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Improving the performance of Escherichia coli KO11 during the fermentation of xylose to ethanol

University of Florida Institutional Repository

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IMPROVING THE PERFORM ANCE OF Escheric hia coli KO11 DURING THE FERMENTATION OF XYLOSE TO ETHANOL By STUART A. UNDERWOOD A DISSERTATI ON PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVE RSITY OF FL ORIDA I N PARTIAL FULFI LL MENT OF THE REQUIREMENTS FO R THE DEGREE OF DOCTOR OF PHIL OSOPHY UNIVERSI TY OF FLORI DA 2003

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Copyright 2003 by Stuart A. Underwood

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This work is dedica ted to my wife, B everly and my family The y ears of their en dless love and support made this work possible.

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iv ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Lonnie O. I ngram, for his insight and guidance in my academic development. His inspiration and gener al enthusiasm for science will have lasting effec ts on the development of my ca reer. My d eepest a pprecia tion is exten ded to the other me mbers of my g raduate committee: Dr. K. T. Shanmugam, Dr. Julie A. Maupin-Furlow, Dr. Greg ory W. L uli and Dr. Jon D. Stewart. Without their guidance and sagacity this work would not have been possible. I am gra teful to Lorraine Yama no, Dr. Shengde Zhou and Dr. F ernando Martinez-Morales for their help in learning the intrica cies of molecular biology Many thanks to Sean York and Alfredo Martinez for their help in learning our fermentation process es. I would also like to tha nk Dr. Ma rian L Buszko f or his gu idance w ith NMR experimen ts. My warmest thanks to the other members of Dr. Ing ram’s laboratory and the biomass re search group f or the ma ny i nsightf ul discuss ions about scientif ic matter s. The text and figures in Chapter 2 and Chapter 3, in part or in full, are reprints of the material as it appears in Applied and Environmental Microbiology (vol. 68, pp. 1071-1081 and pp. 6263-6272, respectively ).

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v TABLE OF CONT ENTS page ACKNOWLEDGMENTS ................................................ iv LI ST OF TABLES ..................................................... viii LI ST OF FIGURES ..................................................... ix ABSTRACT ........................................................... xi CHAPTER 1 INTRODUCTI ON ................................................. 1 Lignocellulose as a Ca rbohydrate Source ............................... 4 Adaptati on to Hig h Sugar Environme nts ................................ 5 Xy lose ver sus Gluco se Metab olism .................................... 8 Pyruvate Dissimilation ............................................. 10 Engine ering E. coli for Ethanol Production ............................. 15 Deleterious Effects of Metabolic Engine ering ........................... 16 Project G oals .................................................... 18 2 FLUX THROUGH CI TRATE SYNTHASE LI MITS THE GROWTH OF ETHANOL OGENI C Escheric hia coli KO11 DURI NG XYL OSE FERMENTATION ................................................ 26 Introduction ..................................................... 26 Materials and Methods ............................................. 27 Microor ganisms and Media ...................................... 27 Fermentation Conditions ........................................ 28 Aerobic Growth Studies ......................................... 29 Analytical Methods ............................................ 29 Genetic Methods .............................................. 30 NAD(P)H /NAD(P) + ratio ........................................ 31 En zym e A ss ays ............................................... 31 Results ......................................................... 32 Macro-nutrient Limitation. ...................................... 32

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vi Energy Limitation. ............................................. 33 Metabolic Imbalance Relieve d by Addition of Py ruvate or Acetaldehy de. .. 33 Pyr uvate as a Source of Carbo n Skeleton s for Bio syn thesis. ............. 35 Whole-cell Fluorescence. ........................................ 37 Citrate Synthase, a L ink Between NADH and 2-Ketog lutarate. .......... 39 Discussion ...................................................... 40 3 GEN ETIC CHA NGE S T O OP TIMIZ E C ARB ON P ART ITION ING IN ETHANOL OGENI C Escheric hia coli KO11 ........................... 53 Introduction ..................................................... 53 Materials and Methods ............................................. 54 Microor ganisms and Media ...................................... 54 Fermentation ................................................. 54 Analytical Methods ............................................ 54 Genetic Methods .............................................. 55 Construction of pLOI 2065 Containing a Removable Tetracy cline Resistance Cassette .................................................. 56 Nucleotide Sequence Accession Number ........................... 56 Construction of SU102 Containing an Insertion Mutation in ackA ........ 56 Construction of SU104 Containing a Deletion in adhE ................. 57 Results and Discussion ............................................ 58 Acetate Addition Stimulates Growth and Ethanol Production by Reducing Net Acetate Productio n During Sugar M etabolism .................... 58 Stimul ation of Gro wth and Eth anol Produ ction by Added Pyruvate Can Be Primarily Attributed to I ncreased Acetate Production. .............. 59 Stimul ation of Gro wth and Eth anol Produ ction by Acetaldehyde Can Be Attributed to Increased A cetyl-CoA. ............................ 62 Stimulation of Growth and Ethanol Production by I nactivation of Nonbiosynthetic Pathway s Which Consume Acetyl-CoA. ............... 64 Conclusions ..................................................... 66 4 A DEFICI T IN PROTECTI VE OSMOLYTES I S RESPONSIBLE F OR THE DECREASED GROWTH AN D ETHANO L PROD UCTI ON DURI NG XYL OSE FERMENTATION ............................................... 77 Introduction ..................................................... 77 Materials and Methods ............................................. 79 Microorganisms and Media. ..................................... 79 Fermentation. ................................................. 79 13C NMR. ................................................... 80 Analy tical Met hods. ............................................ 81 Results and Discussion ............................................ 81

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vii Citrate Synthase Flux L imits the Biosynthesis of Glutamate, a Primary Intracellular Osmoly te. ....................................... 81 Genetic Change s to Optimize Carbon Pa rtitioning Inc reased the Gluta mate Pool. ..................................................... 84 Glutamate Accumulation Functions in Osmoprotection. ................ 85 Replace ment of Gl utamate b y O ther Osmo protect ants. ................. 86 Betaine from Dif co Yeas t Extract Re stores Gr owth in L uria Br oth Fermen tations. ............................................. 87 Conclusions ..................................................... 88 5 GENERAL DISCU SSION AND CONCL USIO NS ...................... 96 Increased A cetyl-CoA Ava ilability Stimulated Growth. ................... 96 Some TCA Intermediates I ncrease Growth. ............................ 98 Citrate Sy nthase–A Unify ing Hy pothesis. .............................. 99 Future Prospects for Metabolic Enginee ring ........................... 104 REFERENCES ....................................................... 106 BIO GRAPHI CAL SK ETCH ............................................. 119

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viii LI ST OF TABLES Table page 2-1. Eff ects of a dditives o n the comp osition of f ermenta tion produ cts ............. 43 2-2. Effects of additives on growth and etha nol production by KO11 .............. 44 3-1. Strains and plasmids used in Chapter 3 .................................. 68 3-2. Eff ects of mu tations an d additive s on cell y ield and e thanol pr oductivity ........ 69 4-1. Intracellular a ccumulation of protective osmolyte s by KO11 ................. 90

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ix LI ST OF FIGURES Figure page 1-1. Gluc ose tran sport by the phosph otransf erase s yst em. ....................... 20 1-2. Gly coly sis. ........................................................ 21 1-3. Xylose transpor t in E. coli ............................................ 22 1-4. Xy lose meta bolism .................................................. 23 1-5. Reactions of the py ruvate dehy drogenase complex ......................... 24 1-6. Fermentation pathway s of E. coli ....................................... 25 2-1. Compa rison of ma ximal cell d ensities ................................... 45 2-2. Comparison of growth and ethanol production from g lucose and xylose ........ 46 2-3. Effects of added py ruvate and acetaldehy de .............................. 47 2-4. I nitial ef fects of added TCA pathway intermed iates ........................ 48 2-5. Effect of metabolites on whole-cell fluoresc ence ........................... 49 2-6. B. subtilis c itZ increases the growth and ethanol produc tion .................. 50 2-7. Relationship between cell y ield and fermentation performance ................ 51 2-8. Fermentation and TCA pathway ........................................ 52 3-1. Allos teric co ntrol of c entral me tabolism ................................. 70 3-2. Plasm ids used to construc t mutations ................................... 71 3-3. Eff ect of me dia addit ions and mu tations ................................. 72 3-4. Effect of media additions and mutations on organic a cid production ........... 73

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x 3-5. Metabolism of added acetaldehy de and pyruva te during fermentation .......... 75 3-6. Part itioning of carb on among competin g pathw ay s ......................... 76 41. Ca rb on fl ow du ri ng fe rm en ta ti on of xyl os e b y E. coli KO11. ................. 91 4-2. Major intracellular osmoly tes accumulated by ethanologenic E. coli during fermentation .................................................... 92 4-3. Effect of osmoprotectants (1.0 mM) on maximum cell concentration ........... 93 4-4. Effects of Betaine and DMSP on gr owth and ethanol production .............. 94 4-5. The chemica l structur e of beta ine and D MSP ............................. 95

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xi Abstract of Dissertation Presented to the Graduate School of the University of F lorida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPROVING THE PERFORM ANCE OF Escheric hia coli KO11 DURING THE FERMENTATION OF XYLOSE TO ETHANOL By Stuart A. Underwood August 2003 Chair: Lonnie O. I ngram Co-Chair: Keelnatham T. Shanmugam Major Department: Microbiology and Cell Science The large-scale conve rsion of lignocellulose to fuel ethanol would grea tly reduce the U.S. dependence on imported oil. To facilitate this need, Escheric hia coli has been geneti cally engine ered fo r the homo fermen tative pr oduction o f ethano l from all constituent sugars of lignocellulose. However high levels of complex nutrients are require d for ra pid ferme ntation of xylos e, the se cond most a bundant su gar in lignocellulose. With low levels of complex nutrients, the rate of xylose fe rmentation was limited by the growth of the biocatalyst. I n a mineral salts medium containing 1% corn steep liquor as a nutrient source (90 g liter -1 xylose), growth wa s limited by an imbalance in the par titioning of carb on betwe en ethan ol produc tion and bi osy nthetic p athway s. Citrate synthase wa s shown to catalyze the spec ific growth-limiting reaction. The allosteric controls of citrate sy nthase regulate carbon flow throug h the oxidizing arm of the TCA pa thway ultimate ly p roducing 2-ketog lutarate and glu tamate. F unctiona lly

