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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.

Permanent Link: http://ufdc.ufl.edu/UFE0021350/00001

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Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.
Physical Description: Book
Language: english
Creator: Kim, Youngnyun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
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theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
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Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Youngnyun Kim.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Shanmugam, Keelnatham T.
Electronic Access: INACCESSIBLE UNTIL 2008-02-29

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Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021350:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021350/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2008-02-29.
Physical Description: Book
Language: english
Creator: Kim, Youngnyun
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Youngnyun Kim.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Shanmugam, Keelnatham T.
Electronic Access: INACCESSIBLE UNTIL 2008-02-29

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021350:00001


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1 METABOLIC ENGINEERING OF Escherichia coli FOR ETHANOL PRODUCTION WITHOUT FOREIGN GENES By YOUNGNYUN KIM 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 2007

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2 2007 Youngnyun Kim

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3 To my parents for their devoted support and encouragement in the pu rsuit of my education

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4 ACKNOWLEDGMENTS I deeply thank Dr. Keelnatham Shanmugam fo r scientific advice, support, and for guiding me to how to approach to scienc e and think rationally in science. Without his expertise, direction and patience, I would not have accomplished my work. I also thank all my committee members, Dr. Lonnie Ingram, Dr. James Preston, Dr. Ju lie Maupin-Furlow and Dr. Shouguang Jin for helping me in achieving my work during my research period. I also thank Mi-Jin Kim for encouraging me during the last moment of my work. I thank all my colleagues: Dr. Adnan Hasona, Dr. Tao Han, Phi Mihn Do, Mark Ou, Dr. Jinwoo Kim, Dr. Munsoo Rhee, Yue Su and Dr. Eunmin Cho fo r their experimental advice and friendship. Also I would like to express my thanks to all th e laboratories in the department for helping me during my Ph.D course. Finally, I thank my parents for their coun tless support and trust ove r the years.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............13 CHAPTER 1 LITERATURE REVIEW.......................................................................................................15 Production of Bioethanol....................................................................................................... .15 Microbial Biocatalysts for Ethanol Production......................................................................17 Native Fermentation Profile, Mixed Acid Fermentation, of E. coli .......................................20 The Importance of Redox Balance.........................................................................................21 Metabolism of Pyruvate......................................................................................................... .22 Pyruvate Dehydrogenase (PDH) Complex.............................................................................25 Ethanologenic Escherichia coli ..............................................................................................26 2 MATERIALS AND METHODS...........................................................................................33 Materials...................................................................................................................... ...........33 Media and Growth Conditions................................................................................................33 Gene Deletions................................................................................................................. .......33 Transformation................................................................................................................. ......34 Co-transduction Frequency.....................................................................................................34 Sequencing DNA................................................................................................................. ...35 Determination of the level of transcription of pdh operon.....................................................35 Construction of P pdh-lac .............................................................................................35 Transduction................................................................................................................... .35 Galactosidase activity measurements..........................................................................36 Quantitative RT-PCR......................................................................................................37 in vitro DNA Mutagenesis......................................................................................................37 Hydroxylamine Mutagenesis...........................................................................................37 PCR Mutagenesis............................................................................................................38 Construction of pTrc99Alpd for Regulated Expression of lpd .............................................38 Construction of pET-15blpd Plasmid for Purification of LPD............................................39 Expression of Dihydrolipoami de Dehydrogenase (LPD).......................................................39 Dihydrolipoamide Dehydr ogenase (LPD) Assay...................................................................40 Purification of Pyruvate Dehydrogenase Complex................................................................40 Pyruvate Dehydrogenase Assay.............................................................................................41

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6 Protein Determination.......................................................................................................... ...42 SDS-Polyacrylamide Gel Electrophoresis..............................................................................42 Fermentation................................................................................................................... ........43 Analysis of Fermentation Products.........................................................................................43 3 RESULTS AND DISCUSSION.............................................................................................51 Isolation of Homo-Ethanol Producing E. coli ........................................................................51 Mapping the Mutation(s) in Strain SE2378............................................................................52 Location(s) of the Mutation(s) in strain SE2378....................................................................54 Confirmation that the Mutation in lpd is Responsible for the Anaerobic Growth Phenotype of Strain SE2378...............................................................................................55 Additional Mutations........................................................................................................... ...56 Metabolic Routes of Pyruvate with th e Mutated LPD in Various Backgrounds....................56 Aerobic and Anaerobic Expression Level of pdh Operon......................................................57 LPD Purification and Characterization...................................................................................58 NADH Sensitivity on Forward Reaction.........................................................................59 NADH Sensitivity of the Reverse Reaction of LPD.......................................................60 PDH Purification and Characterization..................................................................................61 Determination of Kinetic C onstants of PDH Complex...................................................62 Inhibition of PDH activity by NADH.............................................................................62 Fermentation of Sugars to Ethanol.........................................................................................63 Glucose Fermentation......................................................................................................63 Xylose Fermentation.......................................................................................................64 Removal of Trace Amount of Lactic Acid.............................................................................65 Proposed Ethanologenic Fermentation Pathway....................................................................66 LIST OF REFERENCES.............................................................................................................114 BIOGRAPHICAL SKETCH.......................................................................................................127

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7 LIST OF TABLES Table page 2-1 Bacterial strains and plasmids used in this study...............................................................44 2-2 List of primers used in this study.......................................................................................47 3-1 Growth and fermentation profile of the anaerobic (+ ) derivatives of E. coli strain AH242 grown in LB+glucose (0.3 %, w/v) in a batch culture without pH control...........68 3-2 Growth characteristics of ethanologenic E. coli strain SE2378.........................................74 3-3 Anaerobic growth and fermentation profile of E. coli with different lpd alleles...............80 3-4 Fermentation profile of mutant strains with different pyruvate metabolic pathway composition.................................................................................................................... ....82 3-5 Pyruvate dehydrogenase mRNA, transcri ption and protein levels in aerobic andanaerobic E. coli wild type, strain W3110 and et hanologenic mutant, strain SE2378......................................................................................................................... ......83 3-6 Kinetic constants of the native (W 3110) and the mutated (SE2378) LPD .......................88 3-7 Purification of native PDH complex from E. coli strain W3110.......................................93 3-8 Purification of the mutated PDH complex from strain YK176.........................................94 3-9 Kinetics constants of the native (W3110) and the mutated (SE2378) PDH....................100 3-10 Glucose fermentation characteristics of E. coli strain SE2378 and wild type strain W3110.......................................................................................................................... ....106 3-11 Growth and ethanol production by E. coli strain SE2378 grown on glucose or xylosea..............................................................................................................................107 3-12 Xylose fermentation characteristics of E. coli strain SE2378 and wild type strain W3110.......................................................................................................................... ....110 3-13 Fermentation characteris tics of kanamycin-sensitive derivative of ethanologenic strain SE2378, YK1, and its mgsA derivative strainYK96 in LB+glucose (50 g L-1) at pH 7.0 and 37 oC..........................................................................................................112

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8 LIST OF FIGURES Figure page 1-1 Mixed acid fermentation pathways of E. coli ....................................................................29 1-2 Overall reaction cata lyzed by the pyruvate de hydrogenase complex................................29 1-3 Genetic organization of the pdh operon.............................................................................30 1-4 Enzyme reaction diagram of the pyruvate dehydrogenase(PDH) complex.......................31 1-5 Elimination of lactate and formate production in the mixed acid fermentation pathway of E. coli ..............................................................................................................32 2-1 Construction of lpd in pTRC99a for complementation analysis.......................................49 2-2 Construction of LPD expression construct in pET15b with PCR amplified lpd gene.......50 3-1 HPLC analysis of fermentation produc ts. (A) strain W3110, (B) strain SE2378 in LB+glucose (1 %, w/v) in batch fermentations.................................................................70 3-2 Metabolic fates of pyruvate in E. coli ................................................................................71 3-3 Effect of deleting pdh genes in strain SE2378 on anaerobic growth.................................72 3-5 Promoter region and the tr anscription start site of pdhR-aceEF-lpd operon of E. coli K-12........................................................................................................................... ........75 3-6 Comparison of amino acid sequence of PdhR from wild-type (W3110), and three ethanologenic mutants (SE2378, SE2377 and SE2382)....................................................76 3-7 Nucleic acid sequence of intergenic region between pdhR and aceE genes of the ethanologenic mutants and the wild type...........................................................................77 3-8 Comparison of the amino acid sequen ce of LPD among wild-t ype strain (W3110) and 6 isolates................................................................................................................. .....78 3-9 Pyruvate metabolic enzymes in strain SE2378 and their affinity for pyruvate.................81 3-10 SDS-Polyacrylamide gel electrophor esis of purified dihydrolipoamide dehydrogenase (LPD) from wild-type, A) W3110 and B) the ethanologenic strain SE2378......................................................................................................................... ......84 3-11 Linearity of LPD protein concentration vs. activity of the enzyme in the forward reaction....................................................................................................................... ........85 3-12 Native LPD (W3110) activity with various NAD+ concentrations...................................86

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9 3-13 Mutated LPD (SE2378) activity with various NAD+ concentrations................................87 3-14 Inhibition of native LPD (W3110) forward activity by NADH........................................89 3-15 Inhibition of LPD activity by NADH (2.0 mM NAD+) on forward reaction....................90 3-16 Inhibition of mutated LPD (SE2378) by NADH...............................................................91 3-17 Activation of LPD revers e reaction by increasing NAD+/NADH ratio.............................92 3-18 SDS-PAGE of partially purified PDH complex................................................................95 3-19 Native PDH (W3110) activity with various NAD+ concentrations...................................96 3-20 Native PDH (W3110) activity with various pyruvate concentrations...............................97 3-21 Mutated PDH (SE2378) activity with various NAD+ concentrations...............................98 3-22 Mutated PDH (SE2378) activity with various pyruvate concentrations............................99 3-25 Inhibition of PDH comp lex by NADH at a fixed NAD+ concentration of 1.0 mM........103 3-26 Growth and fermentation characteristics of wild type strain W3110 in LB + glucose (50 g L-1) at pH 7.0 and 37 oC.........................................................................104 3-27 Growth and fermentation characteristics of ethanologenic strain SE2378 in LB+ glucose (50 g L-1) at pH 7.0 and 37 oC............................................................................105 3-28 Growth and fermentation characteristics of wild type stra in W3110 in LB+ xylose (50 g L-1) at pH 7.0 and 37 oC..............................................................................108 3-29 Growth and fermentation characteristics of ethanologenic strain SE2378 in LB+ xylose (50 g L-1) at pH 7.0 and 37 oC..............................................................................109 3-30 Fermentation characteris tics of kanamycin-sensitive derivative of ethanologenic strain SE2378,YK1, and its mgsA derivative strain YK96 in LB+glucose (50 g L-1) at pH 7.0, 37 oC................................................................................................................111 3-31 Ethanologenic fermentation pathway..............................................................................113

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10 LIST OF ABBREVIATIONS ACK Acetate kinase ACS Acetyl-CoA synthetase ADH Alcohol dehydrogenase ADP Adenosine diphosphate ATP Adenosine-5-triphosphate BSA Bovine serum albumin bp Base pair cDNA Complementary DNA CoA Coenzyme A CTP Cytosine-5-triphosphate da Dalton DOE Department of energy E1 Pyruvate decarboxylase/dehydrogenase E2 Lipoamide acetyltransferase E3 Dihydrolipoamide dehydrogenase EDTA Ethylenediamine tetraacetic acid EIA Energy information administration EMS Ethylmethane sulfonic acid ETS Electron transport system FAD Flavin adenine dinucleotide FRT Flipase recognition target site GTP Guanosine triphosphate K cat Turnover number kDa Kilo dalton.

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11 Km Kanamycin resistance gene K m Michaelis constant LB Luria broth LDH Lactate dehydrogenase MOI Multiplicity of infection NAD+ Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide reduced NO Nitric oxide NOX NADH oxidase OAA Oxaloacetic acid ONPG O-nitrophenyl-D-galactopyranoside PAGE Polyacrylamide agarose gel electrophoresis PCR Polymerase chain reaction PFL Pyruvate-formate lyase POX Pyruvate oxidase PPC Phosphoenolpyruvate carboxylase PTA Phosphotransacetylase PTS Phosphotransferase system RFA Renewable fuel association rpm Revolution per minute RT-PCR Real time PCR SDS Sodiumdodecyl sulfate SSF Simultaneoul saccharification and fermentation TCA Tricarboxylic acid cycle Tn 10 Transposable element containi ng tetracycline resistance gene

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12 TPP Thiamine pyrophosphate UTP Uridine-5-triphosphate

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy METABOLIC ENGINEERING OF Escherichia coli FOR ETHANOL PRODUCTION WITHOUT FOREIGN GENES By Youngnyun Kim August 2007 Chair: K.T. Shanmugam Major: Microbiology and Cell Science Worldwide dependence on finite petroleum-ba sed energy necessitates alternative energy sources that can be produced from renewable re sources. A successful example of an alternative transportation fuel is bioethanol, produced by micr oorganisms, from corn starch that is blended with gasoline. However, corn, currently the main feedstock for bioe thanol production, also occupies a significant role in human food and animal feed chains. As more corn is diverted to bioethanol, the cost of corn is expected to incr ease with an increase in the price of food, feed and ethanol. Using lignocellulosic bi omass for ethanol production is considered to resolve this problem. However, this requires a microbial biocatalyst that can ferment hexoses and pentoses to ethanol. Escherichia coli is an efficient biocatalyst that can use all the monomeric sugars in lignocellulose, a nd recombinant derivatives of E. coli have been engineered to produce ethanol as the major fermentation produc t. In my study, ethanologenic E. coli strains were isolated from a ldhA, pflBderivative without introducti on of foreign genes. These isolates grew anaerobically and produced ethanol as the main fermentation product. The mutation res ponsible for anaerobic growth and ethanol production was mapped in the lpdA gene and the mutation was identified as E354K in three of the isolates tested Another three isolates carried an lpdA mutation, H352Y. Enzyme kinetic studies revealed that the muta ted form of the dihydrolipoamide dehydrogenase

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14 (LPD) encoded by the lpdA was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity to NADH of the pyruvate dehydrogenase complex in strain SE2378. The net yield of 4 moles of NADH and 2 moles of acetyl-CoA per mole of gluc ose produced by a combination of glycolysis and PDH provided a logical basis to explain the production of 2 mo les of ethanol per glucose. The development of E. coli provides a potential biocatal yst for conversion of pentoses derived from cellulosic biomass to biobased products without the introduction of new genes.

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15 CHAPTER 1 LITERATURE REVIEW Production of Bioethanol Human society is sustained at present by petroleum-based energy, although this fossilbased energy is limited. The proven amount of oil re serves worldwide that can be extracted with existing technology is about 1.3 trillion barrels (EIA, DOE). Taking current annual oil production and consumption of about 30 billion barrels into consideration, this oil reserve will be exhausted in approximately 40 year s. Other estimates of about 6 trillion barrels of oil reserves include estimates of potentially recoverable oil a nd require improvements in oil recovery. If all of these oil deposits, including unconventional oil reserves, ca n be economically tapped, the world-wide reserve can supply e nough petroleum for about 240 years at current rate of use (47, 74, 141). The growth and increase in economy worldw ide is expected to accel erate this rate of depletion of petroleum. United States (as of 2005) consumes each year approximately 30 % of total oil produced in the world (about 30 billion barrels per year) of which 60% is imported (13, 45). Of this total, more than 120 billion gallons of gasoline was consumed as automotive fuels. This equals the net import of petroleum (45, 74). Since the portion of automotive fuel in overall petroleum consumption is significant, alternative resources of fuels for automotive vehicles have received great attention (27, 40, 45, 54, 74, 100, 125, 132). Ethanol has been recognized as one of the immediate and feas ible alternative transportation energy source due to a number of advantages. Most of all, unlike petroleum-based fuels, ethanol can be produced in a renewable manner from biom ass that can be produced continuously in large amounts (101). In the U.S., energy production from re newable biomass can potentially lead to as much as a 30 % reduction in gasoline use (45, 10 1). Ethanol that can be produced from biomass locally also reduces dependence on imported fu els (74). Since biomass is synthesized by

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16 photosynthesis consuming CO2 from the air, biomass-based fuel is a closed loop with no net CO2 release into the atmosphere, mitigating the greenhouse effect of petroleum use. In addition, NO2 emitted from combustion of petroleum-based fuels can be reduced by the use of ethanol due to its 35% oxygen content (5, 142) wh ich also improves air quality. Eventually, as in the U.S., consideration on environment and fuel independence will encourage world-wide development of alternative clean energy sources (40, 54, 60-62, 100, 132). With these advantages of ethanol as a transportation fuel, ethanol production in the US is expanding and in 2006, fuel ethanol production has reached close to 5 billion gallons. With the corn to ethanol plants under construction, this capacity is expected to in crease to over 12 billion gallons per year in the immediate future (45). As mentioned above, one of the most favorable and critical points in ethanol production is the capability of using biomass. Solar energy is the fundamental energy source supporting almost all living organisms on earth and is mainly stored as biomass. This biomass is produced in a renewable manner and only a fraction of this bioma ss, corn starch, is currently used to produce ethanol. Fermentation of corn starch accounts fo r > 93 % of the total ethanol production in US while petroleum-based chemical synthesis ac counts for < 7 % (13, 45, 74). Using corn, an important food and feed source, as a feedstock fo r ethanol production is expected to lead to relatively high cost of ethanol production due to the limitation in the availability of corn and expanding corn starch based etha nol industry (13, 54). An increa se in ethanol production in the future will require more corn to be diverted aw ay from food and feed requirements with further increase in the price of other commodities. This necessitates the need for alternate feedstock that does not compete with food and feed sources, su ch as lignocellulose, fo r ethanol fermentation (45, 74, 78, 125).

