TC44 (h -1 ) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Average Vol. b (mmol L-1 h-1) Max. Spec. c (mmol L-1 h-1 g-1) Betaine 0.09 1 6 2 13 +Betaine 0.29 10 35 16 32 a The data represent an average of two fermentations. b Average volumetric rates. c Maximum specific rates per gram of dry cell weight.

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83 0 6 12 18 24 30 36 42 48 54 60 0.01 0.1 1 10 A GrowthTime (h)Cell Mass (g L-1) 0 6 12 18 24 30 36 42 48 54 60 0 100 200 300 400 500 600 700B GlucoseTime (h)Glucose(mmoles L-1) 0 6 12 18 24 30 36 42 48 54 60 0 100 200 300 400 500 600 700C AcetateTime (h)Acetate (mmoles L-1) Figure 5-1. Effect of 1 mM Betaine on growth and acetate production for TC36. Fermentations were conducted in NBS medium with 10% glucose as the carbon source. Betaine was added to a final concentration of 1 mM when indicated. Fermentations were performed in duplicate. A) Growth. B) Glucose consumption. C) Acetate production. Symbols: Solid squares, + betaine; Open squares, -betaine.

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84 Volumetric and specific pyruvate production rates were dramatically increased 8-fold and 2.5-fold, respectively. In contrast to TC36, betaine improved both growth rate and production rates for TC44. Determination of the Minimal Inhibitory Concentration of Acetate and Pyruvate on E. coli W3110 in NBS Medium There were two possibilities that could explain drastic difference between the fermentation performance of the acetate producing strain, TC36, and the pyruvate producing strain, TC44. One possibility was that TC36 lost the ability to transport betaine by mutation(s) in the proU operon. Although possible, it is unlikely that TC36 lost the ability to transport betaine considering that TC44 was constructed from TC36 (Chapter 4). The more likely conclusion is that betaine was unable to provide the same level of protection for acetate as it did for pyruvate. Therefore, we determined the minimum inhibitory concentration for acetate and pyruvate in E. coli W3110. E. coli W3110 was grown in NBS salts containing 2% glucose and 0-1M acetate or pyruvate as described in the Materials and Methods section. In the absence of betaine the minimum inhibitory concentration for acetate was 300 mM (Fig. 5-3A). The minimum pyruvate concentration necessary to stop growth was about 300 mM, 100 mM greater than that for acetate (Fig. 5-3A). Growth was partially inhibited by both acetate and pyruvate at the lowest concentration (100 mM) tested in the absence of betaine (Fig. 5-3A). Addition of 1 mM betaine did not increase the minimum concentration of acetate necessary to stop growth of W3110 (Fig 5-3B). Betaine may increase the acetate tolerance slightly, of W3110 at low (< 300 mM) concentrations. The minimum pyruvate concentration necessary to completely inhibit growth of W3110 was increased 29% to 700 mM with the addition of 1 mM betaine (Fig. 5-3B). In addition, up to

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85 200 mMpyruvate had a positive effect on the growth of W3110 when betaine was present in the medium. 0 6 12 18 24 30 36 42 48 54 60 0.01 0.1 1 10 A GrowthTime (h)Cell Mass (g L-1) 0 6 12 18 24 30 36 42 48 54 60 0 100 200 300 400 500 600 700 800B Glucose ConsumptionTime (h)Glucose (mmoles L-1) 0 6 12 18 24 30 36 42 48 54 60 0 100 200 300 400 500 600 700 800Time (h)Pyruvate (mmoles L-1)C Pyruvate production Figure 5-2. Effect of 1 mM Betaine on growth and pyruvate production for TC44. Fermentations were conducted in NBS medium with 10% glucose as the carbon source. Fermentations were performed in duplicate. A) Growth. B) Glucose consumption. C) Pyruvate production. Symbols: Solid circles, 1 mM betaine added to the medium; Open circles, no betaine added to the medium.

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86 0 100 200 300 400 500 600 700 800 900 1000 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50Concentration (mM)Growth (OD550) 0 100 200 300 400 500 600 700 800 900 1000 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50Concentration (mM)Growth (OD550)A Acetate betaineB Pyruvate betaine 0 100 200 300 400 500 600 700 800 900 1000 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50Concentration (mM)Growth (OD550) 0 100 200 300 400 500 600 700 800 900 1000 0.00 0.15 0.30 0.45 0.60 0.75 0.90 1.05 1.20 1.35 1.50Concentration (mM)Growth (OD550)A Acetate betaineB Pyruvate betaine Figure 5-3. Effect of 1 mM betaine on the minimum inhibitory concentration of acetate and pyruvate for E. coli W3110. W3110 was grown in 3 mL of NBS salts containing 2% glucose, 0-1 M A) acetate or B) pyruvate. Results plotted from triplicate experiments. Symbols: Solid squares, no betaine; Open squares, 1 mM betaine added to the medium.

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87 Discussion Betaine did not increase acetate productivity by strain TC36. Early in the fermentation, when glucose levels were high and acetate levels were low, incorporating betaine into the medium resulted in a 17% increase in the growth rate. By 24 h the acetate concentration was 300 mM and TC36 was in stationary phase (Fig 5-1A,C). Acetate production was higher for fermentations that included betaine in the medium during the first 24 h. These data are consistant with the studies performed to determine the minimum inhibitory concentration of acetate necessary to stop the growth of E. coli W3110. Acetate was found to be growth inhibitory at 300 mM regardless of the presence of betaine. Growth was slightly elevated at lower acetate concentrations when betaine was present in the medium. This would explain the increased acetate production early during fermentations containing betaine and no benefit once acetate reaches 300 mM. Pyruvate fermentations with TC44 were greatly enhanced when betaine was incorporated into the medium. All of the metabolic parameters measured were increased in fermentations containing 1 mM betaine. Pyruvate production continues until all of the glucose is consumed. These data are consistant with growth inhibition studies that showed 2.3-fold increase in tolerance to pyruvate when 1 mM betaine was in the medium. Why would betaine be beneficial for pyruvate and not for acetate? One explaination could be the difference in pKa values resulting in different concentrations of the protonated species. The protonated, or uncharged, form of weak acids are considered to be toxic because the uncharged form of the acid is able to traverse the cytoplasmic membrane. The charged form cannot freely diffuse across the membrane. Acetate has a pKa of 4.76 and pyruvate has a pKa of 2.49. Applying the Henderson-Hasselbach

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88 equation the ratio of the conjugate acid (HA) to the conjugate base (A ) can be calculated. Although the conjugate acid concentration is very low for both acids at pH 7, the [A ]/[HA] is 187-fold higher for pyruvate than for acetate. Lactic acid, which has a pKa value intermediate (3.86) between pyruvate and acetate, also has a minimum inhibitory concentration (500 mM) between that of pyruvate and acetate ( personal communication with S. Zhou). To date there has been no published study comparing the tolerance of E. coli growing in the presence of weak organic acids when betaine is present or not present in the culture medium.

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CHAPTER 6 CONCLUSIONS E. coli is a facultative anaerobe producing a mixture of organic acids and ethanol from glucose for NAD + regeneration under low oxygen tension. Energy for biosynthesis is derived by oxidative substrate-level phosphorylation (glycolysis and acetate kinase) which results in 2-4 moles ATP produced per mole of glucose consumed. When oxygen tension is high, NADH produced during catabolism is oxidized by shuttling electrons down the electron transport chain. The transfer of electrons is coupled to the extrusion of protons generating a proton motive force. Protons are transported down the concentration gradient by the (F 1 F 0 )H + -ATP synthase with the energy released stored in ATP. A calculated maximum of 25-33 ATP equivalents per glucose (depending on electron fluxes through NADH dehydrogenase I or II and cytochrome bo or cytochrome bd oxidases) are produced under optimal aerobic conditions (Calhoun et al. 1993). When oxygen tension is low, E. coli derives all of the energy for biosynthesis from oxidative substrate level phosphorylation. NADH oxidation is coupled to the reduction of internal metabolites, such as pyruvate and acetyl-CoA. Therefore, fermentative growth of E. coli results in conversion of glucose carbon into reduced products with minimal carbon loss to cell mass and CO 2. In contrast to fermentative growth, aerobic metabolism results in the production of cell mass and CO 2 in an approximate fifty-fifty ratio under optimal conditions (Ingraham et al. 1990). The goal of the work described in this document was to combine aspects of anaerobic and aerobic metabolism in E. coli for the production of chemicals, not limited 89

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90 to reduced compounds, from carbohydrate. The biocatalyst was constructed by making deletions in genes (frdBC, pflB, ldhA, adhE) coding for enzymes that produce the fermentations products (lactate, formate, ethanol, and succinate). Substrate utilization rate was increased by limiting ATP production through oxidative phosphorylation while maintaining cytoplasmic ATPase activity (deletion of atpFH). Substrate loss to CO 2 was limited by blocking the cyclic function of the TCA cycle (deletion of sucA). The strain incorporating all of the mutations mentioned above, efficiently converted glucose to acetate, a compound that is redox neutral relative to glucose. Figure 6-1 outlines the benefits derived each set of mutations. Chromosomal deletions that eliminated the major pathways for organic acid production did not increase acetate production and fermentation time was not significantly increased (Fig. 6-1A,B). Cell mass increased slightly due to the low accumulation of organic acid in the medium (Fig. 6-1A,B). Construction of the atpFH mutation alone resulted in a 4.5-fold increase in acetate production and a reduction in the fermentation time by half compared with W3110 (Fig. 6-1A,C). Combining the mutations that limited organic acid production with the atpFH deletion resulted in a further increase in acetate production over TC40 (Fig. 6-1C,D). Incorporating mutations limiting carbon loss to CO 2 and disruption of the TCA cycle into TC24 (strain TC36) resulted in acetate produced at 70% of the theoretical yield with minimal carbon lost to cell mass or CO 2 (Fig. 6-1E). During glucose fermentations to acetate with TC36, pyruvic acid would accumulate during logarithmic growth and subsequently be consumed when glucose became limiting. The transient accumulation of pyruvate indicated that TC36 could be engineered for

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0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM)TC36(ldhA, focA-pflB, frdBC,atpFH, adhE, sucA)TC24(ldhA, focA-pflB, frdBC, atpFH)TC40(atpFH)W3110 (wild type)SZ47(ldhA, focA-pflB, frdBC)14.0 OD55010.7 OD55013.3 OD55012.9 OD55015.0 OD550AEDCB 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM) 0 5 10 15 20 25 30 0 50 100 150 200 250Time (h)Concentration (mM)TC36(ldhA, focA-pflB, frdBC,atpFH, adhE, sucA)TC24(ldhA, focA-pflB, frdBC, atpFH)TC40(atpFH)W3110 (wild type)SZ47(ldhA, focA-pflB, frdBC)14.0 OD55010.7 OD55013.3 OD55012.9 OD55015.0 OD550AEDCB 91 Figure 6-1. Fermentation profiles for selected strains during the construction of E. coli TC36. Optical densities represent the maximum cell mass attained. A) Wild type fermentation profile. B) Elimination of fermentation products. C) Elimination of oxidative phosphorylation. D) Elimination of fermentation pathways and oxidative phosphorylation. E) Elimination of fermentation pathways, oxidative phosphorylation and limiting CO 2 production. Symbols: Solid squares, glucose; Open squares, acetate; Open circles, total dicarboxylic acids.

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92 pyruvate production by eliminating acetate production pathways, AckA-Pta and PoxB. The major acetate-producing pathway in E. coli is encoded by the ackA (acetate kinase) and pta (phosphotransacetylase) genes. Acetate is produced from acetyl-CoA through the phosphorylated intermediate acetyl-phosphate. Pyruvate oxidase (poxB gene product) produces acetate directly from the decarboxylation of pyruvate. Chromosomal deletions were constructed in ackA and poxB to eliminate acetate production (strain TC44). Strain TC44 was able to convert glucose to pyruvate at 78% of the maximum theoretical yield. Both biocatalysts (TC36 and TC44) experience osmotic stress due to high sugar and salt concentrations during fermentations. Betaine added to the fermentation medium resulted in improved growth and pyruvate production with strain TC44. Betaine had no positive effect on TC36 fermentations. We believe the lack of improvement observed for TC36 fermentations was not due to differences between the strains, TC36 and TC44, but to the different effects of acetate and pyruvate on the cells. At pH 7, 1 mM betaine did not improve the tolerance of E. coli W3110 to acetate but greatly increased the growth when pyruvate was used instead. E.coli TC36 and TC44 are versatile biocatalysts that can be used to convert the carbohydrate fraction of agricultural waste into useful compounds. The biocatalysts are not limited to the production of reduced compounds due to an intact electron transport chain. Tolerance to product may also be improved in medium containing low concentrations of betaine or choline. These biocatalysts will prove useful for the production of chemicals from plant biomass.

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LIST OF REFERENCES Akesson, M., P. Hagander, and J. P. Axelsson. 2001. Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding. Biotechnol. Bioeng. 73:223-230. Altaras, N. E. and D. C. Cameron. 1999. Metabolic engineering of a 1,2-propanediol pathway in Escherichia coli. Appl. Environ. Microbiol. 65:1180-1185. Amarasingham, C. R. and B. D. Davis. 1965. Regulation of -ketoglutarate dehydrogenase formation in Escherichia coli. J. Biol. Chem. 240:3664-3668. Anonymous. Bright development prospect for calcium pyruvate and pyruvic acid. China Chem. Rep. 6 October 2002. Aristidou, A. A., K. San, and G. N. Bennett. 1995. Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction. Biotechnol. Prog. 11:475-478. Asghari, A., R. J. Bothast, J. B. Doran, and L. O. Ingram. 1996. Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11. J. Ind. Microbiol. 16:42-47. Beck, B. J., C. C. Aldrich, R. A. Fecik, K. A. Reynolds, and D. H. Sherman. 2003. Iterative chain elongation by a pikromycin monomodular polyketide synthase. J. Am. Chem. Soc. 125:4682-4683. Berraud, C. 2000. Production of highly concentrated vinegar in fed-batch culture. Biotechnol. Lett. 22:451-454. Blair, G. T. 1999. in Concise Encyclopedia of Chemical Technology, eds. Kroschwitz, J. I., M. Howe-Grant, P. M. Siegel, B. Richman, E. Zayatz, L. Altieri, M. Bickford, and L. Humphreys. (Wiley, New York), p. 1101. Bogachev, A. V., R. A. Murtazina, and V. P. Skulachev. 1996. H + /e stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells. J. Bacteriol. 178:6233-6237. Boyer, P. D. 1997. The ATP synthase -a splendid molecular machine. Annu. Rev. Biochem. 66:717-749. 93

PAGE 106

94 Calhoun, M. W., K. L. Oden, R. B. Gennis, M. J. T. de Mattos, and O. M. Neijssel. 1993. Energetic efficiency of Escherichia coli: Effects of mutations in components of the aerobic respiratory chain. J. Bacteriol. 175:3020-3025. Cameron, D. C., N. E. Altaras, M. L. Hoffman, A. J. Shaw. 1998. Metabolic engineering of propanediol pathways. Biotechnol. Progr. 14:116-125. Campbell, H. D. and I. G. Young. 1983. Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli Biochem. 22:5754-5760. Causey, T. B., S. Zhou, K. T. Shanmugam, L. O. Ingram. 2003. Engineering Escherichia coli W3110 for the conversion of sugar to redox-neutral and oxidized products: Homoacetate production. Proc. Natl. Acad. Sci, USA. 100:825-832. Cayley, S., B. A. Lewis, H. J. Guttman, and M. T. Record, Jr. 1991. Characteization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in Vivo. J. Mol. Biol. 222:281-300. Cayley, S., B. A. Lewis, and M. T. Record, Jr. 1992. Origins of the osmoprotective properties of betaine and proline in Escherichia coli K-12. J. Bacteriol. 174:1586-1595. Chang, D., S. Shin, J. Rhee, and J. Pan. 1999. Homofermentative production of Dor L-lactate in metabolically engineered Escherichia coli RR1. Appl. Environ. Microbiol. 65:1384-1389. Chang, Y.-Y. and J. E. Cronan, Jr. 1983. Genetic and biochemical analysis of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase. J. Bacteriol. 154:756-762. Chang, Y.-Y., A.-Y. Wang, and J. E. Cronan, Jr. 1994. Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor enocoded by the rpoS (katF) gene. Mol. Microbiol. 11:1019-1028. Chao, Y., and J. C. Liao. 1994. Metabolic responses to substrate futile cycling in Escherichia coli. J. Biol. Chem. 269:5122-5126. Cheryan, M., S. Parekh, M. Shah and K. Witjitra. 1997. Production of acetic acid by Clostridium thermoaceticum. Adv. Appl. Microbiol. 43:1-33. Chotani, G., T. Dodge, A. Hsu, M. Kumar, R. LaDuca, D. Trimbur, W. Weyler, and K. Sanford. 2000. The commercial production of chemicals using pathway engineering. Biochim. Biophys. Acta 1543:434-455. Contiero, J., C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe. 2000. Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli. J. Ind. Microbiol. 24:421-430.

PAGE 107

95 Csonka, L. N. 1991. Prokaryotic osmoregulation: genetics and physiology. Annu. Rev. Microbiol. 45:569-606. Cunnungham, C. C. and L. P. Hager. 1975. Reactivation of the lipid-depleted pyruvate oxidase system from Escherichia coli with cell envelope neutral lipids. J. Biol. Chem. 250:7139-7146. Cunningham, L. and J. R. Guest. 1998. Transcription and transcript processing in the sdhCDAB-sucABCD operon of Escherichia coli. Microbiology 144:2113-2123. Danner, H. and R. Braun. 1999. Biotechnology for the production of commodity chemicals from biomass. Chem. Soc. Rev. 28:395-405. Datsenko, K. A. and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645. Dayem, L. C., J. R. Carney, D. V. Santi, B. A. Pfeifer, C. Khosla, and J. T. Kealey. 2002. Metabolic engineering of a methylmalonyl-CoA mutase epimerase pathway for complex polyketide biosynthesis in Escherichia coli. Biochem. 41:5193-5201. Deville-Bonner, D., F. Bourgain, and J. R. Garel. 1991. pH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli. Biochem. 30:5750-5754. Dien, B. S., N. N. Nichols, and R. J. Bothast. 2001. Recombinant Escherichia coli engineered for the production of L-lactic acid from hexose and pentose sugars. J. Ind. Microbiol. Biotechnol. 27:259-264. Dombek, K. M. and L. O. Ingram. 1984. Effects of ethanol on the Escherichia coli plasma membrane. J. Bacteriol. 157:233-239. Donnelly, M. I., C Sanville-Millard, and R. Chatterjee. 1998. Method for construction of bacterial strains with increased succinic acid production. U. S. Patent No. 6159738. Eggeling, L., W. Pfefferle, and H. Sahm. 2001. Amino acids, p 281-304. In C. Ratledge and B. Kristiansen (ed.), Basic Biotechnology, 2 nd edition. Cambridge University Press, Cambridge, U.K. Erni, B., B. Zanolari and H. P. Kocher. 1987. The mannose permease of Escherichia coli consists of three different proteins. J. Biol. Chem. 262:5238-5247. Evans, D. J., Jr. 1969. Membrane adenosine triphosphate of Escherichia coli: activation by calcium ion and inhibition by cations. J. Bacteriol. 100:914-922. Farmer, W. R., and J. C. Liao. 1997. Reduction of aerobic acetate production by Escherichia coli. 1997. Appl. Environ. Microbiol. 63:3205-3210.

PAGE 108

96 Freer, S. N. 2002. Acetic acid production by Dekkera/Brettanomyces yeasts. World J. Microbiol. Biotechnol. 18:271-275. Gimenez, R., M. F. Nuez, J. Badia, J. Aguilar. J. Baldoma. 2003. The gene yjcG, cotranscribed with the gene acs, encodes an acetate permease in Escherichia coli. J. Bacteriol. 185:6448-6455. Henderson, P. J. F. 1990. Proton-linked sugar transport systems in bacteria. J. Bioenerg. Biomembranes. 22:525-568. Hofmeyer, J.-H. S., and A. Cornish-Bowden. 2000. Regulating the cellular economy of supply and demand. FEBS Lett. 467:47-51. Holtmann, G. and E. Bremer. 2004. Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of Opu transporters. J. Bacteriol. 186:1683-1693. Ingledew, W. J. and R. K. Poole. 1984. The respiratory chains of Escherichia coli. Microbiol. Rev. 48:222-271. Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou. 1999. Enteric bacterial catalyst for fuel ethanol production. Biotechnol. Prog. 15:855-866. Ingram, L. O., T. Conway, D. P. Clark, G. W. Sewell, and J. F. Preston.1987. Genetic engineering of ethanol production in Escherichia coli. Appl. Environ. Microbiol. 53:2420-2425. Iuchi, S. and L. Weiner. 1996. Cellular and molecular physiology of Escherichia coli in the adaptation to aerobic environments. J. Biochem. 120:1055-1063. Jaworowski, A., H. D. Campbell, M. I. Poulis, and I. G. Young. 1981. Genetic identification and purification of the respiratory NADH dehydrogenase of Escherichia coli. Biochem. 20:2041-2047. Jensen, P. R. and O. Michelsen. 1992. Carbon and energy metabolism of atp mutants of Escherichia coli. J. Bacteriol. 174:7635-7641. Kang, H.-Y., S. Song, and C. Park. 1998. Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons. Mol. Cells. 8:318-323. Kiley, P. J. and H. Beinert. 1999. Oxygen sensing by the global regulator, FNR: the role of the iron-sulfur cluster. FEMS Microbiol. Rev. 22:341-352. Kirkpatrick, C., L. M. Maurer, N. E. Oyelakin, Y. N. Yoncheva, R. Maurer, and J. L. Slonczewski. 2001. Acetate and formate stress: Opposite responses in the proteomes of Escherichia coli. J. Bacteriol. 183:6466-6477.

PAGE 109

97 Kirschner, M. Acetic acid chemical profile. Chemical Market Reporter. March 31, 2003. Koebmann, B. J., H. V. Westerhoff, J. L. Snoep, D. Nilsson, and P. R. Jensen. 2002. The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 184:3909-3916. Koland, J. G., M. J. Miller, R. B. Gennis. 1984. Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase. Biochem. 23:445-453. Krebs, A. and W. A. Bridger. 1976. On the monomeric structure and proposed regulatory properties of phospoenolpyruvate carboxykinase of Escherichia coli. Can. J. Biochem. 54:22-26 Landfald, B., and A. R. Strm. 1986. Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J. Bacteriol. 165:849-855. Lang, V. J., C. L.-Lantz, and R. A. Cook. 1987. Characterization of the specific pyruvate transport system in Escherichia coli K-12. J. Bacteriol. 169:380-385. Lasko, D. R., N. Zamboni, and U. Sauer. 2000. Bacterial response to acetate challenge: a comparison of tolerance among species. Appl. Microbiol. Biotechnol. 54:243-247. Lee, P. C., and C. Shmidt-Dannert. 2002. Metabolic engineering towards biotechnological production of carotenoids in microorganisms. Appl. Microbiol. Biotechnol. 60:1-11. Li, Y., J. Chen, and S.-Y. Lun. 2001. Biotechnological production of pyruvic acid. Appl. Microbiol. Biotechnol. 57:451-459. Li, Y., J. Chen, S.-Y. Lun, and X. S. Rui. 2001. Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels. Appl. Microbiol. Biotechnol. 55:680-685. Linton, K. J. and C. F. Higgins. 1998. The Escherichia coli ATP-binding cassette (ABC) proteins. Mol. Microbiol. 28:5-13. Lo, T. C. Y., M. K. Rayman, and B. D. Sanwal. 1972. Transport of Succinate in Escherichia coli: Biochemical and genetic studies of transport in whole cells. J. Biol. Chem. 247:6323-6331. Luli, G. W. and W. R. Strohl. 1990. Comparison of growth, acetate production and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl. Environ. Microbiol. 56:1004-1011. Lutgens, M. and G. Gottschalk. 1980. Why a co-substrate is required for the anaerobic growth of Escherichia coli on citrate. J. Gen. Microbiol. 119:63-70.