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xii ex pre ss in g ci tra te s ynth ase II ( citZ ) from Bacillus s ubtilis stimulate d growt h due to its different allosteric and kinetic properties. Ace tyl-CoA server a s an antagonist to the NADH-mediated allosteric inhibition of the E. coli citrate synthase Supplementing the medium with pyruvate, ac etate, acetaldehy de, 2-ketoglutarate or glutama te increased growth and etha nol produc tion by activati ng, re lieving or by passing the allost eric regulation of the E. coli citrate syn thase. Co nservat ion of ac ety l-CoA by mutating acetat e kinase ( ) ackA ) also increased growth and etha nol production, presumably by increasing the avai lability of acet ylCoA (act ivating citrate syn thase). In a ddition to b iosy nthetic needs, large intracellular pools of g lutamate (>20 mM) function as a protective osmoly te. During growth in the high osmotic environment of the c orn steep liquor medium containing 0.6 M xylose, intrace llular glutamate was low (< 10 mM) and cells g rew poorly, consistent with a g lutamate deficiency The addition of glutamate to the medium and all approaches that stimulated citrate sy nthase increased the high intrace llular pool of glutama te during growth in this medi um. Supplem enting w ith other p rotectiv e osmoly tes, such as betaine and dimethy lsulfoniopropionate, restored growth without affecting the intrace llular poo l of glut amate a nd appea r to act d irectly as alter native os moly tes. Thes e results indicate that the poor growth and ethanol production in 1% cor n steep liquor medium (0. 6 M xyl ose), the appare nt requir ement fo r high le vels of nu trients w ithout a specific auxotrophic requirement and the beneficial e ffects of increased intracellular glutamate all result from the requirement for hig h levels of protective osmolyte s. Under these co nditions, th e grow th of the b iocataly st ( E. coli ) and ethanol production are limited by insufficient levels of intra cellular osmoprotectants rather than the sy nthesis of glutamate, per se.

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1 CHAPTER 1 INTRODUCTI ON The prod uction of fuel eth anol fro m renewa ble fee dstocks c ould poten tially decrease the U.S. dependance on imported oil as well as decrease the relea se of fossilized carbon i nto the at mosphere as carb on dioxide ( CO 2 ), a greenhouse ga s. Blends of 95% ethanol with gasoline are effec tive motor fuels, as demonstrated by Brazil’s use of such blends for more than 20 y ears prior to securing inexpensive sources of f ossil fuels. In the year 2002, a pproximately 140 billion gallons of gasoline w ere consumed in the United States, most of which was derived from foreign oil. Approximately 2.9 billion gallons of ethanol are produced annually in the U.S., slightly more than 2% of the gasoline consumed. While the volume of ethanol produced increases ea ch yea r, demands for ethanol a nd energ y a lso incre ase. Fo r example, the phasi ng out of the gas oline oxy genate methyl tertiary -butyl ether (MTB E) over the next several y ears will further increase the demand for fuel ethanol, an alternate oxy genate. A substantial increase in ethanol producti on must be a chieved to repla ce MTBE with 10% e thanol. Today, most of the ethanol de rived from fermentation uses cornstarch as the feedsto ck with y east as th e biocat aly st. Competi ng dema nds for c ornstar ch and va riable crop yields ca use price volatility. Fe edstock is the major contributor to the cost of current ethanol processes. The cost of ethanol production must remain low in order for it to be an economically c ompetitive automobile fuel. The necessity for a less expensive, lower demand feedstock is obvious. Agricultural wastes (c orn stover, sugarcane bag asse, wheat

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2 straw, e tc.) ar e relati vely inexpensiv e source s of car bohy drates th at can be convert ed to ethanol (Arntzen and Dale 1999; Ing ram and Doran 1995; Ing ram et al. 1999; Zaldivar et al. 2002) More tha n 200 billio n gallon s of etha nol could b e produc ed using these lignoce llulosic ma terials, sufficie nt to repla ce all of the gas oline bur ned by automobile s in the United States (Arntzen and Dale 1999). As these ag ricultural wastes have little or no competing uses, they offer long-term solutions to the necessity for inexpensive carbohy drate so urces. However, there is no known organism in nature capable of fermenting all of the various hexose and pentose components of biomass to ethanol. This difficulty is further compounded by the c omplex, polymeric and somewhat varia ble structure of the lignocellulosic biopolymers ( Clarke 1997). Harsh treatments are requir ed to breakdown these sug ar poly mers into s uitable su bstrates for fer mentation During these pr ocesses furfural, hy droxymethy lfurfural, acetate, and many other cytotoxic by products are released into the resulting solutions. An organism must tolerate the environme ntal condition s creat ed by these tre atments to be an ef fective biocata lys t. With adva nces in molecular biology genetically engineering a desira ble microorganism to produce ethanol should be possible. There are essentially two approaches to engineering an organism for the production of ethanol from lignocellulosic residues. Either an e thanol producing microorg anism cou ld be eng ineere d to use al l of the va rious sug ars or a microorg anism already capable of fermenting all of these sugars could be engineer ed to produce exclusively ethanol. The forme r approach has been pursued by many g roups through the engineering of Saccharomyces cerevisiae or Zymomona s mobilis (defic ient in pe ntose

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3 metabolism) to utilize these carbohydrate s by expressing hete rologous transport and metabolic pathway s (Aristidou and Penttila 2000; Chotani et al. 2000; Gong et al. 1999). While hig h produc tivities ha ve been reporte d for both organ isms in optima l condition s, yeasts ca pable of fermenting both xy lose and arabinose have not been reporte d in the literature. Z. mobilis a very f astidious organism, is not environmentally ha rdy, and the harsh co nditions re sulting f rom the pr etreatm ent of the lignoce llulose se verely hinders i ts pr od uc ti vi ty. One of the most studied and characterized org anisms, Escheric hia coli is an excellent candidate for genetic eng ineering. The complete ge netic sequence has been published (Blattn er et al. 1997), a nd much is k nown abou t its phy siology (Neidha rdt et al. 1990). The utility of this orga nism in industrial processes is second only to y east. Typica l of enteric organsism, E. coli is capable of fermenting both the pentoses and he xoses present in lignocellulose. However, E. coli is a mixed acid fermenter, producing lactate, acetate, ethanol, formate and succina te as its major fermentation products. Previous work in our laboratory e ngineered the metabolism of E. coli to produce exclusively ethanol (Ohta et al. 1991). The sugars of hemicellulose hy drolysates, containing mostly xy lose, were fermented by the engineered E. coli strain, with yields approac hing 100% (0.51 g ethanol / g sugar) (Asgha ri et al. 1996; Lawford and Rousea u 1996; Martinez et al. 1999; York and I ngram 1 996a; Yor k and I ngram 1 996b). Ho wever c omplex, expe nsive nutr ients (Lur ia broth) are re quired to obtain the se high y ields. Hig h levels o f inexpens ive nutrie nts are required to replace these rich nutrients (Asghari et al. 1996; L awford and Rouseau 1996; Mar tinez et al 1999; Yor k and I ngram 1 996a; Yor k and I ngram 1 996b), bu t this

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4 creates waste manage ment problems and increases cost. Fermentations which use low levels of complex nutrients or no nutritional supplements would be most desirable for industria l fermen tations. Corn steep liquor (CSL), a by -product from the wet milling of corn, is an inexpensiv e nutrie nt source with demon strated u tility in industri al proce sses. Fermentations of hemicellulose hy drolysate with CSL as the nutrient source exhibited dose-dependent change in etha nol productivities (Martinez et al. 1999). To equal the ethanol productivity achie ved with Difco nutrients (5 g liter -1 yeast extract and 10 g liter -1 tryptone), 50 g liter -1 CSL (w et weig ht; 50% soli ds) were require d. The g oal of this present study is to understand the ba sis of the need for complex nutrients and develop phy siologic al and g enetic s olutions to c ircumve nt this req uiremen t. Lignocellulose as a Ca rbohydrate Source Most of the dry weig ht biomass is lignocellulose, composed of cellulose, hemicellulose, pectin and lignin (Clarke 1997). Cellulose, the most abundant poly mer on the planet, is a homopolymer of c ellobiose ( $ -1,4-glucose) and repre sents 20-50% of the dry weig ht of plant matter. Lignin is a poly mer of aromatic alcohols, comprising 10-20% of the dr y w eight of plant bioma ss. Repre senting only 1-10% of the dry weight pectin i s a methylated homopoly mer of galacturonic acid. He micellulose is a complex, branched polymer of hexoses (g lucose, galactose, mannose, rhamnose, a nd fucose) and pentoses (xylose and arabinose) This polymer repre sents 20-40% of the plant dry weight and is the most easily solubilized component of lignocellulose. The sugars of hemicellulose are r eleased as monomers through a varie ty of hydroly sis procedures, but dilute acid hy drolysis is currently the preferred method