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17 Lignocellulose is a complex substrate composed of cellulose (20 to 50 %), hemicellulose (20 to 40 %), lignin (10 to 20 %) and others (2 to 20 %) (45, 125). Among the components of lignocellulose, cellulose, a -1,4-glucose linear polymer, is formed as a ribbon structure into fibers. The hydrogen bonding betw een cellulose ribbons results in insoluble and anhydrous crystalline structure. This structural comple xity prevents ready access to cellulases toward hydrolysis of the polymer, resulting in the n eed for higher level of enzymes or chemical pretreatments to hydrolyze cellulose into glucose before fermentation to ethanol (5, 60). Unlike cellulose, hemicellulose is composed of heter ogeneous polymers of pentoses, hexoses and sugar acids (60-62). The high concentration of pentoses in hemicellulose is anot her obstacle for ethanol production, because none of the trad itional industrial microbial bi ocatalysts used for ethanol production ferments pentoses (12, 60-62, 74). Thus, converting lignocellulosic biomass to ferm entable sugars and to ethanol is the most challenging part in utilizing th is feedstock. Isolating microorga nisms capable of fermenting all the sugars present in lignocellulosic materials ha s been one of the main issues in achieving the goal of maximizing ethanol production (12, 45, 60, 62, 74). Microbial Biocatalysts for Ethanol Production Historically yeast has been utilized to pr oduce various fermentation products for human use historically and technology for fermentation with yeast is well esta blished (74, 131). In addition to these technical and hi storical advantages, genetic a nd metabolic engineering enabled yeast to become a major microbial biocatalyst for ethanol production as well as other useful fermentation products such as xylitol (12, 88, 131). Since most yeasts do not ferment xylose, the second most abundant saccharide in the hemicellulo se component in biomass, one of the main issues of ethanol production resear ch with using yeast has been th e isolation and construction of

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18 pentose fermenting yeast. Although Pichia stipitis and Candida tropicalis ferment xylose, these microbes have several critical problems such as low ethanol production yield with xylose (45, 62, 63), inability to ferment arabinose (45, 65), and an oxygen requirement for growth (39, 128). In spite of the successful introduction of gene s for xylose metabolism such as the ones encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis into Saccharomyces the recombinant strain still produces low ethanol yi eld from xylose and low growth rate on xylose due to cofactor imbalance compared to glucose (3, 70, 115). Although introduction of heterologous xylose isomerase into Saccharomyces helped overcome cofactor imbalance, this did not improve ethanol yield from xylose (67) Despite improvement in expanding the range of fermentable carbohydrates in yeast (36, 39, 48, 126, 135), the problems caused by genetic engineering, such as differences in internal pH between bacteria and ye asts, unsuitable protein folding, incorrect post-translati onal modification, low ethanol yield, and low specific growth rate, have hampered the development of yeast as efficient biocatalysts fo r fuel ethanol production from lignocellulosic biomass, especially th e hemicellulose component (38, 39, 65, 88, 94). Zymomonas mobilis that metabolizes sugars by En tner-Doudoroff pathway is another extensively studied tradi tional microorganism in ethanol prod uction, with its several advantages, such as homo-ethanol fermentation, high ethanol production yield (up to 120 g/l), high ethanol tolerance and high specific ethanol productivity ( 85, 104, 121). In spite of these attractive points, limited ability of sugar utilization by Z. mobilis has restricted use of this bacterium by the ethanol industry. Therefore, like yeast, genetic engineering of Z. mobilis to improve its ability to metabolize all the sugars in biomass is in progress (32, 35, 143). The recombinant Z. mobilis strains that can ferment xylose or arabinose re quire long fermentation times, despite high ethanol yield (12, 35, 74). As seen with yeast and Z. mobilis expanding the substrat e range of traditional

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19 microbial biocatalysts for optimal ethanol pr oduction at industrial scal e using lignocellulosic biomass as the feedstock is still one of the main challenges (64). While none of the traditional y east or ethanologenic bacteria metabolizes both pentoses and hexoses (57, 61, 74), ente ric bacteria, such as Escherichia coli can use all of these sugars as carbon sources (12, 60). E. coli is used as a work horse by the industry to produce various products because genetic systems as well as physiological aspects of E. coli are well established (8, 44, 118, 137). However, despite these adva ntages, mixed acid production by fermenting E. coli is a hurdle to overcome towards developing a recombinant strain for homo-ethanol fermentation (18, 22, 23, 61, 129). Genetic engineering of E. coli resulted in development of E. coli strain KO11, a most promising ethanol producer that fe rments all the sugars in lignocellulosic biomass to high yield of ethanol. The pyruvate decarboxylase gene ( pdc ) and alcohol dehydrogenase gene ( adhB ) from Z. mobilis were combined into a portable ethanol ope ron cassette (103) and integrated into the chromosomal DNA of E. coli in the construction of stra in KO11 (61, 93). This genetic modification was able to draw all the advantages from E. coli and Z. mobilis and provided an efficient metabolic route for c onverting pyruvate into ethanol. Ho wever, lower ethanol tolerance and specific productivity by E. coli compared to Z. mobilis are being addressed. Enteric bacteria other than E. coli with additional beneficial characteris tics have also been investigated for ethanol production. Integrat ing the PET operon into Klebsiella oxytoca enabled a genetically engineered biocatalyst (strain P2) that metaboliz es cellobiose and cellotriose, hydrolysis products of cellulose, in addition to fermenting monomeri c sugars (89). The best performance obtained using recombinant K. oxytoca strain P2 resulted in successf ul fermentation of glucose or cellobiose to a yield of 45 g/l ethanol within 48 hours (140). When partially hydrolyzed cellulose

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20 was used as a feedstock to utilize the ability of K. oxytoca to ferment cellobiose, the recombinant K. oxytoca strain P2 produced 38.6 g/l ethanol, indi cating a potential to reduce the cost of cellulase supplement in a simultaneous saccharif ication and fermentati on (SSF) process (140). Another approach towards developing a microbial biocatalyst to reduce the cost of SSF was to integrate the genes encoding endoglucanase, a component of cellulase mixtures, into an appropriate host such as K. oxytoca strain P2. Two extracel lular endoglucanase genes, celZ and celY from Erwinia chrysanthemi were integrated into K. oxytoca P2 chromosomal DNA (strain SZ21) and cellulase exporter ( out ) genes were also introduced into this construct with a plasmid (pCPP2006) (144, 145). In SSF of highly crystalline cellulose, Sigmacell 50, with a fixed amount of cellulase Spezyme CP or CE, the ethanol yi eld was higher with strain SZ21 (pCPP2006) as the microbial biocatalyst than that from the parent strain without the cel and out genes (144). Overall, comparison among biocatalysts deve loped for ethanol production mentioned above, E. coli KO11 and its derivative (LY 01) obtained after long-term adaptation for higher ethanol tolerance produced more ethanol in shorter fermentation time than any other biocatalyst, especially on high xylose concentration (140 g L-1) (60, 62, 144). Native Fermentation Profile, Mixed Acid Fermentation, of E. coli Escherichia coli one of the most studied microorgani sms, has mixed acid fermentation as the main fermentation pathway (22) (Figure 1-1) End products of fermentation are a mixture of lactic acid, acetic acid, formic acid, succinic ac id and ethanol, with the possibility of gas formation from formate (CO2 and H2) (6, 7, 87). The pathways to these pr oducts diverge mostly from pyruvate produced by glycolysis from sugars (22, 92). Pyruvate is converted into either lactic acid by lactate dehydr ogenase (LDH) (9, 22, 68) or acet yl-CoA and formate by pyruvate formate-lyase (PFL) (22, 69). A cetyl-CoA is subsequently conve rted to equimolar amounts of acetic acid and ethanol (22). Acetic acid production is carr ied out by phosphotransacetylase

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21 (PTA) and acetate kinase (101) (22, 73) and et hanol is produced via acetaldehyde by alcohol dehydrogenase (ADH) (21, 22, 42). In additi on, phosphoenolpyruvate carboxylase and malate dehydrogenase, fumarase and fuma rate reductase lead to succ inic acid from the oxaloacetate generated from phosphoenolpyruvate (22, 83). This divergence of pyruvate metabolism into these products functions to meet both the ener gy requirement (22) and redox balance depending upon growth condition. Thus, in order to maximi ze the flux from sugar to ethanol, the carbon flux to other fermentation products should be lo wered or blocked while also maintaining redox balance and producing enough energy to s upport cell growth under a given condition. The Importance of Redox Balance As a facultative anaerobe, Escherichia coli grows under both aerobic and anaerobic conditions. One of the key issues in E. coli growth is to mainta in redox balance (NADH/NAD+) in metabolism because some of the metabolic pathways proceed with oxidation-reduction coupled reactions (2, 22). Duri ng glycolysis, when glyceraldeh yde-3-phosphate is oxidized into 1,3-bisphosphoglycerate, 1 mol of NAD+ is reduced to NADH with an introduction of 1 mol of inorganic phosphate (55). This is one of the important steps in the generation of ATP by substrate-level phosphorylation (10, 24, 90). Si nce this oxidation step requires NAD+ as a substrate, and the 1,3-bisphosphogl ycerate leads to ATP producti on, maintaining the level of NAD+ pool is crucial for glycolysis of sugars to pyruvate. Under aerobic conditions, E. coli maintains its redox balance via respiratory elec tron transport systems (ETS) that oxidize NADH produced from glycolysis and TCA cycle, where O2, as the terminal electron acceptor, is eventually reduced to H2O (22, 59). Proton motive force gene rated when NADH is re-oxidized by the ETS is linked to ATP production by ATP synthase (33, 133, 134). However, under fermentative conditions in the absence of external electron acceptors, the cell maintains the redox

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22 balance by producing fermentation products such as lactate and ethanol, due to the nature of the system (Figure 1-1) (22). Metabolism of Pyruvate Pyruvate is the key intermediate in the catabolic pathways of E. coli (58, 107), regardless of growth condition. Under aerobic conditions, the major catabolic pathway from pyruvate is the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 with reduction of NAD+ to NADH catalyzed by pyruvate dehydrogenase (PDH) ( 22, 99, 136) (Figure 1-2). The PDH complex is active in vivo only under aerobic growth condition and is induced by pyruvate. However, despite the expression of PDH under anae robic conditions, the ac tivity of the PDH complex is inhibited during fermentative growth (17, 37, 46, 51, 122-124) The PDH complex consists of 3 subunits that are pyruvate dehydrogenase / decarboxylase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3; LPD), encoded by the aceE aceF and lpd genes, respectively (99). The expression of aceEF-lpd genes is controlled at the transcriptional level by PdhR encoded by pdhR gene that is promoter proximal of the pdhR-aceEF-lpd operon ( pdh operon) (98). In the absence of pyruvate, PdhR binds to the operator region of pdhR gene inhibiting transcri ption of the pdhR-aceEF-lpd genes (51) (F igure 1-3). The lpd is also expressed independently, since LPD is shared with 2-oxoglut arate dehydrogenase. The link between dihydrolipoamide dehydrogenase and 2oxoglutarate dehydrogenase is co-regulation at the transcriptional le vel primarily by ArcA (26, 99, 120). Acetyl-CoA produced by PDH is oxidized by the enzymes of the TCA cycle and subsequently the NADH is oxidized by respiratory ETS (59, 83, 113). During anaerobic growth, pyruvate formate-lyase (PFL) encoded by pflB takes over the role of PDH in pyruvate metabolism converting pyruvate to acetyl-CoA and formate (69, 109, 110). Subsequently, acetyl-CoA produced from pyruvate is converted to acetate by

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23 phosphotransacetylase (PTA) encoded by pta and acetate kinase (101) encoded by ackA or reduced to ethanol by alcohol dehydrogenase (ADH) encoded by adhE (Figure 1-1) (22). The 2 moles of NADH produced during glyc olysis per mole of glucose ar e used to reduce one mole of acetyl-CoA to one mole of ethanol to maintain the redox balance. The other acetyl-CoA from the pyruvate is converted to acetate generating one additional ATP at the acetate kinase step to increase the net ATP yield /glucose to 3.0 (50) Thus, the expected ratio between ethanol and acetate production is 1 (22). In addition to the pflB gene, E. coli genome also has a second putative pyruvate formate-lyase encoded by the pflCD genes. However, this is a silent operon and deleting the pflCD genes has no effect on the PFL activity of the cell (146). The second pathway branching from pyruvate under fermentative cond ition that enables NAD+ to be regenerated is that of lactate produc tion (Figure 1-1). When 1 mol of pyruvate is converted into 1 mol of lactate by lactate dehydrogenase (LDH) encoded by ldhA NADH is oxidized to NAD+. Thus, lactate production alone is suffi cient to maintain the redox balance to support cell growth under fermenta tive conditions (9, 15, 22, 23). Another metabolic pathway originating from pyruvate is direct oxi dation to acetate by pyruvate oxidase (Pox) encoded by the poxAB genes (1, 19, 20). PoxB, a membrane-bound flavoprotein, catalyzes decarboxylation of pyruvate to acetate with the re duction of an enzymebound flavin adenine dinucleotide, FAD. The poxA gene product plays a regulatory role in the expression of poxB (130). The pox genes are primarily induced at the transient phase from the exponential to the stationary phase of growth since the transcription of poxB is dependent on stationary phase sigma factor ( rpoS ) (20). Since pyruvate oxid ase is induced during the stationary phase of growth, the phenotype of a mutant lacking PDH activity is acetatedependence for aerobic growth in glucose minera l salts medium. Although Pox is also expressed

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24 and active during anaerobic growth, its level of expression is lower than that of aerobic conditions. Phosphoenolpyruvate (PEP) is an intermedia te immediately preceed ing pyruvate during glycolysis (22, 107). Therefore, a catabolic pathway branching from PEP should also be considered as a branch of pyruvate during gl ucose fermentation. PEP is converted to small amount of succinate in sequential reactions (22, 66, 119) catalyzed by phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, an d fumarate reductase (22). During this production of succinate, malate dehydr ogenase oxidizes 1 NADH to 1 NAD+. Reduction of fumarate to succinate utilizes a second reduc tant. Hence, 2 NADHs prod uced during glycolysis of glucose to 2 PEP can be oxidize d in this route. However, since 1 PEP is consumed to transport glucose into a cell through the phosphotransferase system (PTS), only one PEP is available for succinate production. Purified fumarate reduc tase failed to use NADH as an electron donor suggesting that in vivo the fumara te reductase utilizes some other form of reductant other than NADH (119). This shortage of PEP combined with the lack of NADH use by fumarate reductase prevents the cell from maintaining redox bala nce by the succinate production pathway alone. Succinyl-CoA required as a precu rsor for amino acid synthesis (81) is made from either succinate or -ketoglutarate by -ketoglutarate dehydrogenase ( -KGDH). Under fermentative conditions, expression of -KGDH, encoded by sucAB is negatively regulated by ArcAB and Fnr proteins (95) so that the comple te TCA cycle is interrupted at the -ketoglutarate dehydrogenase step although a basal amount of -KGDH is maintained to produce succinyl-CoA for growth (22). Based on these results and the observation that a fu marate reductase minus mutant of E. coli does not require succinate implies that succinate production for growth of E. coli under anaerobic conditions can be accomplished by alternate pathways (25). Consumption

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25 of one PEP for transport of glucose by PTS a nd the second for succinate production affects not only the redox balance, but also ATP produc tion (86) because the net ATP production under these conditions is only 1 mole of ATP per mole of glucose (97). Unless another non-ATP (PEP)-dependent glucose transport system is use d, such as transportation by galactose permease (GalP) (52, 53), the succinate production pathway is not the ma in pathway to re-oxidize NADH, due to the shortage of PEP a nd energetic constraints. Based on the metabolic fates of pyruvate re viewed above, lactate production (LDH) and ethanol production (ADH) path ways play key roles in maintaining redox balance under fermentative conditions. When both ldh and pfl genes are deleted, alt hough all the downstream enzymes (ADH and ACK) are present, E. coli can not grow anaerobically due to a decrease in the NAD+ pool. Pyruvate Dehydrogenase (PDH) Complex The structural ratio among each component of the pyruvate dehydrogenase complex, a multimeric protein of 4.6 MDa, is 24:24:12 (E1:E2 :E3), respectively (116). Structural modeling and reconstitution of the pyruvate dehydrogenase complex revealed that dihydrolipoamide transacetylase (E2) plays a central role in forming a multi-subunit complex with enzymatic activity (103, 108). The dimer form of pyruva te dehydrogenase/decarbo xylase (E1) with a molecular mass of 199 KDa is noncovalently bound to E2 (34). The E1 catalyses oxidative decarboxylation of pyruvate leading to acyl-T PP (thiamine pyrophosphate) (28). The acyl group is transferred to CoA by E2, yielding acetyl -CoA. Dihydrolipoamide dehydrogenase (LPD; E3) reducing NAD+ to NADH as a terminal enzymatic subunit in PDH is also homodimeric containing 2 moles of FAD per di mer (103). FAD insertion is critical in forming non-covalent assembly of PDH and activ ity (75) (Figure 1-4).