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98 Martinez-Morales, F., A. G. Borges, A. Martinez, K. T. Shanmugam, and L. O. Ingram. 1999. Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons during construction. J. Bacteriol. 181:7143-7148. McKendry, P. 2002. Energy production from biomass (part 1): overview of biomass. Biores. Technol. 83:37-46. Meinhardt, S. W., K. Matsushita, H. R. Kaback, and T. Ohnishi. 1989. EPR characterization of the iron-sulfur-containing NADH-ubiquinone oxidoreductase of the Escherichia coli aerobic respiratory chain. Biochem. 28:2153-2160. Merlin, C., M. Masters, S. McAteer, and A. Coulson. 2003. Why is carbonic anhydrase essential to Escherichia coli? J. Bacteriol. 185:6415-6424. Miller, J. H. 1992. A short course in bacterial genetics: A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Press, Cold Spring Harbor, NY. Miller, M. J. and R. B. Gennis. 1985. The cytochrome d complex is a coupling site in the aerobic respiratory chain of Escherichia coli. J. Biol. Chem. 260:14003-14008. Nakamura, C.E., A.A. Gatenby, Hsu, A. K.-H., R. D. La Reau, S. L. Haynie, M. Diaz-Torres, D. E. Trimbur, G. M. Whited, V. Nagarajan, M. S. Payne, S. K. Picataggio, and R. V. Nair. 2000. Method for the production of 1,3-propanediol by recombinant microorganisms. U. S. Patent. No. 6,013,494. Neidhardt, F. C., R. Curtiss, III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology, 2 nd Ed. Neidhardt, F. C., J. L. Ingraham, and M. Schaechter. 1990. Physiology of the bacterial cell: A molecular approach. Sinauer Associates, Inc., Sunderland, Mass. Niu, W., K. M. Draths, and J. W. Frost. 2002. Benzene-free synthesis of adipic acid. Biotechnol. Prog. 18:201-211. Ondrey, G. 2001. An enzymatic process to make pyruvic acid. Chem. Eng. 108:21. Park, S.-J., G. Chao, and R. P. Gunsalus. 1997. Aerobic regulation of the sucABCD gene of Escherichia coli, which encode -ketoglutarate dehydrogenase and succinyl coenzyme A synthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter. J. Bacteriol. 179:4138-4142. Patnaik, R., W. D. Roof, R. F. Young, and J. C. Liao. 1992. Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle. J. Bacteriol. 174:7525-7532.

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99 Perroud, B. and D. Le Rudulier. 1985. Glycine betaine transport in Escherichia coli: osmotic modulation. J. Bacteriol. 161:393-401. Pirt, S. J. 1982. Maintenance energy: A general model for energy-limited and energy-sufficient growth. Arch Microbiol. 133:300-302. Pirt, S. J. 1987. The energetics of microbes at slow growth rates: Maintenance energies and dormant organisms. J. Ferment. Technol. 65:173-177. Pos, K. M., P. Dimroth, and M. Bott. 1998. The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts. J. Bacteriol. 180:4160-4165. Posfai, G., M. D. Koob, H. A. Kirkpatrick, and F. C. Blattner. 1997. Versatile insertion plasmids for targeted genome manipulations in bacteria: Isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome. J. Bacteriol. 179:4426-4428. Pozzan, T., V. Miconi, F. DiVergilio and G. F. Azzone. 1979. H + /site, charge/site, and ATP/site ratios at coupling sites I and II in mitochondrial e transport. J. Biol. Chem. 254:10200-10205. Puustinen A., M. Finel, T. Haltia, R. B. Gennis, and M. Wikstrm. 1991. Properties of the two terminal oxidases of Escherichia coli. Biochem. 30:3936-3942. Puustinen, A., M. Finel, M. Virkki, and M. Wikstrm. 1989. Cytochrome o (bo) is a proton pump in Paracoccus denitrificans and Escherichia coli. FEBS Lett. 249:163-167. Rathbun, W. B., and M. V. Betlach. 1969. Estimation of enzymatically produced orthophosphate in the presence of cysteine and adenosine triphosphate. Anal. Biochem. 20:436-445. Rice, C. W. and W. P. Hempfing. 1978. Oxygen-limited continuous culture and respiratory energy conservation in Escherichia coli. J. Bacteriol. 134:115-124. Sambrook, J. and D. W. Russell. 2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NY. Schmid, A., J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, and B. Witholt. 2001. Industrial biocatalysis today and tomorrow. Nature. 409:258-268. Seol, W. and A. J. Shatkin. 1991. Escherichia coli kgtP encodes an -ketoglutarate transporter. Proc. Natl. Acad. Sci., USA. 88:3802-3806. Sorgen, P. L., T. L. Caviston, R. C. Perry, and B. D. Cain. 1998. Deletions in the second stalk of F1F0-ATP synthase in Escherichia coli. J. Biol. Chem. 273:27873-27878.

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100 Spiro, S. and J. R. Guest. 1991. Adaptive responses to oxygen limitation in Escherichia coli. TIBS. 16:310-314. Stanley, G. A., N. G. Douglas, E. J. Every, T. Tzanatos, and N. B. Pamment. 1993. Inhibition and stimulation of yeast growth by acetaldehyde. Biotechnol. Lett. 15:1199-1204. Stanley, G. A. and N. B. Pamment. 1993. Transport and intracellular accumulation of acetaldehyde in Saccharomyces cerevisiae. Biotechnol. Bioeng. 42:24-29. Stouthamer, A. H. 1979. The search for correlation between theoretical and experimental growth yields. Int. Rev. Biochem. 21:1-47. Stouthamer, A. H. and C. W. Bettenhaussen. 1977. A continuous culture study of an ATPase-negative mutant of Escherichia coli. Arch. Miocrobiol. 113:185-189. Stringer, J. 1996. Biologically derived chemicals find niches. Chem. Week. 158:52. Strohl, W. R. 2001. Biochemical engineering of natural product biosynthesis pathways. Metab. Eng. 3:4-14. Styrvold, O. B., P. Falkenberg, B. Landfald, M. W. Eshoo, T. Bjrnsen, and A. R. Strm. J. Bacteriol. 165:856-863. Sun, Y. and J. Cheng. 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technol. 83:1-11. Suzuki, T. 1969. Phosphotransacetylase of Escherichia coli B, activation by pyruvate and inhibition by NADH and certain nucleotides. Biochim. Biophys. Acta 191:559-569. Tao, H., R. Gonzalez, A. Martinez, M. Rodriguez, L. O. Ingram, J. F. Preston, and K. T. Shanmugam. 2001. Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J. Bacteriol. 183:2979-2988. Tomar, A., M. A. Eiteman, and E. Altman. 2003. The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli. Appl. Microbiol. Biotechnol. 62:76-82. Tong, I., H. H. Liao, and D. C. Cameron. 1991. 1,3-propanediol production by Escherichia coli expressing genes from the Klebsiellapneumoniae-DHA regulon. App. Env. Microbiol. 57:3541-3546. Tsai, P. S., M. Nageli, and J. E. Bailey. 2002. Intracellular expression of Vitreoscilla hemoglobin modifies micro-aerobic Escherichia coli metabolism through elevated concentration and specific activity of the cytochrome o. Biotechnol. Bioeng. 79:558-567.

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101 Tseng, C.-P., J. Albrecht, and R. P. Gunsalus. 1996. Effect of microaerophilic cell growth conditions on expression of the aerobic (cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia coli. J. Bacteriol. 178:1094-1098. Underwood, S. A., M. L. Buszko, K. T. Shanmugam, and L. O. Ingram. 2004. Lack of protective osmolytes limits cell yield and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation. Appl. Environ Microbiol. 70:2734-2740. Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano, K.T. Shanmugam, and L. O. Ingram. 2002. Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli. Appl. Environ. Microbiol. 68:6263-6272. United States Department of Energy/Energy Information Agency. 2004. Energy outlook 2004. DOE/EIA-0383(2004). http://www.eia.doe.gov/emeu/plugs/plaeo04.html Varma, A., B. W. Boesch, and B. O. Palsson. 1993. Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates. Appl. Environ. Microbiol. 59:2465-2473. Vemuri, G. N., M. A. Altman, and E. Altman. 2002. Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli. J. Bacteriol. 68:1715-1727. Wang, Chia-wei, Min-Kyu Oh, J. C. Liao. 2000. Directed evolution of metabolically engineered Escherichia coli for carotenoid production. Biotechnol. Prog. 16:922-926. Weidner, U., U. Nehls, R. Schneider, W. Fecke, H. Leif, A. Schmeide, T. Freidrich, R. Zensen, U. Schulte, T. Ohnishi, and H. Weiss. 1992. Molecular genetic studies of complex I in Neurospora crassa, Aspergillus niger, and Escherichias coli. Biochim. Biophys. Acta. 1101:177-180. Weitzman, P. D. J. 1966. Regulation of citrate synthase activity in Escherichia coli. Biochim. Biophys. Acta. 128:213-215. Wikstrm, M. and T. Penttil. 1982. Critical evaluation of the proton-translocating property of cytochrome oxidase in rat liver mitochondria. FEBS Lett. 144:183-189. Wimpenny, J. W. T. and A. Firth. 1972. Levels of nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide in falcultative bacteria and the effect of oxygen. J. Bacteriol. 111:24-32. Yang, Y.-T., G. N. Bennett, and K.-Y. San. 2001. The effects of feed and intracellular pyruvate levels on the redistribution of metabolic fluxes in Escherichia coli. Metab. Eng. 3:115-123.

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102 Yokota, A., Y. Terasawa, N. Takaoka, H. Shimizu, and F. Tomita. 1994. Pyruvic acid production by an F 1 -ATPase-defective mutant of Escherichia coli W1485lip2. Biosci. Biotech. Biochem. 58:2164-2167. Yomano, L. P., S. W. York, and L. O. Ingram. 1998. Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production. J. Ind. Microbiol. Biot. 20:132-138. Young, I. G., B. L. Rogers, H. D. Campbell, A. Jaworoski and D. C. Shaw. 1981. Nucleotide sequence coding for the respiratory NADH dehydrogenase of Escherichia coli. Eur. J. Biochem. 116:165-170. Zaldivar, J., and L. O. Ingram. 1999. Effects of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol. Bioengin. 66:203-210. Zhang, J. and R. Greasham. 1999. Chemically defined media for commercial fermentations. Appl. Microbiol. Biotechnol. 51:407-421. Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam and L. O. Ingram. 2003. Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl. Environ. Microbiol. 69:399-407.

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BIOGRAPHICAL SKETCH Bryce was born in Forrest City, Arkansas on October 3, 1973. He lived in Arkansas until 1989, at which time he moved with his family to Destin, Florida. Bryce graduated from Fort Walton Beach High School in 1992. Shortly after graduation, he joined the United States Army. He served in the field artillary for a little more than two years. After he left the Army Bryce worked for his father and attended Okaloosa-Walton Community College where he obtained an Associate of Arts degree. Bryce then attended the University of Florida where he graduated with honors in 1999 with a BS in microbiology. In August of 1999, Bryce began graduate school in the Department of Microbiology and Cell Science at the University of Florida. Bryce worked on engineering the metabolism of Escherichia coli for the production of fuels and chemicals while studing under the giudence of Dr. Lonnie Ingram. During graduate school, Bryce met his wife, Celeste, also pursuing graduate studies in microbiology. 103


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Permanent Link: http://ufdc.ufl.edu/UFE0005561/00001

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Title: Engineering the Metabolism of Escherichia coli for the Synthesis of Oxidized Products: Acetate and Pyruvate Production
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Copyright Date: 2008

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Material Information

Title: Engineering the Metabolism of Escherichia coli for the Synthesis of Oxidized Products: Acetate and Pyruvate Production
Physical Description: Mixed Material
Copyright Date: 2008

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ENGINEERING THE METABOLISM OF Escherichia coli FOR THE SYNTHESIS OF
OXIDIZED PRODUCTS: ACETATE AND PYRUVATE PRODUCTION















By

THOMAS B. CAUSEY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004


































Copyright 2004

by

Thomas B. Causey





























This dissertation is dedicated to my wife Celeste and my family for their support through
the years.















ACKNOWLEDGMENTS

I would like to acknowledge my Ph.D. committee (Dr. Ingram, Dr. Shanmugam,

Dr. Preston, Dr. Luli and Dr. Stewart) for their guidance. I would also like to

acknowledge Lorraine Yomano and Shengde Zhou for providing some of the strains used

in this study.
















TABLE OF CONTENTS

Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................. ......... .. .. ................. viii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT .............. .................. .......... .............. xi

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

R enew able Fuels and Chem icals .............................................. ........................ .
W hy Use Escherichia coli as a Biocatalyst? ..................................... ............... 2
Overview of Central Carbon Metabolism in Escherichia coli ...................................2
Aerobic versus Anaerobic M etabolism ...................................... ............... 2
A T P and G lycolytic Flux .................. .......... .............................. ................... 10
Combine Beneficial Effects of Aerobic and Anaerobic Metabolism ...............14

2 ENGINEERING THE METABOLISM OF Escherichia coli W3110 FOR THE
CONVERSION OF SUGAR TO ACETATE ...................................................16

In tro d u ctio n ...................................... ................................................ 16
M materials and M methods .............................................. ........................... ........... 17
M icroorganism s and M edia........................................... .......................... 17
G enetic M ethods........... ............................................................ .. .... ...... 20
Fermentation ................ ......... ...................20
ATPase Assay .......................... ...................22
A naly ses ................................................................... 22
Results .............. .. ........ .................. ............................24
Elimination of Fermentation Products (Strain SZ47) .......................................... 24
Disruption of Oxidative Phosphorylation (Strains TC24 and TC57)..................24
Inactivation of Alcohol Dehydrogenase E (Strain TC31)..............................27
Interruption of the Tricarboxylic Acid Cycle (Strain TC32). ..........................27
Elimination of Succinate Requirement (Strain TC36) .....................................30
Disruption of Pyruvate Oxidase (Strain TC42)................................................ 31
Restoration of Pyruvate Formate-lyase Activity (Strain TC51)..........................31
Effects of Gene Disruptions on Growth and Glycolytic Flux .............................32









Production of Acetate and Other Organic Acids.........................................37
Pyruvate Oxidase Contributes to Acetate Production during Stationary Phase..40
Restoration offocA pflB Had No Effect on Acetate Production from Glucose. .41
Elim nation of ATPase A activity ................ ....................... ............... 42
Im proving A cetate Y ields......................................................... ............... 42
Discussion ....................................................44

3 CONVERSION OF ARABINOSE, MANNOSE, SORBITOL AND XYLOSE
TO ACETATE ............... ......... ................. 50

Introduction ......................... ....................... 50
M materials and M methods ....................................................................... ..................5 1
M icroorganism s and M edia.......................................... ........................... 51
F erm entation s ...............................................................5 1
R results ............... ...... ... ..... ............................... .......... .52
Fermentation of Sugars Derived from Hemicellulose (Arabinose, Mannose
an d X y lo se) .............................................................52
Oxidation of Sorbitol to A cetate...................................... ........................ 54
D isc u ssio n ............................................................................................................. 5 4

4 ENGINEERING Escherichia coli FOR THE EFFICIENT CONVERSION OF
GLUCOSE TO PYRUVATE .................................. .....................................57

In tro d u ctio n ...................................... ................................................ 5 7
M materials and M methods .............................................. .................. ............... 59
M icroorganism s and M edia.......................................... ........................... 59
G genetic M ethods............... .................... .. ................ ............ .......... ....... 59
Deletion of Acetate Kinase (Strains TC38 and TC44)......................................60
F erm entation ................................................................................................6 1
A n aly ses ................................................................... 62
R esu lts .................................................................. .................. 62
Pyruvate as a Co-Product during Acetate Fermentations................................62
Acetate Kinase (AackA) Deletion Enhances Pyruvate Production and Inhibits
G lycoly sis (Strain T C 38) ............................................................ ............... .... 65
Pyruvate Oxidase (poxB) Mutation Causes a Modest Reduction in Acetate
Accumulation .................................. .. ............... ........... ............... 65
Combining Mutations for Pyruvate Oxidase (poxB) and Acetate Kinase
(ackA) Improved Pyruvate Production (Strain TC44) ...................................69
The Beneficial Role of a poxB Mutation for Pyruvate Production .................70
Improving Pyruvate Yields and Titers of TC44 by Altering Fermentation
C on edition s ............ .. ................................................... ............... 7 1
D iscu ssio n ........................ ... ............ ... ..........................................7 1

5 EFFECT OF BETAINE ON THE FERMENTATION PERFORMANCE OF
TC44 (PYRUVATE PRODUCTION) AND TC36 (ACETATE PRODUCTION)...78

Intro du action ...................................... ................... ............................ 7 8









M materials an d M eth od s .................................................................... .....................80
M icroorganism s and M edia.......................................... ........................... 80
Acetate and Pyruvate Tolerance.......................... ..................... ............... 80
R e su lts .......................................... ..... ........ .. .. ... ... ...... ......................8 1
Effect of Betaine on Growth and Acetate Production for E. coli TC36 .............81
Effect of Betaine on Growth and Pyruvate Production for E. coli TC44............82
Determination of the Minimal Inhibitory Concentration of Acetate and
Pyruvate on E. coli W3110 in NBS Medium .............................................84
D iscu ssio n ...................................... ................................................. 8 7

6 CON CLU SION S .................................. .. .......... .. .............89

LIST OF REFEREN CES ............................................................................. 93

BIOGRAPHICAL SKETCH ............................................................. ............... 103
















LIST OF TABLES


Table pge

2-1 Sources and characteristics of strains.................................................................... 18

2-2 Sources and characteristics of plasmids ........................................................19

2-3 C om prison of m etabolic rates..................................................................... ...... 34

2-4 Sum m ary of ferm entation products.................................. ..................................... 35

2-5 Effect of atpA or atpFH deletions on metabolic rates...... ......... .................42

3-1 Products formed by TC36 during aerobic catabolism of various sugars .................53

4-1 Sources and characteristics of strains and plasmids.....................................60

4-2 Products formed from glucose catabolism by E. coli strains used in
our study ................................ ..................................... ........... 66



4-3 Comparison of metabolic rates.......................... ...........................69

4-4 Comparison of biocatalysts for pyruvate production ............... ....... ............75

5-1 Comparison of metabolic rates for TC36 in the presence or absence of
1 m M b etain e ............................................... ...................................... ..... 82

5-2 Comparison of metabolic rates for TC44 in the presence or absence of
1 m M b etain e ............................................... ...................................... ..... 82
















LIST OF FIGURES


Figure pge

1-1 Reaction summary for glucose transport......................................... ............... 3

1-2 Reaction summary for the energy consuming reactions of glycolysis.......................3

1-3 Central carbon metabolism in Escherichia coli. ........................................... ............ 4

1-4 R action Sum m ary for glycolysis ........................................ ......................... 5

1-5 Reaction sum m ary of TCA cycle reactions..................................... .....................6

1-6 Electron flow from NADH in Escherichia coli ............. ................................ 7

1-7 Summary of anerobic and aerobic metabolism in E. coli......................................11

1-8 Futile cycles constructed in E. coli............................. ................................. 13

2-1 Reaction summary for the conversion of hexose to acetate.............. ................ 17

2-2 Diagram summarizing genetic modifications used to redirect glucose
m etabolism to acetate ............................................... .. ...... .. ............ 23

2-3 Diagram summarizing plasmid construction..........................................................28

2-4 Effect of selected mutations on E. coli metabolism..............................................33

2-5 Effect of selected mutations on the production of acetate................... ............38

2-6 Comparison of acetate production between TC36 (poxB ) and TC42 (poxB-). .......40

2-7 Effect of restoring PflB activity on growth and acetate production........................41

2-8 Metabolite production in TC36 and TC57. ................................... ............... 43

2-9 Fermentation of 6% glucose to acetate by TC36 in mineral salts medium.............45

3-1 Growth comparison of TC36 with xylose or glucose as the sole carbon
so u rce ..............................................................................5 2









4-1 Genetic modifications used to redirect glucose metabolism for pyruvate
production ................................ .................................... .......... 63

4-2 Effect of oxygen level on pyruvate production by TC36 ......................................64

4-3 Batch fermentation of glucose by mutant strains of E. coli. ................................68

5-1 Effect of 1 mM Betaine on growth and acetate production for TC36...................... 83

5-2 Effect of 1 mM Betaine on growth and pyruvate production for TC44...................78

5-3 Effect of 1 mM betaine on the minimum inhibitory concentration of
acetate and pyruvate for E. coli W 3 110. ........................................ ...............86

6-1 Fermentation profiles for selected strains during the construction of
E coli T C 36. .........................................................................9 1















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENGINEERING THE METABOLISM OF Escherichia coli FOR THE SYNTHESIS OF
OXIDIZED PRODUCTS: ACETATE AND PYRUVATE PRODUCTION

By

Thomas B. Causey

August 2004

Chair: Lonnie O. Ingram
Major Department: Microbiology and Cell Science

To demonstrate a novel approach that combines attributes of fermentative

metabolism and oxidative metabolism into a single biocatalyst for production of oxidized

chemicals, Escherichia coli was genetically engineered for acetate and pyruvate

production. Acetate producing E. coli TC36 was constructed by sequentially assembling

chromosomal deletions to inactivate oxidative phosphorylation (AatpFH), disrupt the

tricarboxylic acid cycle (AsucA), and eliminate fermentation pathways (AfocA-pflB

AfrdBC AldhA AadhE). StrainTC36 produced 572 mM acetate from 6% glucose

(333 mM) and a maximum of 878 mM acetate with glucose excess. Although strain

TC36 produced a maximum of 4 ATP (net) per molecule of glucose as compared to

33 ATP (net) for wild type strains, it grew in glucose minimal medium ([t = 0.49, 82%

of W3110) without supplements. Glycolytic flux in strain TC36 was estimated to be

0.3 [tmol min1 (mg protein)1, twice that of the wild type parent (W3110). Pyruvate

producing strain TC44 (AfocA-pflB AfrdBC AldhA AatpFH AadhE AsucA poxB::FRT









AackA) was constructed from strain TC36 by inserting additional chromosomal deletions

in the acetate kinase (AackA) and pyruvate oxidase (ApoxB) genes. In mineral salts

medium containing glucose as the sole carbon source, strain TC44 converted glucose to

pyruvate with a yield of 0.75 g pyruvate per g glucose (77.9% of theoretical yield; 1.2 g

pyruvate L-1 h-1). The tolerance ofE. coli to such drastic changes in metabolic flow and

energy production implies considerable elasticity in permitted pool sizes for key

metabolic intermediates such as pyruvate and acetyl-CoA. The basic principles

demonstrated using strains TC36 and TC44 can be applied for the production of a variety

of other chemicals, irrespective of their relative oxidation states.