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5 (Ingra m et al. 1999). This procedure uses moderate hea t and low pH to release the sugars of hemicellulose into solution as monomers (Grohmann et al. 1985). The exact ratio of sugars in these h ydr oly sates ca n vary consider ably dependin g on the feedsto ck, but xy lose is the most prevalent sugar in hy drolysates of ha rd woods and grasses (suga rcane, wheat straw, e tc.). Ge neratin g a con centra ted suga r solution d uring hy droly sis is a for midable challenge, but a goal of 100 g liter -1 total sugar monomers in hemicellulose hy drolysates should be achievable. Most of the studies presented here ha ve used 90 g liter -1 xylose as the fermentation substrate. Adaptati on to Hig h Sugar Environme nts Many industrial ferme ntation processes operate as either batch f ermentations (with all required nutrients and substrates supplied initially ) or fed-batch fermentations (multiple additions of nutrie nts; requ ires con centra ted fee d solutions ). As the h ydr oly sis of hemicellulose produces sugar strea ms up to 100 g liter -1 (Ing ram et al 1999), th eir fermentation to ethanol favors a batch ferme ntation process to avoid the additional cost of concentrating these sugar streams and potentially conc entrating growth inhibitory compound s. Howev er, this r elativel y hi gh sug ar conc entratio n requir es E. coli to adapt t o th is hi gh er os mo la ri ty. The rapid accumulation of potassium is the first response of E. coli and related organisms to an increase in the osmotic strength of the medium. Within a minute after an increa se in osmoti c pressu re, glu tamate ( a nega tively -charg ed amino a cid) sy nthesis is increa sed to pro vide cha rge ba lance f or the ac cumulate d potassiu m (McL agga n et al. 1994). The short time between the accumulation of potassium and the biosy nthesis of glutama te, sugg ests that t he onset o f gluta mate biosy nthesis is a result of alloster ic

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6 regulation (<5 min) rather than g enetic induction (10-20 min). Additionally, the accumulation of glutamate in response to osmotic stress was found to be de pendent on the presence of K + in the medium (McLagg an et al. 1994). Escheric hia coli has two biosynthetic pathway s for glutamate. Under a nitroge n limitation (0.1 mM ammonium), glutamate synthase-g lutamine synthetase has be en shown to be the predominant glutamate biosy nthetic pathway ( Pahel et al. 1978). During growth in excess nitrogen, glutamate de hydrog enase (GDH), a pathway that does not consume ATP, is the primary g lutamate biosynthetic pathway (Helling 1994). Ad di ti on al ly, GD H i s a ct iv at ed by K + (Measures 1975). This allosteric regulation of G DH has been proposed to be res ponsible f or osmotic ally activate d glutam ate biosy nthesis (Helling 1994). The intracellular concentration of K + can be as high as 800 mM in E. coli during growth in media o f high os molarity (Cay ley et al. 199 1; Cay ley et al. 199 2). Cells deficie nt in glut amate a ccumula tion have demonstr ated gr owth def ects dur ing osmot ic challen ge (Cso nka 1988; McLa ggan et al. 199 1; Yan et a l 1996) due to an inab ility to maintain s ufficie nt K + (Yan et al. 1996). The large inc reases in intracellular potassium and glu tamate a re tran sient, an d their le vels beg in to decr ease to 2 0-50 mM as trehalo se or other protective osmoly tes accumulate in the cy toplasm (Dinnbier et al.1988; Giaever et al. 198 8). Howe ver, g lutamate pools rem ain elev ated dur ing gr owth the h igher o smotic conditions (Yan et al. 1996). For the long-term adaptation to media of hig h osmolarity, E. coli synthesizes trehalose (Boos et al. 1990; Dinnbier et al. 1988; Giaever et al. 1988) or accumulates other charge-neutra l (zwitterionic) compatible solutes (betaine, proline ectoine,

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7 dimethylsulfonioproprionate, etc.) (Csonka and Hanson 1991). E. coli has a limited capacity for biosynthesis of these c ompounds. Although E. coli is incapable of de novo betaine biosy nthesis, c holine ca n be oxidized to betain e. Howe ver, this process is rest rict ed to aero bic g rowt h (L andf ald a nd Str m 198 6). So me or gan isms s yn thesi ze proline for long-term osmoadaptation (Kawaha ra et al. 1989). However, the ( -g lu ta myl kinase step in proline biosynthesis is subject to strong f eedback inhibition in E. coli prevent ing the b iosy nthesis of this prote ctive osmo lyte (Csonka 1 988; Smith 19 85; Smith et al 1 98 4) T hu s, ma ny o f t he pr ot ec ti ve os mo lyt es ac cu mu la te d b y E. coli must be taken from thei r enviro nment. E. coli and related organisms have two primary transport systems for prote ctive osmolytes during osmotic stress, ProP and ProU (Randa ll et al. 1995). The ProP system uses the p roton gr adient ma intained by t he cell to drive the uptake of osmoprote ctants. This low a ffinity sys tem ( K m for proline is 0.3 mM) also transports many other osmoprotectants (Lucht and B remer 1994). The ProU transport sy stem consists of a periplasmic binding protein with a high affinity for betaine ( K m 1.3 : M), a membrane-spanning component and a membra ne bound enzyme which hy drolyzes ATP for the active transport of betaine (L ucht and Bremer 1994). A hierarchy for osmoprotectants has been empirically established for E. coli primarily for salt-me diated osmotic stress (Randall et al. 1995). Although there have be en conflic ting re ports con cerning the valid ity o f this hier archy for sug ar-med iated osmo tic stress (Glaasker et al. 1998), betaine is ge nerally re garded as the most effective pr otective osmolyte for E. coli. In at least one repor t, the ability of betaine to re store growth during osmotic challenge with different carbon sour ces was dependent on the particular sug ar

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8 (Dulaney e t al. 1968). Thus, the sugar-mediated osmotic stress anticipated for fermentations of hemicellulose hy drolysates (100 g liter -1 sugar) may require the accumul ation of d iffere nt osmoly tes. Xy lose ver sus Gluco se Metab olism The rea ctions inv olved in th e transp ort and me tabolism of glucose are we ll understood and outlined in Figures 1-1 and 1-2. Tra nsport of glucose into the E. coli cy toplasm is m ediated by a phosphotransfe rase sy stem (PTS) The ene rgy and phosp hate required for translocation and phosphory lation of PTS sugars comes from phosphoenolpyruvate ( PEP). An additional ATP is required for the phosphory lation of fructose-6-phosphate to fructose-1,6-bisphosphate. Thus, to metabolize g lucose, an initial investment of 2 ATP equivalents (1 ATP and 1 PEP) is required. Fructose-1,6-bisphosphate is cleaved into dihy droxyacetone-phospha te and gly cerald ehy de-3-ph osphate. These tw o molecul es are i ntercon verted v ia triose -phospha te isomeras e. For th e produc tion of py ruvate, the termi nal produ ct of gl yc oly sis, glyc eraldehy de-3-phosphate is oxidized and phosphorylated to form 1,3-bisphosphoglyce rate by g lycera ldehyde-3phosphate dehydr ogenase. During this step, nicotinamide adenine dinucleotide (NAD + ) is reduced to NADH. The high g roup-transfer potential of the phosphate bond on carbon 1 is used in the production of ATP from ADP in the pro ceeding reactio n cataly zed by phosphog lyc erate k inase. Th e reac tions lead ing to the formation of phosphoenolpyr uvate do not result in any fur ther energy yield or reducin g equiv alents. In converting g lycera ldehyde-3phosphate to PEP, 1 ATP and 1 NADH are produced. The conversion of PEP to py ruvate is either carried out via the

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9 phosphotr ansfer ase sy stem or thr ough an ATP y ielding reactio n cataly zed by a py ruvate kinase. T he net re action of gly coly sis can be written a s follows: glucose + 2NAD + + 2ADP + 2 P i 2 pyruvate + 2NADH + 2H + + 2 ATP The production of pyr uvate from molecule glucose y ields a net of 1 ATP and 1 reducin g equiv alent (N ADH). Re ducing equivale nts are o ften con sidered as pools of both their reduced and oxidized forms, and their ratio is indicative of the metabolic state of the cell (respiration or fermentation) (de Gr aef et al. 1999; Snoep et al. 1990). Though the ratio of the reduced to oxidized form (NADH/NAD + ratio) varies widely with different growth condition s, the abs olute con centra tion of the two forms remains r elativel y constant (de Graef et al. 1999). In contrast to glucose xylose is transported into the cell by either a proton sym po rt pa th wa y ( xylE ) or an ATP dependant transporter ( xylFGH ) (Song and Park 1997; Tao et al. 2001; Fig. 1-3). During f ermentation, the cellular proton gradient is maintained presumably by energy -consuming reactions (F 1 /F 0 ATPase, for example). Thus, a proton symport pathway is fueled indirectly by the hydroly sis of ATP. The transport of each xylose is energized by the hydroly sis of 1 ATP. Once inside the cell, xylose is conver ted into xylulose by xylose isomerase. Xy lulose is then phosphorylated by xylulokinase, utilizing the hydroly sis of a second ATP. Regardless of the pathway xylose uptake and activation (phosphorylation) r equire energy derived from the hy drolysis of 2 ATP molecule s. In contras t, gluco se trans port uses a single ATP equiva lent (PEP) for both transport and activation. Intr acellula r xylu lose-5phosphate is metabol ized by the pento se-phosp hate pathway (F ig 1-4). Through a series of reactions cataly zed by transketolase a nd

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10 transald olase, xy lose is co nverted into inter mediates of gly coly sis (fruc tose-6phosphate and gly ceraldehy de-3-phosphate). For every 6 xyloses consumed (30 carbon a toms), 4 fructose-6-phosphates and 2 gly ceraldehy de-3-phosphates are produced. These molecules are fur ther meta bolized by gly coly sis to ultima tely yie ld 10 molec ules of py ruvate. Thus, all 30 carbon atoms which began in xy lose are converted into py ruvate. The energy (ATP) and reducing equivalents (NAD H) produced from the reactions common to xylose and glucose meta bolism are the same regardless of the or iginal substrate. However, since xy lose is a pentose and requires separate e nergy for transport and activation, growth on xylose results in a relatively low A TP yield. The transport and activati on of 6 xy lose molec ules (30 c arbons) require s 12 ATPs, a ssuming 1 ATP is require d for tra nsport re gardle ss of the p athway In th e conve rsion of th is xylulo se-5-ph osphate t o 10 molec ules of g lyc eralde hyd e-3-pho sphate, a n addition al 4 ATPs are consumed. The conversion of these 10 molecules of glyc eraldehy de-3-phosphate to py ruvate yields 20 ATPs. The ne t gain of energy in the conversion of 6 molecules of xylose to 10 py ruvate is 4 ATPs. An equal amount of glucose on the basis of moles of carbon (5 molecule s; 30 carbon atoms) produces a net of 10 ATPs dur ing con version t o 10 molec ules of py ruvate. The net e nergy gain fo r gluco se catabol ism is 1 ATP pe r py ruvate, 2.5-fold more ATP t han from xylos e catab olism. While ther e is a con siderab le diffe rence i n ATP y ield betw een xy lose and g lucose, o nly one NADH is produced per py ruvate from either glucose and xy lose. Pyruvate Dissimilation A facultative anaerobe, E. coli accomplishes redox balance either by respiration (Gennis and Stewart 1996) or fermentation (Boc k and Sawers 1996). During respiration,