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26 In addition to the regulation at the transcri ption level by PdhR de pending on the presence of pyruvate, the activity of PDH is also regulated at the enzy me level (16, 17, 117). Glycolytic intermediates, such as fructose-6-phosphate, fructose-1,6-diphosphate, and 3-phosphoglycerate, positively regulate PDH activity (114). The enzy me level is inhibited by acetyl-CoA in a competitive manner (112, 114). In contrast to acetyl-CoA, GTP regulates PDH activity noncompetitively (112). ATP, CTP, and UTP have no effect on PDH activity. The inhibition by GTP is reversed by GMP and GDP (112). Sin ce the acetyl-CoA produced by PDH is a key precursor entering the TCA cycle leading to production of NADH and energy through the electron transport system, main taining a higher energy state may be the main reason to negatively regulate PDH activity(112, 114). The activ ity of PDH is also partially inhibited by glucose, acetate, and TCA cycle intermediates. A lacZ fusion study showed that the intracellular ratio between NADH and NAD+ (NADH/NAD+) has a significant effect on PDH level (17) and activity (114). In particular, th e expression or activity of LPD se ems to be highly sensitive to NADH levels (105, 138). Under anae robic conditions, the NADH/NAD+ ratio (0.34) is several fold higher than found for growth under aerobi c conditions (0.19) (aer obic:anaerob ic::0.19:0.34) (49). Values demonstrating an even greater difference were also recently reported by Snoep et al. (aerobic:anaerobic::0.03 :0.70)(121). This sensitivity of LPD to NADH inhibition makes PDH inactive during fermentative growth due to the higher NADH pool and is probably another regulatory mechanism of this critical enzyme. Finally, while mammalian PDH is also regulated by phosphorylation of the E1 component, there is no evidence of phosphorylation of PDH in E. coli and other bacteria (102). Ethanologenic Escherichia coli Ethanol is one of the most promising candida tes as a transportation energy source in the near future. For ethanol to replace petroleum as the fuel of choice, ethanol production by

PAGE 27

27 fermentation should be cost effective. As indica ted above, fermentation of renewable biomass is the best way to produce ethanol in terms of subs trate cost. Based on composition of biomass, the ability of microorganisms to ferment various carb ohydrates present in biomass is one of the key factors. In addition to substr ate range, appropriate metabolic pathways to maximize ethanol production yield is an important requirement for de veloping cost effective microbial biocatalysts for ethanol fermentation. In this connecti on, strain KO11 and other enteric recombinant biocatalysts fulfill the need for a robust biocatalys t that can metabolize all the sugars present in a variety of feedstocks. The presence of Z. mobilis genes in E. coli strain KO11 and other enteric microbial biocatalysts has led to reluctance in wider use of these ethanologens at an industrial scale. Despite the development of significant ne w microbial biocatalysts for fermentation of multiple sugars to ethanol, none of the reported microorganisms (yeast, Z. mobilis entericbacteria) was engineered without the introducti on of foreign gene(s). Is it possible to construct a microbial biocatalyst that ferments all the sugars in biomass to ethanol that also lacks foreign gene input? As mentione d earlier, based on the ability to utilize carbohydrates from various sources, enteric bacteria provide the best platform for further engineering for production of a multitude of compounds. E. coli is the ideal organism of c hoice to address this challenge since this organism already has the metabolic vers atility to ferment all the sugars of biomass and also produces ethanol during fermenta tion, although as a minor product. Towards construction of a microbial biocat alyst lacking foreign genes, production of lactate, acetate and formate produced during fermentation by E. coli must be eliminated. Lactate production and ethanol/acetate production steps are redundant for redox balance maintenance and the lactate pathway can be deleted wit hout a detrimental effect on growth. In an ldhA mutant, ethanol production is the only wa y for the cell to maintain redox balance and removal of this

PAGE 28

28 pathway by deleting the pflB gene, whose product generates acetyl-CoA used for ethanol production prevents growth of the organism ( 22). Although PDH has the ability to produce the required acetyl-CoA and the needed second NADH per acetyl-CoA generated, this enzyme is inactive in the anaerobic cell due to its inhibiti on by NADH. An altered form of PDH that is less sensitive to NADH can support fermentation of suga rs to ethanol (Figure1-5). However, a price for utilizing this pathway would be the loss of an ATP per glucos e that could reduce the growth rate of the bacterium. A net ATP yield of two per glucose may not be that critical for the growth of E. coli with glucose (50).

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29 Figure 1-1. Mixed acid fermentation pathways of E. coli. PPC: phosphoenolpyruvate carboxylase, PKY: pyruvate kinase, LDH: lactate dehydrogenase, PFL: pyruvate formate-lyase, FHL: formate hydrogen-lyase, PTA: phosphotransacetylase, ACK: acetate kinase ADH: alcohol dehydrogenase, MDH: malate dehydrogenase, FUM: fumarase FR: fumarate reductase, CS: citrate synthase, ACN: aconitase, IDH: isocitrate dehydrogenase. Figure 1-2. Overall reacti on catalyzed by the pyruvate dehydrogenase complex. Glucose PEP Pyruvate Formate + Acetyl-CoA Acetyl phosphate Acetaldehyde Acetate Ethanol Lactate OAA Malate Fumarate Succinate NADH NAD+ NADH NAD+ADP ATP NADH ATP CO2H2 ADP NADH NAD+ 2[H] LDH PFL FHL PTA ADH ACK ADH PPC MDH FUM FR NAD+ PKY NADH NAD+ ADP ATP Citrate Isocitrate K eto g lutarate CS ACN IDH NADPH NADP+ Pyruvate + NAD+ + CoASH Acetyl-CoA + NADH + H+ + CO2 PDH

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30 Figure 1-3. Genetic organization of the pdh operon. pdhR : regulator gene, aceE : pyruvate decarboxylase, aceF : dihydrolipoamide acetyltransferase, lpdA : dihydrolipoamide dehydrogenase, KGDH : -ketoglutarate dehydrogenase. pdhR aceE aceF lpdA P1 P2 transcript for PDH transcript for theLPD component of KGDH P1:Promoter fo r the entire p dh o p eron P2:Promoter for the trascri p tion of l p dA for KGDH

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31 Figure 1-4. Enzyme reaction diagram of the pyruvate dehydrogenase(PDH) complex. TPP: thiamine pyrophosphate, E1: pyruvate decarboxylas e/dehydrogenase, E2: dihydrolipoamide acetyltransferase, E3: dihydrolipoamide dehydrogenase. Pyruvate CO2 TPP Acyl-TPP Lipoamide Dihydrolipoamide CoA-SH Acetyl-CoA NAD+ FADH2 FAD NADH+H+ Acyl-lipoamide E1 E2 E3

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32 Figure 1-5. Elimination of l actate and formate production in the mixed acid fermentation pathway of E. coli PKY: pyruvate kinase, PPC: phosphoenolpyruvate carboxylase, MDH: malate dehydrogenase, FU M: fumarase, FR: fumarate reductase, LDH: lactate dehydrogenase, PFL: pyruvate-formate lyase, FHL: formate-hydrogen lyase, PTA: phosphotransacetylase, ACK: acetate kina se, ADH: alcohol dehydrogenase, CS: citrate synthase, ACN: aconitase, IDH: isocitrate dehydrogenase, PDH: pyruvate dehydrogenase. Glucose PEP Pyruvate Formate + Acetyl-CoA Acetyl phosphate Acetaldehyde Acetate Ethanol Lactate OAA Malate Fumarate Succinate NADH NAD+ NADH NAD+ADP ATP NADH ATP CO2H2 ADP NADH NAD+ 2[H] LDH PFL FHL PTA ADH ACK ADH PPC MDH FUM FR NAD+ PKY NADH NAD+ ADP ATP Citrate Isocitrate -KG CS ACN IDH NADH NAD+ X X PDH Acetyl-CoA + CO2 NADH NAD+

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33 CHAPTER 2 MATERIALS AND METHODS Materials Biochemicals were purchased from Sigma-Al drich Chemicals Co. Or ganic and inorganic chemicals were purchased from Fisher Scientific Co. and were analytical grade. DNA restriction endonucleases, T4 DNA ligase and DNA polymerases were obtained from New England Biolabs Inc., Invitrogen or Clontech Laboratories. Real-time PCR reagents were from Bio-Rad Laboratories, Inc. Plasmid extraction and DNA ge l-extraction kits were from Qiagen Inc. Media and Growth Conditions Luria broth was prepared as described previously (96). Glucose-minimal medium contained Na2HPO4 (6.25 g), KH2PO4 (0.75 g), NaCl (2.0 g), FeSO4 7H2O (10 mg), Na2MoO4 2H2O (10 mg), MgSO4 7H2O (0.2 g), and (NH4)2SO4 (1 g) in 1 L of deionized water. Sugars were added after autocl aving the medium at a final co ncentration of glucose at 3 g.L-1 for aerobic growth and 10 g.L-1 for anaerobic growth. Aerobic li quid cultures were grown in a shaker at 200 RPM and routine anaerobic culture s were grown in screw-cap tubes filled to the top and incubated without mixing. Solid medium was prepared by the addition of agar (15 g.L-1) into liquid medium. Top-agar for growing phage contained 0.7 % agar instead of 1.5 %. Media used for propagation of phage P1, and transduction were as per Miller (84). Gene Deletions Construction of gene disruption in E. coli was as described by Datsenko et al. (29). Genes disrupted were amplified by PCR and cloned into TOPO pCR2.1 plasmid vector (Invitrogen). After removal of the gene, a DNA cassette cont aining kanamycin-resistance gene, flanked by FRT sequences, was integrated into the deleted area. The antibiotic resistance gene with the flanking E. coli DNA was PCR-amplified and the P CR product was transformed into E. coli

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34 strain BW25113 (pKD46) that was pre-grown in LB+arabinose (0.3% w/v) to O.D. 0.6 at 420 nm. Transformants with the deletion were sele cted on the corresponding antibiotic markers and the deletion was verified by PCR with the primers flanking the antibiotic marker gene inserted in the deleted gene. The deletion mutation was transduced by phage P1 to other genetic backgrounds before use (84). Transformation Chemical transformation was carried out as de scribed previously with minor modification (79). Cells inoculated from an overnight cu lture (0.1 ml) were grown in a 125 ml flask containing 5 ml LB medium at 37 oC for about 1.5 hours. After harvest by centrifugation at room temperature (1,200 x g; 5 min) cells were resuspended in 0.5 ml of cold 0.1 M CaCl2. Plasmid DNA was added to 0.2 ml of the cell suspension and the cell-DNA mixture was incubated on ice for 20 min. After heat-shock (1 min, 42 oC), the mixture was incubated on ice for an additional 5 min. One ml of LB was added to the reaction mixture and incubated for 2 hours at 37 oC, standing. Transformants were selected on appropri ate antibiotic medium. Electroporation of cells for plasmid transformation was as recommende d by Bio-Rad (MicroPulser electeroporation apparatus operating instructions and applications guide). Co-transduction Frequency Co-transduction frequency of two genes was calculated by a formula described by Sanderson et al (106) (Eqn. 2-1). C = 1[d / L]3 (Eqn. 2-1) C, cotransduction frequency; d, distance be tween the two genes; L, size of the DNA fragment transferred (usually the averag e DNA size packaged by phage P1; about 95 kbp).

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35 Sequencing DNA Nucleotide sequence of DNA was determined by the Interdisciplinary Center for Biotechnology Research, DNA Sequenci ng Core Facility at the Univer sity of Florida. Invitrogen and Sigma-Genosys synthesized oligonucleotide pr imers used for sequencing and the primers are listed in Table 2-2. Determination of the leve l of transcription of pdh operon Construction of P pdh-lac The promoter DNA with operator corresponding to the pdh operon (-326 to -15 of the pdhR with the A in the start c odon ATG as +1) was prepared by digestion of the DNA from plasmids pKY10 (SE2378 P pdh ) and pKY13 (W3110 P pdh ) with BlpI and AflII endonucleases. Ends of the digested DNA products were filled in by Klenow-fragment of DNA polymerase and cloned into SmaI restriction site of plasmid pTL61t (77) 208 base pairs upstream of promoterless lacZ gene. The plasmid constructs (pKY15 for SE2378 P pdh and pKY17 for W3110 P pdh ) were selected afte r transformation of E. coli Top10 as blue colonies on LBampicillin medium with X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside, 40 g.ml-1). The cloned pdh promoter DNA was sequenced to confirm the sequence. The plasmid constructs were transformed into strain LE392, and the tran sformants were grown in LB+maltose (0.3 %). Strain LE392 with plasmid was infected with RZ5, mixed with 2.0 ml of top-agar and poured as an overlay over LB-agar. Phage was propagated, at 37 oC for about 6 hours, collected and stored for further studies. Transduction An overnight culture of strain SE2366 was inoc ulated (1% v/v) into 3 ml of LB+ maltose in a 125 ml flask and incubated in a shaker (200 RPM) for about 2 hrs. Cells from 1 ml of the culture were harvested by centrifugation at room temperature and resuspended in 0.4 ml of 10

PAGE 36

36 mM MgSO4. Five l of phage stock (0.2 of MOI) was adde d to 0.2 ml of the cell suspension and the mixture was incubated at room temperature for 20 min. MOI is the average number of phage per bacterium. After adding 1.8 ml of LB, th e transduction mixture was incubated at 37 oC for 2 hours. Transductants were selected for resist ance to ampicillin and blue color on LB-agar containing ampicillin and X-Gal. Transductants with the lowest galactosidase activity, presumptive single lysogens, were used in pdh operon transcription studies. Galactosidase activity measurements Transductants carrying P pdh-lac fusion were grown in LB+glucose under aerobic or anaerobic conditions for 4 hours. Cells were harvested by centrif ugation, washed with 2.0 ml of cold Z-buffer (60 mM Na2HPO4.7H2O, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4.7H2O, 50 mM mercaptoethanol, pH 7.0) and resuspended in 1.0 ml of Z-buffer. Optical density of culture was adjusted to 2.4 (420 nm; Beckman DU 640) in 1.0 ml as a final volume. To 1.0 ml of this cell suspension, 54 l of chloroform and 27 l of 0.1 % SDS was added. After the suspension was mixed for 10 seconds in a vortex mi xer to permeabilize the cells for the substrate ONPG, the cells were kept on ice. Permeabilized cel ls (0.1 ml) were added to 0.9 ml of Z-buffer. Z-buffer (1.0 ml) only was used as a control. The tubes were placed in a water bath at 28 oC. After 2 min, 0.2 ml of ONPG (4 mg ml-1) was added to each tube and incubation was continued until the appearance of yellow color. Sodium car bonate (1 M) solution (0.5 ml) was added to stop the reaction For calculation of galactosidase activity, 2.4 O.D. units at 420 nm represented 350 g protein ml-1 and one nmole of O-nitrophenol ml-1 had an absorbance of 0.0045 at 420 nm (1 cm light path ). The specific activity of -galactosidase was expressed as nmoles min-1 mg cell protein-1.