CHAPTER 1
INTRODUCTION

Renewable Fuels and Chemicals

The current trend toward increasing demand for foreign petroleum in the United

States is forecast to continue, reaching 70% imported petroleum in 2025 as opposed to

37% in 1980 (USDOE/EIA 2004). Subordination to foreign countries, some of which

demonstrate mercenary ideals toward US interests, for such a critical component of the

day-to-day functioning of society is a national security problem. The use of microbial

biocatalysts for the conversion of renewable plant biomass to fuels and chemicals can

significantly reduce the United States' dependence on imported petroleum.

The commercial production of commodity chemicals by microbial fermentation has

been limited to reduced and redox neutral compounds such as ethanol and lactic acid,

respectively, under anaerobic conditions where substrate loss to cell mass or CO2 are

minimal and product yields are high. In aerobic processes, Escherichia coli is widely

used as a biocatalyst for production of higher value products such as recombinant

proteins (Akesson et al. 2001; Aristidou et al. 1995; Contiero et al. 2000; Luli and Strohl

1990) and amino acids (Chotani et al. 2000; Eggeling et al. 2001). Traditional chemical

companies have begun to develop infrastructure for the production of compounds using

biocatalytic processes (reviewed in Schmid et al. 2001; Stringer 1996; Strohl, 2001).

Considerable progress has been reported toward new processes for commodity chemicals

(Danner and Braun 1999) such as ethanol (Ingram et al. 1999; Underwood et al. 2002),

lactic acid (Chang et al. 1999; Dien et al. 2001; Zhou et al. 2003), 1,3-propanediol









(Nakamura et al. US Patent 6,013,494; Tong et al. 1991), and adipic acid (Niu et al.

2002). In addition, advances have been made in the genetic engineering of microbes for

high-value specialty compounds such as polyketides (Beck et al. 2003; Dayem et al.

2002) and carotenoids (Lee and Shmidt-Dannert 2002; Wang et al. 2000).

Why Use Escherichia coli as a Biocatalyst?

Escherichia coli is a facultative anaerobe, possessing a robust metabolism, capable

of growing on mineral salts media with only the addition of a carbon source. The

capability E. coli to respire, exploiting a variety inorganic electron acceptors, and

ferment, using internal metabolites as an electron sink, provide the metabolic engineer

with flexibility to explore the production of many compounds regardless of redox state.

Escherichia coli is capable of catabolizing hexose and pentose sugars which constitute

the majority of carbohydrates in plant biomass commonly used as feedstock for

fermentation processes. The efficient conversion of substrate into product is essential if a

process is to be economically viable. The physiology and biochemistry ofE. coli has

been well documented, affording numerous tools for genetic manipulation. Many years of

laboratory and industrial experience has demonstrated that E. coli is safe and reliable.

Therefore, the broad substrate range and robust metabolism combined with years of

technical experience makes E. coli an attractive biocatalyst for the production of fuels

and chemicals.

Overview of Central Carbon Metabolism in Escherichia coli

Aerobic Versus Anaerobic Metabolism

Escherichia coli is a heterotrophic, facultative anaerobe. Hexose and pentose sugars

are transported into the cell using either phosphoenolpyruvate:carbohydrate

phosphotransferase system (PTS), ATP-binding cassette (ABC) transporter (the energy









for transport provided by ATP) or cation (H and Na ) symporters (Linton and Higgins.

1998; Neidhardt et al. 1996). The PTS system transports sugars, such as glucose, using

phosphoenolpyruvate (PEP) as the phosphoryl donor and as the energy source needed for

translocation (Fig. 1-1).


PTS
PEPin + Glucoseout PTS Pyruvatein + Glucose-6-Pin

Figure 1-1. Reaction summary for glucose transport

Glucose-6-P can then be used as a carbon and energy source (glucose is also a source of

oxygen for biosynthesis). Glucose catabolism can be directed through various pathways

dependent on the biosynthetic needs of the cell. The research described in this document

is primarily related to central carbon metabolism, therefore only these pathways are

discussed in detail.

The first stage in glucose-6-P metabolism is the transformation of glucose-6-P to

fructose-6-P followed by phosphorylation. (Fig. 1-3A). The additional phosphate is

supplied by ATP, therefore the first stage of glycolysis results in an investment of two

ATP equivalents (one from PEP and the other from ATP). The net reaction for stage 1 is

summarized in Figure 1-2.


PEPn + Glucoseot PTS Pyruvatei + Glucose-6-Pin

Glucose-6-P + ATP -- Fructose-1,6-diP + ADP

Figure 1-2. Reaction summary for the energy consuming reactions of glycolysis













Lactate
A
NAD'
NAD dhA
NADH---


NAD p
PEP pykA
P0
po
CO Cytc;
2 Cyt,,(red)
ppc CO4

Aceta
Glutamate
Aspartate aspC Oxaloacetate
NADH
2-Ketoglutarate NAD_ mdh
Acetyl-CoA
\. Malate .


CO +
NAD* NADH


SNH g ac Citrate NAD'
asp4 mABC H
Glyoxylate acn
SF rate aceA B ADP ATP
QH2 Isocitrate p
FADH, ---
d sdhABCD /NADP+
frdABCD, icdA
/\ UQ
NADPH
FAD Succinate co
2-Oxoglutarate
sucDC sucAB NAD
IpdA
NADH
P Succinyl-CoA l DN
CO2

Figure 1-3. Central carbon metabolism in Escherichia coli. Dashed arrows represent
fermentative pathways. A) Central carbon metabolism B) Oxidative
phosphorylation. Genes: aceA, isocitrate lyase; aceB, malate synthase B;
aceEF, Elp and lipoate acetyltransferase/dihydrolipoamide acetyltransferase
component of the PDHc; ackA, acetate kinase; acnB, aconitase; adhE,
alcohol/aldehyde dehydrogenase pyruvate formate-lyase deactivase;
atplBEFHAGDC, (FiFo)H+-ATP synthase; fdABCD, fumarate reductase;
glcB, malate synthase G; gltA, citrate synthase; icdA, isocitrate
dehydrogenase; IdhA, lactate dehydrogenase; IpdA, dihydrolipoate
dehydrogenase/dihydrolipoamide dehydrogenase component of the PDHc and
2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase; poxB, pyruvate
oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II; pykF, pyruvate
kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-oxoglutarate
dehydrogenase; sucDC, succinyl-CoA synthetase.


I
I
I









Glyceraldehyde-3-P oxidation to pyruvate is the energy yielding stage (stage 2) of

glycolysis. Glyceraldehyde-3-P dehydrogenase incorporates an inorganic phosphate with

the concomitant reduction ofNAD+ and the formation of 1,3-diphosphoglycerate. The

acyl phosphate is then used to phosphorylate ADP to ATP. The incorporation of the

inorganic phosphate results in the net gain in ATP from glycolysis. Another ATP is

formed from PEP during the production of pyruvate. A reducing equivalent is produced

during the oxidation of glyceraldehyde-3-P (Fig. 1-3A andl-4).


Glucose + 2ADP + 2 Pi + 2 NAD+

2 Pyruvate + 2 ATP + 2 NADH + 2 H+


Figure 1-4. Reaction Summary for glycolysis

Glycolysis is not only an energy generating pathway but also provides precursors for the

biosynthesis of several amino acids, lipids, polysaccharide, and components of

peptidoglycan.

The fate of pyruvate produced during glycolysis is dependent upon oxygen

available in the environment. When E. coli is growing in an aerobic environment,

pyruvate is oxidatively decarboxylated to acetyl-CoA by the pyruvate dehydrogenase

complex (PDHc) with the concomitant production of NADH. Acetyl-CoA can then enter

the tricarboxylic acid cycle (TCA cycle), serve as the substrate for acetate production

(phosphotransacetylase and acetate kinase reactions) yielding an ATP by substrate level

phosphorylation, or serve as the basic building block for fatty acid biosynthesis. Entry

into the TCA cycle is the major route for acetyl-CoA during aerobic metabolism which,

like glycolysis, serves energetic and biosynthetic functions. The cyclic functioning of the

TCA cycle results in the production of four reducing equivalents, two carbon dioxides,









and one GTP (Fig 1-3A and 1-5). Therefore, four ATP equivalents are produced by

substrate-level phosphorylation from catabolism of one glucose molecule by glycolysis

and the TCA cycle.

Acetyl-CoA + 2 H20 + GDP + Pi + FAD + NADP + 2 NAD

2CO2 + GTP +FADH2 +NADPH + 2 NADH +3H+ + CoASH

Figure 1-5. Reaction summary of TCA cycle reactions

The reducing equivalents produced during aerobic growth are re-oxidized via the

electron transport chain with molecular oxygen as the final electron acceptor (Fig. 1-3B

and 1-6). Electrons from NADH enter the electron transport chain by reduction of

ubiquinone (UQ) to ubiquinol (UQH2). The NADH dehydrogenases, NDH I and NDH II,

catalyze the reduction of UQ to UQH2 (Campbell and Young 1983; Ingledew and Poole

1984; Jaworowski et al. 1981; Meinhardt et al. 1989; Weidner et al. 1992; Young et al.

1981). There are two terminal cytochrome oxidases, cytochrome bo oxidase and

cytochrome bd oxidase, in E. coli that transfer the electrons from UQH2 to oxygen

(Ingledew and Poole 1984; Koland et al. 1984; Miller and Gennis 1985; Neidhardt

et al.1990; Puustinen et al. 1991; Rice and Hempfing 1978). Cytochrome o oxidase is

predominant under aerobic conditions and has a low affinity, with a Km for oxygen of

0.20 tM (Rice and Hempfing 1978). Cytochrome bo oxidase functions as a scalar

transporter of protons as well as a proton pump (Neidhardt et al. 1996; Puustinen et al.

1989; Puustinen et al. 1991). Therefore, the H+/e- for cytochrome bo oxidase is 2. The

high affinity cytochrome bd complex, which is predominant under micro-aerobic

conditions (Tseng et al. 1996), has a Km for oxygen of 0.024 tM (Rice and Hempfling

1978). The respiratory efficiency of E. coli depends on the stoichiometry of the NADH

dehydrogenases and cytochrome oxidases (Fig. 1-6). Cytochrome bd oxidase can only


















Pool 02 ---2H20


Cytochrome bd
NDH II (ndh) oxidase
0 H+/e (cydAB)
1 H+/e-



Figure 1-6. Electron flow from NADH in Escherichia coli (figure adapted from
Neidhardt, F. C. et al. 1996). NADH is oxidized by the NADH deydrogenases,
nuo and ndh. Quinones serve as intermediate electron carriers which are
oxidized by the terminal cytochrome oxidases, cyoABCDE and cydAB.

transport protons by scalar chemistry resulting in a H/e- of 1 (Neidhardt et al. 1990;

Puustinen et al. 1991). Figure 1-6 is a simplified diagram of the process and summarizes

the H /e- for each redox reaction.

The NADH dehydrogenases, like the cytochrome oxidases, differ in the number of

protons that are extruded (H /e-) on reduction of the substrate (UQ or 02, respectively).

NDH I has a calculated H /e of 1.5-2.0 (Bogachev et al. 1996; Calhoun et al. 1993) and

NDH II does not contribute to the membrane potential gradient (Young et al. 1981). The

number of protons extruded by NDH I is yet to be established. Difficulty in obtaining

evidence for the actual H/e- arises from the many respiratory chain linked

dehydrogenases present in E. coli (Ingledew and Poole 1984). Oxygen pulse experiments

performed under anaerobic conditions conducted by Calhoun et al. (1993) and Bogachev

et al. (1996) have indicated a H/e- = 2 and H/e- = 1.5, respectively. Since, a H/e- = 1.5

is unlikely, two protons transported per electron is a reasonable assumption. Another line









of evidence which suggests the H /e- = 2 is that NDH I is the homolog of the

mitochondrial complex I (Meinhardt et al. 1989; Weidner et al. 1992) which has been

demonstrated to extrude two protons per electron (Pozzan et al. 1979; Wikstrom and

Penttila 1982).

The stoichiometry of the NADH dehydrogenases and cytochrome oxidases, which

depend on the growth conditions (Spiro and Guest 1991), result in 1 H+/e- to as many as

4 H /e- delivered to the periplasm under aerobic conditions (Fig. 1-6). The greater the

H /e- the higher the respiratory efficiency i.e., increasing the membrane potential

difference results in more ATP produced per mole of oxygen consumed (P/O). If all of

the electrons originating from glucose catabolism are transported through NDH I and

cytochrome bo oxidase then a maximum of 33 ATP can be produced. However, growth

yield experiments conducted by Stouthamer (1979) have demonstrated that a maximum

of 26 ATP are produced per molecule of glucose when E. coli is grown under optimal

conditions. This lower value is the result of 1) differential electron flux through the

NADH dehydrogenases and terminal oxidases, 2) the maintenance energy requirement

(Pirt 1982; Pirt 1987) and 3) proton leakage reducing the energized state of the

cytoplasmic membrane (Stouthamer and Bettenhaussen 1977). The large amount of ATP

produced and the high respiratory efficiency ofE. coli, grown under aerobic conditions

and limiting glucose, result in the production of carbon dioxide and cell mass in a 50:50

ratio (Calhoun et al. 1993; Neidhardt et al. 1990).

When E. coli is grown anaerobically on glucose with out an exogenous terminal

electron acceptor, NAD is regenerated by reduction of internal organic compounds

(Fig. 1-3A). The major end products of glucose fermentation are acetate, ethanol, lactate,









and format in a 1:1:2:2 ratio (Neidhardt et al. 1996). Ten percent of the glucose

consumed is converted to succinate and cell mass (Neidhardt et al. 1996). In contrast to

the large amount of ATP produced during aerobic metabolism, only two net ATP are

produced (by substrate-level phosphorylation) during fermentation of glucose to pyruvic

acid (Fig. 1-3A). An additional ATP is produced with the formation of acetate through

acetate kinase. In addition to the low ATP yield and low cell mass, fermentative

metabolism also differs from aerobic metabolism in that the TCA cycle functions in a

non-cyclic manner. The transcription of 2-oxoglutarate dehydrogenase (sucAB gene

product) is repressed under anaerobic conditions (Amarasingham and Davis 1965;

Cunningham and Guest. 1998; Park et al. 1997) which seperates the TCA cycle into

reductive and oxidative branches. The anaerobic TCA cycle functions only in a

biosynthetic capacity.

During fermentative growth, many of the genes involved in aerobic metabolism

are repressed and expression of genes involved in anaerobic metabolism are activated.

Two global regulatory systems play a major role in the transition to fermentative

metabolism. The ArcAB (aerobic respiratory control) two-component signal transduction

pathway (ArcA is the response-regulator and ArcB is the sensor kinase) and the redox-

sensitive protein, FNR (fumarate and nitrate reduction) function to regulate gene

expression in response to oxygen availability (Iuchi and Weiner 1996; Kiley and Beinert

1999). The ArcAB system functions mainly to repress expression of aerobic genes under

anaerobic conditions (Spiro and Guest 1991; luchi and Weiner 1996). Under micro-

aerobic conditions the expression of the cyoABCDE cytochromee bd, the high affinity

terminal oxidase) operon is enhanced by ArcAB (Iuchi and Weiner. 1996). The









expression of both oxidases is repressed by FNR under anaerobic conditions (Kiley and

Beinert 1999; Spiro and Guest 1991). FNR is known to activate 22 operons and repress

eight operons under anaerobic conditions (Spiro and Guest 1991). When E. coli is

growing fermentatively, FNR activates the transcription ofpflB, pyruvate formate-lyase.

The products of pyruvate formate-lyase are acetyl-CoA and format (Fig 1-3A). There

are no reducing equivalents produced during the decarboxylation of pyruvate under

fermentative conditions. The lipoamide dehydrogenase (lpdA) protein, shared by PDHc

and 2-oxoglutarate dehydrogenase, is repressed by ArcAB under anaerobic conditions

(Cunningham and Guest 1998). Transcription of sucAB, which codes for the El and E2

subunits of 2-oxoglutarate dehydrogenase, is repressed by FNR from the sdhC promoter

(Park et al. 1997). The repression of sucAB under anaerobic conditions results in the non-

cyclic functioning of the TCA cycle mentioned above.

In summary, high growth rate and conversion of glucose into high levels of cell

mass and large amounts of carbon dioxide, are characteristic of aerobic metabolism in E.

coli. During anaerobic metabolism, approximately 95% of the glucose is converted to

organic acids and ethanol, while less than 5% is converted to cell mass. Figure 1-7 is a

simplified diagram of carbon flow in E. coli during aerobic and anaerobic metabolism.

Typically, the growth rate is also decreased during anaerobic conditions.

ATP and Glycolytic Flux

Studies conducted by Patnaik et al. (1992) and Chao and Liao (1994) have

demonstrated that creation of futile cycles in E. coli, which consume ATP, cause a two-

fold increase in glycolytic flux. Over-expression of phosphoenolpyruvate synthase (pps)

(Fig. 1-8A) by 30-fold resulted in a two-fold increase in glycolytic flux (Patnaik et al.

1992). Low induction levels of Pps resulted in increased respiration to compensate for








A Anaerobic (- oxygen) B Aerobic (+ oxygen)

Glucose, C6H1206 Glucose, C6H1206




SCell Mass L Cell Mass
5% of Carbon 50% of Carbon


Up to 95 % of carbon converted to 50% of carbon converted to CO2
products (low CO2 production) 33 ATP (max.) expected
2 ATP expected

Figure 1-7. Summary of anerobic and aerobic metabolism in E. coli. A) Anaerobic
metabolism. B) Aerobic metabolism
the loss of ATP. High levels of Pps induction resulted in both increased respiration and

increased glycolytic flux. The authors explained the observation at high Pps induction

levels by 1) saturation of the respiratory chain and 2) increased glucose uptake due to a

high phosphoenolpyruvate/pyruvate ratio resulting in an increase in the phosphorylated

forms of enzyme I, Hpr, and enzyme IIIglc of the PTS (Patnaik et al. 1992). Although the

increase in glucose transport may contribute to the increased glycolytic flux in the

mutant, it is unlikely the only determinant. Phosphoenolpyruvate is an allosteric inhibitor

of phosphofructokinase which may become a "bottleneck" at high phosphoenolpyruvate

levels. The Pyk-Pps futile cycle resulted in a net loss of ATP concomitant with elevated

respiration (Patnaik et al. 1992), indicative of a reduction in respiratory efficiency.

Therefore, the increased glycolytic flux may be due to decreased ATP production

coupled with increased availability of ADP, an allosteric activator of

phosphofructokinase (Deville-Bonne et al. 1991), under aerobic conditions.









Introduction of a futile cycle between phosphoenolpyruvate and oxaloacetate by

over-expression of phosphoenolpyruvate carboxykinase (pck) and phosphoenolpyruvate

carboxylase (ppc) also resulted in an increase in glycolytic flux (Fig. 1-8B) (Chao and

Liao 1994). Increasing the magnitude of the futile cycle for ATP consumption was

followed by (in order) an increase in respiration rate, increase in glucose consumption

and excretion of acetate and pyruvate (Chao and Liao 1994). These results suggest that E.

coi compensates for decrease in ATP by first, increasing respiration to form ATP at a

higher rate, secondly, by increasing glycolytic flux and the formation of ATP by

substrate-level phosphorylation. Another interesting observation was that inactivation of

2-oxoglutarate dehydrogenase in addition to the futile cycle resulted in

even greater increase in glycolytic flux than the futile cycle alone (Chao and Liao 1994).

This is a result of the decreased amount of electrons available for electron transport and

oxidative phosphorylation. The authors comment that, "cells with an incomplete TCA

cycle cannot effectively compensate for the energy stress caused by the induced futile

cycle" (Chao and Liao 1994).Our observations with the atpFH mutant are contrary to

those of Chao and Liao and will be discussed in Chapter 2.

Deletion of the (FiFo)H+-ATP synthase has also been found to increase glycolytic

flux as well as acetate production (Jensen and Michelsen 1992). A two-fold increase in

glycolytic flux was observed in an E. coli strain carrying a deletion in the

atplBEFHAGDC operon, consistent with the experiments using artificially induced futile

cycles. By inactivating the (FiFo)H+-ATP synthase E. coli can no longer derive ATP from

the proton motive force generated during oxidative respiration. The growth rate for the

(FiFo)H+-ATP synthase mutants was decreased to -74% of the wild type level and the




















2Pi


AMP + PPi


ATP


ADP,


pck


ATP


Pyr
Pyr cetyl-CoA


I Citrate


OAA


Pyk-Pps futile cycle


Figure 1-8. Futile cycles constructed in E. coli. A) The pps gene was cloned into a
plasmid behind an IPTG-inducible bacteriophage T7 promoter modified to
overlap two lac operators. Over-expression ofpps during growth on glucose
results in net loss of ATP due to formation of a futile cycle involving Pps and
Pyk. Abbreviations: ppc, phosphoenolpyruvate carboxylase; pps,
phosphoenolpyruvate synthase; pykA and pykF, major and minor pyruvate
kinases, respectively (adapted from Patnaik, R. et al. 1992). B) The structural
genes ofppc and pck onto a plasmid behind the IPTG inducible tac promoter.
Simultaneous expression ofppc and pck result in a net loss of ATP forming a
futile cycle. Abbreviations: pck, phosphoenolpyruvate carboxykinase; ppc,
phosphoenolpyruvate carboxylase (Chao, Y., and J. C. Liao. 1994).

ATP/ADP ratio was decreased from 19 in the wild type to 7 in the mutant strain (Jensen

and Michelsen 1992). Deletion of the atp operon also resulted in increased respiration

proceeded by accumulation of acetate. The following observations comparing the growth

characteristics between wild type E. coli and the AatplBEFHAGDC mutant:

1. increased respiration
2. increased glycolytic flux
3. accumulation of acetate


B Pck-Ppc futile cycle
Glucose







CO2 PEP


Co2



"PC

Pyr Acety-CoA



_a Citrate


t AA


/-^A


I









led the investigators to conclude that E. coli will oxidize NADH by respiration even if it

is not coupled to ATP synthesis leading to enhanced substrate level phosphorylation

(Jensen and Michelsen 1992).

The approaches described above demonstrate that limiting ATP by enhancing futile

cycles or suppression of the oxidative phosphorylation results in increased glycolytic

flux. Recently, Koebmann et al. (2002) describe a genetic approach that induces ATP

hydrolysis to elucidate the nature of control on glycolytic flux. The genes atpAGD,

encoding the Fi subunit of the (FiFo)H+-ATP synthase, were cloned behind synthetic

constituative promoters of different strengths. These investigators discovered that control

of glycolytic flux varies with ATP/ADP ratio (Koebmann et al. 2002). During growth of

wild type E. coli on glucose minimal medium the anabolic (ATP consuming) reactions

exert a majority of the control on glycolytic flux. When the ATP/ADP increases flux

control shifts to the catabolic (ATP producing) reactions. These findings are consistent

with mathematical models that indicate greater than 75% of glycolytic flux is controlled

by ATP/ADP in E. coli (Hofmeyer and Cornish-Bowden. 2000).