PAGE 23

11 reducing equivalents are oxidized when their electrons a re donated to the primary oxido-reductases of the electron transport sy stem. These electrons are passed betwe en the various proteins of the electron transport chain and ultimately used to reduce a terminal electro n accep tor (oxy gen dur ing aer obic res piration, for examp le). The energ y f rom these reducing equivalents is preserved in the for m of a proton gradient established by the concomitant translocation of H + from the c yto sol to the p eriplasm This prot on gra dient is used to produce ATP via the F 1 /F 0 ATPase. The py ruvate d ehy droge nase com plex (PDH) cataly zes the oxida tive meta bolism of py ruvate t o acety l-Coenzy me A (ac ety l-CoA ) a nd CO 2 with the formation of 1 reducin g equiv alent (N ADH). Th is complex c onsists of t hree ac tivities: py ruvate decarboxylase ( aceE ), acety ltransferase ( aceF ), and lipoate dehy drogenase ( lpd ) (Fig. 1-5). Th e decar boxyl ation of p yr uvate to a n enzy me-bound acety l moiety by t he py ruvate decarboxylase of this complex requires a thiamine pyrophosphate (TPP) cofactor, a carrier of the “active” a cetaldehy de. The acy l moiety is then transferr ed to an acy l-carrier through the reduction of a disulfide bond. The resulting thioester has a high group transfe r potential and is transferred to Coenzy me A, an acety ltransferase reaction. The disulfide which accepts the acetaldehy de from the TPP must be regenerated throug h an oxidation/reduction reaction. NAD + is reduc ed to NAD H as the su lfhy dry l group i s oxidized, forming the required disulfide bond. This reaction is subject to strong feedba ck inhibition by NADH (Hansen a nd Henning 1966). During aerobic growth, the tr icarboxylic acid (TCA) c ycle is responsible for the total oxidat ion of ac ety l-CoA to CO 2 (Cronan Jr. and L aPort 1996 ). The fi rst step in this cycle, c itrate synthase, is also the r ate controlling step (Le e et al. 1994; Walsh and

PAGE 24

12 Koshland, Jr. 1985). This enzyme cataly zes the condensation of acety l-CoA and oxaloacetate to form citrate (Weitzman 1981). Citrate sy nthase is primarily reg ulated by allosteric controls, activated by acetyl-CoA a nd inhibited either by NADH a nd 2ketoglutarate (Gram-neg ative) or ATP (Gram-positive, archea a nd eukaryote s) (Weitzman 1981). This provides a link between the energe tic needs of the cell and the gener ation of reducing equivalents (and ultimately ATP) through the TCA cy cle. The TCA cyc le is also a source of carbon skeletons for biosy nthesis. More than half of th e amino a cids made by t he cell a re deri ved from intermed iates of t he TCA cy cle (Neidhardt et al. 1990). Oxaloacetate must be reg enerated for the continued cy clic action as intermediates are drawn into biosy nthesis. This anapleurotic reaction is cataly zed by phosphoenolpyruvate c arboxylase in E. coli. The biosy nthetic n eeds and metabolic state of the cell dictate the activity of this reaction through allosteric control. Acety l-CoA, fructos e-1,6-b isphospha te and GT P are ac tivators o f this enzy me (I zui et al. 19 81), while malate and aspartate (products of oxaloacetate utilizing reactions) are inhibitors (Izui et al. 1981). Acety l-CoA and oxaloacetate are co-substrates for c itrate synthase. Thus, the allosteric activation of phosphoenolpy ruvate carboxyla se and citrate sy nthase by acetyl-CoA links the ava ilability of the two co-substrates for citrate synthase During fermentation, no external terminal electron a cceptors are available for respiration, resulting in the accumulation of NADH to hig her levels than during respiration (de Graef et al. 1999). F or redox balance to be maintained, intracellular metabolites serve as electron acceptor s. As NAD + regeneration becomes diffic ult, NADH gener ation is no t favore d. The fo rmation of acety l-CoA fro m the py ruvate d ehy droge nase reaction is inhibited by the hig h NADH/NAD + ratio (Hansen and Henning 1966),

PAGE 25

13 necessitating an alternate, non-oxidative route to ace tyl-CoA ge neration. The non-oxidat ive clea vage o f py ruvate t o acety l-CoA and formate is cataly zed by pyr uvate formate-lya se (PFL; Knappe and Saw ers 1990). PFL activity is relative to the metabolic state of the cell, similar to PDH and citrate synthase However, PFL a ctivity is regula ted by post-translational modification enzy mes which are allo sterica lly c ontrolled The pro tein is tra nslated in an inact ive form. An oxy gen-la bile fre e radic al is plac ed on a g lyc ine resid ue by a PFL -activa se enzy me ( pflA ) forming the active PFL enzyme (Conradt et al. 1984) To protect the enzyme f rom irreversible inactivation by oxygen, the multi functional alcohol dehy drogenase ( adhE ) also has a PFLdeactiv ase act ivity to remove the oxy gen-la bile fre e radic al (Kess ler et al 1991). The PFL-dea ctivase activity of adhE is inhibited by NADH (Kessler et al. 1992), linking the activation state of PFL to the meta bolic state of the cell as described by the NADH/NAD + ratio. Whe n oxyg en supply is limited, N ADH acc umulates a nd inhibits PDH and the PFL deactivase activity. This causes a shift in flux to acetyl-CoA from oxidative pyruvate cleava ge (PDH) to the non-oxidative cleavage (PFL). In contrast to respiration, ac etyl-CoA is an elec tron acceptor during fermenta tion. The twostep red uction of acety l-CoA to e thanol is c ataly zed by alcohol d ehy droge nase ( adhE ; Fig. 1-6), regene rating 2 NAD + Glycoly sis produces only one NA DH per pyr uvate. T hus, the na tive alco hol produc tion pathw ay results in an NADH deficit. This is overcome by converting one of the acety l-CoA to acetate, producing an additional ATP by substrate-leve l phosphorylation. I n E. coli grown under anaerobic f ermentation conditions with glucose as the carbon and energ y source, e qual amounts of acetate and ethanol are produced.

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14 Lac ti c a ci d i s o ft en pr od uc ed by E. coli during f ermenta tion in add ition to ac etate and ethanol, primarily a s active growth slows and stationary growth. Pyr uvate is reduced in a single-step reaction cataly zed by lactate de hydrog enase (LDH, ldhA gene p roduct; Bunch et al. 1997), resulting in the re-oxidation of 1 NADH per lactate produced. The pathway for lactate production in E. coli is controlled by allosteric re gulation, activated by py ruvate (Tarmy and Kaplan 1968). In c onditions of surplus supply of py ruvate, the lactate pathway is activa ted. The re is an a ssociate d energ etic loss a s a resul t of lacta te producti on compar ed to the c o-produc tion of ac etate a nd ethano l, as no AT P is made in the reduction of py ruvate to lactate. In c ontrast to respira tion, the T CA cy cle is inte rrupted at 2-ket oglutar ate dehydrog enase due to transcriptional regulation during fermentation (Iuchi a nd Lin 1988). The resulting pathway has two sides, the reductive (leading to succinate production) and the oxidative (stopping at 2-ketoglutarate). For suc cinate production during fermentation, the anapleurotic pathway for oxaloacetate production (PPC) is the first step. As described previous ly, there a re multipl e alloste ric eff ectors o f this enzy me which c ontrol its phy siologic al activi ty. Oxaloace tate is co nverted to malic a cid throu gh the r everse activity of the malate dehy drogenase, rege nerating 1 NAD + (Bock a nd Sawer s 1996). F umarase ca ta lyz es th e c on ve rs io n o f m al at e t o f um ar at e. Fu ma ra te is re du ce d t o s uc ci na te by a fermentation specific fumarate reduc tase ( frdBACD gene products) with the oxidation of a reduc ed menaq uinone (C ronan, Jr. and L aPort 1996 ). The oxida tive side o f the TCA pathway provide s carbon skeletons for biosy nthesis (Neidhardt et al. 1990).