PAGE 37

37 Quantitative RT-PCR For isolation of total RNA from aerobic cultures cells were grown in 10 ml of LB in a 250 ml flask at 37 oC with shaking at 200 RPM. For anaerobic cultures, cells were grown in 9 ml of LB medium in a 13x100 mm screw-cap tube filled to the top. Cells were ha rvested at early to mid exponential phase of growth. Total RNA was ex tracted by a hot phenol method (4). Isolated RNA was further processed with Qiagen RNA cleaning kit as per the Manufacturers protocol. Treating with ribonuclease-free DNase (Sigma) removed contaminating DNA from the RNA. Reverse transcription to genera te cDNA was carried out as described previously (4) using a primer based on the aceE sequence (72 to 240 from A of start codon, ATG, within coding sequence). The 20 l reaction mixture contained 4 l of iScript reaction mix (5x), 1 l of iScript reverse transcriptase, dH2O and 2 g RNA sample. The reaction was conducted at 25 oC for 5 min, at 42 oC for 30 min and at 85 oC for 5 min. Quantitative RT-PCR was carried out with SYBR-Green-490 as per the Manufacturers instructions (Bio-Rad) with the aceE cDNA as the template and a set of aceE specific primers (forward pr imer, CATCCGTGAAGAAGGTGTTG; reverse primer, GCGTTCCAGTTCCAGATTAC). The PCR conditions were 3.5 min at 95 oC followed by 65 cycles of 30 sec at 95 oC, 50 sec at 55 oC, 20 sec at 72 oC. in vitro DNA Mutagenesis Hydroxylamine Mutagenesis The lpd+ gene in plasmid pKY32, was mutagenized in vitro as described by Davis et. al. (30) with minor modification. A 200 l reaction mixture containing 7.5 g plasmid DNA (30 l), 40 l phosphate-EDTA buffer (0.5 M K-phosphate, pH 6.5; 5 mM EDTA), 80 l of freshly prepared hydroxylamine-hydrochloride, pH 6.0 (1.0 M NH2OH-HCl, 0.45 M NaOH) and 50 l deionized H2O was incubated at 37oC for 18 hours. DNA was purified by 3 successive phenol

PAGE 38

38 extractions and 3 successive chloroform extr actions. DNA was precipita ted with 100 % ethanol and dissolved in dH2O. PCR Mutagenesis The lpd gene was also mutagenized by erro r-prone PCR as described by Ichiro et al (82) with plasmid pKY32 as the template. PCR reacti on was conducted with the same primer set as used for cloning the lpd gene into plasmid pET15b. Mutage nic buffer contained 8 mM dTTP, 8 mM dCTP, 48 mM MgCl2 and 5 mM MnCl2 in addition to Taq DNA polymerase and its supplied buffer. PCR reaction was pe rformed using the following cycle: 1 min at 95oC followed by 5 cycles of 1 min at 95 oC, 30 sec at 45 oC, 2 min at 72 oC followed by 30 cycles of 1 min at 95 oC, 30 sec at 55oC, 2 min at 72 oC, followed by 15 min at 72 oC. The PCR product was purified and stored at 4 oC. Construction of pTrc99Alpd for Regulated Expression of lpd The lpd gene was amplified by PCR from the genomic DNA of E. coli wild type, strain W3110 or SE2378. The forward primer (5GCGACCATGG AGAAGGAGATATACCATGAGTACT-3) contained an NcoI restriction site at the 5 end (underlined), and the reverse primer (5GCGAAAGCTT TTACTTCTTCTTCGCTTTCG-3) contained a HindIII restriction site at 5 end (underlined). A Shine-Dalgarno sequen ce (ribosomal binding site) was located 10 nucleotides upstream of the st art codon (ATG). Both the P CR product and plasmid pTrc99A were digested with NcoI and HindIII restricti on enzymes and ligated together to construct plasmids pKY32 and pKY33 containing wild-type lpd and lpd gene with mutation, respectively (Figure 2-1). The cloned DNA sequence was verified by sequencing the DNA.

PAGE 39

39 Construction of pET-15blpd Plasmid for Purification of LPD The lpd gene was amplified by PCR with th e forward priming oligonucleotide (5GAGCCTCGAG ATGAGTACTGAAATC-3) and reverse priming oligonucleotide (5GCGTGGATCC TTACTTCTTCTTCG-3). The forward primer contains XhoI restriction site at the 5 end and the reverse primer has BamHI restriction site at the 5 end. PCR product digested with XhoI and BamHI was ligated with plasmi d pET-15b also digested with XhoI and BamHI (Figure 2-2). E. coli TOP10 competent cells were transfor med with the ligation product and the transformants were selected for resistance to am picillin on LB medium. The plasmid constructs (pKY36 with lpd from W3110 or pKY37 with the lpd from SE2378) were used for protein purification. Expression of Dihydrolipoamide Dehydrogenase (LPD) Dihydrolipoamide dehydrogenase (LPD ) was produced in strain JM109( DE3) transformed with pKY36 or pKY37 enc oding LPD. A 500 ml LB+ampicillin (100 g ml-1) culture was grown at 37oC with shaking at 250 rpm in a 2.8 liter Fernbach flask to an O.D. of 0.6 at 420 nm (Beckman DU 640 spectrophotomete r). The T7-RNA polymerase was induced by addition of arabinose (1.5 %) (91) After 4 hours of incubation at room temperature with shaking, cells were harvested by centrifugation (12,000 xg; 15 min; 4 oC), washed with 50 mM potassium phosphate buffer (pH 8.0) (Buffer A) and resuspe nded in 5 ml of the same buffer. Cells were passed through a French pressu re cell at 20,000 psi. The cr ude extract was clarified by centrifugation (30,000 x g; 45 min), and the supernatant was filtered through a 0.22 m filter. The filtered protein solution was loaded onto a Hi Trap chelating column (5 ml; General Electric) that was pre-washed with 0.1 M NiCl2 in the Buffer A. Unadsorbed and loosely-bound proteins were washed with 5 volumes of Buff er A followed by 5 volumes of Buffer A with 50

PAGE 40

40 mM imidazole. (His)6-tagged LPD protein was eluted with an imidazole gradient of 0.05 M to 0.5 M in K-phosphate buffer (pH 8.0). All the frac tions containing LPD activity were combined. The His-tag was cleaved off the protein by in cubation with Thrombin (25 unit mg protein-1; General Electric) at 4 oC, overnight. Thrombin and the small peptide were removed by gel filtration through a Sephacryl S200 HR column (2.6/60 cm; Genera l Electric). The protein was eluted with Buffer A contai ning 0.1 M NaCl at 0.5 ml.min-1 flow rate. All the fractions with LPD activity were combined and dial yzed against 50 mM potassium phosphate buffer, pH 8.0. Purity of the protein was confirmed by 12 % SDS-PAGE. Dihydrolipoamide Dehydrogenase (LPD) Assay Dihydrolipoamide dehydrogenase wa s assayed as described by Wei et al. (136). The standard reaction mixture for fo rward reaction contained 0.1 M KH2PO4 (pH 8.0), 3 mM NAD+, 3 mM DL-dihydrolipoic acid, and 1.5 mM EDTA in 1 ml, at room temperature. The enzyme uses both dihydrolipoamide and dihydrolipoic acid as s ubstrates and replacement of dihydrolipoamide with dihydrolipoic acid only re duces the LPD activity by 20 % (136). One unit of enzyme activity is defined as th e amount of NADH produced ( mol NADH min-1mg protein-1). The reverse reaction mixture included 0.1 M KH2PO4 (pH 8.0), 0.1 mM NAD+, 0.1 mM NADH, 3 mM DL-lipoamide, and 1.5 mM EDTA in 1 ml at room temperature. Both the forward and reverse reactions were carried ou t at room temperature. One unit of activity is defined as the amount of NADH oxidized ( mol NADH min-1 mg protein-1). Purification of Pyruvate Dehydrogenase Complex Pyruvate dehydrogenase (PDH) complex was purified from strains YK175 (native form) and YK176 (mutated form) as described by Bisswange r (11) with minor modification. Cells were cultured in six liters of glucose-minimal medi um (1 L per 2.8 L Fernbach flask). When cell

PAGE 41

41 density reached a density of approximately 2.0 (O.D. 420 nm; Beckman DU640), cells were harvested by centrifugation (10,000 g 15 min, 4oC ) and washed with 100 ml 50 mM Kphosphate buffer (pH 8.0; referred to as Buffer A). Cells were lysed by passage through a French pressure cell (20,000 psi) in the presence of protease inhibitor cocktail (5 ml / 20 g cell wet weight) (Sigma). DNase I and RNase I were added to the extract at 100 g.ml-1 each and incubated at 37 oC for 1 hour with gentle mixing in a cen trifuge tube to reduce the viscosity. After nuclease treatment, cell extract was centrifuged at 12,000 g for 30 min to remove cell debris and all operations from this point on were at 4C. The supernatant was further centrifuged at 150,000 g for 4 h to sediment the PDH complex. The supernatant was immediately decanted and the pellet was dissolved in 6 ml of phosphate buffer for 2 h with gentle mixing with a rocker. The protein solution was centrifuged at 12,000 g for 15 min to remove particulate that did not dissolve. The supernatant was passed through a hydroxyapatite column (15 x120 mm; Bio-Rad) that was equilibrated with Buffer A. The PDH comp lex was eluted from the column with a linear gradient of 50 mM to 500 mM Kphosphate (pH 8.0) at 0.15 ml min-1 flow rate. All the fractions containing PDH activity were combined, dial yzed in Buffer A and concentrated. The concentrated protein solution was further purifie d with a gel filtration column (Sephacryl S200HR, 2.6 X 60 cm) with Buffer A as the el uant at a flow rate of 0.5 ml min-1. All the active fractions were pooled and used im mediately for the enzyme assay. Pyruvate Dehydrogenase Assay Activity of pyruvate dehydrogenase was determin ed in both crude extracts as well as in partially purified protein. Two different assay conditions were used to determine the pyruvate dehydrogenase activity in crude extracts. A standard assay for determination of the activity of the pyruvate dehydrogenase complex in crude extrac t or purified protein was based on pyruvate-

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42 dependent reduction of NADH at 340 nm ( cm-1) at room temperature as described by Hinman (56). One ml reaction mixture co ntained 0.2 mM thiamine pyrophosphate, 0.1 mM CoA 1.0 mM MgCl2 0.3 mM dithioth reitol 2.5 mM NAD+ 100 g.ml-1 of BSA and crude extract or purified protein in Buffer A. The reaction was started with the addition of 5 mM pyruvate. The K m for NAD+ was estimated from a double reciprocal (Lineweaver-Burk) plot of substrate concentra tion and velocity (76). Effect of NADH on enzyme activity was determined in the same reaction mixture with the addi tion of various concentrations of NADH. A reaction that only determined the activity of the E1 component of the PDH complex contained 12.5 mM MgCl2 0.18 mM thiamine pyrophosphate 0.175 mM CoA 2.0 mM NAD+ 5.0 mM pyruvate and 1.0 mM pota ssium ferricyanide in one ml of 50 mM sodium phosphate buffer (pH 7.0). The reaction was based on the pyr uvate-dependent reduction of ferricyanide at 430 nm ( =1,030 M-1cm-1). The reaction was started with the addition of pyruvate. Protein Determination Protein concentration was determined us ing Coomassie Blue G-250 as described by Bradford (14) with bovine serum albumin as the standard. SDS-Polyacrylamide Gel Electrophoresis SDS-PAGE was performed with 12.5 % gels as described by Laemmli (72). Broad molecular weight standards used in SDS-PAGE were myosin (200,000 da), -galactosidase (116,250 da), phosphorylase b (97,400 da), bovine serum albumin (66,200 da), ovalbumin (45,000 da), carbonic anhydrase (31,000 da), soybean trypsin inhibitor (21,500 da), lysozyme (14,400 da) and aprotinin (6,500 da) (Bio-Rad). Prot eins were visualized with Coomassie blue R250.

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43 Fermentation Batch fermentation without pH control was carried out in 13 x 100 mm screw-cap tubes filled to the top with appropriate medium. Inocul um (1% v/v) for these fermentations was grown aerobically for about 16 h. pH-contro lled fermentation was conducted at 37 C in a 500 ml vessel containing 250 ml of corresponding medium in a cu stom-made pH-stat as described before (96). Culture pH was maintained by the addition of 1 N or 2 N KOH (80) unless sp ecified otherwise. Analysis of Fermentation Products Sugars and fermentation products were anal yzed by HPLC (Hewlett Packard 1090 series II equipped with a filter photometric detector (210 nm) and a refractive index detector in series) fitted with a Bio-Rad Aminex HPX-87H ion excl usion column as described previously (129).

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44Table 2-1 Bacterial strains and plasmids used in this study Strains Relevant Genotype Reference or Source W3110 Wild type ATCC 27325 BW25113 lacIq rrnBT14 lacZW116 hsdR514 araBADAH33 rhaBADLD78 B. Wanner AH218 BW25113 ( focA-pflB )-FRT-Km-FRT This study AH241 W3110 ldhA Lab collection AH242 AH241 ( focA-pflB )-FRT-Km-FRT ldhA AH241 X P1(AH218) SE2377 AH242 Anaerobic-(+) Isolate SE2378 AH242 Anaerobic-(+) Isolate SE2382 AH242 Anaerobic-(+) Isolate SE2383 AH242 Anaerobic-(+) Isolate SE2384 AH242 Anaerobic-(+) Isolate SE2385 AH242 Anaerobic-(+) Isolate YK1 SE2378-FRT KmS This study YYC186 ( aroP-aceF ) yac-284 ::Tn 10 lacZ608 (Am)Chang et al YK2 YK1 ( aroP-aceF ), yac -284::Tn 10 YK1 X P1(YYC186) CAG12025 zad-220:: Tn 10 l rph -1 CGSC YK29 AH242-FRT KmS This study YK55 SE2378 TcR Anaerobic (+) SE2378 X P1(CAG12025) YK93 YK1 aceFFRT-Km-FRT This study YK95 BW25113, mgsA -FRT-Km-FRT This study YK96 YK1, mgsA -FRT-Km-FRT This study YK98 BW25113 lpdFRT-Km-FRT This study YK99 YK1 lpd -FRT-Km-FRT YK1 X P1(YK98) YK100 YK29 lpd -FRT-Km-FRT YK29 X P1(YK98) YK111 YK100, KmS, aerobic+ YK100 X P1(YK1) YK112 YK101-P1(YK55), TcR YK101-P1(YK55) YK121 W3110-P1YK(112) W3110-P1(YK112) YK122 SE2377 TcR Anaerobic (+) SE2377 X P1(CAG12025)

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45Table 2-1 Continued. Strains Relevant Genotype Reference or Source YK123 SE2382 TcR Anaerobic (+) SE2382 X P1(CAG12025) YK126 YK100 TcR Anaerobic (+) YK100 X P1(YK123) YK127 YK100 TcR Anaerobic (+) YK100 X P1(YK122) YK128 YK100 (+pKY32) This study YK129 YK100 (+pKY33) This study YK130 YK99 (+pKY32) This study YK131 YK99 (+pKY33) This study YK132 W3110 (+pKY32) This study YK133 W3110 (+pKY33) This study YK134 W3110 lpd -FRT-Km-FRT W3110 X P1(YK98) YK135 YK134 (+pKY32) This study YK136 YK134 (+pKY33) This study YK137 YK101 (+pKY32) This study YK138 YK101 (+pKY33) This study YK139 YK100, KmS, anaerobic (+) YK100 X P1(SE2377) YK140 YK134 KmS, aerobic+ YK134 X P1(SE2377) YK141 YK100, KmS, anaerobic+ YK100 X P1(SE2382) YK142 YK134 KmS, aerobic+ YK134 X P1(SE2382) YK143 AH241, lpd -FRT-Km-FRT AH241 X P1 (YK98) YK146 YK143, KmS, aerobic+ YK143 X P1 (SE2382) YK149 YK96, KmS, AmpS This study YK150 JM109( DE3)(+pKY36) This study YK151 JM109( DE3)(+pKY37) This study YK152 YK29, KmR, aerobicYK29 X P1(YK93) YK153 W3110, KmR, aerobicW3110 x P1(YK93) YK154 YK93, KmS, aerobic+ YK93 X P1(W3110) YK156 YK93, KmS, aerobic+ YK93 X P1(SE2378)