Combine Beneficial Effects of Aerobic and Anaerobic Metabolism.

It is possible to combine the beneficial effects of aerobic and anaerobic metabolism

into a single biocatalyst for the production of oxidized and redox neutral compounds.

This can be accomplished by combining the high rate of glycolysis and low cell yield of

fermentative metabolism with the high growth rate and availability of an external electron

acceptor of aerobic metabolism. The advantages for production of oxidized compounds

are expected to be increased product yield and rate of product formation combined with

decreased production of carbon dioxide and cell mass. The major advantage for

production of reduced compounds would be the increase in process stability due to the






15


elimination of the energy advantage of NADH oxidation by the electron transport chain.

To demonstrate this concept, the metabolism of Escherichia coli was engineered for the

production of acetate and pyruvate.














CHAPTER 2
ENGINEERING THE METABOLISM OF Escherichia coli W3110 FOR THE
CONVERSION OF SUGAR TO ACETATE

Introduction

In all aerobic and anaerobic processes, acetate production by the native E. coli

pathway (phosphotransacetylase and acetate kinase) has been generally regarded as an

undesirable consequence of excessive glycolytic flux (Akesson et al. 2001; Aristidou

et al. 1995; Contiero et al. 2000; Farmer and Liao 1997; Underwood et al. 2002; Zhou

et al. 2003). Although acetate is equivalent to glucose in redox state, pathways in E. coli

that produce acetate also produce CO2 resulting in a combined redox state of carbon more

oxidized than in glucose with the production of NADH. To facilitate expansion into more

oxidized commodity chemicals, such as acetate, we developed a novel genetic approach

that combines the attributes of fermentative metabolism and oxidative metabolism (rapid

growth, external electron acceptor for NADH oxidation) into a single biocatalyst.

The biological production of acetic acid is currently limited to food uses.

Petrochemical routes have largely displaced biological processes as uses expanded to

plastics, solvents, and road de-icers (Berraud 2000; Cheryan et al. 1997; Freer 2002).

There are two major petrochemical routes for commercial acetic acid production;

methanol carbonylation and liquid-phase oxidation of n-butane. Approximately 35% of

commercial acetic acid is recovered from other processes, most significantly, from the

production of cellulose acetates and polyvinyl alcohol (Kirschner 2003). Synthesis of

acetate in the United States is estimated at 7.8 billion pounds per year, with a projected









growth of 2% per year through 2008 (Kirschner 2003). In the current biological process,

sugars are fermented to ethanol by Saccharomyces. Ethanol in the resulting beer is

subsequently oxidized to acetic acid by Acetobacter sp. under aerobic conditions

(Berraud 2000; Cheryan et al. 1997; Freer 2002). This process can summarized as

follows:

C6H1206 + 2 H2O 2 2 C2H402 + 2 CO2 + 8 [H]

8 [H] + 2 02 4 H20

Figure 2-1.Reaction summary for the conversion of hexose to acetate

In our study, we have genetically modified E. coli W3110 to produce acetic acid as

the primary product from glycolysis. Although ethanol was eliminated as an intermediate,

the stoichiometry of the reaction remained as shown above. The resulting biocatalyst

(TC36) contains multiple chromosomal alterations (Fig. 2-2A and B) that direct carbon

flow to acetate and minimize carbon loss to cell mass, CO2, and alternative products. This

strain is devoid of plasmids, foreign genes, and antibiotic resistance markers and grows

well in mineral salts medium without complex nutrients.

Materials and Methods

Microorganisms and Media

Strains and plasmids used in our study are listed in Table 1. Working cultures of E.

coli W3110 (ATCC 27325) derivatives were maintained on minimal medium containing

mineral salts (per liter: 3.5 g KH2PO4; 5.0 g K2HPO4; 3.5 g (NH4)2HPO4, 0.25 g MgSO4

* 7 H20 15 mg CaC12 2 H20, 0.5 mg thiamine, and 1 mL of trace metal stock),

glucose (2% in plates; 3% in broth), and 1.5% agar. The trace metal stock was prepared

in 0.1 M HC1 (per liter: 1.6 g FeC13, 0.2 g CoC12 6 H20, 0.1 g CuC12, 0.2 g










Table 2-1. Sources and characteristics of strains
Strains Relevant Characteristics
W3110 K12 wild type
TOP10F' lacP (episome)
LYO1 E. coli B,frdpfl::pdozM adhEzM cat
LY74 W3110, ApoxB::FRT-tet-FRT
LY117 W3110, ycaC: :FRT-aap-FRT: :ycaD
SE2279 MG1655,pflB ldhA::TnlO
SZ33 W3110, IdhA::Tnl0
SZ40 W3110, A(focA-pflB): :FRT AfrdBC
SZ46 W3110, A(focA-pflB)::FRT AfrdBC IdhA::Tnl 0
SZ47 W3110, A(focA-pflB): :FRT AfrdBC AldhA
TC20 W3110, AadhE: :FRT-tet-FRT

TC21 W3110, AatpFH::FRT-tet-FRT
TC23 W3110, A(focA-pflB): :FRT AfrdBC AldhA
Aatp(FH): :FRT-tet-FRT
TC24 W3110, A(focA-pflB): :FRT AfrdBC AldhA
Aatp(FH)::FRT
TC25 W3110, AsucA::FRT-tet-FRT
TC30 W3110, A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT-tet-FRT
TC31 W3110, A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT
TC32 W3110, (Succ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT-tet-FRT
TC35 W3110, (Succ+), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA: :FRT-tet-FRT
TC36 W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
TC41 W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
ApoxB: :FRT-tet-FRT
TC42 W3110, (Succ+), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
ApoxB::FRT
TC45 W3110, AatpA::FRT-tet-FRT
TC49 W3110, (Succ ), ycaC::FRT-aap-FRT::ycaD focA+
pflB+ AfrdBC AldhA Aatp(FH)::FRT AadhE::FRT
AsucA::FRT
TC51 W3110, (Succ), ycaC::FRT::ycaDfocA+ pflB
AfrdBC AldhA Aatp(FH)::FRT AadhE::FRT
AsucA::FRT
TC55 W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AatpA: :FRT-tet-FRT
TC56 W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AatpA::FRT


Reference
ATCC 27325
Invitrogen
Yomano et al. 1998
Causey et al. 2004
This study
Causey et al. 2003
Causey et al. 2003
Zhou et al. 2003
Causey et al. 2003
Causey et al. 2003
Underwood et al.
2002
Causey et al. 2003
Causey et al. 2003

Causey et al. 2003

Causey et al. 2003
Causey et al. 2003

Causey et al. 2003

Causey et al. 2003

Causey et al. 2003

Causey et al. 2003

Causey et al. 2004


Causey et al. 2004


This study
This study


This study


This study


This study










Table 2-2. Sources and characteristics of plasmids


Plasmids
pCR2.1-TOPO
pFT-A

pKD46


pLOI2065

pLOI2075
pLOI2078
pLOI2080
pLOI2403

pLOI2745
pLOI2800
pLOI2801
pLOI2802

pLOI2803
pLOI2805
pLOI2820
pLOI2821
pLOI3421
pLOI3430
pLOI3439
pLOI3447


Relevant Characteristics
bla kan, TOPOTM TA cloning vector
blaflp low-copy vector containing recombinase and
temperature-conditional pSC101 replicon
bla y 0 exo low-copy vector containing red
recombinase and temperature-conditional pSC 101
replicon
bla, Smal fragment with FRT flanked tet gene

bla kan poxB
bla poxB
bla poxB::FRT-tet-FRT
bla

kan temperature conditional replicon
bla kan sucA
bla kan sucA::FRT-tet-FRT
bla kan adhE

bla kan adhE::FRT-tet-FRT
bla kan atpEFH
bla kan atpA
bla kan atpA::FRT-tet-FRT
EcoRI fragment containing FRT flanked aap gene
bla kan ycaCycaD
kan ycaC ycaD temperature conditional replicon
bla kan ycaC FRT-aac-FRT ycaD used for
repairing focA pflB deletion


Reference
Invitrogen
Posfai et al. 1997

Datsenko and
Wanner 2000

Underwood et al.
2002
Causey et al. 2004
Causey et al. 2004
Causey et al. 2004
Martinez-Morales
et al. 1999
This study
Causey et al. 2003
Causey et al. 2003
Underwood et al.
2002
Causey et al. 2003
Causey et al. 2003
This study
This study
This study
This study
This study
This study


ZnCl2 e 4 H20, 0.2 g NaMoO4, and 0.05 g H3BO3). MOPS (0.1 M, pH 7.1) was added to

both liquid and solid media (filter-sterilized) when needed for pH control, but was not

included in medium used for 10 L fermentations. Minimal medium was also prepared

using succinate (1 g L1) as a sole source of carbon (nonfermentable substrate). Succinate

(1 g L1) was also added as a supplement to glucose-minimal medium when needed.

During plasmid and strain construction, cultures were grown in Luria-Bertani (LB) broth

or on LB agar plates (1.5% agar) (Miller 1992). Glucose (2%) was added to LB medium

for all strains containing mutations in (F1FO)H+-ATP synthase. Antibiotics were

included as appropriate (kanamycin, 50 mg L-1; ampicillin, 50 mg L-1; and tetracycline,









12.5 or 6.25 mg L-1). Fusaric acid plates were used to select for loss of Tnl0-encoded

tetracycline resistance (Zhou et al. 2003).

Genetic Methods

Standard methods were used for plasmid construction, phage P1 transduction,

electroporation, and polymerase chain reaction (PCR) (Miller 1992; Sambrook and

Russell 2001). Chromosomal DNA from E. coli W3110 (and derivatives) served as a

template to amplify genes using primers complementary to coding regions (ORFmers)

purchased from the Sigma-Genosys, Inc. (The Woodlands, TX). PCR products were

initially cloned into plasmid vector pCR2.1-TOPO. During plasmid constructions,

restriction products were converted to blunt ends using either the Klenow fragment of

DNA polymerase I (5' overhang) or T4 DNA polymerase (3' overhang) as needed.

Integration of linear DNA was facilitated by using pKD46 (temperature conditional)

containing an arabinose-inducible Red recombinase (Datsenko and Wanner 2000).

Integrants were selected for tetracycline resistance (6.25 mg L-1) and screened for

appropriate antibiotic resistance markers and phenotypic traits. At each step, mutations

were verified by analyses of PCR products and fermentation products. FRT-flanked

antibiotic resistance genes used for selection were deleted using a temperature-

conditional plasmid (pFT-A) expressing FLP recombinase from a chlortetracycline-

inducible promoter (Martinez-Morales et al. 1999; Posfai et al. 1997).

Fermentation

Acetate production was examined in glucose-minimal medium containing 167 mM

glucose using a New Brunswick Bioflow 3000 fermentor (New Brunswick Scientific,

Edison, NJ) with a 10 L working volume (370C, dual Rushton impellers, 450 rpm).

Dissolved oxygen was maintained at 5% of air saturation (unless otherwise stated) by









altering the proportion of N2 and 02. Broth was maintained at pH 7.0 by the automatic

addition of 11.4 M KOH. During fed batch experiments, additional glucose was added

from a sterile 60% stock. Three fed batch regimes were investigated: A. 3% glucose

initially with the addition of 3% after 12 h (6% total); B. 6% glucose initially with the

addition of 4% glucose after 16 h (10% total); C. 3% glucose initially with multiple

additions to maintain glucose levels above 100 mM. Five liter fermentations (7 L vessel,

350 rpm using dual Rushton impellers) were used to examine the effects of deletion of

atpFH and atpA or the effects) of restoring pyruvate formate-lyase activity. For these

fermentations the dissolved oxygen was allowed to fall from 100% to 5% of air saturation

at which time 02 was mixed with air to maintain a dissolved oxygen concentration higher

than 5%.

Seed cultures were prepared by inoculating colonies from a fresh plate (48 h) into

3 mL of glucose-minimal medium (13 x 100 mm tube) containing 0.1 M MOPS. After

incubation for 14 h (120 rpm rotator), cultures were diluted 400-fold into 1 L baffled

flasks containing 200 mL of mineral salts medium (370C, 280 rpm). When cells reached

1.5-2.2 OD550nm, sufficient culture volume was harvested (5000 x g, 25C) to provide an

inoculum of 33 mg dry cell weight L-1 in the 10 L working volume.

Broth samples were removed to measure organic acids, residual glucose, and cell

mass. Volumetric and specific rates were determined from measured values for glucose

and acetate using GraphPad Prism (GraphPad Software, San Diego, CA). A smooth curve

was generated with 10 points per min (Lowess method) to fit measured results. The first

derivative (acetate or glucose versus time) of each curve served as an estimate of









volumetric rate. Specific rates (mmoles L-1 h-1 [mg dry cell weight]-1) were calculated by

dividing volumetric rates by respective values for cell mass.

ATPase Assay

Cells were grown for enzyme assays as described above for seed cultures. Upon

reaching 0.75-1.0 OD550nm, cultures were chilled on ice and harvested by centrifugation

(8000 x g, 5 min at 40C). Cell pellets were washed 5 times with 0.1 M Tris-HCl (pH

7.55), resuspended in 1 mL of this buffer, and broken using a model W220F ultrasonic

cell disruptor (Heat Systems Ultrasonics, Plainview, NY). Total ATPase activity in

disrupted cell preparations was assayed at pH 7.55 essentially as described by Evans

(Evans 1969). Inorganic phosphate was measured by the method of Rathbun and Betlach

(Rathbun and Betlach 1969). Results represent an average of three cultures for each

strain. Specific activity is expressed as [tmol Pi released min1 (mg protein)-.

Analyses

Organic acids and glucose concentrations were determined using a Hewlett Packard

HPLC (HP 1090 series II) equipped with a UV monitor (210 nm) and refractive index

detector. Products were separated using a Bio-Rad HPX-87H column (10 [l injection)

with 4 mM H2SO4 as the mobile phase (0.4 mL min-1, 450C). Cell mass was estimated by

measuring OD550nm (1.0 OD550nm is equivalent to 0.33 g dry cell weight L-) using a

Bausch & Lomb Spectronic 70 spectrophotometer with 10 x 75 mm culture tubes as

cuvettes. Protein concentration was determined using the BCA Protein Assay Kit from

Pierce (Rockford, IL).










Lactate


NAD'


H* O0+e H20


FADH,


p ATP


ATP ADP
+ PI


sucDC
~ Succinyl-CoA
-P


Figure 2-2. Diagram summarizing genetic modifications used to redirect glucose
metabolism to acetate. Bold arrows mark principal metabolic routes in TC36.
Dashed arrows represent fermentative pathways. Reactions that have been
blocked are highlighted. A) Central carbon metabolism. Acetate is the
principal product from sugar metabolism with the net production of four ATP
equivalents per glucose molecule. B) Oxidative phosphorylation. The
(FiFo)H+-ATP synthase is inactive in TC36, although the electron transport
system remains functional as the primary route for NADH oxidation. C) F1-
ATPase. TC36 lacks the essential subunits coupling the ATP synthase to the
membrane and thus defective in ATP synthesis but retains a functional
cytoplasmic Fi-ATPase. Genes: aceA, isocitrate lyase; aceB, malate synthase
B; aceEF, Elp and lipoate acetyltransferase/dihydrolipoamide
acetyltransferase component of the PDHc; ackA, acetate kinase; acnB,
aconitase; adhE, alcohol/aldehydrogenase pyruvate format lyase deactivase;
atplBEFHAGDC, (FiFo)H+-ATP synthase; frdABCD, fumarate reductase;
glcB, malate synthase G; gltA, citrate synthase; icdA, isocitrate
dehydrogenase; IdhA, lactate dehydrogenase; IpdA, dihydrolipoate
dehydrogenase/dihydrolipoamide dehydrogenase component of the PDHc and
2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase; poxB, pyruvate
oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II; pykF, pyruvate
kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-oxoglutarate
dehydrogenase; sucDC, succinyl-CoA synthetase.









Results

Elimination of Fermentation Products (Strain SZ47)

Inspection of native pathways in E. coli (Fig. 2-2A) indicated that the production of

acetate and CO2 as sole metabolic products from glucose would require an external

electron acceptor such as oxygen. Due to low oxygen solubility, however, it is difficult to

satisfy the oxygen demand from active E. coli metabolism. Typically a portion of

substrate is converted into fermentation products (Tsai et al. 2002; Varma et al. 1993).

This problem was eliminated by combining deletions in genes encoding lactate

dehydrogenase, pyruvate formatelyase, and fumarate reductase as follows. The

IdhA::Tnl0 mutation in E. coli SE2279 was transduced into E. coli W3110 using phage

P1 to produce strain SZ33. P1 phage grown on SZ33 was used to transfer this mutation

into SZ40(A(focA-pflB)::FRT AfrdBC) to produce SZ46. Tetracycline-sensitive

derivatives of SZ46 were selected using fusaric acid medium (Zhou et al. 2003). One

clone was designated SZ47 (A(focA-pflB)::FRT AfrdBC AldhA). The AldhA mutation in

SZ47 was confirmed by the absence of lactate in fermentation broth, inability to grow

anaerobically in glucose-minimal medium, and by PCR analysis using IdhA ORFmers

(1.0 kbp for the wild type IdhA as compared to 1.1 kbp for SZ47). The slightly larger size

of the amplified product from SZ47 is attributed to remnants of Tnl0.

Disruption of Oxidative Phosphorylation (Strains TC24 and TC57).

Growth under oxidative conditions is characterized by conversion of up to 50% of

substrate carbon to cell mass (Neidhardt et al. 1990). To reduce the potential drain of

substrate into cell mass, a mutation was introduced into SZ47 that deleted portions of two

subunits in (FiFo)H+-ATP synthase required for assembly to the plasma membrane

(Sorgen et al. 1998), disrupting oxidative phosphorylation while preserving the hydrolytic









activity of F1-ATPase in the cytoplasm. To construct this deletion, the atpEFH coding

region of the atplBEFHAGDC operon was amplified by PCR using primers (ORFmers)

complementary to the 5'- end of the atpE gene and the 3'- end of the atpH. The amplified

fragment (1.3 kbp) was cloned into pCR2.1-TOPO and one clone was selected in which

the atpEFH genes were oriented to permit expression from the lac promoter (pLOI2805;

Fig. 2-3). The atpF gene and 117 nucleotides at the 5' end of atpH gene were removed

from pLOI2805 by digestion with Hpal and BstEII (Klenow-treated). This region was

replaced with a 1.7 kbp Smal fragment from pLOI2065 containing the FRT-tet-FRT

cassette to produce pLOI2807 (Fig.2-3). After digestion with Scal, pLOI2807 served as a

template for amplification of the atpEA(FH)::FRT-tet-FRT region (2.4 kbp) using the 5'

atpE and 3' atpH primers. Amplified DNA was precipitated, digested again with Scal to

disrupt any residual plasmid, and purified by phenol extraction. This DNA was

introduced into E. coli W3110(pKD46) by electroporation while expressing Red

recombinase. Plasmid pKD46 was eliminated by growth at 42C. Recombinants (double

cross-over) were identified using antibiotic markers (tetracycline resistant; sensitive to

ampicillin and kanamycin) and by the inability to grow on succinate-minimal plates in

the absence of glucose (fermentable carbon source). Integration was further confirmed by

PCR analysis using the 5' atpE primer and the 3' atpH primer (1.3 kbp fragment for

W3110; 2.3 kbp fragment for mutants). One clone was selected and designated

TC21(Aatp(FH): :FRT-tet-FRT).

Phage P1 was used to transduce the Aatp(FH)::FRT-tet-FRT mutation in TC21 to

SZ47 and produce TC23. The tet gene was removed from TC23 by the FLP recombinase

(pFT-A). After elimination of pFT-A by growth at 420C, the Aatp(FH)::FRT mutation









was further confirmed by PCR analysis using the 5' atpE primer and the 3' atpH primer

(0.8 kbp for deletion and 1.3 kbp for SZ47). The resulting strain was designated TC24

(A(focA-pflB)::FRTAfrdBC AldhA Aatp(FH)::FRT). TC57 was unable to grow in

minimal medium without a fermentable carbon source (substrate level phosphorylation)

but retained the ability to oxidize NADH by the electron transport system.

In order to demonstrate the effectiveness of preserving the hydrolytic activity of the

F1 subunit, a deletion was inserted into the atpA gene which codes for the alpha subunit of

the (FiFo)H+-ATP synthase. To construct this deletion, the atpA coding region of the

atplBEFHAGDC operon was amplified by PCR using primers (ORFmers)

complementary to atpA The amplified fragment (1.5 kbp) was cloned into pCR2.1-

TOPO and one clone was selected in which the atpA gene was oriented to permit

expression from the lac promoter (pLOI2820). A 249 bp section was removed from the

central region of atpA by digestion with SmaI (blunt). This region was replaced with a 1.7

kbp Smal fragment from pLOI2065 containing the FRT-tet-FRT cassette to produce

pLOI2821. After digestion with HindIII and Pvull, pLOI2821 served as a template for

amplification of the atpA::FRT-tet-FRT region (2.9 kbp) using the atpA primers.

Amplified DNA was gel-purified using the QIAquick gel extraction kit (Qiagen,

Valencia, CA). This DNA was introduced into E. coli W3110(pKD46) by electroporation

while expressing Red recombinase. Plasmid pKD46 was eliminated by growth at 420C.

Recombinants (double cross-over) were identified using antibiotic markers (tetracycline

resistant; sensitive to ampicillin and kanamycin) and by the inability to grow on

succinate-minimal plates in the absence of glucose (fermentable carbon source).

Integration was further confirmed by PCR analysis using atpA primers (1.5 kbp fragment









for W3110; 2.9 kbp fragment for mutants). One clone was selected and designated TC45

(AatpA: :FRT-tet-FRT).

Phage P1 was used to transduce the AatpA::FRT-tet-FRT mutation in TC45 to

TC36 and produce TC55. The tet gene was removed from TC45 by the FLP recombinase

(pFT-A). After elimination of pFT-A by growth at 420C, the AatpA::FRT mutation was

further confirmed by PCR analysis using the atpA primers (1.2 kbp for deletion and 1.5

kbp for TC36). The resulting strain was designated TC57 (A(focA-pflB)::FRTAftdBC

AldhA AadhE: :FRT AsucA: :FRT AatpFH::FRT AatpA: :FRT).

Inactivation of Alcohol Dehydrogenase E (Strain TC31)

The native bifunctional alcohol/aldehyde dehydrogenase (adhE) in E. coli catalyzes

the reduction of acetyl-CoA to ethanol, a reaction that directly competes with the

conversion of acetyl-CoA to acetate by phosphotransacetylase and acetate kinase (Fig. 2-

2A). To eliminate this activity, phage P1 was used to transduce the AadhE::FRT-tet-FRT

mutation in TC20 to TC24 and produce TC30. Chromosomal integration was confirmed

by PCR analysis using adhE primers (2.7 kbp for TC24 and 3.2 kbp for the AadhE::FRT-

tet-FRTmutant). The tet gene was deleted from TC30 by FLP recombinase using pFT-A.