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15 Engine ering E. coli for Ethanol Production The ente ric bac terium E. coli can use all of the sugar constituents of ligno cellu lose, whil e the n atural ethan ol pro ducin g Saccharomyces cerevisiae and Zymomona s mobilis are limited to growth on hexoses,. Wild-type E. coli produces ethanol from the two step reduction of acety l-CoA oxidizing two NADH to NAD + As a result, a cetate (no furt her red uction re quired) is made in a pproximate ly e qual amou nts to ethanol. However, Z. mobilis and S. cerevisiae produce ethanol from py ruvate through a pathway whic h only re-oxidizes one NADH. The irr eversible, non-oxidative cleavage of pyr uvate int o aceta ldehy de and c arbon dio xide is cata lyze d by pyr uvate de carboxy lase (PDC). Ac etaldeh yde is reduc ed to etha nol, oxidizing 1 NADH. T hus, for e ach py ruvate that is con verted t o ethanol via this pa thway one NAD H is re-o xidized. With th is stoichiometry, all of the py ruvate generated by glyc olysis can be conve rted to ethanol without the necessity of other oxidized products to maintain redox balance. Previous studies demonstrated that the Z. mobilis genes involved in ethanol production are expressed well in E. coli (Ing ram and Co nway 1988). Th ese ge nes ( pdc and adhB ) were used to construct a sy nthetic operon which was integrated into the chromosome for increased ge netic stability of the rec ombinant strain (Ohta et al. 1991). A deletion was introduced in fumarate reductase to decrease succinate production, problematic in xylose fermentation. The r esultant strain, designated KO11, fermented both pentoses and hexoses to ethanol with yields approa ching 100% of total sugars present (0.51 g ethanol/ g suga r = 100% t heoreti cal y ield) dur ing fer mentation in laboratory me dia containing excess complex nutrients. In addition to the alterations to the fermen tation pro file, the re were some nota ble eff ects on g rowth phy siology In b roth

PAGE 28

16 cultures, comparatively high cell y ields were achieved. On solid media, colonies exhibited a raised mo rpholog y, s imilar to y east. Deleterious Effects of Metabolic Engine ering The eng ineerin g of met abolic pa thway s for the producti on of indus trial che micals as an alt ernativ e to chem ical sy nthesis ha s been pe rformed for a va riety of chemi cals (Chotani et al. 2000). Metabolic engineering for renewable chemicals such as e thanol (Ingra m et al. 1999), acetate (Causey et al, 2003), lactate (Bianchi et al. 2001; Chang et al. 1999a; Dien et al 2001; Ky la-Nikkila et al. 2000; Zhou et al. 2002; Zhou et al. 2003), propanediol (Nakamura et al. 2000; Tong e t al. 1991), adipic acid (Niu et al. 2002) and succinate (Donnelly et al 1998a; Donnelly e t al. 1998b; Vemuri et al. 2002) have focused primarily on product y ields. The metabolic engineering of these new products has often resulted in unexpected changes which incre ased the need for complex nutrients and decreased potential utility (Bunch et al. 1997; Chang et al. 1999a; Chao and L iao 1994; Chao et al. 1993; Martinez et al. 1999). Undesirable changes such as r educed growth, decrea sed glyc olytic flux and low volumetri c produc tivity are ge nerally attribute d to a lac k of ATP (G okarn et al. 2000; X ie et al. 200 1), cre ation of f utile cy cles (Cha o and L iao 1994; C hao et al 1993; Patn aik et al. 1992), c hange s in intrac ellular m etabolite pools or a metabolic imbalanc e (Arist idou et al 1992; Bunch et al. 1997; Chang et al. 1999b; Contiero et al. 2000; L iao et al. 1996; Yang et al. 199 9a; Yang et al. 199 9b; Zhou et al. 200 2; Zhou e t al. 2003) Often, these detrimental effects are masked by abundant complex nutrients in laboratory media and are only appare nt in mineral salts or low-nutrient media (Bunch et al. 1997; Chao and L iao 1994; Chao et al. 1993; Martinez et al. 1999).

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17 Salmonella typhimurium was engineered for succ inate production by incr easing the expression of pyc encod ing pyruva te carb oxylase (Xie et al. 20 01). A ltho ugh succinate production increased, gr owth rate declined by 18% and gly colytic flux decreased by 40%. Similar results were reported for an ana logous construction in E. coli (Gokarn et al. 2000). Donnelly and coworkers (1998a and 1998b) isolated E. coli mutants which produced 5-times more succinate than the par ent strain, and again growth ra te was impai red. Gr owth r ate and cell yiel d were a lso d ecreas ed by engin eering E. coli for the production of 3-deoxy-D-a rabinoheptulosonate 7-phosphate (DAHP) by over-expression of pps (phospho enolpy ruvate s ynt hase) ( Patnaik a nd Lia o 1994; Patn aik et al. 1992). Th is inhibition of growth was more pronounced in minimal medium (Chao and L iao 1994). Acetate production during the aerobic growth of E. coli on sugars has been correlated with a decline in metabolic activity and reduced expression of heterologous genes (Aristidou et al. 1995; Bauer e t al. 1990; Chang et al. 1999b; Luli and Strohl 1990). Many approac hes have been emp loy ed to dec rease a cetate producti on and inc rease recombinant products (Aristidou et al. 1995; Barbosa and I ngram 1994; Bauer et al. 1990; Chang et al. 1999b; Contiero et al. 2000; Yang et al. 1999a) Mutations in the primary acetate pathway ( pta, phosphotr ansace tyla se; ackA, acetate kinase) increased the yield of recombi nant prot eins, but u sually reduce d cell g rowth. Th e detrime ntal eff ect on g rowth was attributed to the accumulation of metabolic intermediates such as ac etyl-CoA or acetyl-phospha te. An alternative approach, channe ling pyruva te away from acetate by expressing the Bacillus s ubtilis alsA gene encoding ac etolactate syntha se, also reduced acetat e produc tion by 80% and in crease d produc t yie lds, but ag ain redu ced cel l growth (Yang et al. 1999a). Other attempts to dec rease acetate production by increased

PAGE 30

18 expression of ldhA (lactat e dehy droge nase) w ere ine ffectiv e in rich medium (Y ang et a l. 1999b). In mineral salts medium, over-expression of ldhA was accompanied by a severe growth limitation (Bunch et al. 1997). Lactate dehy drogenase ( ldhA ) has been expressed to divert carbon away from acetate accumulation. Despite the relatively high K m for pyruva te, lactate production increa sed by 50% (Ya ng et al 1999b). Inte resting ly, the amoun t of ace tate pro duced in these fermentations was not altered. However these studies were conducted in a rich laborat ory medium. I n a miner al salts me dium (M9), expressio n of L DH resul ted in severe growth defec ts (Bunch et al. 1997). These growth de fects were attributed to a decrease in py ruvate availability ne cessary for growth. Project G oals Though strain KO11 is prototrophic, high levels of complex nutritional suppleme nts are r equired for the r apid fer mentation of suga rs to etha nol (Mar tinez et al 1999). For example, during the fermentation of 90 g liter -1 xylose to ethanol, the addition of CSL as a nutritional supplement (0-50 g liter -1 ) had a dose dependent effec t on final cell con centra tion. With the increa se in bioc ataly st conce ntration, there wa s a proportional increase in fermentation rate and de crease in required fermenta tion time. Although the addition of 50 g liter -1 CSL is not cost-prohibitive, the handling of this much materia l on the sc ale of a n industria l fermen tation co uld be pro blematic and ge nerate excessive cost for waste dis posal. These studies will examine the basis for the high nutrient requirement for KO11 for the rapid conversion of sugar to e thanol. As a starting point, growth and ethanol production will be evaluated in a medium containing 1% CSL 90 g liter -1 xylose, and

PAGE 31

19 mineral salts. Physiological a nd genetic approaches will be used to cha racterize the growth limitation. Solutions for solving this limitation will be presented. Further work will demonstrate the basis for a specific biosy nthetic pathway unde r the conditions tested. Knowledge gained in these studies will have a pplications in the further development of the commercial production of ethanol from plant biomass by metabolically eng ineered E. coli and will significantly c ontribute to the field of metabolic engineering by emphasizing the importance of metabolic intermediates down-stream of the product forming node.

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20 Figure 1-1. Glucose transport by the phosphotransferase sy stem. The phosphate from PEP passes through a cascade of enzymes and ultimately to intracellular glucose. The hyd roly sis of 1 AT P equivale nt (PEP) is u sed to ene rgize tr ansport a nd activa te glucose.

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21 Figure 1-2. Gly coly sis. The e nzym es and g enes tha t cataly ze the con version o f gluco se to pyruvate a re as follows: (1) phosphoglucose isomerase, pgi ; (2) phosphofructokinase, pfkA ; (3) fructose-6-phosphate aldolase, fba ; (4) trio se phosphate isomerase, tpi ; (5) gly ceraldehy de-3-phosphate dehy drogenase, gapA ; (6) phosphogly cerate kinase, pgk ; (7) phosphoglyc erate mutase, gpmA or pgmI ; (8) enolase, eno ; (9) phosphotransferase sy stem; (10) pyruvate kinase, pykA or pykF

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22 Figure 1-3. Xylose transpor t in E. coli In contrast to glucose the transport and activation of xyl os e i s n ot co up le d, ea ch re qu ir in g e ne rg y.

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23 Figure 1-4. Xy lose meta bolism. Xy lose is met abolized to intermed iates of g lyc oly sis (in bold) by the pento se-phosp hate pat hway For the sake of c arbon ba lance, 6 xylos e are con verted t o 4 fruct ose-6-p hosphate and 2 gl yc eralde hyd e-3-pho sphate. N ote that neith er ATP nor reducin g equiv alents a re produ ced or c onsumed in this pa th wa y.

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24 Figure 1-5 R eac ti ons of t he p yruv ate deh ydro gen ase com pl ex En zyme I ( aceE ) catalyzes the oxidativ e decar boxyl ation of p yr uvate thr ough a p yr uvate de hyd rogen ase activity The thia mine py rophosph ate bound activate d aceta ldehy de is pass ed to the lipoa te transa cety lase (En zyme 2; aceF ), and ultimately to Coenzy me A. To regen erate th e reduc ed lipoat e, the F AD bound t o Enzy me 3 (dihy drolipoa te dehy droge nase; lpd ) is reduced. NAD + is reduced to NADH by this FADH 2 allowing for another cy cle.