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46Table 2-1 Continued. Strains Relevant Genotype Reference or Source YK157 YK152, KmS, aerobic+ YK152 X P1(W3110) YK158 YK152, KmS, aerobic+ YK152 X P1(SE2378) YK161 JM109( DE3)(+pKY38) This study YK167 YK142, ( focA-pflB )-FRT-Km-FRT YK142 X P1(AH240) YK168 YK134, KmS, AmpS This study YK169 YK168, ( focA-pflB )-FRT-Km-FRT YK168 X P1(AH240) YK170 YK169 (+pKY33) This study YK175 AH241, ldhA adhE -FRT-Km-FRT AH241 X P1(YK87) YK176 YK141, adhE -FRT-Km-FRT YK141 X P1(YK87) Top 10 FmcrA ( mrr hsdRMS mcrBC ) 80 lacZ M15 lacX74 deoR nupG Invitrogen recA1 araD139 ( ara-leu ) 7697 galU galK rpsL (StrR) endA1 Plasmids Reference or Source pTrc99A pTrc expression vector, AmpR Lab collection pET15b T7 expression vector, AmpR Novagen pTL61t transcriptional fusion vector Linn et al pKY32 W3110 lpd in pTRC99a This study pKY33 SE2378 lpd in pTRC99a This study pKY36 W3110 lpd in pET15b This study pKY37 SE2378 lpd in pET15b This study pKY38 SE2377 lpd in pET15b This study Phage P1 Tn 9 CmR clr-100 Lab collection bla lacZ lacY+ Lab collection

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47Table 2-2 List of primers used in this study. Primer list Oligonucleotide sequence Use pdhR -P1 AATGGTATGCGGCAGCGAAT pdhR -P2 GGCGTTCCAGTTCCAGATTA pdhR -P3 TGATCCTCGAAGGCACTCTC pdhR sequencing pdhR -P4 CAGATGGCGATGCGATGCTT pdhR -P5 TCGCTTCGAGACGTTGAATC pdhR -P6 CGCCAGAACTTCGAATTGCT P1 GAAGCATCGCATCGCCATCT P1 to R17 F2 AGGTGTTGAGCGTGCTCAGT aceEF-lpd sequencing primers F3 AGTTCACGGCAATGGCCTCT F4 GGTCACCGGTAACGGCAAGA P2 GCACACCGTCCATGTTCATT F6 CGTGCTCTGAACGTGATGCT F7 CGAAGGTCTGCAGCACGAAG P3 GGTGGTCACGATCCGAAGAA F9 CCGGCGCACTGATTATGATT F10 TACCGGCTCGCTGATTATGG P5 TTCGCTGAGCAGGTCCGTA F12 CTGCAGCTCTTGAGCAGATG F14 ACTCAGGTCGTGGTACTTGG F15 CCGCATGAAGATCCGCGTAT F16 GTACCAACGTACCGCACATC P2 GCACACCGTCCATGTTCATT R2 GCGATAGCACGACCAGAAGC R3 TACCAGCACGGCGTCGTAAC R4 TGCCGTTCTCACCTTCAACT R5 CAACGGAAGGCAGCGGAGTA P1 to R17 P4 TTGTCGCCTTCTACGGTGAT aceEF-lpd sequencing primers

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48Table 2-2 Continued. Primer list Oligonucleotide sequence Reference R7 TTCCGGCGCGTTCACAATCG P6 TCAGGTTCGCACCAGAGATT R9 GCTTATGTTCACGCGACTCC R10 CATAATCAGCGAGCCGGTAG P8 CGCTTGCTACCTTGCTGC R12 ACGGACCTGCTCAGCGAACA R13 CACCGTACATACGCTCCAGA P9 CGCCGTCTGGTGATGTAAGTA P10 CGAGCAACGGTCAGCAGTAT R14 CGGCAGATTGTTGGTGCTGT R16 TGCCTTCCAGTTCGTTGATG R17 CCAGCTGCTCCTGAGTCAGA mgsA -F ACAAATGCTGATGAGCTGGGTGGAACGGCATCAA mgsA deletion CCGTTACTGGAGTGCTGCAAGGCGATTAAGT mgsA -R AGAATATCGACCGCGTC GTTGAAATGCGGCGACT GGATTATGAAGGATTACGAATTCCGGTCTCC lpd -pTrc99a-NcoI GCGACCATGGAGAAGGAGATATACCATGAGTACT lpd for pTrc99a construct GAAATCAAAACTC lpd -pTrc99a-HindIII GAGCAAG CTTTTACTTCTTCTTCGCTTTCG lpd -pET15b F-XhoI GAGCCT CGAGATGAGTACTGAAATC lpd for pET15b construct lpd -pET15b F-BamHI GCGTGGATCCTTACTTCTTCTTCG adhE -F ATCACCGCACTGACTATACTCTCG adhE -R CCTGTTGTGGAAGCCGTTAT

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49 Figure 2-1 Construction of lpd in pTRC99a for complementation analysis. lpd lipoamide dehydrogenase gene; bla -lactamase; lacI, lac repressor; oriR, or igin of replication; rrnB, transcription termination sequence. pTRC99A4176 bps HindIII Ptrc rrn bla oriR lacIq AAGCTT lpd CCATGG lpdA in pTRC99a 5599 bps Ptrc lpd rrnB bla oriR lacIq ligation PCR product of lpdA gene digested with NcoI and HindIII. N coI

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50 Figure 2-2 Construction of LPD expression construct in pET15b with PCR amplified lpd gene. pET15B 5708 bps B amH I X ho I lac I ori bla T7 promoter T7 transcription start CCTAG TCGAG lpd PCR product of lpd gene digested with XhoI and BamHI lpd in pET15b 7134 b p s B amH I XhoI H is-lpd lacI ori bla ligation

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51 CHAPTER 3 RESULTS AND DISCUSSION Isolation of Homo-Ethanol Producing E. coli Production of either lactate or ethanol catalyzed by lact ate dehydrogenase or alcohol dehydrogenase (encoded by ldhA or adhE respectively), is a key fermentation pathway that allows E. coli maintain redox balance under fermentative growth conditions. In a pflB mutant, although adhE gene is intact, pyruvate is not converted to ethanol due to the absence of acetylCoA, the substrate for alcohol dehydrogenase. An ldhA pflB double mutant is defective for anaerobic growth due to a shortage of NAD+ for the activity of gl yceraldehyde-3-phosphate dehydrogenase, a key enzyme of glycolysis Isolating a mutant starting from a pflB ldhA deletion strain that can grow under anaerobic co nditions would help identify new pathways by which NADH is reoxidized by E. coli to maintain the overall redox balance. Towards this objective, strain AH242 ( pflB ldhA ) was mutagenized by EMS (ethylmethane sulfonate) and 31 mutant strains that grew anaerobically on LB+glucose solid medium were isolated. All the isolates produ ced ethanol as the primary fermentation product (Table 3-1). The level of succinate produced by these isolates was sim ilar to that of the wild type strain, W3110, indicating that the succinate produc tion pathway is not responsible for the observed anaerobic growth of the isolates. Although the genes encoding PTA and ACK for acetate production from acetyl-CoA were not deleted in the isolates, these isolates produced very low levels of acetate. These results suggest that ethanol production is not due to activation of the second PFL enzyme encoded by the silent pflCD operon or another PFLlike protein. In addition, the absence of formate among the fermentation pr oducts also suggests that the ethanol produced by these isolates did not originat e from PFL activity. Interestingly, a trace amount of lactate was observed in the fermentation broth, despite the deletion of ldhA gene in this mutant, (Table 3-1;

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52 Figure 3-1). One isolate that grew faster than others, strain SE 2378, was used in further studies (Table 3-1). Mapping the Mutation(s) in Strain SE2378 Based on the fermentation profile and grow th pattern, ethanol production is the new pathway that helps maintain redox bala nce in the mutants derived from the ldhA, pflB double mutant. Since lactate yield wa s minimal and formate was not detected, lactate dehydrogenase and pyruvate-formate lyase activity were not restored in strain SE2378. This suggests an induction of a new anaerobic pathway in strain SE2378 and other similar isolates for ethanol production that is responsibl e for oxidation of NADH to NAD+ under fermentative conditions. The known pathway for production of ethanol as the major fermentation product from glucose utilizes pyruvate decarboxylase and alcohol dehydrogenase, found in Z. mobilis and a few other bacteria (Figure 3-2). This enzy me activity was not detected in E. coli and a gene corresponding to pdc was not found in the annotated genome of E. coli An alternate pathway is to generate acetyl-CoA from pyruvate and reduce this to et hanol in two steps by alcohol dehydrogenase. Pyruvate oxidase and acetyl-CoA synthetase c ould serve this function (Figure 3-2). However, pyruvate oxidase is reported to be only produced during the stationary pha se of growth, and ACS is an ATP-dependent reaction. A fe rmenting cell is not likely to have the ATP needed to support ethanol production by this latter pathway. In addition, the ADH-E dependent reduction of acetylCoA requires two NADH per acetyl-CoA and glyc olysis followed by the POX pathway does not generate the needed two NADH per acetyl-CoA. An alteration of pyruvate dehydrogenase (PDH) complex that is not normally active in an anaer obic cell is of high pr obability in strain SE2378 (Figure 3-2) (17). To evaluate the role of PDH complex in supporting anaerobic growth of strain SE2378, ( aroP aceF ), zac ::Tn 10 was transduced into strain SE2378. The cotransduction frequency of

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53 ( aroP aceF ) with Tn 10 was close to 100%. Transductants were first selected for resistance to tetracycline on rich medium with tetracycline. Since a pdh mutant required acetate for optimal aerobic growth in glucose-minimal medium, all th e tetracycline-resistant transductants were tested for growth on glucose-minimal medium. All the tested transductant s required acetate for growth on glucose-minimal medium (Figure 3-3). These transductants lost the anaerobic growth positive phenotype of the parent, strain SE 2378. When the same deletion mutation was transduced into wild type strain, W3110, the ( aroP aceF ), zac::Tn 10 transductants grew normally under anaerobic conditions (Figure 3-4) These results show that the PDH is not required for anaerobic growth of E. coli but is only required for the anaerobic growth of strain SE2378 that lacks both ldhA and pflB However, the possibility that a mutation in another gene that is near the pdh operon is responsible fo r the anaerobic growth of strain SE2378 can not be ruled out from these experiments. To distinguis h between the two altern atives, a part of the aceF gene of the pdh locus ( pdhR-aceEF-lpd ) was deleted in strain SE2378 and the phenotype of the new derivative (strain YK93) was determined. Strain YK93 was acetate-dependent for aerobic growth in minimal medium and anaerobic growth defective in all media tested (Figure 3-4). Similar results were also obtained when a lpd mutation was introduced into strain SE2378. Restoration of aerobic growth phenotype of strain YK93 by transducing the aceF+ gene from strain SE2378 (strain YK158) also restored an aerobic growth. Although the anaerobic growth rate of strain YK158 was about 50% of that of strain W3110, this growth rate was comparable to that of strain SE2378 (Table 3-2). Strain YK158 also produced ethanol as the major fermentation product. In contrast, when aerobic grow th was restored by transducing the aceF+ gene from the wild type strain, W3110, about 93 % of the tran sductants still retained the anaerobic growth defect of the recipient (Table 3-2). However, the anaerobic growth of the 7% of the transductants

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54 carrying the aceF+ from the wild type suggests that the mutation that supported anaerobic growth of strain SE2378 is not located in the aceF gene but in a gene within 0.05 minute (about 2.25 kbp) (based on co-transduction frequency) of aceF in the E. coli genome. While the aerobic growth of strain YK152 was restored with the aceF+ from wild type strain, W3110, none of the transductants grew anaerobically. Location(s) of the Muta tion(s) in strain SE2378 The genetic analysis revealed that a mutation in the pdh-aceEF-lpd operon or in a closely linked gene in strain SE2378 is responsible fo r the anaerobic growth of SE2378. In order to explore the nature of the mutation( s), the DNA sequence of the entire pdh operon of strain SE2378 and 2 other isolates (SE2377, SE2382) wa s determined. This included the upstream promoter region also (Figure 3-5) (99). None of the three mutant strains carried a mutation in the promoter region or further upstream of the pdh operon (up to -243). The DNA sequence revealed 2 mutations within th e coding region of pdhR gene of strain SE2378 (T to C of 34th nucleotide & insertion of TGC between 352th and 353th nucleotide from th e start codon ATG of pdhR ), which lead to an amino acid s ubstitution (S12P) and an amino acid insertion (leucine as 118th amino acid) (Figure 3-6). A single nucleotide substitution (G to A) was also found in the intergenic region between pdhR and aceE in SE2378 at position -59 from the A of start codon ATG of aceE gene. However, no mutation was found in pdhR gene or the intergenic regi on of the other two isolates (Figures 3-6 and 3-7). Since strains SE2377 and SE2382 also grew anaerobically, a mutation in another gene in the pdh operon other than the pdhR appears to be responsible for the anaerobic growth phenotype (Table 3-1). The genes aceE and aceF encode E1 component (pyruvate decarboxylase / dehydrogenase) and E2 component (dihydrolipoamide transacetyla se), respectively, of pyruvate dehydrogenase

PAGE 55

55 complex. The DNA sequence of these two genes wa s unaltered in all the strains tested. These three strains carried a single nucleotide substi tution (G to A at position +1,060 or C to T at position +964 from A) in the lpd This nucleic acid change led to an amino acid substitution from glutamate to lysine (E354K ) or histidine to tyrosine (H322Y) (Figure 3-8). Since the lpd mutation in the three mutants was in one of two amino acid positions, the lpd genes in three other mutants (SE2383, SE2384 and SE2385) were also se quenced. Mutation in these three mutants was also in the amino acid position 322 or 354 as with the other three ethanologenic mutants (Figure 3-8). In concert with mapping experi ments, the sequencing results provide high probability that pyruvate dehydrogenase plays an essential role in restoring anaerobic growth of SE2378 and other similar derivatives. Confirmation that the Mutation in lpd is Responsible for the Anaerobic Growth Phenotype of Strain SE2378 DNA sequence analysis identified a si ngle amino acid substitution in the lpd in the six mutants that grew anaerobically in a mutant lack ing LDH and PFL (Figure 3-8). The role of this mutation in lpd gene in supporting the an aerobic growth phenotype of these mutants was examined directly by complementation analysis. For these experiments, an lpd deletion mutation was introduced into a kanamycin-sensitive deriva tive of strain AH242, the parent of strain SE2378 and other anaerobic-plus isolates. The resulting strain, YK100, was transformed with plasmids that express the lpd gene either from the wild type or strain SE2378 from a lac promoter. Since strain AH242 is the parent strain of SE2378, this strain sh ould be free of any of the mutation(s) responsible for the anaerobicplus phenotype of stra in SE2378 (Table 3-3). Complementation of the lpd mutation with the native lpd+ gene (strain YK128) supported aerobic growth in minimal medium but not anae robic growth. Complementation of the same lpd mutation in strain YK100 with the lpd gene alone from SE2378 supported both aerobic and

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56 anaerobic growth with ethanol as the main fermentation product (Table 3-3). These results indicate that the mutation found in the lpd gene is sufficient to suppor t anaerobic growth of strain SE2378 and other isolates. Additional Mutations In order to further confir m that the mutation in the lpd is sufficient for the observed ethanologenic phenotype of the ldhA, pflB mutant, the lpd+ gene from wild type strain, W3110, was mutagenized by either hydroxylamine or by error-prone PCR amplification. When plasmid pKY32 with the wild type lpd+ gene was mutagenized with hydroxylamine hydrochloride and transformed into strain YK100 ( ldhA, pflB, lpd ), 5 additional independent mutations were also obtained. All 5 transfor mants carrying the altered lpd gene grew aerobically and anaerobically and produced et hanol as the main fermentation product. Sequencing the lpd gene in these 5 plasmids with the mutations showed that the amino acid chan ge (E354K) is the same as that of the lpd gene in strain SE2378. Under similar conditions, error-prone PCR mutagenesis did not yield any mutation and was not pursued fu rther. These results further confirm that a single mutation, such as the E353K, in the lpd gene is sufficient to support anaerobic growth of a ldhA, pflB mutant and the etha nologenic phenotype of E. coli Metabolic Routes of Pyruvate with th e Mutated LPD in Various Backgrounds In the presence of the lpd mutation ( lpd* ), the PDH is active under anaerobic growth conditions. Transduction of this lpd* mutation into wild type E. coli is expected to provide a third alternative route for pyruvate metabolism, in addition to lact ate dehydrogenase and pyruvate formate-lyase (Figure 3-9). Of the thr ee enzymes, PDH has the highest affinity for pyruvate ( K m of 0.4 mM). Based on its lower K m for pyruvate, the PDH would be the expected primary enzyme metabolizing pyruvate rather th an LDH or PFL. As seen with strain SE2378, metabolism of pyruvate through the PDH pathway leads to ethanol as the main fermentation