After elimination of pFT-A by growth at 420C, a clone was selected and designated

TC3 (A(focA-pflB)::FRT AfrdBC AldhA Aatp(FH)::FRT AadhE::FRT).

Interruption of the Tricarboxylic Acid Cycle (strain TC32).

During oxidative growth, up to 50% of substrate carbon can be released as CO2

(Neidhardt et al. 1990). This loss of carbon can be attributed in large measure to the high

efficiency of the tricarboxylic acid cycle and the electron transport system (NADH

oxidation). During fermentative metabolism, the production of CO2 and NADH are























< .. El


SnaBI


, -PvuI


SmaI


pLO 12065

4356 bps

_..i FA


SmaI


PvuI \ SnaBI


PvuI .
ScaI


SmaI


- ..I El
S FRT


pLO 2801

7259 bps

n
U i'
sucA .

\FRT


PvuI


Figure 2-3. Diagram summarizing plasmid construction


PvuI-
ScaI --


pLO 12800

673C t.pc


BstEII


atpE
<..I El at '


pLOI2805
52.- tl .s


BstEII (Klenow)


S pLO12807

6254 bps


ScaI-









reduced primarily by repression of sucAB encoding 2-oxoglutarate dehydrogenase

(Cunnungham and Guest 1998; Park and Gunsalus 1997), disrupting the cyclic function

of the tricarboxylic acid cycle. We have genetically imposed a similar restriction in

carbon flow during oxidative metabolism by deleting part of the sucA gene.

The sucA coding region was amplified using ORFmers. The resulting 2.8 kbp PCR

product was cloned into pCR2.1-TOPO to produce pLOI2800 (Fig. 2-3) in which the

sucA coding region was oriented to permit expression from the lac promoter. A 1.1 kbp

fragment was removed from the central region of sucA by digestion of pLOI2800 with

SnaBI and AccI (Klenow-treated). This region was replaced with a 1.7 kbp Smal

fragment containing the FRT-tet-FRT cassette from pLOI2065 to produce pLOI2801

(Fig. 2-3). Plasmid pLOI2801 was digested with Pvul and Scal and used as a template to

amplify the 3.3 kbp region containing sucA::FRT-tet-FRTusing sucA ORFmers.

Amplified DNA was precipitated, digested with Pvul and Scal to disrupt any residual

circular plasmid, and extracted with phenol. Purified DNA was electroporated into E. coli

W3110(pKD46) while expressing Red recombinase. Plasmid pKD46 was eliminated by

growth at 420C. Disruption of sucA was confirmed by PCR analysis using sucA ORFmers

(2.8 kbp fragment for wild type and 3.3 kbp for sucA::FRT-tet-FRT mutants) and

designated TC25.

Phage P1 was used to transduce the sucA::FRT-tet-FRT mutation from TC25 into

TC31. Transfer of this mutation was verified by PCR analysis (2.8 kbp for wild type

sucA and 3.3 kbp for sucA::FRT-tet-FRT mutants) and phenotype (Succ-). Inactivation of

2-oxoglutarate dehydrogenase (AsucA) in this AfrdBC background resulted in an

undesirable auxotrophic requirement for succinate (Succ ) during growth on glucose-









minimal medium. The resulting strain was designated TC32(Succ A(focA-pflB)::FRT

AfrdBC AldhA Aatp(FH)::FRT AadhE::FRT AsucA::FRT-tet-FRT).

Elimination of Succinate Requirement (Strain TC36)

Spontaneous mutants of TC32, that did not require succinate in the growth medium

(Succ+), were readily obtained after serial transfers in glucose-minimal broth containing

decreasing amounts of succinate (4 mM to 0.4 mM) followed by selection on glucose

minimal plates without succinate. Over 170 clones were recovered per mL of culture after

enrichment (approximately 3% of viable cells). Ten clones were tested and all grew in

glucose minimal broth without succinate and produced acetate as the dominant product.

One was selected (TC35) for deletion of the tet gene using the FLP recombinase. This

deletion was confirmed by analysis of PCR products using sucA primers (3.3 kbp for

TC35 and 1.8 kbp after tet deletion). The resulting strain was designated TC36 (Succ+;

A(focA-pflB)::FRT AfrdBC AldhA Aatp(FH)::FRT AadhE::FRT AsucA::FRT). Although

the genetic change that eliminated the succinate requirement was not identified, likely

candidates include mutations which provide a low constitutive expression of aceAB

(glyoxylate shunt) or citC (citrate lyase).

Total ATPase activity was examined in disrupted cell extracts of TC36 and W3110

(wild type). The activity in TC36 (0.355 U [mg protein]-1) was equivalent to 71% of the

unmodified parent (0.502 U [mg protein]-'), confirming that F1-ATPase was not

inactivated by the AatpFH::FRT mutation. This is similar to the levels of ATPase

reported for an atpH mutant of E. coli which blocked membrane assembly and coupling

to oxidative phosphorylation (Sorgen et al. 1998).









Disruption of Pyruvate Oxidase (Strain TC42)

A single derivative of pCR2.1-TOPO was selected in which the amplified poxB

gene was oriented in the same direction as the lac promoter (pLOI2075). To eliminate

extraneous BsaBI sites in the vector, the small EcoRI fragment containing poxB was

ligated into the corresponding site of pLOI2403 to produce pLOI2078. The Smal

fragment from pLOI2065 containing a tet gene flanked by FRT sites was ligated into the

unique BsaBI site inpoxB to produce pLOI2080. Plasmid pLOI2080 served as a template

for the amplification ofpoxB::FRT-tet-FRT (3.4 kbp) using poxB primers. Amplified

DNA was integrated into E. coli W3110(pKD46) as previously described (Causey et al.

2003) to produce LY74. The poxB::FRT-tet-FRT mutation in LY74 was transduced into

TC36 to produce TC41. The tet gene was removed using FLP recombinase and the

resulting strain designated TC42 (AfocA-pflB::FRT AfdBC::FRT A IdhA AatpFH::FRT

AadhE::FRT AsucA: :FRTpoxB::FRT).

Restoration of Pyruvate Formate-lyase Activity (Strain TC51)

Strain LY117 was constructed for restoring pyruvate formate-lyase activity by a

colleague, Lorraine Yomano, in which an apramycin marker flanked by FRT sites was

inserted downstream of thefocA-pflB operon as follows. The ycaC and ycaD region

(2.1 kbp), located 2.6 kbp downstream from pflA, was PCR amplified and cloned into

TOPO-pCR2.1 using the 3'ycaC primer (ORFmer) and the 3'ycaD primer (ORFmer) to

generate plasmid pLOI3430. In order to limit the background after electroporation the

EcoRI fragment from pLOI3430 containing ycaC ycaD was ligated into the EcoRI site of

pLOI2745 generating a new plasmid, pLOI3439, which contains a temperature

conditional replicon. The EcoRI fragment, containing an apramycin resistance marker

flanked by FRT sites (1.9kbp), was treated with the Klenow fragment of DNA









polymerase I to generate blunt ends and ligated into the XmnI site of pLOI3439. The new

plasmid contained a ycaC::FRT-aap-FRT::ycaD EcoRI fragment (3.5 kbp) and a

temperature conditional replicon.

Plasmid pLOI3447 was digested with EcoRI and the 3.5 kbp ycaC::FRT-aap-

FRT::ycaD fragment was purified by gel extraction (Qiagen, Valencia, CA). The DNA

was integrated into E. coli W3110(pKD46) as previously described (Causey et al. 2003)

to produce LY117 (ycaC::FRT-aap-FRT: :ycaD). The ycaC::FRT-aap-FRT: :ycaD

mutation in LY117 was transduced into TC36 to produce TC49. The aap gene was was

removed using FLP recombinase and the resulting strain designated TC51 (focA+pflB

AfrdBC::FRT A IdhA AatpFH::FRT AadhE::FRT AsucA: :FRTpoxB::FRT). ThefocA

pflB+ genotype was verified by PCR and pyruvate formate-lyase activity was verified by

observing format in fermentation broths.

Effects of Gene Disruptions on Growth and Glycolytic Flux

Strain TC36 was genetically engineered for the production of acetate from

carbohydrates such as glucose (Fig. 2-2A, B and C). Batch fermentations with pH control

were used to compare the performance of this strain with W3110 (wild type) and two

intermediate strains used in the construction, SZ47(A(focA-pflB) AfrdBC AldhA) and

TC24(A(focA-pflB) AfrdBC AldhA AatpFH). Based on preliminary experiments, 5%

oxygen saturation and 3% glucose (370C) were selected as test conditions. Broth pH was

maintained at 7.0 to minimize toxicity from undissociated acids.

Disruption of oxidative phosphorylation and the cyclic function of the tricarboxylic

acid cycle, elimination of the primary fermentation pathways, and the production of

acetate as the primary endproduct from glycolysis had relatively little effect on the

growth of E. coli. The maximum growth rates for strains W3110 (wild type) and SZ47










(lacking the three native fermentation pathways) were similar although the cell yield for

SZ47 was higher (Fig. 2-4A; Table 2 and Table 3). Inactivation of oxidative

phosphorylation (AatpFH) in SZ47 resulted in a modest reduction (12%) in growth rate


A



(C)




S125
O


0
175 ---


S100


( 50
25
0


/2

9 12 15 18 21 24 27 30
Time (h)





i.


-w \


0 3 6 9 12 15 18 21 24 27 30
Time (h)


0 3 6


9 12 15 18 21 24 27 30
Time (h)


Figure 2-4. Effect of selected mutations on E. coli metabolism. A) growth B) glucose
utilization C) KOH consumption. Solid squares, W3110 (wild type); Open
squares, SZ47 (A[focA-pflB]::FRT AfrdBC AldhA); Open circles, TC24
(A[focA-pflB]::FRT AfrdBC AldhA Aatp[FH]::FRT); Solid circles, TC36
(A[focA-pflB]::FRT AfrdBC AldhA Aatp[FH]::FRT AadhE::FRT
AsucA: :FRT).









(TC24). Cell yield for strain TC24 was equivalent to that of W3110. Strain TC36,

containing additional mutations in 2-oxoglutarate dehydrogenase (AsucA) and alcohol

dehydrogenase (AadhE), had a lower cell yield and growth rate; approximately 80% of

the unmodified parent W3110.

Rates for glucose utilization (maximum specific and average volumetric) were

higher for TC36 and TC24 than for W3110 and SZ47 (Table 2). This increase in

metabolic activity can be primarily attributed to the AatpFH mutation. ATP levels serve

as an allosteric regulator of several key glycolytic enzymes (Neidhardt et al. 1990), and

acetate kinase (Suzuki 1969). In strains containing the AatpFH mutation, increased

glycolytic flux and substrate level phosphorylation could partially compensate for the

loss of ATP production from respiration-coupled phosphorylation. Differences between

strains were particularly evident when comparing incubation times required to complete

sugar metabolism (Fig. 2-4B). With TC36 and TC24, glucose was exhausted in 16-18 h

as compared to 26 h for SZ47 and 30 h for W3110. The maximum specific rate of

glucose utilization (glycolytic flux) was 9 mmole h-1 (g dry cell weight)-' in the

unmodified parent (W3110), 20 mmole h-1 (g dry cell weight)-' in TC24, and 18 mmole

h-1 (g dry cell weight)-1 in TC36.

Table 2-3. Comparison of metabolic rates
Glucose consumption rate" Pyruvate production rate
Average Vol.b Max. Spec.b Average Vol.b Max. Spec.b
Strain g (h-1) (mmol L1 h-') (mmol L-1 h-1 g-') (mmol L-1 h-') (mmol L-1 h-1 g-')
W3110 0.60 7 9 4 10
SZ47 0.55 9 11 4 10
TC24 0.53 10 20 10 16
TC36 0.49 11 18 12 16
a Data are derived from averages of two fermentations.
b Average volumetric rates for glucose consumption and acetate production or maximum specific rates (dry
cell weight basis) for glucose consumption and acetate production.














Table 2-4. Summary of fermentation products


Strain Conditions

W3110 3% glucose
5% DO
SZ47 3% glucose
5% DO
TC24 3% glucose
5% DO
TC36 3% glucose
5% DO
TC36 3% glucose
15% DO


Cell
yield
(g L-')


Fermentation Productsa (mmoles L-1)


Acetate 2-Oxoglutarate


4.5 30

5.3 6

4.4 156
3.5+0. 14
224+14
2
3.2 190


39

11

1

16+6

24


Fumarate Lactate Pyruvate

0.8 33 < 1


1.0 <1 <1

0.4+0.1 < 1 0+0.5

<1 <1 <1


Yield
Succinate (%b)


2 47


3 57


Carbon recovery
(% substrate C")

60

20

66

89

88


TC36 3%glucose 2.5 220 31 <1 < 1 < 1 10 66 95
5% DO N-limited
DO, dissolved oxygen.
a Concentrations in broth after all glucose had been depleted, except as noted.
b Yield expressed as a percentage of the maximal theoretical yield (0.67 g acetate per g glucose).
Carbon recovery represents the percentage of substrate carbon recovered. Recovered carbon was calculated as the sum of carbon in cell mass, fermentation
products, and CO2 (C02 calculated as the difference of [acetate + 2-oxoglutarate]-[succinate + fumarate]).
d In the final sample, 44 mM glucose was present.
e Excess glucose (9.5%) was added to maintain levels above 100 mM; 107 mM glucose was present in the final sample.














Table 2-4. Continued


Cell Fermentation Productsa (mmoles L 1)


yield


Yield
C_- ^


Carbon recovery


Strain Conditions (g L-) ^cetate z-uxogutarate rumarate Lactate ryruvate Succinate (%) (% substrate Cc)
TC36 3+3% glucose 3.8 523 21 <1 3 14 2 78 95
5% DO
3+3% glucose
TC36 3+3%glucose 3.0 572 33 < 1 < 1 < 1 6 86 102
5% DO N-limited
TC36 6% glucose 4.2 415 47 0.3 < 1 46 7 62 92
5% DO
6+4% glucose
TC36 % glucose 4.5 767 37 0.5 < 1 72 5 72 97
5% DOd
Fed batch
TC36 F ed 4.1 878 33 3.4 <1 <1 25 75 88
5% DOe
DO, dissolved oxygen.
a Concentrations in broth after all glucose had been depleted, except as noted.
b Yield expressed as a percentage of the maximal theoretical yield (0.67 g acetate per g glucose).
Carbon recovery represents the percentage of substrate carbon recovered. Recovered carbon was calculated as the sum of carbon in cell mass, fermentation
products, and CO2 (CO2 calculated as the difference of [acetate + 2-oxoglutarate]-[succinate + fumarate]).
d In the final sample, 44 mM glucose was present.
e Excess glucose (9.5%) was added to maintain levels above 100 mM; 107 mM glucose was present in the final sample.


)


n__LL_ ~T\~~IL_~_L~ ~~~_~_L~ T_~LL~ n__L~









The slightly lower glycolytic flux in TC36 as compared to TC24 may be related to the

increase in ATP yield resulting from improvements in acetate yield (1 ATP per acetate).

Assuming protein represents 55% of dry cell weight, maximal glycolytic flux in TC36 is

approximately 0.55 moless glucose min1 (mg protein)1.

Production of Acetate and Other Organic Acids

A substantial portion of glucose carbon was not recovered in the carbon balance

(Table 3) for W3110 (40%) and SZ47 (80%). This loss is attributed to production of

volatile products by high flux through the tricarboxylic acid cycle (CO2) but may also

include the reduction of acetyl-CoA to acetaldehyde and ethanol (Fig. 2-2A). Although

ethanol was absent in broth samples from all pH-controlled fermentations (sparged at 1 L

min'), a small amount of ethanol (6 mM) was found in seed cultures of W3110 (shaken

flasks). No ethanol was present in seed cultures of TC36, which contains a mutation in

alcohol dehydrogenase E. The native electron transport system (5% dissolved oxygen)

and fermentation pathways in W3110 (Table 3) serve as complementary routes for

NADH oxidation. Eliminating the fermentation pathways (SZ47) doubled the loss of

carbon as volatile products (Table 3) and increased cell yield but decreased the rate of

acetate production in comparison to W3110 (Table 2 and Table 3).

Inactivation of oxidative phosphorylation (AatpFH) in SZ47 to produce TC24

resulted in a 26-fold increase in acetate yield and a 3-fold improvement in carbon

recovery (Table 3, Fig. 2-5A). Acetate yield and carbon recovery increased by another

30% with the introduction of the sucA and adhE mutations (TC36). TC36 produced an

average of 224 mM acetate in 16 h with small amounts of other products. This represents

68% of the maximum theoretical yield using native pathways (2 acetates per glucose),

remaining carbon being divided between cell mass, dicarboxylic acids, and CO2. The















200 -

, 150 /

I 100
U,
o C
< 50:



0 3 6 9 12 15 18 21 24 27 30

Time (h)


E 30
-

20
10-
10


0 3 6 9 12 15 18 21

Time (h)


24 27 30


16
-

E 12
12

CU
8-
n

0. 4


0-


0 3 6 9 12 15 18 21

Time (h)


24 27 30


Figure 2-5. Effect of selected mutations on the production of acetate. A) Acetate B)
dicarboxylic acids (2-oxoglutarate, succinate and fumarate) C) pyruvate.
Symbols: Solid sqaures, W3110 (wild type); Open squares, SZ47 (A(focA--
pl/B)::FRTfrdBC AldhA); Open circles, TC24 (A(focA--plfB)::FRTfrdBC
AldhA Aatp(FH)::FRT) Solid circles, TC36 ((A(focA--plB)::FRTAfrdBC
AldhA Aatp(FH)::FRT AsucA: :FRT AadhE::FRT).









maximum specific and average volumetric rates of acetate production were

approximately 1.6-fold and 2.5-fold higher, respectively, for TC24 and TC36 than for

SZ47 and W3110 (Table 2), a difference which can be attributed solely to the mutation in

the (FiFo)H+-ATP synthase. This mutation eliminated ATP production by oxidative

phosphorylation while retaining cytoplasmic F1-ATPase for the gratuitous consumption

of ATP. Strain W3110 accumulated the highest levels of dicarboxylic acids (primarily

succinate and 2-oxoglutarate) during glucose metabolism, approximately 3-fold that of

the engineered strains (Fig. 2-5B). The order of appearance of dicarboxylic acids in the

broth correlated with growth rate and the order of entry into stationary phase for the four

strains examined. Dicarboxylic acids were partially consumed as glucose levels declined,

and may represent spillover products from excessive glycolysis during the transition from

exponential to stationary phase. It is interesting to note that pyruvate levels in the broth of

TC36 also increased (16 mM at 12 h) during this transition (Fig. 2-5C). Although this

pyruvate was subsequently metabolized, the excretion of pyruvate indicates that glucose

uptake and glycolysis per se may not be limiting for acetate production. No significant

accumulation of pyruvate was observed for the three other strains (W3110, SZ47 or

TC24).

The consumption of base to maintain pH 7.0 provides an overall measure of total

organic acid production (Fig. 2-4C). Higher rates and maxima for TC24 and TC36 are

consistent with more rapid glucose metabolism. In general, variations in glucose

utilization were accompanied by corresponding changes in base utilization. The

exponential nature of the early time points reflects growth of the biocatalysts.










Pyruvate Oxidase Contributes to Acetate Production during Stationary Phase

Pyruvate can be metabolized to acetate by the membrane-bound protein pyruvate oxidase

using the electron transport system to couple oxygen as a terminal electron acceptor. The

poxB gene is typically repressed during exponential growth but is induced by stress or

entry into stationary phase (Chang and Cronan 1983; Chang et al. 1994). An isogenic

Pyruvate oxidase mutant of TC36 was constructed by introducing stop codons in the

central region of the gene to produce strain TC42. Introducing thepoxB mutation had no

effect on glucose utilization (Fig. 2-6). No difference was observed in the rate of acetate

production for the poxB mutant during logarithmic growth (Fig. 2-6). Upon entry into late

logarithmic/stationary phase the rate of acetate production by thepoxB mutant declined

resulting in a 20% decrease in final acetate concentration (Fig. 2-6).




10- 250-
A B
_1 200-

0
S1 E
E 150-
U, w
U,
1 1 1 1 1 100"







Time (h) Time (h)
0
2 50


0.017
0 4 8 12 16 20 0 4 8 12 16 20
Time (h) Time (h)

Figure 2-6. Comparison of acetate production between TC36 (poxB ) and TC42 (poxB-).
Data from triplicate fermentations. A) Growth. Symbols: Solid squares, TC36;
Open circles, TC42. B) Glucose consumption and acetate production.
Symbols: Solid squares, TC36 glucose; Solid circles, TC42 glucose; Open
squares, TC36 acetate; Open circles, TC42 acetate.











Restoration offocA pflB Had No Effect On Acetate Production from Glucose

Pyruvate formate-lyase catalyzes the non-oxidative decarboxylatation of pyruvate


to format and acetyl-CoA under fermentative conditions. Under the 5% dissolved


oxygen E. coli W3110 produced 33 mM lactate (Table 2-4) suggesting the cells


experience fermentative conditions once the cell density is greater than 2 g L-1 dry weight


(data not shown). Therefore PFL activity was restored to provide more acetyl-CoA for


the phosphotransacetylase reaction in an attempt to increase acetate production. Pyruvate


formate-lyase had no effect on growth rate, final cell mass, glucose consumption, or


acetate production (Fig. 2-7A and B).


10-
A






0






0 3 6 9 12 15
Time (h)
220
S200- B
180E
S160-
E 140-
120-
100-
80-
60-




0 3 6 9 12 15
Time (h)

Figure 2-7. Effect of restoring PFL activity on growth and acetate production. A) Growth.
B) Glucose consumption and acetate production. Symbols: Circles, TC51;
Squares, TC36; Solid symbols represent glucose and open symbols represent
acetate in part B.









Elimination of ATPase Activity

The efficacy of the atpFH deletion by constructing an isogenic strain containing an

additional deletion in the atpA gene (TC57). The atpA gene codes for the alpha subunit of

the (F1Fo)H+-ATP synthase, an essential protein for catalysis (Boyer 1997). Growth rate

and volumetric glucose consumption were reduced by 20% and 25% respectively, for the

atpA mutant compared to the atpFH mutant (Table 2-5). The most striking difference

between the two strains was the 62% reduction in the specific glucose consumption rate

for TC57. Unfortunately, the elevated glucose consumption rate did not result in an

increased rate of acetate production, demonstrated by the modest increase in acetate

production rate for TC36 (Table2-5). Where did the rest of the carbon go? Inspection of

Figure 2-8 A and B reveals that the increased glucose consumption rateresults in

excretion of pyruvate by TC36. The pyruvate is converted to acetate once glucose levels

diminish. The increase in pyruvate excretion in TC36 may be due to elevated ADP levels

which would allow for increased flow though glycolysis.