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25 Figure 1-6. Fermentation pathway s of E. coli (1) pyruva te kinase, pykA or pykF or PTS sugar transport; (2) py ruvate formate-ly ase, pflB ; (3) phosphotransacety lase, pta ; (4) acetate kinase, ackA ; (5) PFL-deac tivase / alcohol dehydr ogenase / acetaldehy de dehydrog enase, adhE ; (6) PEP carboxylase, ppc ; (7) mala te dehydrog enase, mdh ; (8) fumarase, fumB ; (9) fumarate reductase, frdABCD ; (10) lactate dehy drogenase, ldhA

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26 CHAPTER 2 FLUX THROUGH CI TRATE SYNTHASE LI MITS THE GROWTH OF ETHANOL OGENI C Escheric hia coli KO11 DURING XYL OSE FERMENTATION Introduction Our laboratory has previously eng ineered E. coli strain B for the production of ethanol from pentose-rich, hemicellulose sy rups by expressing hig h levels of Zymomonas mobilis pdc (pyruvate decarboxylase) and adhB (alcoho l dehy droge nase) ( Ing ram et al 1999; Ohta et al. 199 1). This st rain was chosen f or metab olic eng ineerin g beca use of its hardiness, wide substrate range, and a bility to grow well in minera l salts medium without organ ic nutrie nts (Alte rthum et a l. 1989; L uli and Str ohl 1990). During xylos e fermentation, ATP yield in E. coli is lo w (~0. 67 ATP per x ylose) du e to se parate energy require ments for uptake a nd phospho ry lation (T ao et al. 2001). Un like most g enetica lly engineered strains of E. coli KO11 g rew to hig her dens ities than the pare nt in both mineral s alts and c omplex media (Martine z et al. 199 9). I nitial stud ies with L uria bro th demonstrated rapid and efficient conversion of sug ars to ethanol by KO 11, with yields approaching 95% of the theoretica l maximum. However, volumetric productivity and ethanol yields wer e considerably lower in mineral salts medium without complex nutrients (Lawford and Rousea u 1996; Martinez et al. 1999; Moniruzzaman and Ingram 1998; York and Ingr am 1996a; York and Ing ram 1996b). Supplemen ting mine ral salts medium with complex nut rients sig nifican tly increased ethanol production. The least expensive complex nutrient, corn steep liquor, supporte d growt h rates a nd ethano l product ivities ne ar those for L uria bro th but only

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27 when provided at high concentrations (5% w/v) Although not prohibitively expensive, the addition of high levels of complex nutrients adds to the cost of ethanol production and increases the requirements for waste tr eatment. The lowe r rate o f ethano l product ion (volum etric pr oductivity ) in minima l media (compared to Luria br oth) resulted from low cell densities and reduced expression of recombi nant pdc and adhB (lower metabolic activity ). Inorga nic components did not appear to be limiting and no specific auxotrophic requirements could be identified (Martine z et al. 199 9). Reduc ed expres sion of he terolog ous gen es was a ttributed to “biosy nthetic b urden”, the compe titive re duction in syn thesis of h eterolo gous pr oducts due to derep res si on o f na ti ve g ene s fo r bi osyn th eti c en zyme s (M art in ez et a l. 199 9). In this study, I have used a mineral salts medium containing 1% CSL and investigated the basis of the requirement for higher leve ls of nutrients during xylose fer mentation. Four hypotheses wer e examined as the basis for the decreased g rowth in the CSL medium: 1) availab ility of macr o-nutrie nts; 2) loss of a biosy nthetic p athway due to met abolic engine ering; 3) insuff icient AT P during xylos e ferme ntation; a nd 4) an im balance in centra l metaboli sm. Materials and Methods Microor ganisms and Media E. coli B (ATCC 1 1303) an d an etha nologe nic deri vative, s train KO 11 (Ohta et al. 1991), were used in all fermentation experiments. KO11 contains a dele tion in the frd region (anaerobic fumar ate reductase) which eliminates succinate pr oduction. Genes encoding the Zymomona s mobilis et ha no l p at hw ay ( pdc, adhB ) and chloramphenicol acetyltra nsferase ( cat ) were integrated into the pfl gene ( chromoso me) by a single

PAGE 40

28 cross-over event resulting in a functional, full leng th pfl gene downstream. Both E. coli B and KO11 are prototrophic. Stock cultures were store d in glyce rol at -75C. Working cul tu res wer e tr ans fer red dai ly on sol id med iu m co nt ain in g mi ner al s alt s an d 1% CS L. Xylose (2%) and chloramphenicol (alternating betwe en 40 and 600 mg liter -1 ) were included in solid media for K011. A citrate syntha se mutant, E. coli W620 ( glnV 44, gltA6, galK30, pyrD36, spdL129, thi-1 ), was obtained from the E. coli Genetic Stock Center (CGSC # 4278) and used to test expression of the B. subtilis c itZ gene ( citrate syn thase). This stra in contai ns a gltA 6 mutation (citrate synthase ) that prevents growth on M9 medium containing thy mine and glucose (Herbert and G uest 1968). Corn steep liquor medium (CSL+X) contained (pe r liter in distilled water): 10 g of corn steep liquor (~50% solids), 1 g of KH 2 PO 4 0.5 g of K 2 HPO 4 3.1 g of (NH 4 ) 2 SO 4 0.4 g of Mg CL 2 •6H 2 O, and 20 m g of Fe Cl 3 C 6H 2 0. A one-liter stock solution of CSL was prepared by dilution of 200 g with distilled water, adjustment to pH 7.2 with 50% NaOH and stea m steriliza tion. Be fore use the ster ile stock s olution of CSL wa s aseptic ally clarified by centrifugation (10,000 x g, 5 minutes). Mineral solutions were prepa red as described previously (Martinez et al. 1999). Broth cultures and fermenta tions contained 9% (w/v) xylos e medium, unless ind icated o therwise In s ome exper iments, L uria bro th containing xylose was include d for comparison. Fermentation Conditions Seed cult ures (10 0 ml in 250 mlflask) w ere gr own 14-16 hours at 3 5C with agitation (120 rpm). Cells were harvested by centrifugation (5,000 x g, 5 min) and used as an inoculum to provide an initial concentration of 33 : g ml -1 dry weig ht (0.1 OD 550nm ).

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29 Fermentation vessels contained a total volume of 350 ml ( 35C, 100 rpm). Cultures were maintained at pH 6.5 by the a utomatic addition of 2N KOH (Moniruzzaman and Ing ram 1998). For strain B, 6 N KOH was used to maintain pH af ter the initial 24 h. Supplements were added with distilled water as necessary (10 ml total volume). Organic acids and amino acids wer e neutralized with NaOH, sterilized by f iltration and added a t a final c oncentr ation of 2 mg ml -1 Acetaldehy de was added at a final concen tration of 0.25 mg ml -1 or 0.5 mg ml -1 Cell mass, ethanol, organic acids and suga rs were monitored at 24 h intervals. Aerobic Growth Studies Cells wer e grow n with aer ation in 25 0 ml, baff led flask (35C, 220 rpm) containing 50 ml of CSL+X medium. A rang e of sugar concentrations was te sted (0.5% 5%) to determine maximal cell density under conditions of sugar excess. Media were inoculat ed direc tly u sing ce lls grow n on solid me dia (1824 h). Eth anol and c ell mass were measured after 16 h. F or comparison, Luria broth conta ining 5% (w/v) xylose wa s also included. Analytical Methods Cell mass was estimated as OD 550nm using a Baush & L omb Spectronic 70 (1 OD 550 = 0.33 mg ml -1 dry cell we ight). Ethanol was measured by gas chromatography using a Varian model 3400 CX as described pre viously (Moniruzzaman and I ngram 199 8). Org ani c ac id s an d su gar s we re m eas ure d by H PLC usi ng a HP 109 0 S eri es II chromat ograp h equippe d with a B ioRad Amin ex HPX-87H ion exclusio n column ( 45C, 4 mM H 2 SO 4 0.5 ml min -1 10 l injection) and dual detectors (refractive index monitor and UV detector at 210 nm).

PAGE 42

30 Fermentation products were also analy zed by NMR to confirm the identities of HPLC peaks. Broth samples wer e centrifuged to remove ce lls. Supernatants (0.9 ml) were mixed with de uterium oxid e (0.1 ml) and sodium 3-(trime thy lsily l)propio nate (10 mM internal standard) in 5 mm sample tubes. Proton spectra were obtained using a modified Nicolet NT300 spectrometer in the Fourier transf orm mode (Buszko et al. 1998) as follows: frequency 300.065 MHz; excitation pulse width, 5 s; pulse repetition delay, 3 s; spectral width, 3.6 KHz. A minimum of 100 acquisitions were obtained for each sample. Genetic Methods The citZ gene enc odin g B. subtilis citrate syn thase I I ha s been pr eviously described (Jin and Sonenshein 1994). This gene was amplified by PCR (forward primer, 5'-TGTGCTCTTCCATGTTTTTACAACACTGTTAAAG-3' ; reverse primer, 5'-TTG CTCTTCGTT AGGCTCTT TCTTCAAT CG-3') using g enomic DN A from B. subtilis strain YB 886 as the template (Barb osa and I ngram 1 994). Prim ers wer e added to the Taq PCR Master mix (Qiagen) as recommended by the manufacturer. Conditions of thermal cyc ling were as follows: 1) two initial cy cles with denaturation at 94C (60 s), anneali ng at 50 C (60 s) a nd elong ation at 6 8C (90 s) ; 2) twent yeight c yc les with denaturation at 94C (10 s), annealing at 70C (60 s), and e longation at 68C (90 s); and 3) a fina l elonga tion step a t 72C (10 mi n). The PCR product ( 1.5 kbp) w as clone d into pCR2.1-TOPO (Invitrogen) using ampicillin (50 : g ml -1 ) for selection. Colonies were screened for size and ability to complement the gltA mutation of E. coli W620 on glucose-minimal medium (Herbert and Guest 1968). The citZ gene was also confirmed by DNA sequencing using a L I-COR model 4000L sequencer (Middendorf et al. 1992).