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57 product to maintain redox balance. These observa tions suggest that the fermentation profile of strain W3110 with the lpd* mutation will shift from mixed acid to mostly ethanol. Surprisingly, replacing the lpd+ gene of wild type strain, W3110, with the lpd* from strain SE2378 did not alter the fermentation profile and the primar y product was lactic acid (Table 3-4). Similar results were obtained with strain YK144 producing only LDH and PDH activities (Table 3-4). These results show that the PDH ev en with its 18-fold higher affinity for pyruvate failed to compete with LDH (127, 139). A dist inguishing characteristic of anaerobic E. coli is the higher NADH/NAD+ ratio (31). This lower NAD+ concentration, the need ed substrate of PDH, coupled with a higher NADH concentration, the s ubstrate of LDH, is apparently the deciding factor in the flow of pyruvate in the anaerobic cell. Furthermor e, LDH is activated by pyruvate and NADH (127). In the presence of only PDH and PFL activities ( ldhA ) (strain YK146), the fermentation products were acetate, formate and ethanol during th e initial growth phase. With a decline in the growth rate and at the stati onary phase of growth, PDH becam e the main pyruvate-metabolizing enzyme as seen by an increase in ethanol duri ng this phase. During the anaerobic growth phase, PFL pathway is favored because of the additi onal ATP per glucose pr oduced at the acetate kinase step (Figure 1-1) to s upport a higher growth rate. As the culture reaches the stationary phase, PDH takes over pyruvate metabolism, because the additional ATP produced by ACK is apparently not needed to maintain the lower gr owth rate. These results indicate that lactate dehydrogenase pathway needs to be deleted in or der for the mutated PDH to direct carbon flux from glucose to ethanol. Aerobic and Anaerobic Expression Level of pdh Operon In wild type E. coli cells, pyruvate dehydrogenase is re sponsible for oxidation of pyruvate only under aerobic conditions. Sin ce the mapping of the mutation in the ethanologenic isolates

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58 revealed that pdh is responsible for anaerobic growth, it was of interest to evaluate the expression level of pdh operon in the mutant duri ng aerobic and anaerobic grow th. The relative level of aceE gene mRNA of wild type strain W3110 was not significantly different under either aerobic or anaerobic growth conditions, indi cating that the transcription of pdh operon is independent of the presence of O2 (Table 3-5). Independence of pdh transcription to O2 in the growth medium was also observed with the et hanologenic strain SE2378. These results were confirmed by the level of -galactosidase activity (P pdh lacZ ) produced by the cultures grown with or without O2. Despite the same level of transcription of pdh operon in aerobic a nd anaerobic cultures of W3110, the activity of PDH measured as E1 com ponent activity in the ex tracts of W3110 cells grown anaerobically was only about 50 % of that of aerobically grown cells. It is possible that the inactive PDH complex rapidly turned over in the anaerobic cell. In contrast to W3110, the mutant, strain SE2378, had similar levels of PDH enzyme activity regardless of the presence of O2. The observed lower level of PDH activity in strain SE2378, compared to strain W3110, is in accordance with the lower mRNA levels in the muta nt. These results show that the lack of PDH activity in the wild type E. coli (W3110) during anaerobic growth is attributable to lower protein level and inhibition of enzyme activity. LPD Purification and Characterization The dihydrolipoamide dehydrogenase (LPD ), E3 component, in the pyruvate dehydrogenase (PDH) complex plays an important role not only in the cata lytic activity of the complex but also in regulating PDH activity depending on the NADH/NAD+ ratio of the cell. It is known that the activity of E3 is inhi bited by NADH (105, 111, 138). The mutation in the lpd gene in strain SE2378 and the other isolates appa rently reversed the inhibitory effect of NADH on PDH activity. To evaluate this possibility both the LPD and PDH were purified and the

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59 kinetic characteristics of these enzymes, especi ally the inhibition of activity by NADH, were determined. NADH Sensitivity on Forward Reaction The LPD from W3110 and SE2378 was purified to homogeneity for determination of enzyme activity (Figure 3-10). {Move this se ntence to the Methods section. The LPD activity was linear up to 80 ng of the native protein a nd 200 ng of the mutated enzyme from strain SE2378 (Figure 3-11). For determination of the kine tic parameters of the enzyme, 53.8 ng of the native LPD and 200 ng of the mutated LPD were used per reaction. Both the native and the mutated LPD exhibited typical Michaelis-Menten type kinetics (Figure 3-12, 3-13). Kinetic constants for the native and the mutated LPD are pr esented in Table 3-6. The affinity of the two enzymes for NAD+ (0.4 mM) was the same. Turnover number for the mutated form of the enzyme was about 4-fold lower than the native enzyme and reduced the catalytic efficiency ( K cat/ K m) by about 4 fold, compared to that of the native LPD. One of the distinctive char acteristics of LPD is its high sensitivity to NADH/NAD+ (105, 111, 136, 138). In order to examine whether the mutation in lpd in strain SE2378 altered the sensitivity of the enzyme to NADH inhibition, LPD activity was determined in the presence of NADH. The activity of the native LPD was progressively inhibited by increasing NADH concentration (Figure 3-14; 3-15). The nativ e LPD was completely inhibited at an NADH concentration of 0.16 mM. This con centration relates to an NADH/NAD+ ratio of 0.08. The mutated LPD (E354K) was significantly more sensit ive to inhibition at a lower concentration of NADH (0.02 mM) than the native LPD (Figur e 3-16). Increasing the NADH concentration reversed the inhibitory eff ect on the mutated LPD. Beyond an NADH concentration of 0.06 mM, increasing NADH concentration had minimal e ffect on the activity of the mutated enzyme. The physiological concentrations of NAD+ and NADH in E. coli growing under aerobic

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60 conditions are 2.54 mM and 0.08 mM, respectively (NADH/NAD+ of 0.03) (31, 71). Both the native and the mutated LPD was inhibite d equally at this ratio of NADH/NAD+ of 0.03 (Figure 3-15). The physiological significan ce of this in vitro observati on with a PDH component to in vivo physiology is not clear. The NADH/NAD+ ratio of an anaerobic E. coli is about 0.7 (31). Since the native enzyme is inhibited by as low an NADH/NAD+ ratio as 0.08 (Figure 3-15), it is expected that the native LPD would have no activity under anaerobic growth conditions. In contrast, the mutated LPD still retained about 35% of the control activity at 0.16 mM NADH and the effect of NADH concentrations higher than this were not evaluated due to experimental difficulty. These results clearly show that the mutated LPD is less sensitive to NADH inhibition. NADH Sensitivity of the Reverse Reaction of LPD The reduced sensitivity of mutated LPD to NADH can be more readily seen by following the reverse reaction that transfers elec trons from NADH to lipoamide producing dihydrolipoamide and NAD+. Although this is not the native reaction, this reaction is also inhibited by NADH although one of th e substrates of the reaction is NADH. For this reaction to proceed, NAD+ is a required activator (105, 138). This requirement was also observed for the native LPD (Figure 3-17) and th e reverse reaction of the enzy me was maximal only above an NAD+/NADH ratio of 1.5 or higher. Activat ion of the native LPD activity by NAD+ was biphasic. At a lower NAD+/NADH (up to 1.0), only about 35 % of the enzyme was activated and in a second higher phase, increasing the ratio fr om 1.0 to about 1.5 increased the activity to almost 100 %. This is in agreement with the previous report by Schmincke-Ott et al. (111), suggesting that LPD may have two NAD+ binding sites. In contrast, the mutated LPD retained about 80 % of its maximum specific ac tivity even in th e absence of NAD+. Maximal activity of the mutated enzyme was reached at an NAD+/NADH ratio of about 0.2. 0.2. (Figure 3-17). These results suggest that the E354K mutation overcame the need for binding of NAD+ to one of the

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61 two sites for activation of the enzyme. In doing so, the mutated LPD apparently increased its catalytic capacity at higher NADH/NAD+ normally found in the cell under anaerobic conditions. An alternative possibility that the muta ted form of the LPD has a tightly bound NAD+ in one of the activation sites can not be ruled out. It is expected that this re duced sensitivity of LPD to NADH inhibition would be associated with reduced sensitivity of the pyruvate dehydro genase complex of the mutant, strain SE2378, at supporting the activity of the enzyme at NADH/NAD+ ratios that is inhibitory to the native pyruvate dehydrogenase complex (136, 138). PDH Purification and Characterization The native PDH is composed of three enzymes including the LPD and has a molecular mass of about 4x106 Da. This complex was purified from strain YK175 ( ldhA adhE ) and the mutated form of the enzyme was purified fr om strain YK176. Since neither strain grew anaerobically, cultures were grow n aerobically for purification and kinetic characterization of the native and mutated PDH complex. Biochemical properties of purified enzyme are not expected to differ based on the growth conditions. Purification of the two forms of the complex is summarized in Table 3-7 (native PDH) and in Table 3-8 (mutated PDH). The ultracentrifugation step significantly removed most of the smaller proteins. While the recove ry of mutated PDH was about 80 % after the ultracentrifugation step, onl y about 50 % of the native PDH was obtained after this step. Despite significan t loss of activity in each of th e steps, PDH was purified with only one contaminating protein (Figure 3-18). Th is contaminating protein (about 38,000 Da) copurified with the PDH complex from both st rains and had no dete ctable NADH oxidation activity including LDH, ADH, and NADH oxidase activity. The nature of the contaminating protein is not known.

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62 Determination of Kinetic Constants of PDH Complex Both forms of the enzyme fit the Michaelis-Menten kine tics (Figures 3-19, 20, 21, 22). Both forms of the PDH complex had similar affi nity for the two substrates. Affinity for NAD+ by the mutated form of PDH was similar to that of th e native PDH (Table 3-9). This is in agreement with the observed similar affinity of the LPD component for NAD+ (Table 3-6). However, the K m for NAD+ by the PDH complex was significantly lo wer than the isolated LPD component (0.1 mM vs. 0.4 mM for the LPD). These results indicate that the conformational changes associated with the formation of the comple x form of PDH increased the affinity for NAD+ in both cases. Turnover number and ca talytic efficiency of the two enzymes were also comparable. These results indicate that the mutated form of PDH complex functions as efficiently as the native PDH complex converting pyruvate and NAD+ to acetyl-CoA and NADH. Inhibition of PDH activity by NADH Under anaerobic conditions, the NADH/NAD+ ratio of E. coli is significantly higher (about 0.70) than that of an aerobic ce ll (about 0.03) (31). Since the mu tated form of LPD was found to be less sensitive to NADH, especially at the hi gher concentrations of NADH, it is possible that this effect is transferred to the PDH comple x since the site of NAD H inhibition is the LPD component. The native PDH was more severely inhibited by NADH than the mutated PDH and the progressive inhibition of the native PDH by NADH was observed as in the native LPD (Figures 3-14 and 3-23). Although the mutated PDH was also inhibited by NADH, its sensitivity to NADH inhibition was less than that of th e native PDH (Figure 3-24). At a fixed NAD+ concentration of 1.0 mM NAD+ (10 times higher than K m for NAD+), while the native LPD was inhibited by about 60 % at an NADH/NAD+ of about 0.03 (Figure 3-15), the PDH activity was reduced only by about 10 % at the same NADH/NAD+ ratio (Figure 3-25). Inhibition of the native PDH activity by NADH was biphasic and about 80 % of its activity was inhibited at 0.1

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63 mM NADH (NADH/NAD+ of 0.1) (Figure 3-25). In contra st, the mutated PDH still retained about 80 % of its activity at the same concen tration of NADH (0.1 mM) (Figure 3-25). This difference in NADH sensitivity of the two enzymes is reflected in the K i value for NADH of 1 M for native enzyme and 10 M for the mutated form (Table 3-9). It is interesting to note th at both the native PDH comple x and purified LPD component were inhibited by about 70% in the presence of 0.1 mM NADH (Figures 3-15 and 3-25). However, the NADH inhibition characteristics of the PDH complex from the mutant, strain SE2378, differed from that of the LPD com ponent from the same mutant. The observed sensitivity of the mutated LPD to low level of NADH was absent in the PDH complex indicating that the inhibition by 0.02 mM NADH was not physiological since the LPD only functions as part of the PDH complex in the cell. Discounting this part of the inhibition curve, the PDH complex and the LPD appear to be inhibited by about the same level by NADH. These results clearly demonstrate that the E354K mutation in lpd gene of strain SE2378 that reduced the severity of NADH inhibition of the LPD component is transferred to the PDH complex and is the main reason for the anaerobic grow th phenotype of strain SE2378. Fermentation of Sugars to Ethanol Glucose Fermentation The ability of strain SE2378 and similar isolates to produce ethanol as the main fermentation product provided an opportunity to evaluate these isolat es as ethanologenic microbial biocatalysts that lack foreign genes for fermentation of sugars to ethanol. When wild type E. coli was cultured in a pH-controlled medium containing 50 g glucose L-1, strain W3110 grew at a specific growth rate of 0.44 h-1 ( max ), producing lactate, formate, acetate and ethanol as fermentation products (Figure 3-26; Table 3-10). Strain SE2378 grew after a lag of about 6 h

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64 with a specific growth rate of 0.46 h-1 that is comparable to that of the wild type (Figure 3-27; Table 3-10). While wild type consumed almost all of the glucose in 24 h, strain SE2378 required about 72 h to consume the same amount of glucose. This difference can be attributed to the cell density of the culture at the e nd of the growth phase (2.4 mg ml-1 dry wt for the wild type vs. 1.7 mg ml-1 dry wt for the mutant) and also the highest rate of glucose consumption by wild type was slightly higher than that of strain SE2378 (4.1 to 3.26 g glucose h-1 g cells-1; Table 3-11). The highest concentration of ethanol produced by SE2378 with 5 % glucose was about 480 mM (22 g L-1) and this accounted for 88 % of total fermenta tion products (Table 3-10). Considering the fermentation yield of ethanol from 1 mole of gl ucose is 2 moles of ethanol, ethanol yield of strain SE2378 with 50 g L-1 glucose was about 81 % (Table 3-10). Xylose Fermentation Since xylose is the major sugar present in he micellulosic biomass, a microbial biocatalyst is required to ferment xylose efficiently. In a pH-controlled culture containing 50 g xylose /L, both the wild type and strain SE2378 grew at similar growth rate although strain SE2378 exhibited a longer lag time (about 6 hours) before the beginning of growth ( max, 0.37 h-1 vs. 0.38 h-1; Figure 3-28, Figure 3-29;Table 3-12). This lo wer growth rate in xylose medium is in agreement with previous reports that anaerobic growth rate of E. coli on xylose is lower than that of E. coli on glucose (50). Also, wild type strain re quired more than 72 hours to ferment 50 g/L xylose while the same strain fermented the same amount of glucose with in less than 36 hours. Since the cell mass of strain SE2378 at the end of the growth phase wa s the same as that of strain W3110, this difference in fermentation time is probably due to the higher rate of xylose consumption by strain SE2378. As expected, xylose fermentation led to an increase in acetate production by the wild type E. coli than in glucose fermentation, in agreement with the previous

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65 report (50) that lower ATP generation on xylos e directed more xylos e to acetate production. While wild type produced about 190 mM et hanol in 72 hours, strain SE2378 produced approximately 440 mM ethanol within 48 hours. Ethanol yield by strain SE2378 was as 2.4fold higher than that of the wild t ype (0.82 vs. 0.34; Table 3-12). It is interesting to note th at the specific ethanol productiv ity of wild type and the lpd mutant was higher on xylose than on glucose (T able 3-11). This is probably a reflection of energy output of xylose fermentation. For wild ty pe, the net ATP yield from xylose is only about 1.5 per xylose compared to 3.0 per glucose. This w ould require that the ce lls utilize more xylose to generate the same amount of energy. However, the specific rate of xylose consumption by the wild type was only slightly higher than that of gl ucose (4.93 vs. 4.10 g sugar h-1 g cells-1; Table 3-11) accounting for the lower cell yield and lo nger fermentation time compared to glucose fermentation (Figure 3-26, Figure 3-28). In contra st, strain SE2378 lacks pyruvate formate-lyase, an enzyme critical for xylose fermentation in mi nimal medium (50). Due to this mutation, the net calculated ATP yield from xylose fermentation in strain SE2378 is only 0.67 per xylose. It is apparent that this lower ATP yield is drivi ng the high xylose flux in this ethanologen. These results suggest that the engineered ethanologenic E. coli without foreign genes has the potential to increase specific ethanol productivity in gluc ose fermentation by decreasing the net ATP yield from glucose. Removal of Trace Amo unt of Lactic Acid During glucose fermentation, small amounts of l actic acid were also produced by strain SE2378 (Table 3-10). Since the ldhA gene is deleted in this strai n, it is unlikely that the LDH is responsible for this lactic aci d. It is known that the metabo lic pathways responsible for detoxification of methylglyoxa l produced from dihydroxyacetone phosphate (DHAP) result in lactic acid formation (41, 43). By dele ting the methylglyoxal synthase gene ( mgsA ), the first

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66 enzyme in the methylglyoxal to lactic acid path way, this trace amount of lactic acid could be eliminated, if this pathway is responsible for this lactic acid. Introduc ing a deletion into the mgsA in the chromosome of the et hanologenic strain SE2378, YK96, eliminated the lactic acid production during glucose fermentation (Fi gure 3-30; Table 3-13). In addition, the mgsA mutation also increased the ethanol yield to about 88 % of the glucose fermented without reduction in growth rate or cell yield (Table 3-13). Proposed Ethanologenic Fermentation Pathway In the ethanologenic mutant E. coli strain SE2378 isolated and described in this study, pyruvate dehydrogenase complex is active under fermentative conditions because a specific mutation in the LPD reduced the sensitivity of th e enzyme to inhibition by high concentration of NADH associated with the anaerobic cell. This altered PDH complex supports anaerobic growth of E. coli without external electron acceptors even in the absence of LDH and PFL activity (Figure 3-31). The production of one acetyl-CoA per pyruvate yiel ds an extra NADH, leading to a total of 4 NADHs per glucose. Since the glyc olysis and PDH yields a total of 4 NADHs along with 2 acetyl-CoAs, the redox bala nce can be easily restored by th e reduction of the 2 acetylCoAs with the 4 NADHs to 2 ethanols. For construction of an ethanologenic E. coli using the PDH system, the ldhA needs to be eliminated. Since the shift from PFL to PDH is accomplished by physiological means, a mutation in pflB is not required and i ndeed the presence of pflB may be beneficial to increasing the gr owth rate and higher cell yield es pecially with pentoses leading to a reduction in fe rmentation duration. The PDH from almost all organisms is known to be inhibited by NADH to varying degrees and is probably a control mechanism that prev ents excess production of NADH that cannot be oxidized under O2 limitation conditions. Thus, an increase in NADH/NAD+ ratio is also a signal used by the cell to shift the metabolism from oxidative to fe rmentation. Introducing a mutation

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67 similar to that of E352K in the LPD of other bacteria may exert a profound physiological change. Therefore, the development of biocatalyst, st rain SE2378, provides a sign ificant potential to avoid introduction of foreign genes for conversio n of pentoses derived from cellulosic biomass to not only bioethanol but al so other biobased products.