Improving Acetate Yields

Dicarboxylic acids and cell mass were the dominant competing co-products from

glucose. A limited set of experiments was conducted to evaluate the potential for process

Table 2-5. Effect of atpA or atpFH deletions on metabolic rates
Glucose consumption rate" Pyruvate production rate
Average Vol.b Max. Spec.c Average Vol.b Max. Spec."
Strain g (h-) (mmol L-1 h-) (mmol L'- h-1 g-) (mmol L-1 h-) (mmol L-1 h-1 g-1)
TC36 0.51+
TC36 0 10.70.9 29.73.5 10.71.6 31.94.7
0.01
TC57 0.41 8.0 11.3 9.2 22.1
a Five liter fermentations were conducted in 7 L vessels. Dissolved oxygen was allowed to fall from 100%
to 5% of air saturation. The data for TC36 represent an average of three fermentations and the data for
TC57 represent and average of two fermentations.
b Average volumetric rates.
' Maximum specific rates per gram of dry cell weight (dcw).













225 A Acetate


0 3 6 9 12 15 18 21 24

Time (h)


90 B Pyruvate
80

70

60

50

40

30

20


0 3 6 9 12 15 18 21 24

Time (h)

Figure 2-8. Metabolite production in TC36 and TC57. A) Comparison of acetate
production between strains. B) Comparison of pyruvate production between
strains. Symbols: Solid squares, TC36. Open squares, TC57.









changes to improve acetate yield (Table 2-4). Acetate yield was not improved by

increasing the dissolved oxygen level from 5% to 15% of air saturation, reducing

ammonia nitrogen (2 g L-1 ammonium phosphate) by 40% to limit growth, or increasing

the initial concentration of glucose from 3% to 6% (Table 2-4). However, a simple two-

step batch feeding strategy was beneficial. A second addition of 3 % glucose at the end of

the growth phase (12 h) was metabolized to completion and produced 523 mM acetate

with minimal loss to cell mass (Fig. 2-9). Acetate yield for this one-step addition (6%

total glucose) was 78 % of the theoretical maximum as compared to 68% for 3% glucose.

The highest acetate yield, 86% of the theoretical maximum, was obtained by combining

the one-step addition of 3% glucose with the nitrogen limitation to further limit loss of

carbon into cell mass (Table 2-4). Additional fed-batch experiments were conducted in

which multiple additions were made to glucose levels above 100 mM. With this

approach, 878 mM acetate was produced representing 75% of the maximum theoretical

yield (Table 2-4).

Discussion

E. coli was genetically engineered to combine the attributes of fermentative

metabolism (low cell mass, high product yields) and oxidative metabolism (external

electron acceptor) into a single biocatalyst (strain TC36) for the production of oxidized

chemicals such as acetate (Fig. 2-2). Chromosomal deletions were used instead of point

mutations to maximize stability. All antibiotic resistance genes and auxotrophic

requirements were eliminated to permit growth in simple mineral salts medium. During










5
500

4
E Glucose Added
S400-

S3
0300

06 k /<
S200 2
0 CD

100- -


0- 0
0 6 12 18 24 30 36
Time (h)
Figure 2-9. Fermentation of 6% gluocse to acetate by TC36 in mineral salts medium.
Fermentation was initiated at 3% glucose followed by a second addition of 3%
glucose after 12 h. Symbols: Open squares, cell mass; Solid circles, acetate.

oxidative metabolism, up to half of the substrate carbon can be converted to roughly

equal amounts of cell mass and CO2 (Contiero et al. 2000; Neidhardt et al. 1990; Varma

et al. 1993) with minimal carbon flow into alternative products. To reduce the

opportunity for excessive growth during oxidative metabolism, ATP production from

NADH oxidation oxidativee phosphorylation) was eliminated by deleting the portion of

(FiFo)H+-ATP synthase involved in membrane assembly while preserving a functional

cytoplasmic F1-ATPase to provide gratuitous hydrolysis of ATP. With this mutation, a

maximum of 4 ATP molecules (net) can be produced per glucose (assumes all pyruvate is

metablized to acetyl-CoA and acetate) as compared to a theoretical maximum of 33 ATP

molecules for wild-type strains ofE. coli. Excessive oxidation of substrate to CO2 and

production of NADH were eliminated by disrupting the cyclic function of the

tricarboxylic acid cycle (AsucA) with the added benefit of reducing oxygen demand for









NADH oxidation. Additional mutations were introduced to eliminate all major

fermentation pathways as alternative routes for NADH oxidation, minimizing the

formation of alternative products. The resulting strain, TC36, has absolute requirements

for a fermentable carbon source (substrate level phosphorylation) and for an external

electron acceptor that can couple to the electron transport system during growth in

mineral salts medium to maintain redox balance. With genetic blocks in all major

fermentation pathways and oxidative phosphorylation, this strain is relatively insensitive

to variations in dissolved oxygen.

The (FIFo)H+-ATP synthase and 2-oxoglutarate dehydrogenase mutations

introduced into TC36 to miminize the levels of ATP and NAD(P)H produced from

glucose under oxidative conditions would also be expected to promote glycolysis through

native allosteric controls (Neidhardt et al. 1990; Underwood et al. 2002a; Underwood

et al. 2002b), providing a mechanism for the observed 2-fold increase in glycolytic flux

as compared to W3110 (wild type). With additional mutations in fermentation pathways,

further metabolism of pyruvate was limited primarily to small biosynthetic needs and

conversion to acetyl-CoA by the pyruvate dehydrogenase complex. Although pyruvate

dehydrogenase is activated by low NADH, acetyl-CoA production may be limited by the

availability of free CoA (note pyruvate accumulation in TC36 broth between 9 h and 15

h; Fig. 5C). Resulting increase in pyruvate pools would serve as an allosteric activator of

phosphotransferase (Suzuki 1969), the first committed step for acetate production from

acetyl-CoA. Gratuitous ATP hydrolysis by F1-ATPase should ensure the availability of

ADP for the final step in acetate production catalyzed by acetate kinase (Fig. 2-2A).

Excess pyruvate can also be directly oxidized to acetate by pyruvate oxidase (poxB), an









enzyme that is induced during the later stages of growth and by environmental stress

(Chang et al. 1994). This enzyme may also contribute to acetate production by TC36.

Eliminating oxidative phosphorylation while preserving (Fi) H+ATPase resulted in

a 2-fold increase in glycolytic flux (TC24 and TC36). Previously, Chao and Liao (1994)

and Patnaik et al. (1992) demonstrated a similar 2-fold stimulation of glycolytic flux in E.

coli using plasmids to express genes that created futile cycles to consume ATP. Recently,

Koebmann et al. (2002) independently concluded that glycolytic flux is limited by ATP

utilization during the oxidative metabolism of glucose. In their studies, flux increased in a

dose-dependent manner with controlled expression of F1-ATPase genes from a plasmid.

Our results with TC24 and TC36 containing a chromosomal deletion (AatpFH) provide

further support for this hypothesis. Thus, glycolytic flux appears to be regulated by the

economy of supply and demand as proposed by Hofmeyer and Comish-Bowden (2000).

During the oxidative metabolism of glucose, glycolytic flux is limited by the metabolic

ability to utilize ATP (availability of ADP) rather than by glucose transport or the

catalytic capacities of central glycolytic enzymes. With this in mind, similar strategies

that delete subunits essential for membrane assembly of the (FiFo)H+-ATP synthase,

create futile cycles for ATP consumption, or increase cytoplasmic levels of the ATPase

activities may prove useful to decrease cell yield, increase metabolic flux, and increase

product yield in many other bioconversion processes.

Strain TC36 can be used as a biocatalyst platform for the efficient production of

oxidized products. Under conditions of excess glucose, strain TC36 produced a

maximum of 878 mM acetate, 75% of the maximum theoretical yield (Table 2-4) or 0.50

g acetate per g glucose. Only cell mass and small amounts of organic acids were









produced as co-products with acetate. It is likely that 878 mM acetate approaches the

upper limit of tolerance for metabolism in TC36. Concentrations as low as 50 mM acetate

have been shown to induce a stress response in E. coli (Kirkpatrick et al. 2001). The

minimal inhibitory concentration for growth has been previously reported as 300 400

mM acetate at neutral pH (Lasko et al. 2000; Zaldivar and Ingram 1999). Oxygen transfer

often becomes limiting during aerobic bioconversion processes, promoting the

accumulation of reduced products (Tsai et al. 2002; Varma et al. 1993). Synthesis of

reduced products was eliminated by mutations in genes (AfocA-pflB AfrdBC AldhA

AadhE) encoding the four major fermentation pathways. Excessive oxygen demand and

NADH production were also reduced by a deletion in 2-oxoglutarate dehydrogenase

(AsucA). The resulting strain, TC36 (AfocA-pflBAfrdBC AldhA AatpFH AsucA AadhE)

metabolizes sugars to acetate with the efficiency of fermentative metabolism, diverting a

minimum of carbon to cell mass biocatalystt) and CO2. By replacing the acetate pathway,

a variety of alternative oxidized products can be produced using the mutational strategies

employed for the construction of TC36.

Commercial production of acetate with biocatalysts involves a 2-step process in

which sugar is first fermented to ethanol by yeasts followed by aerobic oxidation to

acetate by Acetobacter (Berraud 2000; Cheryan et al. 1997). Although titers of around

650 mM are typically produced, higher titers can be readily achieved by the addition of

complex nutrients in fed-batch processes requiring 60-120 h. Overall, yields for

commercial processes have been estimated to be 76% of the theoretical maximum (2

acetate per glucose; 0.67 g acetic acid per g glucose). Genetically engineered E. coli

TC36 can produce acetate in a simpler, single step process using glucose with titers and









yields equivalent or higher than current batch processes. Although maximum titers with

TC36 were lower than can be achieved by ethanol oxidation using Acetobacter (Berraud

2000), acetate production rates from glucose by TC36 were almost two-fold higher than

ethanol oxidation and required only mineral salts as nutrients. E. coli TC36 offers a

unique set of advantages over currently employed biocatalysts for the commercial

production of acetate:

1. The fermentation is a single step process using sugars as substrates.
2. Strain TC36 has high rates of acetate production, high acetate yields and a simple
nutritional requirement (mineral salts plus carbon source).
3. Robust metabolism permitting the bioconversion of hexoses, pentoses, and many
disaccharides and sugar alcohols.

Derivatives of this strain may prove useful as biocatalysts for the production of other

compounds that are equivalent to, or more oxidized than the substrate.














CHAPTER 3
CONVERSION OF ARABINOSE, MANNOSE, SORBITOL AND XYLOSE TO
ACETATE

Introduction

One advantage to employing E. coli as a biocatalyst is its ability to consume hexose

and pentose sugars, the carbohydrate constituents of lignocellulosic plant biomass. The

plant cell wall is primarily composed of two major carbohydrate polymers: cellulose and

hemicellulose (Ingram et al. 1999; McKendry 2002). The carbohydrate composition of

the cell wall differs with respect to the type of plant (hardwood, softwood, grass, etc.).

Generally, cellulose constitutes 20-55% of agricultural residues and hemicellulose

constitutes 20-40% (Sun and Cheng 2002). Pectin, a methylated polymer of galacturonic

acid, typically constitutes a small percentage (2-4%) of agricultural residues (Ingram et al

1999). Cellulose is a polymer of glucose (cellobiose) therefore once treated with fungal

cellulase the monomers become available for degredation. Hemicellulose is a complex

carbohydrate polymer composed of xylose and arabinose in hardwoods and crop residues

(Asghari et al. 1996; Ingram et al 1999). Xylose, galactose and mannose are the major

sugars in softwoods (Asghari et al. 1996; Ingram et al 1999). The stoichiometry of

hemicellulose sugars vary depending on the type of plant.

Our laboratory is interested in the conversion of biomass, such as sugarcane

bagasse, into fuels and chemicals. The hemicellulose fraction of sugarcane is composed

mostly of xylose (89%) with minor amounts of arabinose and glucose (Asghari et al.

1996). In this chapter the engineered E. coli strain TC36 is shown to efficiently convert









all major sugar constituents of plant biomass into acetate. In addition, strain TC36 also

metabolized sorbitol, a sugar alcohol, into acetate.

Materials and Methods

Microorganisms and Media

Escherichia coli TC36 and TC51 (described in Chapter 1) were used for our study.

The NBS salts described in Chapter 1 were used in minimal salts agar and for

fermentations as described in Chapter 1. Arabinose, mannose, sorbitol, or xylose was

added (2% in plates and 3% in broth) as the sole carbon and energy source, when

appropriate, from a 50% stock solution. When needed for pH control, 3-[N-

morpholino]propanesulfonic acid (0.1 M, pH 7.4) was added, but was not included in pH-

controlled fermentations.

Fermentations

Fermentations (5 L) were conducted at 370C (dual Rushton impellers, 350 rpm; pH

7.0) in Bioflow 3000 fermentors (New Brunswick Scientific, Edison, NJ). Dissolved

oxygen levels were 100% of air saturation at the time of inoculation and allowed to fall to

5% of air saturation during cell growth with continuous air sparging (0.2 vvm). This level

of oxygen was maintained by mixing 02 with air at a constant flow rate of 1.0 L min1.

Broth pH was controlled with 11.4 M KOH.

Seed cultures were started as described in Chapter 1. Fermentors were inoculated at

an initial OD550 of 0.05 except for xylose fermentations. For xylose fermentations an

initial OD550 was not sufficient for logarithmic growth therefore these fermentations were

inoculated at an initial OD550 of 0.5.









Results

Fermentation of Sugars Derived from Hemicellulose (Arabinose, Mannose and
Xylose)

Pentose sugars constitute the majority of sugars found in hemicellulose. Xylose is

the most abundant sugar found in sugarcane derived hemicellulose. Escherichia coli

TC36 converted 180 mM xylose into 205 mM acetate, 69% of the maximum theoretical

yield (Table 3-1). All of the xylose carbon was accounted for by HPLC analysis of the

fermentation broth (Table 3-1). Minor quantities of glycolytic and TCA cycle

intermediates were observed in the fermentation broth, 2-oxoglutarate being the most

abundant (Table 3-1). Acetate accounted for 88% of the organic acids in the broth. The

maximum specific growth rate of TC36 grown on xylose is 75% of the maximum specific

growth rate when grown on glucose under the same conditions (Fig. 3-1). The growth

rate reduction may be attributed to decreased ATP production on xylose compared to

glucose (see discussion).

10-





Glucose
S1. L= =0.44
Xylose
S: ^= =0.33




0.1
O 2 4 6 8 10 12 14 16
Time (h)
Figure 3-1. Growth comparison of TC36 with xylose or glucose as the sole carbon
source. Fermentations were begun at an initial OD550 of 0.5. Symbols: Solid
rectangles, xylose; Open rectangles, glucose.













Table 3-1. Products formed by TC36 during aerobic catabolism of various sugars
Concentration mmoles L-la
Cell mass ,max Carbon Acetate 2-Oxo-
Conditionb (g L1) (h-'l) Balance (%) Yield (%)d Acetate Pyruvate glutarate Succinate Malate
Arabinosee 4.01 0.46 94 63 220 <1 21 3 4
Glucosef 3.47 0.51 87 62 250 <1 19 14 <1
Mannosee 3.26 0.23 83 59 187 <1 13 2 2
Sorbitolf 3.44 0.30 87 61 195 <1 15 2 <1
Xylosef 3.85 0.33 102 69 205 7 19 2 <1
a Final concentrations at the end of fermentation. Fumarate was not included in Table 3-1 because the concentrations was observed to be less than 1 mM for all
fermentations.
b All fermentations were conducted with 3% initial sugar concentration. Arabinose, mannose, and sorbitol fermentations were inoculated at an initial OD550 of
0.05 and the xylose fermentations were inoculated at an initial OD550 of 0.5.
' Maximum specific growth rate.
d Acetate yield as a percentage of the maximum theoretical yield (0.67 g of acetate per g of glucose).
e Results from one fermentation.
f Averages of two fermnetations.









Arabinose constitutes the second most abundant sugar in hemicellulose derived

from sugarcane. E. coli TC36 converted 207 mM arabinose into 220 mM acetate, 63% of

the maximum theoretical yield (Table 3-1). Again, acetate was the dominant (89%)

organic acid recovered from the fermentation broth. The final cell mass and maximum

specific growth rate were equivalent to those of TC36 grown on glucose under the same

conditions (Tables 2-3 and 3-1).

Mannose is an optical isomer of glucose and constitutes a small fraction of the

sugars found in hemicellulose. E. coli TC36 converted mannose to acetate with a

maximum theoretical yield of 59% (Table 3-1). The specific growth rate (0.23 h-1) and

final cell density on mannose was the lowest of all sugars tested (Table 3-1). Acetate

accounted for 92% of all organic acids recoved, although 17% of the substrate carbon

was not recovered (Table 3-1).

Oxidation of Sorbitol to Acetate

Sorbitol is a six carbon sugar alcohol which is more reduced than glucose. One

more NADH is generated from the oxidation of sorbitol than from glucose. The specific

growth rate on sorbitol was 59% of the growth rate on glucose and the final cell density

was 99% of glucose grown cultures (Table 3-1). The maximum theoretical acetate yield

attained was 61%, which is comparable to that acheived on glucose under the same

conditions.

Discussion

Arabinose and xylose are pentose sugar constituents of hemicellulose. Both are

transported into the cell by high affinity ABC (ATP binding cassette) transporters

specific for the respective sugar (Neidhardt et al. 1996). There are also low affinity H -

linked symporters involved in transport of the pentoses (Henderson 1990). Once in the









cell, arabinose and xylose are catabolized through the pentose phosphate pathway (PPP).

Arabinose enters the PPP at the level of ribulose-5-phosphate and xylose enters at the

level of xylulose-5-phosphate. Net ATP yield is much lower for the catabolism of the

pentoses (0.67 per xylose or arabinose) than for glucose (2 per glucose) with lactate as

the end-product (Tao et al. 2001). The NADH produced during xylose and arabinose

catabolism is the same as that produced from glucose catabolism.

Acetate yield and final cell density were equivalent for the pentose fermentations

and glucose fermentations. If ATP was to become limiting during pentose fermentations

one would expect greater flux to acetate to generate more ATP or a reduction in the final

cell mass. These results imply that net ATP is not limiting the growth of TC36 on

arabinose and xylose during aerobic growth. One may expect reduced maximum specific

growth rate on xylose due to a slower rate of ATP production, although the maximum

specific growth rate for arabinose grown cells is 1.4-fold greater than that of xylose

grown cells. If the rate of ATP production was limiting growth rate, one would expect

arabinose and xylose cultures to exhibit similar rates. Arabinose was demonstrated to be

used preferentially by E. coli K12 when xylose or ribose is present in minimal medium

(Kang et al. 1998). The regulatory mechanism was determined to be at the transcriptional

level (Kang et al. 1998). Preferential use of arabinose suggests that E. coli derives some

thermodynamic or catalytic advantage from catabolism of arabinose over the other

pentoses. Unfortunately, the mechanism has not been documented.

Mannose was the poorest carbon source examined for growth and acetate yield.

Growth on mannose was characterized by a 53% reduction in specific growth rate, as

compared to glucose grown cells, and an acetate yield of 59%. Mannose is transported









into the cell by a PTS-linked mannose permease (Erni et al. 1987) and isomerized to

fructose-6-phosphate. The growth rate reduction observed for mannose may reflect a

slower rate of transport into the cell or isomerization (decreased Vmax) to fructose-6-

phosphate for mannose than for glucose.

Sorbitol is a polyalcohol commonly found in ripe berries. Commercial production

of sorbitol uses high-pressure hydrogenation or electrolytic reduction of glucose. Sorbitol

was used as a substrate for TC36 in order to demonstrate the ability of the organism to

convert a reduced substrate into an oxidized product, acetate. Strain TC36 was able to

converted sorbitol efficiently to acetate and maintain a growth rate 61% that of glucose-

grown cultures. Alteration of the process conditions, such as increasing the dissolved

oxygen above 5%, providing more electron acceptor for respiration, may improve growth

rate and/or acetate yield. Conversion of sorbitol to acetate demonstrates the utility of the

mutations constructed in TC36. This biocatylist is not limited to the production of

compounds reduced or neutral relative to the substrate.

In this chapter, the ability ofE. coli TC36 to efficiently convert the major sugars

found in hemicellulose into acetate is demonstrated. In addition, strain TC36 is capable of

converting of substrate carbon into an oxidized product, such as acetate and CO2.














Chapter 4
ENGINEERING Escherichia coli FOR THE EFFICIENT CONVERSION OF
GLUCOSE TO PYRUVATE

Introduction

There is a growing trend in the commodity and specialty chemicals industries

towards the use of microorganisms as biocatalysts. Many commodity chemicals (ethanol,

lactate, 1,3-propanediol, and adipic acid) and specialty chemicals (polyketides and

carotenoids) can be made directly or indirectly from the same central metabolite, pyruvic

acid.

The metabolic fate of pyruvate, a key branch point in central carbon metabolism, is

regulated in large part by the redox state of the cell. During the fermentative metabolism

of glucose, NADH levels are high, and > 95% of pyruvate is used as terminal electron

acceptor for NADH oxidation; < 5% is partitioned into biosynthesis (see Figure 1-3 for

an overview). During oxidative metabolism, pyruvate is oxidatively decarboxylated by

the pyruvate dehydrogenase complex (PDHc) to produce CO2 and acetyl-CoA. Oxygen

or other external compounds serves as the terminal electron acceptor, and NADH levels

remain relatively low. Up to half of the glucose carbon is used for biosynthesis, the

remainder being fully oxidized by the tricarboxylic acid cycle to CO2. Construction of an

E. coli strain that excretes pyruvate as the major product of glucose catabolism requires

that pyruvate utilizing pathways be eliminated (Fig. 4-1A) and carbon accumulation in

the form of cell mass be minimized.









Pymvic acid is currently manufactured for use as a food additive, nutriceutical, and

weight control supplement (Li et al. 2001a; Anonymous 2002). Pyruvic acid can also be

used as a starting material for the synthesis of amino acids such as alanine, tyrosine,

phenylalanine, and tryptophan (Li et al. 2001a) and for acetaldehyde production (Ingram

and Conway 1987). Pyruvate is produced commercially by both chemical and microbial

processes (Li et al. 2001a). Chemical synthesis from tartrate involves toxic solvents and

is energy-intensive (Ondrey 2001; Blair 1999) with an estimated production cost of

$8,650 per ton of pyruvate (Li et al. 2001a). Microbial pyruvate production is based

primarily on two microorganisms, a multi-vitamin auxotroph of the yeast Torulopsis

glabrata (Li et al. 200 b) and a lipoic acid auxotroph of Escherichia coli containing a

mutation in the F1 subunit of the (FiFo)H+-ATP synthase (Yokota et al. 1994). Both

strains require precise regulation of media composition during fermentation and complex

supplements. Estimated production costs for pyruvate production with these strains is

14.5% ($1,255 per ton pyruvate) that for chemical synthesis (Li et al. 2001a). Recently,

Tomar et al. (2003) have described a new E. coli mutant for pyruvate production from

glucose and acetate in complex medium. This strain contains mutations in

phosphoenolpyruvate carboxylase (ppc), pyruvate dehydrogenase (aceF), and alcohol

dehydrogenase (adhE).