PAGE 43

31 NAD(P)H /NAD(P) + ratio Whole cel l fluores cence w as used a s a rela tive meas ure of r educed nucleoti des in situ (Tartakosvsky e t al. 1996; Trivedi and Ju 1994). Since only the reduce d form of NAD(P)H fluoresces at 460 nm, an immediate decre ase in the fluorescence of fer menting cells is interpreted as a decline in the level of NAD( P)H and the NAD(P)H/NAD(P)+ ratio. Cells were grown for 12 h in CSL +X medium, harvested by centrifugation (5,000 x g, 5 min) and washed 3 times in mineral salts. The pellet was then suspended in minera l salts solution at a concentration of 1.0 OD 550 Emission a t 460 nm (e xcitation a t 340 nm) was recorded at 5 s intervals using an AmincoBowman Series 2 Luminesce nce Spectrometer. Cells were energ ized by the addition of 1% xy lose resulting in an immediate increase in fluorescence, pr imarily due to the incre ase in NADH/NAD+ ratio. Test comp ounds wer e added at a fina l concen tration of 2 mg ml -1 (organic acids, amino acids) o r 0.25 mg ml -1 (acetaldehy de) using distilled water as a control. Results for each test compound were expressed relative to the xy lose-dependent increase in fluoresce nce. Control experiments confirmed that quenching of cellular fluoresc ence did not occur when additives were mixed with energy -deficient cells (without xylose). En zym e A ss ays Citrate synthase wa s assayed using a modification of the method described previously (Evans e t al. 1993, Faloona and Srere 1969). Cultures were g rown in one-liter flasks (250 ml Luria broth) for 16 h a t 35C (150 rpm). Cells were harvested by centrifugation, washed 3 times in buffer conta ining 50 mM Tris-Cl (pH 8.0) and 20% glyc erol and suspended in 2 volumes of same buffer. Cell-fre e preparations were made by two passa ges thr ough a F rench Pr essure c ell (20,0 00 psi) fo llowed by treatme nt with

PAGE 44

32 ~100 g ml -1 deox yribonuc lease I. C ell de bris w as rem oved b y centrifu gation (15,0 00 x g, 1h, 4C). The supernatant was dialy zed against 20 mM Tris-Cl and 20% gly cerol. Each assay (1 ml) con tained 20 mM Tris-Cl ( pH 8.0), 1 0 mM KCl, 1 mM 5',5'-dithio-bis-(2-nitrobenzoic acid), 10 mM oxaloacetate a nd 0.5 mM acetyl-CoA Reaction s were in itiated by the addit ion of ce ll ly sate and monitored for 300 s a t 412 nm. Specific activity was rep orted as mol of re duced c oenzy me A prod uced pe r min per m g protein. Results Macro-nutrient Limitation. Previous s tudies ha ve shown t hat up to 5% CSL is ne eded to su pport ana erobic growth and ethanol production at a rate ne ar that of Luria broth (Ma rtinez et al. 1999). Based on a comparison of E. coli elemental composition (Taylor 1946), the macro-nutrient salts in CSL+X medium should provide sufficient nitrog en and phosphor us to suppor t the gr owth of a t least 5 mg ml -1 dry weight During anaero bic growth in pH-sta ts with CSL +X (Fig 2-1), th e maximal c ell densit y f or KO11 w as only about 1 mg ml -1 33% lowe r than the parent E. coli B (1.5 mg ml -1 ) in CSL+X medium and only 25% of tha t reach ed by KO11 (4 mg ml -1 ) in Luria broth plus sugar ( Martinez et al. 1999, York and Ing ram 1996b York an d Ing ram 1996b ). During aerobic growth with the same nutrients howeve r, KO11 g rew to a m aximum densi ty o f 2.7 mg m l -1 This is almost two-fold higher than E. coli B under the same conditions and 2.7-fold higher tha n KO11 dur ing ana erobic g rowth. To gether these r esults indi cate tha t the ana erobic g rowth of KO11 in 1% CSL with 9% xy lose is not l imited by the avai lability of macr o-nutrie nts (ie. N, P, etc.) or by t he inact ivation of a biosy nthetic p athway due to g enetic

PAGE 45

33 manipula tion. Howe ver, met abolic e nginee ring of the etha nol pathw ay does app ear to contribute to the reduced growth of KO11 in this medium under a naerobic conditions. Energy Limitation. The separate energ y require ment for uptake (ATP-dependent transporter or proton symport) and phosphory lation (xylulokinase) results in a low net y ield of ATP from xylose fermentation (0.67 ATP per xy lose), 33% of the y ield from glucose (2 ATP per glucose) (Tao et al. 2001). An additional ATP ca n also be produced from py ruvate by the acetat e pathwa y. Si nce KO1 1 produc ed less a cetate than stra in B (Ta ble 2-1) the gr owth of KO11 could be limited by the a vailability of ATP. To test this hy pothesis, I compared the growth of KO11 in 1% CSL conta ining 9% xylose with grow th in 1% CSL containing 9% gluc ose (Fig 2-2A an d Fig. 2 -2B). G lucose w as ferme nted to et hanol at a higher rate than xylose. However, the c ell yield of KO11 wa s identical for both sugars, despite the 3-fold difference in net ATP production. Although ce ll densities were low, cells remained metabolically ac tive for at least 96 h and produced most of the ethanol after growth had ceased. Metabolic Imbalance Relieve d by Addition of Py ruvate or Acetaldehy de. Pyruvate serve s a dual role during fermentation, as a sourc e of carbon skeletons for biosynthesis and as a source of electron acceptors ( acetaldehy de) to allow continued ATP produ ction by gly coly sis. Durin g suga r ferme ntation to e thanol, on e NADH is produced per py ruvate. Each NADH must be oxidized by r educing an electron acc eptor such as a cetalde hyd e or by biosy nthetic r eaction s (Mat-Jan et al. 198 9). I n a gro wing wil d type E. coli partitioning of pyr uvate between biosy nthesis and redox needs is presumed to be balanced for optimal growth. Metabolic eng ineering of the ethanol pathway

PAGE 46

34 contribu ted to the reduce d growt h of KO11 only under fe rmentati ve condit ions, consiste nt with a me tabolic im balance resulting from unc ontrolled utilization o f py ruvate for etha nol produc tion. This p ossibility was conf irmed by the addit ion of py ruvate t o CSL+X medium. Py ruvate a ddition re sulted in a dose-de pendent i ncreas e in cell g rowth and ethanol production that was particularly evident after 24 h (Fig. 2-3A a nd Fig. 2-3B). With 2 mg ml -1 of added pyr uvate, growth and ethanol production were twice that of the control w ithout py ruvate a ddition (T able 2-2 ). Supplem enting w ith py ruvate d id not cau se a buildup of TCA intermediates or acidic fermentation products (Ta ble 2-1). Note that formate was produced in all fermentations, confirming the that the pfl gene encoding pyruvate f ormate-lya se remains functional in KO11. The addition of pyruva te to media has been shown to increase the intrace llular pyruvate pool in E. coli (Yang et al. 2001), increasing the ratio of potential electron acceptors for the oxidation of NADH (from gly colysis). When added at a level of 2 mg ml -1 pyruvate wa s metabolized concurrently w ith cell growth during the first 24 h after inoculat ion (Tabl e 2-1). T he py ruvatedepende nt incre ase in ce ll mass (~1 mg ml -1 ) was roughly equivale nt to half o f the add ed py ruvate (Table 2-2, Fig. 2-3A). Remaining pyr uvate is p resumed to be meta bolized to a cetalde hyd e by recombi nant Z. mobilis pyr uvate de carboxy lase. Sinc e aceta ldehy de has be en previ ously shown to sti mulate growth and ethanol production by yeasts (WalkerCaprioglio et al. 1985) and Z. mobilis (Stanley et al. 199 7), it see med possib le that the stimulation of cell g rowth by pyr uvate could be med iated in par t by an inc rease i n aceta ldehyde fr om pyruva te (Tab le 2-2 Fig. 2-3C and F ig. 2-3D ). Conce ntrations of acet aldehy de above 0.50 mg ml -1 were toxic. With lower concen trations o f aceta ldehy de (0.25 and 0.50 m g ml -1 ), cell growth and

PAGE 47

35 ethanol p roductio n were in crease d. Lik e py ruvate, added a cetalde hyd e was fu lly metabolized during the initial 24 h after inoculation (Table 2-1). A nea r optimal level of acetal dehy de was pr ovided by 2 addition s of 0.25 mg ml -1 each to CSL+X medium (initially and afte r 12 h). T his was al most as ef fective as py ruvate ( 2 mg ml -1 ) in stimulating ethanol production and also caused a 65% increa se in cell mass. The basis for the increase in cell growth is not readily explained by the limited routes for ac etaldehyde metabolism in E. coli as compa red to tho se for py ruvate, a key centra l metaboli te. These results pr ovide evi dence th at the be neficia l effec t of adde d py ruvate r esults pri marily from an in crease in electr on acce ptors. Pyr uvate as a Source of Carbo n Skeleton s for Bio syn thesis. The pyruva te-stimulated increase in cell growth ref lects a two-fold increase in the flow of carbon into biosynthe sis. Pyruvate and upstrea m metabolites in glycoly sis are used for the biosy nthesis of approxima tely half of c ellular c onstituen ts. Pools fo r these upstream intermediates may increase when py ruvate is added, increasing ava ilability for biosynthesis. Py ruvate (and phosphoenolpy ruvate) is also converted to a series of biosynthetic intermediates by the TCA pathway a nd linking reactions. The TCA pathway provides half of the carbon skeletons for cell prote in. None of the TCA intermediates can be produ ced rea dily from ac etaldeh yde by b iosy nthetic r eaction s. Note tha t the TCA pathway is not cy clic during fermentation. This pathway is interrupted between 2-ketog lutarate and succ inate by ArcAB -mediate d repre ssion of g enes ( sucAB ) encoding 2-ketoglutarate dehy drogenase (I uchi and Lin 1988). One side of the TCA pa thway produce s 2-ketog lutarate the pre cursor f or the g lutamic a cid famil y of amino ac ids, poly amines, a mong othe rs. Prec ursors su ch as oxalo acetat e on the ot her side of the TCA