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68Table 3-1. Growth and fermentation profile of the anaerobic (+) derivatives of E. coli strain AH242 grown in LB+glucose (0.3 %, w/v) in a batch culture without pH control. O.D. (420 nm) at culture time (hrs)Fermentation Products (mM) Isolate 24 48 72 91 Succinate Lactate Formate Acetate Ethanol 1 0.0 0.7 0.9 2.8 3.1 31.1 2 0.8 2.1 3.1 28.4 3 0.0 0.4 0.8 2.7 3.6 30.6 4 0.1 0.9 5.4 30.7 5 0.5 0.9 3.3 3.9 29.4 (SE2382) 6 0.1 0.9 2.9 3.0 30.8 7 0.9 1.7 4.8 30.3 8 0.1 0.7 2.8 0.5 3.3 3.4 26.8 9 0.9 2.4 3.1 30.4 10 0.0 0.1 0.2 2.0 2.6 5.8 11 0.6 0.9 2.8 3.2 32.0 12 0.1 0.9 3.3 0.2 3.0 30.7 13 0.1 0.9 2.5 0.2 2.4 32.4 14 1.0 2.3 2.9 33.3 15 0.2 0.9 2.6 3.1 32.7 (SE2383) 17 0.8 1.8 2.4 24.6 18 +/0.9 2.3 2.7 4.0 31.2 (SE2377) 19 +/0.9 2.6 2.7 33.1 (SE2378) 20 0.8 0.9 7.6 2.2 11.4 21 1.0 1.9 2.6 31.9 22 0.2 0.5 0.8 2.5 4.9 2.9 28.2 23 0.2 0.9 2.6 2.9 33.2 24 0.3 0.9 2.5 3.8 3.0 29.2 25 0.0 0.4 0.9 3.1 2.7 32.6 (SE2384) 26 0.5 0.9 2.4 4.5 2.7 27.4 27 0.0 0.9 2.1 0.2 2.5 32.0 28 0.5 0.5 0.6 3.3 0.6 3.5 4.9 26.3

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69Table 3-1. Continued. O.D. (420 nm) at culture time (hrs)Fermentation Products (mM) Isolate 24 48 72 91 Succinate Lactate Formate Acetate Ethanol Reference 29 0.1 0.1 0.9 3.1 3.6 32.2 30 0.0 0.0 0.7 2.1 2.8 23.5 31 0.1 0.9 2.3 2.8 32.8 SE2385 32 0.4 0.9 2.9 3.0 32.0 AH242 W3110 0.6 4.2 18.5 2.6 9.3 7.3 wild type -, No detectable growth in fermentation products indicate s undetectable level of the product.

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70 Figure 3-1. HPLC analysis of fermentation products. (A) strain W3110, (B) strain SE2378 in LB+glucose (1 %, w/v) in batch fermentations. Cells were harvested after 36 hours. See Methods for HPLC analysis. min 15 17.5 20 22.5 25 27.5 30 32.5 35 mAU 600 800 1000 1200 1400 1600 1800 18.574 Succinate 19.812 Lactate 21.733 Formate 23.717 Acetate Ethanol W3110 min 15 17.5 20 22.5 25 27.5 30 32.5 35 mAU 600 800 1000 1200 1400 1600 18.581 Succinate 19.810 Lactate 23.717 Acetate Ethanol SE2378 (A) (B) RI detector response (mAU) RI detector response (mAU)

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71 Figure 3-2. Metabolic fa tes of pyruvate in E. coli X indicates deletion of the gene. adhE alcohol dehydrogenase

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72 Figure 3-3. Effect of deleting pdh genes in strain SE2378 on anaerobic growth. TcR P1 {YYC186, ( aro P -aceF ) za c284 ::Tn 10 } YK1 (SE2378, Kms) LB-Glc; anaerobic (+) (-) 100% 0% aroP pdhR aceE lpdA zad aceF zac-284:: Tn 10 Deletion (Anaerobic Growth Defective) (acetate) (+ acetate) No growth 100 % growth (strain YK2) Relevant genotype of strain YK2 (aerobic; glucose-minimal medium)

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73 Figure 3-4. Growth characteristics of various deletion deri vatives of wild type W3110 and strain SE2378. Aerobic growth was tested in glucose-minimal medium. Growth (+) only with acetate s upplementation (1 mg.ml-1). ; wild type genes, ; mutant genes, ; deletion YK34 Anaerobic Growth aroP pdhR aceE aceF lpdA + YK98 YK93 YK62 + Wild type + Aerobic Growth + SE2378 + + -* + YK2

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74 Table 3-2. Growth characte ristics of ethanologenic E. coli strain SE2378 with a mutation in aceF and its transductants. Specific growth rate (h-1) Aerobic Anaerobic Strain Relevant genotype LB Minimal LB Minimal W3110 wild type 1.31 1.05 0.98 0.51 AH242 ldhA ( focA-pflB ), KmR 1.21 0.97 SE2378 AH242, anaerobic+ 1.18 0.51 0.46 NG* YK153 W3110, aceF 0.46 NG (0.55) 1.07 0.44 YK152 AH242, aceF 0.83 NG (0.54) NG NG YK157 YK152, aceF+(W3110) 1.32 0.96 NG NG YK158 YK152, aceF+(SE2378) 1.17 0.51 0.45 NG* NG no growth. Values in parenthesis represent the specific growth rate in glucose minimal medium with acetate (1 mg ml-1) under aerobic conditions. Growth in the presence of glutamate (100 g/ml).

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75 Figure 3-5. Promoter region and th e transcription start site of pdhR-aceEF-lpd operon of E. coli K-12. start codon for PdhR transcription start (-35) (-10) P pdh PdhR -131 TAAAGTCTAC ATTTGTGCAT AGTTACAACT TTGAAACGTT ATATATGTCA -81 AGTTGTTAAA ATGTGCACAG TTTCATGATT TCAATCAAAA CCTGTATGGA -31 CATAAGGTGA ATACTTTGTT ACTTTAGCGT CA CAGACATG AAATTGGTAA +1 20 GACCAATTGA CTTCGGCAAG TGGCTTAAGA CAGGAACTCA TGGCCTACAG 70 CAAAATCCGC CAACCAAAAC TCTCCGATGT GATTGAGCAG CAACTGGA

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76 W3110 PdhR 1 MAYSKIRQPKL S DVIEQQLEFLILEGTLRPGEKLPPERELAKQFDVSRPS SE2377 PdhR 1 .................................................. SE2378 PdhR 1 ........... P ...................................... SE2382 PdhR 1 .................................................. W3110 PdhR 51 LREAIQRLEAKGLLLRRQGGGTFVQSSLWQSFSDPLVELLSDHPESQYDL SE2377 PdhR 51 .................................................. SE2378 PdhR 51 .................................................. SE2382 PdhR 51 .................................................. W3110 PdhR 101 LETRHALEGIAAYYAAL-RSTDEDKERIRELHHAIELAQQSGDLDAESNA SE2377 PdhR 101 .................-................................ SE2378 PdhR 101 ................. L ................................ SE2382 PdhR 101 .................-................................ W3110 PdhR 150 VLQYQIAVTEAAHNVVLLHLLRCMEPMLAQNVRQNFELLYSRREMLPLVS SE2377 PdhR 150 .................................................. SE2378 PdhR 151 .................................................. SE2382 PdhR 150 .................................................. W3110 PdhR 200 SHRTRIFEAIMAGKPEEAREASHRHLAFIEEILLDRSREESRRERSLRRL SE2377 PdhR 200 .................................................. SE2378 PdhR 201 .................................................. SE2382 PdhR 200 .................................................. W3110 PdhR 250 EQRKN SE2377 PdhR 250 ..... SE2378 PdhR 251 ..... SE2382 PdhR 250 ..... Figure 3-6. Comparison of amino acid sequence of PdhR from wild-type (W3110), and three ethanologenic mutants (SE2378, SE2377 and SE2382).

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77 W3110 1 CAACGAAAGAATTAGTGATTTTTCTGGTAAAAATTATCCAGAAGATGTTG SE2377 1 .................................................. SE2378 1 .................................................. SE2385 1 .................................................. W3110 51 TAAATCAAGCGCATATAAAAGCGCGGCAACTAAACGTAGAACCTGTCTTA SE2377 51 .................................................. SE2378 51 .................................................. SE2385 51 .................................................. W3110 101 TTGAGCTTTCCGGCGA G AGTTCAATGGGACAGGTTCCAGAAAACTCAACG SE2377 101 .................................................. SE2378 101 ................ A ................................. SE2385 101 .................................................. W3110 151 TTATTAGATAGATAAGGAATAACCC ATG TCAGAACGTTTC SE2377 151 ........................................ SE2378 151 ........................................ SE2385 151 ........................................ Figure 3-7. Nucleic acid sequence of intergenic region between pdhR and aceE genes of the ethanologenic mutants and th e wild type. The mutated nu cleotide is underlined and the translation start codon of aceE gene is in bold.

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78 W3110 LPD 1 MSTEIKTQVVVLGAGPAGYSAAFRCADLGLETVIVERYNTLGGVCLNVGC SE2377 LPD 1 .................................................. SE2378 LPD 1 .................................................. SE2382 LPD 1 .................................................. SE2383 LPD 1 .................................................. SE2384 LPD 1 .................................................. SE2385 LPD 1 .................................................. W3110 LPD 51 IPSKALLHVAKVIEEAKALAEHGIVFGEPKTDIDKIRTWKEKVINQLTGG SE2377 LPD 51 .................................................. SE2378 LPD 51 .................................................. SE2382 LPD 51 .................................................. SE2383 LPD 51 .................................................. SE2384 LPD 51 .................................................. SE2385 LPD 51 .................................................. W3110 LPD 101 LAGMAKGRKVKVVNGLGKFTGANTLEVEGENGKTVINFDNAIIAAGSRPI SE2377 LPD 101 .................................................. SE2378 LPD 101 .................................................. SE2382 LPD 101 .................................................. SE2383 LPD 101 .................................................. SE2384 LPD 101 .................................................. SE2385 LPD 101 .................................................. W3110 LPD 151 QLPFIPHEDPRIWDSTDALELKEVPERLLVMGGGIIGLEMGTVYHALGSQ SE2377 LPD 151 .................................................. SE2378 LPD 151 .................................................. SE2382 LPD 151 .................................................. SE2383 LPD 151 .................................................. SE2384 LPD 151 .................................................. SE2385 LPD 151 .................................................. W3110 LPD 201 IDVVEMFDQVIPAADKDIVKVFTKRISKKFNLMLETKVTAVEAKEDGIYV SE2377 LPD 201 .................................................. SE2378 LPD 201 .................................................. SE2382 LPD 201 .................................................. SE2383 LPD 201 .................................................. SE2384 LPD 201 .................................................. SE2385 LPD 201 .................................................. W3110 LPD 251 TMEGKKAPAEPQRYDAVLVAIGRVPNGKNLDAGKAGVEVDDRGFIRVDKQ SE2377 LPD 251 .................................................. SE2378 LPD 251 .................................................. SE2382 LPD 251 .................................................. SE2383 LPD 251 .................................................. SE2384 LPD 251 .................................................. SE2385 LPD 251 .................................................. Figure 3-8. Comparison of the amino acid sequ ence of LPD among wild-type strain (W3110) and 6 isolates. The mutated nucleotides are in bold charac ter and underlined.

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79 W3110 LPD 301 LRTNVPHIFAIGDIVGQPMLA H KGVHEGHVAAEVIAGKKHYFDPKVIPSI SE2377 LPD 301 ..................... Y ............................ SE2378 LPD 301 .................................................. SE2382 LPD 301 .................................................. SE2383 LPD 301 ..................... Y ............................ SE2384 LPD 301 ..................... Y ............................ SE2385 LPD 301 .................................................. W3110 LPD 351 AYT E PEVAWVGLTEKEAKEKGISYETATFPWAASGRAIASDCADGMTKLI SE2377 LPD 351 .................................................. SE2378 LPD 351 ... K .............................................. SE2382 LPD 351 ... K .............................................. SE2383 LPD 351 .................................................. SE2384 LPD 351 .................................................. SE2385 LPD 351 ... K .............................................. W3110 LPD 401 FDKESHRVIGGAIVGTNGGELLGEIGLAIEMGCDAEDIALTIHAHPTLHE SE2377 LPD 401 .................................................. SE2378 LPD 401 .................................................. SE2382 LPD 401 .................................................. SE2383 LPD 401 .................................................. SE2384 LPD 401 .................................................. SE2385 LPD 401 .................................................. W3110 LPD 451 SVGLAAEVFEGSITDLPNPKAKKK SE2377 LPD 451 ........................ SE2378 LPD 451 ........................ SE2382 LPD 451 ........................ SE2383 LPD 451 ........................ SE2384 LPD 451 ........................ SE2385 LPD 451 ........................ Figure 3-8. Continued.