Nutrients in culture medium often represent a major cost associated with

commercial fermentations. The use of a mineral salts medium and an inexpensive carbon

source offer the potential to improve the economics of many biological processes by

reducing the costs of materials, product purification, and waste disposal (Zhang and

Greasham 1999). A microbial platform that combines attributes of aerobic and









fermentative metabolism for the production of compounds that are redox neutral or

oxidized relative to glucose, described in Chapter 2, was developed by our laboratory

(Causey, PNAS. 2003). In order to demonstrate further the flexibility of the biocatalyst

with regard to the variety of compounds that can be produced, we have engineered

Escherichia coli W3110 for the efficient production of pyruvic acid as the major end

product of glucose catabolism.

Materials and Methods

Microorganisms and Media

E. coli W3110 (ATCC 27325) and derivatives (Table 4-1) were grown on mineral salts

medium (Causey et al. 2003) containing, glucose (2% in plates; 3% in broth). When

needed for pH control, 3-[N-morpholino]propanesulfonic acid (0.1 M, pH 7.4) was

added, but was not included in pH-controlled fermentations. During plasmid and strain

construction, cultures were grown in Luria-Bertani (LB) broth or on LB plates (1.5%

agar) (Miller 1992). Glucose (2%) was added to LB medium for all strains containing

mutations in (FiFo)H -ATP synthase. Antibiotics were included as appropriate

(kanamycin, 50 mg L-1; ampicillin, 50 mg L-1; apramycin, 50 mg L-1; and tetracycline,

12.5 or 6.25 mg L1).

Genetic Methods

Standard methods were used for plasmid construction, phage P1 transduction,

electroporation, and polymerase chain reaction (PCR) (Miller 1992; Sambrook and

Russell 2001). Coding regions for ackA and poxB were amplified using ORFmer primers

(Sigma-Genosys, Inc., The Woodlands, TX) and initially cloned into pCR2.1-TOPO.

Chromosomal integration of mutated genes was facilitated by pKD46 containing an

arabinose-inducible Red recombinase (Datsenko and Wanner 2000).










Table 4-1. Sources and characteristics of strains and plasmid
Strains/Plasmids Relevant Characteristics
Strains


W3110
TOP OF'
SZ61
TC36


TC37


TC38


TC42


TC43


TC44


Plasmids
pCR2.1-TOPO
pFT-A

pKD46


K12 wild type
lacP (episome)
W3110, AackA: :FRT-tet-FRT
W3110, (Succ+), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AackA: :FRT-tet-FRT
W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AackA: :FRT
W3110, (Succ ), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
ApoxB: :FRT
W3110, (Succ+), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AackA: :FRT ApoxB: :FRT-tet-FRT
W3110, (Succ+), A(focA-pflB)::FRT AfrdBC AldhA
Aatp(FH)::FRT AadhE::FRT AsucA::FRT
AackA: :FRT ApoxB: :FRT

bla kan, TOPOTM TA cloning vector
blaflp low-copy vector containing recombinase and
temperature-conditional pSC101 replicon
bla y 0 exo low-copy vector containing red
recombinase and temperature-conditional pSC 101
replicon


Reference

ATCC 27325
Invitrogen
Zhou et al. 2003
Causey et al. 2003

Causey et al. 2004


Causey et al. 2004


Causey et al. 2004


Causey et al. 2004


Causey et al. 2004



Invitrogen
Posfai et al. 1997

Datsenko and
Wanner 2000


Integrants were selected using antibiotic resistance, screened for appropriate antibiotic

resistance markers and phenotypic traits, and verified by analyses of PCR products and

fermentation profiles. FRT-flanked antibiotic resistance genes were deleted using FLP

recombinase (Martinez-Morales et al. 1999; Posfai et al. 1997).

Deletion of Acetate Kinase (Strains TC38 and TC44)

The AackA::FRT-tet-FRT mutation was transduced from SZ61 into TC36 and TC42

to produce strains TC37 (AfocA-pflB::FRTAfrdBC::FRT A IdhA AatpFH::FRT

AadhE::FRT AsucA::FRT AackA::FRT-tet-FRT) and TC43(AfocA-pflB::FRT

AfJdBC::FRT A IdhA AatpFH::FRT AadhE::FRT AsucA: :FRTpoxB::FRT AackA: :FRT-









tet-FRT), respectively. After removal of the tet gene using FLP recombinase, resulting

strains were designated TC38 (A(focA-pflB::FRT AfrdBC::FRT AldhA AatpFH::FRT

AadhE::FRT AsucA: :FRT AackA ::FRT) and TC44 (AfocA-pflB::FRT AfrdBC::FRT AldhA

AatpFH: :FRT AadhE: :FRT AsucA: :FRTpoxB: :FRT AackA: :FRT), respectively.

Fermentation

Fermentations (5 L and 10L) were conducted at 370C (dual Rushton impellers, 350

- 450 rpm; pH 7.0) in Bioflow 3000 fermentors (New Brunswick Scientific, Edison, NJ).

Unless stated otherwise, dissolved oxygen levels were 100% of air saturation at the time

of inoculation and allowed to fall to 5% of air saturation during cell growth with

continuous air sparging (0.2 vvm). This level of oxygen was maintained by mixing 02

with air at a constant flow rate of 1.0 L min-. Broth pH was controlled with 11.4 M

KOH. For fed-batch studies, glucose was added from a sterile 4 M stock. Two fed batch

regimes were investigated: 1) 3% initial glucose followed the addition of 3% glucose

after 15 h (6% total); 2) 3% initial glucose with the addition of 590 mL of 4 M glucose at

a constant rate over a 20-h period (9.8% total glucose).

Seed cultures prepared as previously described (Causey et al. 2003) were used to

provide an inoculum of 16.5 mg dry cell weight L-1. Broth samples were removed to

measure organic acids, residual glucose, and cell mass. Volumetric and specific rates

were estimated from measured values for glucose and acetate using GraphPad Prism

(GraphPad Software, San Diego, CA). A smooth curve was generated with 10 points per

min (Lowess method) to fit measured results. The first derivative (acetate or glucose

versus time) of each curve served as an estimate of volumetric rate. Specific rates

(mmoles L-1 h-1 [mg dry cell weight]-1) were calculated by dividing volumetric rates by

respective values for cell mass.









Analyses

Organic acids and glucose were measured using a Hewlett Packard HPLC (HP

1090 series II) equipped with a UV monitor (210 nm) and refractive index detector

(Causey et al. 2003). Cell mass was estimated using a Bausch & Lomb Spectronic 70

spectrophotometer (1.0 OD550nm is equivalent to 0.33 g dry cell weight L-1).

Results

Pyruvate as a Co-Product during Acetate Fermentations

Escherichia coli TC36 (AfocA-pflB AfrdBC AldhA AatpFH AadhE AsucA) was

previously engineered from W3110 (wild type) for the production of acetate (Fig. 3-1A)

by combining chromosomal deletions that minimize cell yield, oxygen consumption, CO2

evolution, and reduced fermentation products (Causey et al. 2003). In this strain,

glycolytic flux was higher than the parent due to a deletion of genes (AatpFH) encoding

two membrane proteins that couple the Fi and Fo components of the FiFo(H )ATP

synthase complex. This mutation eliminated ATP production by oxidative

phosphorylation and created an active, cytoplasmic FiATPase (Fig. 3-1B and 3-1C).

Glycolytic flux in TC36 exceeded the capacity for acetate production under our initial

test conditions (5% air saturation at inoculation and during fermentation), resulting in

transient accumulation of approximately 16 mM pyruvate near the end of exponential

growth (Fig. 3-2). The peak level of pyruvate was increased to 81 mM (Fig. 4-2) by

inoculating the fermentor at an initial dissolved oxygen level of 100% air saturation

(rather than 5% of saturation) with continuous air sparging until the oxygen level

declined to 5% air saturation, then adding oxygen to maintain 5% of air saturation. Under

this condition, pyruvate yield at the peak was 25% of the maximum theoretical yield and

11% of the maximum theoretical yield at the end of fermentation (Fig. 4-2; Table 4-2).













Lactate


NAD*


CO2
NAD NADH


U2 pta Acetyl-CoA ------- adh -- Ethanol


SAcetate
-P
I F -- H+ H+ 02+e -- H20
NADH Oxaloacetate A
NAD gltA out
Acetyl-CoA Fo Electron Transpor
Malate A system
a at glcB aceB Citrate Sy
fumABC atplBEFHAGD
F rate Glyoxylate eA acnB I lNAD NA
ace- AD+ AD.
FADH / UQH2 Isocltrate
rBC sdhABCD NADP
uQ i cdAt ATP ADP F
H +NCDPH
FAD Succinate NADPH P,
S2-Oxoglutarate 2
sucDC NAD
NADH ATP
Succinyl-CoA 4 P,
CO2



Figure 4-1. Genetic modifications used to redirect glucose metabolism for pyruvate
production. Metabolic steps that have been blocked by mutation or deletion of
genes are highlighted. A) Central carbon metabolism. B) Oxidative
phosphorylation. C) Cytoplasmic Fi ATPase subunit (active). Genes: aceA,
isocitrate lyase; aceB, malate synthase B; aceEF, Elp and lipoate
acetyltransferase/dihydrolipoamide acetyltransferase component of the PDHc;
ackA, acetate kinase; acnB, aconitase; adhE, alcohol/aldehydrogenase
pyruvate format lyase deactivase; atplBEFHAGDC, (FiFo)H+-ATP synthase;
frdABCD, fumarate reductase; glcB, malate synthase G; gltA, citrate synthase;
icdA, isocitrate dehydrogenase; IdhA, lactate dehydrogenase; IpdA,
dihydrolipoate dehydrogenase/dihydrolipoamide dehydrogenase component of
the PDHc and 2-oxoglutarate dehydrogenase; pflB, pyruvate formate-lyase;
poxB, pyruvate oxidase; pta, phosphotransacetylase; pykA, pyruvate kinase II;
pykF, pyruvate kinase I; sdhABCD, succinate dehydrogenase; sucAB, 2-
oxoglutarate dehydrogenase; sucDC, succinyl-CoA synthetase.






64


We have explored additional genetic modifications of TC36 to increase the

efficiency of pyruvate production from glucose. Although many metabolic routes lead to

acetate, two primary routes are present in E. coli (Fig. 4-1A): 1) conversion of

acetyl-CoA to acetate by phosphotransacetylase (pta) and acetate kinase (ackA); and 2)

direct oxidation of pyruvate to acetate by pyruvate oxidase (poxB) (Fig. 4-1A).

Derivatives of TC36 were constructed with mutations in both pathways.

200 100

180 90

S160- 80 3

140- 70 <

S120- 0
E 1Glucose (5% DO) /
E 100- 50 3
Glucose (100 5% DO) o
a( 80- 40
80 Pyruvate (5% DO)
o 60- Pyruvate (100 5% DO) 30
40 20

20- 10
0- 0 n
0 2 4 6 8 10 12 14 16 18 20
Time (h)

Figure 4-2. Effect of oxygen level on pyruvate production by TC36. Cells were
inoculated into fermentation broth at 100% air saturation and continuously
sparged with air until the oxygen levels declined to 5% saturation. At this
time, oxygen was blended to maintain 5% 02 saturation during the remaining
period of incubation (open symbols). Alternatively, media was sparged with a
mixture of air and nitrogen to provide 5% air saturation prior to inoculation.
Sparging was switched to air and oxygen as needed to maintain 5% air
saturation (closed symbols).









Acetate Kinase (AackA) Deletion Enhances Pyruvate Production and Inhibits
Glycolysis (Strain TC38)

To block acetate production by the acetate kinase pathway, the central region of the

ackA gene was deleted in TC36 to produce TC38. Acetate production was reduced by

85% (Fig. 4-3C; Table 4-2), consistent with the acetate kinase pathway being the

dominant route for acetate production in TC36. However, this deletion also reduced net

ATP production (estimated) by 30% (Fig. 4-1A), cell yield by 36% (Fig. 4-3A; Table 4-

2), and maximum specific growth rate by 45% (Table 4-3). Glycolytic flux was reduced

by 45% (Table 4-3) increasing the time required to complete fermentations from 18 h for

TC36 to 24 h for TC38 (Fig. 4-3B). Although both volumetric and specific rates of

glucose metabolism were lower for TC38 (Table 4-3), pyruvate yield was 5.5-fold higher

(Table 4-2; Fig. 4-3D) and the specific rate of pyruvate production was 4-fold higher

(Table 4-3) than for TC36. Small amounts of 2-oxoglutarate, succinate, and fumarate

were produced by both strains. From 10% to 15% of the carbon was not recovered as cell

mass or acidic fermentation products and is presumed to be lost as CO2 due to metabolite

cycling. With strain TC38, the pyruvate yield (195 mM) was 58% of the theoretical

maximum. Acetate (28.9 mM) remained as the second most abundant product.

Pyruvate Oxidase (poxB) Mutation Causes a Modest Reduction in Acetate
Accumulation

Pyruvate can be metabolized to acetate by the membrane-bound protein pyruvate

oxidase using the electron transport system to couple oxygen as a terminal electron

acceptor. The poxB gene is typically repressed during exponential growth but is induced

by stress or entry into stationary phase (Chang and Cronan 1983; Chang et al. 1994).

Pyruvate oxidase was inactivated in TC36 by constructing three stop codons, one in each

reading frame, in the central region to produce TC42.













Table 4-2. Products formed from glucose catabolism by E. coli strains used in our study
Product Concentrations (mmoles L ')a
Carbon Pymvate
Cell mass Balance Yield 2-Oxo-
Strain Condition Replicates (g L ') (%) (%)b Pyruvate Acetate glutarate Succinate Fumarate
3%
W3110 32 4.13 80.6 5.9 20.8 180.0 8.3 13.7 0.9
glucose
3%
TC36 33 3.470.23 89.02.7 10.57.9 38.127.2c 197.721.1 16.616.2 13.7+13.2 1.40.2
glucose
3%
TC38 3 2.210.09 84.35.2 57.52.6 194.59.1 28.916.7 10.51.9 8.19.1 0.80.7
glucose
3%
TC38 3 d 2 2.40 84.7 68.8 241.9 7.0 7.9 <2.0 <0.2
glucose
TC42 32 3.40 86.8 29.1 79.0 178.4 76.2 24.3 1.7
glucose
a Unless stated otherwise the concentrations represent measurements at the time of glucose exhaustion.
b Maximum theoretical yield is 2 moles pyruvate per mol glucose (0.978 g pyruvate per g glucose)
c The pyruvate concentrations during glucose fermentations ranged from 14.9 to 111.9 mM. Pyruvate production was very sensitive to dissolved oxygen
fluctuations.
d Dissolved oxygen was allowed to fall from 100% to 50% of air saturaiton.
e Three percent initial glucose followed by the addition of 208 mL of a 4 M glucose solution.
f Three percent initial glucose followed by the addition of 590 mL of a 4 M glucose solution over a period of 20 h.













Table 4-2. Continued


Product Concentrations (mmoles L ')a


Strain
TC44
TC44


Condition
3% glucose
3% glucose,
2 Nitrogen
2 o/_ -IO/


Cell mass Carbon Pyruvate 2-Oxo-
Replicates (g L ') Balance (%) Yield (%)b Pyruvate Acetate glutarate
3 2.360.10 88.50.6 69.31.5 252.56.2 11.61.2 3.61.2


2


2.02


73.6


38.8


125.2


50.3


30.0


Succinate Fumarate
16.80.7 1.10.2
7.7 2.9


TC44 "e 2 2.63 86.7 72.3 479.8 39.8 31.7 10.9 0.7
glucose
TC44 6% glucose 2 1.95 94.8 588.9 46.0 26.1 <2.0 0.7
excess
TC44 excess 2 2.51 -- 749.0 62.4 45.3 14.7 3.3
glucose 2
a Unless stated otherwise the concentrations represent measurements at the time of glucose exhaustion.
b Maximum theoretical yield is 2 moles pyruvate per mol glucose (0.978 g pyruvate per g glucose)
The pyruvate concentrations during glucose fermentations ranged from 14.9 to 111.9 mM. Pyruvate production was very sensitive to dissolved oxygen
fluctuations.
d Dissolved oxygen was allowed to fall from 100% to 50% of air saturaiton.
e Three percent initial glucose followed by the addition of 208 mL of a 4 M glucose solution.
f Three percent initial glucose followed by the addition of 590 mL of a 4 M glucose solution over a period of 20 h.






























- TC36
STC38 (ackA-)
- TC42 (poxB )
-TC44 (ackA poxB-)


250-
-j
S200


E 150
a)
(U)
o 100-

(3


0 4 8 12 16 20 24 28 0 4 8 12 16
Time (h) Time (h)


- TC36
STC38 (ackA-)
- TC42 (poxB )
STC44 (ackA poxB-)


250
-j
) 200-
a)
0
E
E 150-
a
> 100

5o
50-


0-


--TC36
TC38 (ackA-)
- TC42 (poxB )
-TC44 (ackA- poxB-)











20 24 28


-TC36
TC38 (TC36 ackA-)
-TC42 (TC36 poxB-)
- TC44 (TC36 poxB ackA-)


0 4 8 12 16
Time (h)


20 24 28 0 4


12 16
Time (h)


20 24 28


Figure 4-3. Batch fermentation of glucose by mutant strains of E.

D) Pyruvate Production.


coli. A) Cell Growth B) Glucose utilization C) Acetate production


-i


u)

01


0.01


300


250

-j
* 200
a)

E
E 150


o -
<









In contrast to the ackA deletion (TC38), the poxB mutation (TC42) caused a

relatively small decease in acetate and pyruvate (Table 4-2; Fig. 4-3C and 4-3D)

consistent with a minor role for the POX pathway. Pyruvate yield with TC42 was less

than 30% of the theoretical maximum. Unlike the mutation in ackA (TC38), inactivation

ofpoxB (TC42) did not reduce the rate of growth or glucose metabolism (Table 4-3).

Combining Mutations for Pyruvate Oxidase (poxB) and Acetate Kinase (ackA)
Improved Pyruvate Production (Strain TC44)

Strain TC44 (AfocA-pflB AfrdBC AldhA AatpFH AadhE AsucA poxB::FRT AackA)

was constructed in which both the acetate kinase and pyruvate oxidase mutations were

combined in the TC36 background. Inactivation ofpoxB was beneficial for growth and

pyruvate production (Fig. 4-3A; Table 4-2 and Table 4-3) in comparison to TC38

(isogenic strain containing functional poxB). Both volumetric and specific rates of

glucose metabolism were higher for TC44 than for TC36 or TC38 (Table 4-3). Acetate

production by TC44 was half that of TC38 and pyruvate yield was 17% higher.

Table 4-3. Comparison of metabolic rates
Glucose consumption rate Pyruvate production rate
Average Volb Max. Spec.c Average Vol.b Max. Spec.c
Strain g (h-') (mmol L-1 h-') (mmol L'1 h-1 g-') (mmol L-1 h-') (mmol L-1 h-1 g-')
W3110 0.69 11.9 17.4 nd nd
TC36d 0.490.03 10.12.6 17.61.5 nd nd
TC36e 0.510.01 10.70.9 29.73.5 3.83.0 5.33.1
TC38e 0.280.01 6.70.6 16.32.2 8.30.7 21.1+3.7
TC38f 0.21 6 28 8 28
TC42e 0.55 10 17 6 10
TC44e 0.340.02 9.70.7 27.24.1 13.10.3 40.47.4
a Standard deviations are included for data from three or more fermentations; others represent an average of
two fermentations.
b Average volumetric rates.
' Maximum specific rates. dcw, dry cell weight.
d Dissolved oxygen controlled at 5% of air saturation by adjusting the ratio of 02 to N2.
e Dissolved oxygen allowed to fall from 100% to 5% of air saturation during growth with air sparging; 02
added to maintain 5% air saturation.
f Dissolved oxygen allowed to fall from 100% to 50% of air saturation during growth with air sparging; 02
added to maintain 50% air saturation.









The specific rate of pyruvate production by TC44 was 8-fold that of TC36 and twice that

of TC38 (Table 4-3). The time required to complete fermentation with TC44 was 30%

shorter than with TC38 (Fig. 4-3B). Broth containing 3% glucose (167 mM) was

metabolized to 2.2% pyruvate (252 mM) after 18 h in mineral salts medium (Fig. 4-3D).

Although acetate levels were substantially reduced by combining thepoxB and ackA

mutations (Fig. 4-3C), acetate (12 mM) and dicarboxylic acids (20 mM) remained as

minor products.

The Beneficial Role of a poxB Mutation for Pyruvate Production

Although eliminating the primary route for acetyl-CoA dissimilation (AackA) in

TC38 increased pyruvate production, this was accompanied by detrimental effects on

growth and fermentation rates (Fig. 4-2; Table 4-2 and Table 4-3) that were substantially

reduced by inactivation of PoxB (TC44). Pyruvate oxidase competes with NADH

oxidation for oxygen as a terminal electron acceptor and represents a potential

mechanism for this action. Oxygen transfer rates are frequently limiting during aerobic

fermentations at relatively high levels of saturation (Varma et al. 1993), and may be even

more problematic under our fermentation conditions (5% of air saturation). Increased

PoxB activity resulting from larger pyruvate pools in the acetate kinase mutant (TC38)

would be expected to decrease the availability ofNAD+ for glycolysis and to increase the

levels of NADH, an allosteric inhibitor of key biosynthetic enzymes such as citrate

synthase (Weitzman 1966) and phosphoenolpyruvate carboxylase (Krebs and Bridger

1976). This hypothesis was tested in part by examining the effect of increasing the

oxygen level during fermentation with TC38.

Increasing oxygen saturation from 5% to 50% during TC38 fermentations (Table 4-

2 and Table 4-3) was beneficial, consistent with our hypothesis. Cell yield, pyruvate









yield, and the specific rate of glucose metabolism were 8% to 41% higher for TC38 at

50% air saturation than at 5% air saturation. These results were very similar to those

observed for the isogenicpoxB mutant, TC44, during fermentation at 5% air saturation.

Increasing the oxygen saturation during TC38 fermentations also decreased the final

concentrations of acetate to a level equivalent to TC44 at 5% air saturation and decreased

the production of dicarboxylic acids (Table 4-2).

Improving Pyruvate Yields and Titers of TC44 by Altering Fermentation
Conditions

Prior experience with the acetate-producing strain, TC36, demonstrated that yields

could be improved by simple changes in fermentation conditions (Causey et al. 2003).

Unlike TC36, decreasing the ammonia level by half did not increase product yields for

TC44 (Table 4-2). However, doubling the initial concentration of glucose or providing a

second addition of glucose (3% plus 3%) increased pyruvate yields by 11% and doubled

the final pyruvate titer. With excess glucose, 749 mM pyruvate was produced. This may

represent the limit for pyruvate tolerance. Addition of 600 mM pyruvate to mineral salts

medium inhibited the growth of wild type W3110 (data not included).

Discussion

Pyruvate occupies a key branch point in central carbon metabolism, its fate

dependent on the redox state of the cell. During respiration, pyruvate is oxidatively

decarboxylated by the pyruvate dehydrogenase complex (PDHc) to form NADH, CO2

and acetyl-CoA. During fermentative metabolism, pyruvate acts as a final electron

acceptor for NADH oxidation and is converted to reduced fermentation products such as

lactic acid. During fermentation, approximately 5% of the metabolized pyruvate is

converted to acetyl-CoA and format (pyruvate formate-lyase; pflB gene) for









biosynthesis. Construction of an E. coli strain that excretes pyruvate at high yield during

glucose catabolism required the elimination of non-essential pathways for pyruvate

utilization and mutations that reduce the utilization of pyruvate for cell growth.