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80 Table 3-3. Anaerobic growth and fermentation profile of E. coli with different lpd alleles. Anaerobic growth and fermentation were in LB+g lucose in batch fermentations without pH control. See text for other details. 68.0 3.6 0.0 0.8 11.9 YK100 (+p lpd *) YK129 No growth + YK100 (+p lpd+) YK128 No growth YK29 l p dA YK100 No growth ldh pf l, K m S YK29 No growth ldh pf l, K m R AH242 Acetate Formate Lactate Succinate Anaerobic Growth Relevant Genotype Strain Fermentation products (mM) Ethanol

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81 Figure 3-9. Pyruvate metabolic enzymes in st rain SE2378 and their affinity for pyruvate. Pyruvate Acetyl-CoA + Formate Acetyl-CoA+ CO2 PDH D-Lactate LDH PFL K m, 2 mM K m, 7 mM K m, 0.4 mM NADH NAD NADH NAD

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82Table 3-4. Fermentation profile of mutant strains w ith different pyruvate metabolic pathway compositiona. Enzyme(s) Fermentation Products (mM) % of Strain Relevant Genotype present Growth Succinate Lactate Formate Acetate Ethanol ethanol in products W3110 wild type PFL, LDH + 4.2 18.5 2.6 9.3 7.3 22.8 YK142 l pd, lpd* PFL, LDH, PDH* + 3.6 15.8 3.7 8.8 8.7 23.6 AH240 ( focA-pflB ) LDH + 3.9 24.9 5.1 0 YK144 ( focA-pflB ), lpd* LDH, PDH* + 3.8 24.4 4.5 0 AH241 ldhA PFL + 2.2 0.6 4.7 5.5 10.8 56.5 YK146 ldhA lpd* PFL, PDH* + 2.3 1.2 7.2 6.9 49.1 82.5 AH242 ldhA ( focA-pflB ) No Growth SE2378 ldhA ( focA-pflB ), lpd* PDH* + 4.1 0.8 0 4.7 107.3 91.5 a Fermentation was conducted in LB+glucose in batch fermentations. Pyruvate dehydrogenase with the mutated LPD (lpd*)

PAGE 83

83 Table 3-5. Pyruvate dehydrogena se mRNA, transcription and pr otein levels in aerobic and anaerobic E. coli wild type, strain W3110 and et hanologenic mutant, strain SE2378. Strain Relative mRNA levela -galactosidase Activityb PDH Activityc + O2 O2 + O2 O2 + O2 O2 W3110 1.00 0.98 600 630 370 185 SE2378 0.71 0.77 570 680 240 200 a Relative mRNA levels were determined by quantitative RT-PCR and the level of aceE mRNA in cells grown under aerobic conditions was taken as 1.0 and the relative level of aceE mRNA for the other growth conditions and strain SE2378 was determined. b -galactosidase activity of pdhR-lacZ fusion is presented as nmoles min-1 (mg protein)-1. c PDH activity represents the pyruvate deca rboxylase (E1) activity of the PDH complex nmoles ferricyanide reduced min-1 (mg protein)-1. See Methods section for details.

PAGE 84

84 (A) (B) Figure 3-10. SDS-Polyacrylamide gel electroph oresis of purified dihydrolipoamide dehydroge nase (LPD) from wild-type, A) W3110 and B) the ethanol ogenic strain SE2378. Lane 1, Molecular weight markers in KDa, lane 2, cells; lane 3, crude extract; lane 4, after affinity chromatography (Ni2+ column); lane 5, after Thrombin treatment; lane 6, af ter gel filtration. 1 2 3 4 6 5 200 45 31 1 2 3 4 6 5 200 116 97.4 66.2 45 31 21.5 116 97.4 66.2 21.5 LPD LPD

PAGE 85

85 Figure 3-11. Linearity of LPD protein concentration vs. activity of the enzyme in the forward reaction. Enzyme Activity ( mole NADH.min-1) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 20406080Protein (ng) LPD (W3110) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 050100 150200 250Protein (ng) Enzyme Activity ( mole NADH.min-1) LPD (SE2378) 100

PAGE 86

86 Figure 3-12. Native LPD (W3110) activity with various NAD+ concentrations. 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0 0.5 1.0 1.5 2.0 2.5 3.0 [NAD+] (mM) Enzyme Activity mole NADH.min-1) R2 = 0.9989 0 10 20 30 40 50 60 -5 -3 -1 1357911 1/[NAD+] 1/ v K m, 0.4 mM

PAGE 87

87 Figure 3-13. Mutated LPD (SE 2378) activity with various NAD+ concentrations. 0.00 0.01 0.02 0.03 0.04 0.05 0 1.02.03.0 [NAD+] (mM) Enzyme Activity ( mole NADH.min-1) R2 = 0.9961 0 10 20 30 40 50 60 70 80 -4 -2 0246 1/[NAD+] 1/v K m, 0.4 mM

PAGE 88

88Table 3-6. Kinetic constants of the nativ e (W3110) and the mutated (SE2378) LPD K m K i K cat K cat /K m NAD+ (mM) NADH (mM) S-1 M-1S-1 Native LPD 0.4 5.2 1.7 x 103 4.2 x 106 Mutated LPD 0.4 not determined 4.3 x 102 1.1 x 106

PAGE 89

89 Figure 3-14. Inhibition of native LPD (W3110) forward activity by NADH. Relative activity was calculated from specific activity, moles NADH min-1 (mg protein)-1. 0 20 40 60 80 100 120 0 1 2 3 4 5 6[NAD+] (mM)Relative Activity w/ NADH no NADH 0.02 mM 0.1 mM 0.14 mM 0.16 mM

PAGE 90

90 Figure 3-15. Inhibition of LP D activity by NADH (2.0 mM NAD+) on forward reaction. 0 20 40 60 80 100 0.00 0.020.040.060.080.10 0.120.140.16 [NADH] (mM)% Specific Activity native LPD mutated LPD

PAGE 91

91 Figure 3-16. Inhibition of mu tated LPD (SE2378) by NADH. Relative activity was calculated from specific activity, moles NADH min-1 (mg protein)-1. no NADH 0.02 mM 0.1 mM 0.14 mM w/ NADH 120 100 8 6 4 2Relative Activity 0 012 3456[NAD+] (mM)

PAGE 92

92 Figure 3-17. Activation of LPD re verse reaction by increasing NAD+/NADH ratio. Top panel, NAD+/NADH; Bottom panel, NADH/NAD+. NADH (0.1 mM) and various concentrations of NAD+ were used in the determination of LPD activity by the reverse reaction. Specific activity, moles NADH min-1 (mg protein)-1. 0 20 40 60 80 0 123 [NAD+]/[NADH] Specific Activit y Native Mutated [NADH]-0.1 mM 0 20 40 60 80 100 0 1 23456 [NADH]/[NAD+] Specific Activit y N ative LPD Mutated LPD [NADH]-0.1 mM

PAGE 93

93Table 3-7. Purification of native PDH complex from E. coli strain W3110. Volume Protein Conc. Total protei n Specific yield purification Purification step (ml) (mg/ml) (mg) activitya Total activityb (%) fold Crude extract 25 15.41 385.3 1.6 628.0 100 1 Ultra centrifugation 6 13.68 82.1 3.8 309.4 49 2.3 Hydroxyapatite 15 0.91 13.7 6.0 81.2 13 3.7 Gel filtration 20 0.14 2.8 8.9 24.8 4 5.4 a mole NADH min-1mg protein-1 b mole NADH min-1

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94Table 3-8. Purification of the muta ted PDH complex from strain YK176. Volume Protein Conc. Total Protei n Specific yield Purification Purification step (ml) (mg/ml) (mg) Activitya Total Activityb (%) fold Crude extract 20 27.4 548.0 1.2 657.6 100 1 Ultra centrifugation 5 22.6 112.9 4.6 523.9 80 3.9 Hydroxyapatite 10 1.45 14.5 7.6 109.5 17 6.3 Gel filtration 25 0.29 7.3 9.4 68.2 10 7.8 a mole NADH min-1mg protein-1 b mole NADH min-1

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95 1 2 3 4 5 6 7 8 9 Figure 3-18. SDS-PAGE of partia lly purified PDH complex. 1,6-crude extract; 2,7-after ultracentrifugation; 3, 8-after hydroxyapatite chromatography; 4, 9-after gelfiltration; 5-molecular weight marker. sizes, top to bottom: 200, 116, 97.4, 66.2, 45, 31, 21.5 (KDa). Lane 1-4, Native enzyme, Lane 6-9, Mutated form of the enzyme. Arrows indicate the components of the PDH complex from top to bottom, E1 (AceE), E2 (AceF), and E3 (LPD).

PAGE 96

96 Figure 3-19. Native PDH (W3110) activity with various NAD+ concentrations. Enzyme activity is measured with 1.4 g protein.ml-1. 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0 0.5 1.0 1.5 2.0 2.5 [NAD+] (mM) R 2 = 0.997 0 50 100 150 200 250 300 01020304050 1/[NAD+] 1 / v K m, 0.1 mM Enzyme Activity ( mole NADH.min-1 ) 3.0

PAGE 97

97 Figure 3-20. Native PDH (W3110) activity wi th various pyruvate concentrations. Enzyme activity is measured with 1.4 g protein.ml-1. Enzyme Activity ( mole NADH.min-1 ) 0.000 0.003 0.006 0.009 0.012 0.015 0 1 2 3 4 5 6 [Pyruvate] (mM) R2 = 0.9969 0 50 10 15 20 25 30 0246 1/[Pyruvate 1/ K m, 0.4 mM

PAGE 98

98 Figure 3-21. Mutated PDH (SE 2378) activity with various NAD+ concentrations. Enzyme activity is measured with 2.9 g protein.ml-1. 0.000 0.005 0.010 0.015 0.020 0.025 0.0 0.5 1.01.5 2.02.5 3.0 [NAD+] (mM) R 2 = 0.9993 0 50 10 150 20 25 515253545 1/[NAD+] 1/ K m, 0.1 mM Enzyme Activity ( mole NADH.min-1 )

PAGE 99

99 Figure 3-22. Mutated PDH (SE2378) activity with various pyruvate concentrations. Enzyme activity is measured with 2.9 g protein.ml-1. Enzyme Activity ( mole NADH.min-1 ) 0.000 0.005 0.010 0.015 0.020 0.025 0 1 2 3 4 5 6 [Pyruvate] (mM) R2 = 0.9993 0 50 100 150 200 250 300 -0510 1 20 25 1/[Pyruvate 1/ K m, 0.3 mM

PAGE 100

100Table 3-9. Kinetics constants of the nativ e (W3110) and the mutated (SE2378) PDH. K m K ca t K cat /K m K i NAD+ (mM) Pyruvate (mM) S-1 M-1S-1 NADH ( M) Native PDH 0.1 0.4 4.5 x 102 4.6 x 106 1.0 Mutated PDH 0.1 0.3 3.0 x 102 3.1 x 106 10.0

PAGE 101

101 Figure 3-23. Inhibition of native PDH (W310) activity by NADH. Specific acitivity, mole NADH min-1 mg Protein-1. 0 1 2 3 4 5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 [NAD+] (mM) no NADH 0.04 mM 0.05 mM 0.06 mM native PDH w/ NADH Specific Activity

PAGE 102

102 Figure 3-24. Inhibition of mu tated PDH (SE2378) by NADH. Specific activity, mole NADH min-1 mg Protein-1 0 1 2 3 4 5 0 0.5 1 1.5 2 2.5 [NAD+] (mM) 0.05 mM 0.06mM w/ NADH mutated PDH no NADH 3 Specific Activity

PAGE 103

103 Figure 3-25. Inhibition of PDH complex by NADH at a fixed NAD+ concentration of 1.0 mM. 0 20 40 60 80 100 120 0.00 0.02 0.040.060.080.10 0.12 [NADH] (mM)% Inhibitio n mutated PDH native PDH

PAGE 104

104 Figure 3-26. Growth and fermentation characteristics of wild type strain W3110 in LB + glucose (50 g L-1) at pH 7.0 and 37 oC. W3110-Glucose 0 24 48 72 96 0 100 200 300 400 500 600 Growth Ethanol Glucose Lactate Acetate 0 2 4 6 8 10 12Time (hrs) Glucose / Products (mM) O.D. (420nm)

PAGE 105

105 Figure 3-27. Growth and fermentation characteri stics of ethanologenic strain SE 2378 in LB+ glucose (50 g L-1) at pH 7.0 and 37 oC. SE2378 -Glucose 0 24 48 72 96 0 100 200 300 400 500 600 Growth Ethanol Glucose Lactate Acetate 0 2 4 6 8 10 12Time (hrs) Glucose / Products (mM) O.D. (420nm)

PAGE 106

106Table 3-10. Glucose fermenta tion characteristics of E. coli strain SE2378 and wild type strain W3110. Glucose Product Concentration (mM) Ethanol Strain consumed (mM) h Succinate Lactate Formate Acetate Ethanol Yield W3110 298.4 19.0 0.44 18.4 0.7 206.1 10.5 205.6 11.4 162.4 6.4 142.4 6.6 0.24 0.01 SE2378 295.7 4.0 0.46 26.6 2.3 12.7 2.1 0.0 27.4 2.3 478.4 15.4 0.81 0.02

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107 Table 3-11. Growth and ethanol production by E. coli strain SE2378 grown on glucose or xylosea. W3110 SE2378 Glucose Xylose Glucose Xylose Max 0.44 0.37 0.46 0.38 YX/S 0.04 0.04 0.04 0.04 QS 2.94 1.58 1.29 1.65 QP 0.50 0.36 0.61 0.53 YP/S 0.12 0.18 0.41 0.42 qS 4.10 4.93 3.26 5.33 qP 0.49 0.89 1.34 2.24 Max specific growth rate, h-1 YX/S g cells (g substrate)-1 QS g sugar consumed L-1 h-1 QP g ethanol L-1 h-1 YP/S g ethanol (g substrate)-1 qS g sugar consumed (g cells-dry weight)-1 h-1 qP g ethanol (g cells-dry weight)-1 h-1 aGlucose and xylose fermentations by strains W3110 and SE2378 in LB broth are presented in Tables 3-10 and 11 and Figur es 3-26, 27, 28 and 29. The repor ted values are calculated maximum values.

PAGE 108

108 Figure 3-28. Growth and fermentation characte ristics of wild type strain W3110 in LB+ xylose (50 g L-1) at pH 7.0 and 37 oC. W3110-Xylose 0 24 48 72 96 0 100 200 300 400 500 Growth E t h a n o l Xylose Lactate Acetate 0 2 4 6 8 10 12Time (hrs) Xylose / Products (mM) O.D. (420nm)

PAGE 109

109 Figure 3-29. Growth and fermentation characteri stics of ethanologenic strain SE2378 in LB+ xylose (50 g L-1) at pH 7.0 and 37 oC. SE2378-Xylose 0 24 48 72 96 0 100 200 300 400 500 Growth Ethanol X y lose Lactate Acetate 0 2 4 6 8 10 12Time (hrs) Xylose / Products (mM) O.D. (420nm)

PAGE 110

110Table 3-12. Xylose fermentation characteristics of E. coli strain SE2378 and wild type strain W3110. Xylose Product Concentration (mM) Ethanol Strain consumed (mM) h-1 Succinate Lactate Formate Acetate Ethanol Yield W3110 332.5 8.3 0.37 57.2 1.0 32.4 2.5 248.3 52.5 214.7 10.0 190.8 7.4 0.34 0.00 SE2378 324.7 1.5 0.38 32.9 5.4 0.0 0.0 24.9 2.3 444.0 9.17 0.82 0.01

PAGE 111

111 Figure 3-30. Fermentation characte ristics of kanamycin-sensitiv e derivative of ethanologenic strain SE2378,YK1, and its mgsA derivative strain YK96 in LB+glucose (50 g L-1) at pH 7.0, 37 oC. 0 100 200 300 400 500 600 024 48 72 96Time (hrs) Glucose / Ethanol ( mM ) Ethanol -YK96 Ethanol -YK1 Glucose -YK96 Glucose -YK96

PAGE 112

112Table 3-13. Fermentation characteri stics of kanamycin-sensitive derivative of ethanologeni c strain SE2378, YK1, and its mgsA derivative strainYK96 in LB+glucose (50 g L-1) at pH 7.0 and 37 oC. Glucose Product Concentration (mM) Ethanol Carbon Strain consumed (mM) h-1) Succinate Lactate Formate Acetate Ethanol Yield Recovery YK1 263.8 0.45 35.2 12.7 0 42.4 390.2 0.79 0.91 YK96 270.3 0.46 12.5 0 0 10.5 476.1 0.88 0.92

PAGE 113

113 Figure 3-31. Ethanologenic fermentation path way. (A) Mixed acid fermentation and (B) et hanologenic fermentation pathway of stra in SE2378. 2 NAD+2 NADH 2 NADH 2 NAD+ 2 Gly-3-P 2 3-PGA 2 Pyruvate 2 Acetyl-CoA 2 Acetaldehyde 2 Ethanol Glucose PDH* ADH ADH 2 NADH 2 NAD+ 2 NADH 2 NAD+ 2 NAD+2 NADH 2 Gly-3-P 2 3-PGA 2 Pyruvate 2 Acetyl-CoA Acetaldehyde Ethanol Glucose ADH ADH NADH NAD+ NADH NAD+ 2 NAD 2 NADH 2 Lactate LDH 2Formate 2H2+2CO2PFL Acetylphosphate Acetate PTA ACK (A) (B)

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127 BIOGRAPHICAL SKETCH Youngnyun Kim was born in Seoul, Korea, in 1971. He attended Shin-sa Middle school and Kujung High School. He received his bach elors degree and masters degree in biotechnology from Ajou University He worked in Dr. Ryus labor atory for his masters degree. After he received his masters degree, he work ed in the environmental engineering field in Hyosung Co. before he came to the USA. He was accepted to the Ph.D program in the in the Department of Biological Engineering at Utah State University. He transferred to th e Department of Microbiology and Cell Science at the University of Florida in 2002, to work in Dr. Shanmugams laboratory. He was employed by Amyris Biotechnology before his graduation.