Previously, we described a derivative of Escherichia coli W3110 (strain TC36) that

combines many of the attributes of aerobic and fermentative metabolism and efficiently

converts glucose to acetate, a redox neutral product (Causey et al. 2003). This strain was

used as a microbial platform to engineer TC44 for the conversion of glucose to pyruvate,

a more oxidized product, by mutating genes encoding acetate kinase (ackA) and pyruvate

oxidase (poxB). Strain TC44 converted glucose to pyruvate with a yield of 0.75 g

pyruvate g-1 glucose (77.9% of theoretical yield) under optimal conditions in minimal

medium. Under conditions of glucose excess, a maximum of 749 mM pyruvate was

produced.

Strain TC44 contains multiple mutations that ensure the rapid and efficient

conversion of sugars into pyruvate. The minimal effect of these multiple mutations on

growth rate was surprising. Indeed, glycolytic flux and maximal growth rate for TC44

were only 20% lower than the unmodified W3110 under similar growth conditions

(unpublished results). The pyruvate oxidase mutation was surprisingly beneficial for

growth and pyruvate production. Introduction of this mutation into the acetate-producing

strain (TC36) resulted in a small improvement in growth rate in contrast to the mutation

of acetate kinase that decreased growth rate by half (Table 4-3). The decreased growth

rate of the acetate kinase mutant, TC36, cannot be explained by the predicted 30%

reduction in ATP yield since growth rate was substantially restored by adding a

subsequent mutation in pyruvate oxidase. These results suggested that the reduction in









growth rate by the acetate kinase mutation may be due to increased PoxB activity

associated with a larger pyruvate pool.

Pyruvate oxidase transfers electrons from pyruvate to ubiquinone upon the

decarboxylation of pyruvate (Cunningham and Hager 1975 ). An increase in pyruvate

metabolized by pyruvate oxidase would increase oxygen demand and reduce the oxygen

available for the oxidation of NADH. Since the total nicotinamide adenine dinucleotide

pool is relatively constant (Wimpenny and Firth 1972) and the NAD+/NADH ratio is

responsive to changes in oxygen availability (Wimpenny and Firth 1972), increased

pyruvate oxidase activity would also reduce the level of NAD+ available for glycolysis.

High NADH levels also serve as an important allosteric regulator of key enzymes such as

citrate synthase (Weitzman 1966) and phosphoenolpyruvate carboxykinase (Krebs and

Bridger 1976). Allosteric inhibition of these enzymes in minimal medium would limit

both glycolytic flux and the partitioning of carbon into biosynthesis. Oxidation of NADH

is also essential to provide the stochiometric levels ofNAD+ required for glycolysis.

Thus, inactivating pyruvate oxidase may be expected to improve growth by eliminating

the increase in oxygen demand, restoring the NAD+ pools for glycolysis, and decreasing

the level of NADH. This hypothesis was supported by further experiments in which the

availability of oxygen was increased from 5% of air saturation to 50% of air saturation

during fermentation with the acetate kinase mutant, TC38. The increase in oxygen (50%

air saturation) substantially reversed the negative impact of the acetate kinase mutation

on growth rate and metabolism, analogous to the effect of adding a pyruvate oxidase

mutation. A similar increase in oxygen was of no benefit for TC44 in which both acetate

pathways had been mutated (data not included).









During the construction of TC44 from the acetate-producing strain TC36,

mutations in acetate kinase and pyruvate oxidase were tested individually prior to

constructing the combination. These intermediate strains allowed an assessment of the

relative roles of each pathway for acetate production in TC36. The acetate kinase

pathway represents the dominant route for acetate production since deletion of the ackA

gene decreased acetate production by 85% (Table 4-2). The pyruvate oxidase mutation

decreased acetate production by 10% while the combination of both mutations decreased

acetate production by 94%. The low level of acetate produced by TC44 containing both

mutations is presumed to reflect a combination of multiple minor pathways and remains

as a potential target for future improvement in pyruvate yield.

Pyruvate can be produced by a variety of microorganisms including mutants of yeasts

and bacteria (Table 4-4). Typical production rates for these biocatalysts are around

1 g L-1 h-1 with yields exceeding half the weight of substrate. Torulopsis glabrata, a yeast

strain currently used for the commercial production, can achieve pyruvate titers of

69 g L-1 although yields are somewhat lower than for TC44 and more elaborate process

controls are required. T. glabrata strains used in the commercial process are multivitamin

auxotrophs requiring tight regulation of vitamin concentrations which result in complex

vitamin feeding strategies during fermentation (Li et al. 2001a). Previous E. coli strains

constructed for pyruvate production were cultured in complex media and have been

plagued by low titers and yields (Tomar et al. 2003; Yokota et al. 1994). In contrast to

previous biocatalysts where vitamins and other complex nutrients are required for

effective production of pyruvate by fermentation, E. coli TC44 requires only mineral

salts, glucose and very simple process controls. The lack of a requirement for vitamin












Table 4-4. Comparison of biocatalysts for pyruvate production


Strain
Candida
lipolytica
AJ 14353
Debaryomyces
hansenii
Y-256
Torulopsis
glabrata ACII-
3
Torulopsis
glabrata
WSH-IP 303
Escherichia
coli TBLA-1
Escherichia
coli
CGSC7916

Escherichia
coli TC44


Relevant
genotype/
phenotype


Carbon
Source


Ferm-
Nitrogen entation
Source Time (h)


B1 Met- glucose NH4N03


Bi Bio-


Bi' Bio- B6
NA-
acetate
leaky
Bi- Bio- B6-
NA-
lipA2 bgf
atpA401
aceFfadR
adhEppc

pflBfrdBC
IdhA atpFH
adhE sucA
ackA poxB


glucose Peptone


Soy
glucose hydrolysate
(NH4)2S04

glucose NH4Cl


glucose Poly-peptone

glucose Tryptone
acetate (NH4)2-HP04


glucose (NH4)2HP04


[Pyruvate]
(gL-1)


Vol. Prod.
(g L-1 h-1)


Pyruvate
yield
(g g-1)


0.61


0.44


0.44


0.42


0.68



0.62


0.60


0.65



0.76


Ref.

Li et al.
2001a

Li et al.
2001a


Li et al.
2001b


Li et al.
2001b
Yokota et
al. 1994
Tomar et
al. 2003


Causey et
al. 2004


1.28



1.23


1.25


0.97



1.21









supplements, complex nutrients or complicated process controls for TC44 should provide

a substantial savings in production costs. In addition, the lack of complex nutrients in the

fermentation broth should also reduce costs associated with product purification and

waste disposal. Based on this comparison, E. coli TC44 appears to represent a

competitive alternative to the current pyruvate-producing biocatalysts due to high yields,

high product titers, simple fermentation conditions, and the ability to grow well in

mineral salts medium with glucose as the sole carbon source.

The remarkable tolerance ofE. coli to drastic changes in metabolic flow that allows

the production of acetate or pyruvate as alternative products of metabolism implies

considerable elasticity in permitted pool sizes for key metabolic intermediates such as

pyruvate and acetyl-CoA. It is interesting to note that pyruvate transiently accumulated

in broth during fermentations with TC36, but was rapidly co-metabolized with glucose

during the latter stages of fermentation. Similar transient accumulation and re-utilization

was previously observed in minimal medium for other central metabolites carboxylicc

acids and acetate) in other derivatives of W3110 (Causey et al. 2003). Exogenously

added pyruvate was shown to be rapidly mixed with intracellular pyruvate followed by

dissimilation (Yang et al. 2001). Carboxylic acids (Lutgens and Gottschalk 1980;

Underwood et al. 2004) and acetate (Causey et al. 2003; Underwood et al. 2002) are also

co-metabolized with sugars. Active transport systems have been identified in E. coli for

pyruvate (Lang et al. 1987), acetate (Gimenez et al. 2003), and dicarboxylic acids (Lo

et al. 1972; Pos et al. 1998; Seol and Shatkin 1991). Thus for pyruvate and perhaps any

other compound, the extracellular milieu may be reasonably regarded as a reservoir for

the temporary expansion of metabolic pools. A similar argument can be made for









CO2(Merlin et al. 2003) and for acetaldehyde during ethanol production (Dombek and

Ingram 1984; Stanley and Pamment 1993; Stanley et al.1993). The transient

accumulation of metabolites in the aqueous milieu may be of evolutionary advantage.

This accumulation could arguably regarded as a form of metabolic conditioning of the

environment, as a storage reservoir, or as an expanded metabolic pool to relieve a

temporary imbalance in metabolic flux. Enzymes are relatively stable in E. coli.

Substantial remodeling of metabolism is primarily regulated at the level of transcription

with dilution during subsequent growth. Transient storage of products from imbalanced

metabolism in the extracellular milieu could increase the metabolic flexibility ofE. coli

during adaptation to a changing environment














CHAPTER 5
EFFECT OF BETAINE ON THE FERMENTATION PERFORMANCE OF TC44
(PYRUVATE PRODUCTION) AND TC36 (ACETATE PRODUCTION)

Intoduction

The membranes that encompass bacterial cells are permeable to water but provide a

barrier against most solutes. High osmolarity of the extracellular environment would

result in dehydration unless the cell is able to increase the intracellular solute

concentration comparablee solutes). Compatable solutes may be taken up from the

environment (K+, proline, betaine, etc.) or synthesized by the organism (glutamate and

trehalose). Escherichia coli compensates for hyperosmotic stress, first, by increasing

intracellular K+ and glutamate concentrations and, second, synthesis of trehalose (Csonka

and Hanson 1991). In minimal medium, trehalose is synthesized and replaces K and

glutamate as the major comparable solute in the cytoplasm (Csonka and Hanson 1991).

Other osmoprotectant compounds, such as betaine, are also used for osmoprotection

when they are available (Perroud and Le Rudulier 1985).

Betaine (and glycine betaine) is an effective osmoprotectant not synthesized by E.

coli. Betaine has been demonstrated to protect bacteria against hyperosmotic stress as

well as heat (reviewed in Csonka and Hanson 1991; Holtmann and Bremer 2004). In

addition, the growth of ethanologenic E. coli KO11 was shown to be improved by the

presence of betaine or choline in the growth medium (Underwood et al. 2004). Transport

of betaine into the cytoplasm is through an energy dependent proton symport system

(Perroud and Le Rudulier 1985; Styrvold et al. 1986). The transport protein, ProU, is









membrane bound and is transcribed from the proU promoter (Csonka and Hanson 1991).

The proV and proW gene products are the membrane-associated proteins and the proX

gene product is the periplasmic binding protein (Csonka and Hanson 1991). Transport

does not depend on the solute causing osmotic stress but on the magnitude of the osmotic

shock (Perroud and Le Rudulier 1985). E. coli can also transport choline into the cell and

oxidize the choline to glycine betaine (Landfald and Strom 1986). Betaine cannot be used

as a carbon or nitrogen source (Perroud and Le Rudulier 1985). Betaine is effective as an

osmoprotectant under both aerobic and anaerobic conditions but choline is only effective

under aerobic conditions (Landfald and Strom 1986).

Cayley et al. (1991) measured the cytoplasmic osmolytes ofE. coli grown in

MOPS-buffered glucose minimal medium, similar to the NBS medium used in our

studies, with or without betaine. In the absense of betaine, K+ accounted for 53% of the

total osmolytes measured in the cytoplasm (Cayley et al. 1991; Cayley et al. 1992).

Glutamate, trehalose and MOPS were present in minor quantities. Addition of betaine

resulted in a modest increase in total cytoplasmic osmolyte pool (- 10%), betaine

accounted for 58% of the measured osmolytes (Cayley et al. 1991; Cayley et al. 1992).

Potassium and glutamate were the only other osmolytes in the cytoplasm at

concentrations above the limits of detection.

In this chapter the effects of betaine on the growth and product formation of TC36

and TC44 are described. Growth of E. coli W3110 in glucose minimal medium

supplemented with increasing concentrations of acetate or pyruvate in the presence or

absence of betaine will also be discussed.









Materials and Methods

Microorganisms and Media

Escherichia coli W3110 (wild type) was used to determine the minimum inhibitory

concentration of acetate and pyruvate in the presence and absence of 1 mM betaine. The

acetate producing strain TC36 (Table 2-1) and the pyruvate producing strain TC44 (Table

4-1) were used in 5 L fermentations. Strains were carried on NBS 2% glucose minimal

plates as described in Chapter 2. Acid tolerance experiments were conducted with NBS

2% glucose minimal broth containing 0-1 M acetate or pyruvate. Seed culture and Batch

fermentations were conducted as described in Chapter 4 (5 L working volume, 37C, 5%

dissolved oxygen, 0.5 vvm gas flow and 350 rpm agitation) except that 10 % final

glucose concentration was used. Duplicate fermentations were conducted.

Acetate and Pyruvate Tolerance

Acetate and pyruvate tolerance studies were conducted using NBS 2% glucose

medium at pH 7 or pH 8 containing 0-1 M acetate or pyruvate. Betaine HC1 was added to

a final concentration of 1 mM or excluded from the broth. One millimolar betaine was

previously determined to be the optimum concentration for protection against osmotic

shock (Perroud and Le Rudulier 1985). Ten microliters of a logarimically growing

culture of E. coli W3110 (- 1 OD550) was inoculated into 3 mL of the appropriate

medium in a 13 x 100 mm test tube (triplicate cultures). The cultures were grown in a

37C water bath with shaking at 120 rpm. After incubation for 24 h the OD550 was read

using a Bausch & Lomb Spectronic 70 spectrophotometer.









Results

Effect of Betaine on Growth and Acetate Production for E. coli TC36

Betaine has previously been shown to protect E. coli from osmotic shock (reviewed

in Csonka 1991; Underwood et al. 2004).

Acetate fermentations with TC36, conducted using excess glucose, have acetate

concentrations greater than 800 mM resulting in a 1.6 osmolar increase of the broth after

neutralization with KOH. It was expected that addition of 1 mM betaine to the growth

medium would improve growth rate by protecting TC36 from the osmotic stress imposed

on the organism by 10% glucose. In addition, betaine was expected to increase acetate

productivity by reducing the effect of the rising potassium acetate concentration

throughout the fermentation.

E. coli TC36 fermentations were conducted in minimal medium containing 10%

glucose with or without 1 mM betaine. The maximum specific growth rate was increased

2.8-fold by the addition of 1 mM betaine to the culture medium (Table 5-1) although both

cultures entered stationary phase at approximately 24 h (Fig. 5-1A). Betaine had no effect

on volumetric consumption of glucose or acetate production rates (Table 5-1; Fig. 5-

1B,C). The maximum specific glucose consumption rate was reduced by 19% when

betaine was incorporated into the medium (Table 5-1). Initially, the specific rate of

acetate production was higher in the betaine-grown cultures (Fig. 5-1C) resulting in an

increase in the maximum specific acetate production rate of 17% (Table 5-1). The rate of

acetate production was lower during stationary phase with betaine in the medium (Fig. 5-

1C). Overall, betaine did not improve the performance of TC36.









Table 5-1. Comparison of metabolic rates for TC36 in the presence or absence of 1 mM
betaine
Glucose consumption rate" Acetate production rate
E. coli g Average Vol.b Max. Spec.c Average Vol.b Max. Spec.c
TC36 (h-) (mmol L1 h-) (mmol L-1 h-1 g-') (mmol L-1 h-) (mmol L-1 h-1 g-1)
-Betaine 0.15 10 16 10 20
+Betaine 0.42 11 13 11 24
a The data represent an average of two fermentations.
b Average volumetric rates.
Maximum specific rates per gram of dry cell weight.


Effect of Betaine on Growth and Pyruvate Production for E. coli TC44

Similar to acetate fermentations with TC36, strain TC44 experiences hyperosmotic

environments when glucose and pyruvate concentrations are high. It was expected that

addition of 1 mM betaine would at least partially alleviate the osmotic stress imposed on

TC44. The maximum specific growth rate was increased 3.2-fold by the addition of

betaine (Table 5-2). Cell mass reached 2 g L-1 by 24 h, more than double the cell mass at

60 h when betaine was not present (Fig. 5-2A). Betaine had a dramatic positive effect on

both glucose consumption and pyruvate production (Fig. 5-2B,C). All of the glucose was

consumed in 60 h when betaine was present, yet in the absence of betaine 90% of the

glucose remained after 60 h (Fig. 5-2B). The average volumetric rate of glucose

consumption was increased 10-fold and the specific rate of glucose consumption was

increased 5.8-fold by the incorporation ofbetaine in the medium (Table 5-2).

Table 5-2. Comparison of metabolic rates for TC44 in the presence or absence of 1 mM
betaine
Glucose consumption rate" Pyruvate production rate
E. coli g Average Vol.b Max. Spec.c Average Vol.b Max. Spec.c
TC44 (h-) (mmol L1 h-) (mmol L- h-1 g-') (mmol L-1 h-) (mmol L1 h-1 g-1)
-Betaine 0.09 1 6 2 13
+Betaine 0.29 10 35 16 32
a The data represent an average of two fermentations.
b Average volumetric rates.
c Maximum specific rates per gram of dry cell weight.


















A Growth


0 6 12 18 24 30 36
Time (h)


42 48 54 60



B Glucose


0 6 12 18 24 30 36 42 48 54 60
Time (h)


700


C Acetate


600


500
-I

S400
E
300


< 200


100


0 6 12 18 24 30 36
Time (h)


42 48 54 60


Figure 5-1. Effect of 1 mM Betaine on growth and acetate production for TC36.

Fermentations were conducted in NBS medium with 10% glucose as the

carbon source. Betaine was added to a final concentration of 1 mM when

indicated. Fermentations were performed in duplicate. A) Growth. B) Glucose

consumption. C) Acetate production. Symbols: Solid squares, + betaine; Open

squares, -- betaine.


001



700


600


500
-J

S400


S300


S200


100









Volumetric and specific pyruvate production rates were dramatically increased 8-fold and

2.5-fold, respectively. In contrast to TC36, betaine improved both growth rate and

production rates for TC44.

Determination of the Minimal Inhibitory Concentration of Acetate and Pyruvate on
E. coli W3110 in NBS Medium

There were two possibilities that could explain drastic difference between the

fermentation performance of the acetate producing strain, TC36, and the pyruvate

producing strain, TC44. One possibility was that TC36 lost the ability to transport betaine

by mutation(s) in the proUoperon. Although possible, it is unlikely that TC36 lost the

ability to transport betaine considering that TC44 was constructed from TC36

(Chapter 4). The more likely conclusion is that betaine was unable to provide the same

level of protection for acetate as it did for pyruvate. Therefore, we determined the

minimum inhibitory concentration for acetate and pyruvate in E. coli W3110.

E. coli W3110 was grown in NBS salts containing 2% glucose and 0-1M acetate or

pyruvate as described in the Materials and Methods section. In the absence of betaine the

minimum inhibitory concentration for acetate was 300 mM (Fig. 5-3A). The minimum

pyruvate concentration necessary to stop growth was about 300 mM, 100 mM greater

than that for acetate (Fig. 5-3A). Growth was partially inhibited by both acetate and

pyruvate at the lowest concentration (100 mM) tested in the absence of betaine (Fig. 5-

3A). Addition of 1 mM betaine did not increase the minimum concentration of acetate

necessary to stop growth of W3110 (Fig 5-3B). Betaine may increase the acetate

tolerance slightly, of W3110 at low (< 300 mM) concentrations. The minimum pyruvate

concentration necessary to completely inhibit growth of W3110 was increased 29% to

700 mM with the addition of 1 mM betaine (Fig. 5-3B). In addition, up to













200 mMpyruvate had a positive effect on the growth of W3110 when betaine was present


in the medium.


A Growth


0 6 12 18 24 30 36 42 48 54 60
Time (h)

B Glucose Consumption


0 6 12 18 24 30 36 42 48 54 60
Time (h)
800
C Pyruvate production
700

600-

S500-
0
E 400

300-

IL 200-

100


0 6 12 18 24 30 36 42 48 54 60
Time (h)


Figure 5-2. Effect of 1 mM Betaine on growth and pyruvate production for TC44.

Fermentations were conducted in NBS medium with 10% glucose as the
carbon source. Fermentations were performed in duplicate. A) Growth. B)

Glucose consumption. C) Pyruvate production. Symbols: Solid circles, 1 mM
betaine added to the medium; Open circles, no betaine added to the medium.
















A Acetate betaine


0 100 200 300 400 500 600 700 800 900 1000
Concentration (mM)


B Pyruvate betaine


0 100 200 300 400 500 600 700 800
Concentration (mM)


900 1000


Figure 5-3. Effect of 1 mM betaine on the minimum inhibitory concentration of acetate
and pyruvate for E. coli W3110. W3110 was grown in 3 mL of NBS salts
containing 2% glucose, 0-1 M A) acetate or B) pyruvate. Results plotted from
triplicate experiments. Symbols: Solid squares, no betaine; Open squares,
1 mM betaine added to the medium.


a,
o,
P
n









Discussion

Betaine did not increase acetate productivity by strain TC36. Early in the

fermentation, when glucose levels were high and acetate levels were low, incorporating

betaine into the medium resulted in a 17% increase in the growth rate. By 24 h the acetate

concentration was 300 mM and TC36 was in stationary phase (Fig 5-1A,C). Acetate

production was higher for fermentations that included betaine in the medium during the

first 24 h. These data are consistent with the studies performed to determine the minimum

inhibitory concentration of acetate necessary to stop the growth of E. coli W3110.

Acetate was found to be growth inhibitory at 300 mM regardless of the presence of

betaine. Growth was slightly elevated at lower acetate concentrations when betaine was

present in the medium. This would explain the increased acetate production early during

fermentations containing betaine and no benefit once acetate reaches 300 mM.

Pyruvate fermentations with TC44 were greatly enhanced when betaine was

incorporated into the medium. All of the metabolic parameters measured were increased

in fermentations containing 1 mM betaine. Pyruvate production continues until all of the

glucose is consumed. These data are consistent with growth inhibition studies that

showed 2.3-fold increase in tolerance to pyruvate when 1 mM betaine was in the

medium.

Why would betaine be beneficial for pyruvate and not for acetate? One explanation

could be the difference in pKa values resulting in different concentrations of the

protonated species. The protonated, or uncharged, form of weak acids are considered to

be toxic because the uncharged form of the acid is able to traverse the cytoplasmic

membrane. The charged form cannot freely diffuse across the membrane. Acetate has a

pKa of 4.76 and pyruvate has a pKa of 2.49. Applying the Henderson-Hasselbach






88


equation the ratio of the conjugate acid (HA) to the conjugate base (A-) can be calculated.

Although the conjugate acid concentration is very low for both acids at pH 7, the [A-

]/[HA] is 187-fold higher for pyruvate than for acetate. Lactic acid, which has a pKa

value intermediate (3.86) between pyruvate and acetate, also has a minimum inhibitory

concentration (500 mM) between that of pyruvate and acetate ( personal communication

with S. Zhou). To date there has been no published study comparing the tolerance of E.

coli growing in the presence of weak organic acids when betaine is present or not present

in the culture medium.