PAGE 48

36 pathway a re derived from phosphoenolpy ruvate. Oxaloacetate is used for sy nthesis of the aspartic acid fa mily of amino a cids, etc The add ition of py ruvate c ould poten tially increa se the flo w of car bon into bot h sides. TCA pathw ay intermed iates we re teste d as addit ives to CSL +X medium Util izat ion o f thes e addi tives was in vesti gated u sing HP LC and NM R (Tab le 2-2 Fig. 2-4). All except two, succinate (100% remaining) a nd isocitrate (78% remaining), were metabolized efficiently during the initial 24 h of fermentation (Table 2-1). Additions of malate a nd fumar ate res ulted in a similar sma ll increa se in fuma rate, bu t did not stim ulate growth or ethan ol produc tion. Desp ite the po tential in terconv ersion of these intermediates, fumarate did not accumulate when oxaloace tate was added. Addition of aspartate, the transamination product of oxaloacetate, wa s similarly ineffective. I ndeed, addition of oxaloacetate, malate, fumarate and aspar tic acid reduced growth and e thanol production. In contrast, 2-ketog lutarate was almost as effective as py ruvate in stimulating growth and ethanol production by KO11. A similar stimulation was also observed for glutamate, the transamination product of 2-ketog lutarate. TCA intermediates that are immediate precursors of 2ketoglutarate were not beneficial. Isocitra te was not readily metabolized. Citrate was metabolized but had no effect on growth and ethanol produc tion. Growth with added citrate was accompanied by an accu mulation of fumara te and a h igh ace tate/for mate rat io similar t o that with pyr uvate (Table 2 -1). Note that this r atio is nea r unity for othe r ferme ntations w ith TCA intermed iates, pr oviding a clue to th e ineff ectiven ess of ci trate. T he additi on of citr ate may induce c itrate ly ase (Fu rlong 19 87; Lu tgens a nd Gottsch alk 1980; Sc hneider et al. 2000), an enzyme that cle aves citrate into an equimolar mixture of oxaloacetate and

PAGE 49

37 acetate. Oxaloacetate is readily metabolized to furmarate. Both fumarate and ac etate were higher in ferme ntations w ith added citrate than with 2 -ketog lutarate and other TCA intermed iates, c onsistent w ith the indu ction of c itrate ly ase. I nduction o f this enzy me is presume d to block t he bene ficial e ffects o f this TCA in termedia te for bio syn thesis. When considered together, studies with added TCA intermediates pr ovide evidence that the beneficial effe ct of pyruva te for growth and ethanol production by KO11 in CSL+X medium results in large pa rt from an increase in the flow of car bon skeletons into 2-ketoglutarate and subsequent products of biosy nthesis. However, investig ations wit h added p yr uvate an d aceta ldehy de provid ed evide nce that an incre ase in electron acceptors was arg uably of primary importance for the beneficial effe ct of pyr uvate. F or both to b e possible both must b e mediate d by a common m echanis m. Whole-cell Fluorescence. The ratio of NAD(P)H/NAD(P)+ has bee n shown to alter cellular patterns of metabolic flux (de Graef et al. 1999). NAD(P)H is an allosteric inhibitor of many enzy mes includ ing py ruvate d ehy droge nase (G raham e t al. 1989) phosphot ransac ety lase (Suzuki 1969), malate dehydrog enase (Sanwal 1969) and citrate sy nthase (Faloona and Srere 19 69; Weitzman 1966). I n KO11, th e additio n of ace taldehy de or py ruvate (metabolized to acetaldehy de by rec ombinant pyruvate dec arboxylase) would be e xpected to decrease the level of NAD(P)H a nd the NAD(P)H/NAD(P)+ ratio by increasing the pool of ac etaldeh yde availab le for re duction to ethanol. This has be en invest igated in non-growing cells by examining the effects of these additives on whole-ce ll fluorescence. (Fig. 2-5A and Fig. 25B).

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38 Fluorescence chang es in responses to additives were immediate and stable as shown for acetaldehy de (Fig. 2-5A). Relative fluoresc ence increased when fer mentation was initiated by the addition of xy lose, and decreased immediately upon the addition of acetaldehy de (alcohol dehy drogenase) and py ruvate (py ruvate decarboxy lase plus alcohol dehydrog enase), consistent with expected changes in the oxidation of NADH. The fluorescence of energ ized cells also decreased immediately upon the addition of 2-ketog lutarate and oxaloa cetate (Fig. 2-5B). The appa rent dec line in NA D(P)H in response to these two TCA pathway intermediates may be due to reductive amination (oxaloac etate a nd 2-keto glutar ate). Ad dition of th e respe ctive ami no acid pr oducts, glutamic acid and aspartic acid, did not cause a similar change. With added oxaloacetate, malate dehydr ogenase provides an additional opportunity for NADH oxidation. Additions of malate, fumarate, succinate, citrate a nd isocitrate did not significantly a lter whole cell fluorescence. Tog ether, these data demonstrate that three compounds which increased the growth and f ermentation of KO11 in CSL+X medium (aceta ldehy de, py ruvate a nd 2-keto glutar ate) als o decre ased the NAD(P)H /NAD(P)+ ratio in cells. C ompounds w hich did no t decre ase this r atio wer e not bene ficial. O xaloacet ate was an e xception. A lthough t his compou nd decre ased the NAD(P)H /NAD(P)+ growt h and fermentation were retarde d. The negative effects of a dded oxaloacetate may be attribute d to the ind uction of pyr uvate ca rboxy kinase. T ogethe r with phosphoe nolpy ruvate c arboxy lase, this enzy me crea tes a futi le cy cle for ATP (Chao et al. 1994; Chota ni et al. 2 000 ). ATP yie lds are lo w for xy lose and A TP wasted by t his futile cycle ma y offset any potential benefits from increased oxidation of NADH.

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39 Citrate Synthase, a L ink Between NADH and 2-Ketog lutarate. In E. coli ( gltA ) as in most Gram-ne gative bacter ia, citra te sy nthase is allosterically inhibited by NADH and activated by acetyl-CoA ( Weitzman 1981). The activity of this enzy me serves to regulate the flow of c arbon into the 2-ketoglurate side of the TCA pathway, linking the cellular abundance of NADH a nd acetyl-CoA to the production of 2-ketoglutarate for biosy nthesis (Faloona and Srere 1969; L ee et al. 1994; Walsh and Koshland, Jr. 1985). This enzyme integra tes both beneficial effects of added pyr uvate, in crease d electr on acce ptors (a cetalde hyd e) and in crease d carbon skeleton s in the 2-ketoglutarate arm of the TCA pathwa y (2-ketog lutarate). The allosteric control of this enzyme by NADH could restrict the flow of carbon into the biosy nthesis of 2-ketoglutarate and other products. This hy pothesis can be readily tested by expressing a n NADH-insensitive recombinant citrate sy nthase gene in KO11. The primary c itrate synthase in Gr am-positive bacteria is allosterically regulated by ATP and rela tively insensitive to NADH (Jin and Sonenshein 1996). Since an over-abundance of ATP is not anticipated during xylose fermentation (Tao e t al. 2001), expression of B. subtilis c itZ in KO11 would be expected to increase carbon flow into the oxidizing arm of the TCA pathway. Primers we re used to clone the citZ gene (including ribosomal binding site) into pCR2.1-TOPO to produce pLOI 2514. Plasmid pLOI2514 was found to complement a gltA mutation in E. coli W620 on plat es conta ining M9 minimal media supplemented with glucose and thy mine. Citrate synthase ac tivity (0.08 U mg prote in -1 ) was also confirmed in strain W620(pLOI 2514) and absent in strain W620 lacki ng citZ

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40 Expression of citZ in KO11(pLOI 2514) increased growth and etha nol production by approximately 75% (Fig. 2-6) in comparison to the control with vector a lone, KO11(pCR2.1-TOPO). The low level of NADH-insensitive citrate sy nthase produced from pL OI25 14 was al most as ef fective as py ruvate, acetal dehy de and 2ketoglu tarate additions in stimulati ng gr owth. Thus the allost eric re gulatio n of the na tive citr ate syn thase by high NA DH appe ars to limit the flow o f carbo n skeleto ns into bios ynt hesis in CSL+X medium. Discussion The rate of ethan ol produc tion and e thanol y ield are important factor s in determining the cost of large-sc ale fermentation processes. For KO11, both of these are directly related to the exten t of gro wth of the biocata lys t (Fig. 2-7). I n CSL+ X medium, more than half of the ethanol was produced af ter cells entered stationary phase (Fig. 2-1). By incr easing cell densities, fermentation times can be re duced without sacrificing ethanol yield. Howe ver, previous studies with KO11 have shown that high levels of complex nutrients were needed for cell gr owth and rapid ethanol production (Martinez et al. 1999; York and Ing ram 1996b; York and Ing ram 1996b). This apparent requirement for high levels of complex nutrients now appears to reflec t a regulatory error in the partitioning of pyr uvate skeletons between competing require ments for the oxidation of NADH and biosynthesis. Our study demonstrates that the g rowth of KO11 was not limited by nutrients a lack o f biosy nthetic e nzym es, or ins ufficie nt ATP fro m xylos e metabolism (0.67 ATP per xylose). During the fermentation of 9% xylose, g rowth was limited by a lack of ca rbon skeletons for the biosynthe sis of products derived from

PAGE 53

41 2-ketoglutarate. Growth and ethanol produc tion were increased by the addition of pyruvate or 2-ketoglutarate, but not by the addition of oxaloacetate, malate or fumarate. The appa rent star vation fo r carbo n skeleto ns to produ ce 2-ke togluta rate wa s also alleviated by the a ddition of acetaldehy de, consistent with an involvement of NADH or NADH/NAD + ratios. The ratio of NADH/NAD + is typically higher during fermentation than during oxidative metabolism (de Graef et al. 1999). Hig h levels of NADH serve as an allosteric inhibitor of citrate sy nthase, the first committed step for the production of 2-ketog lutarate and a like ly b ottlenec k for the biosy nthesis of many amino ac ids (Walsh and Koshland, Jr. 1985). Addition of acetaldehy de decreased the NADH/NAD + ratio by increasing the pool of electron ac ceptors, potentially incre asing the function of the native citrate synthase in vivo This hypothesis was confirmed, in pa rt, using the B. subtilis c itZ gene encoding an NA DH-insensitive citrate sy nthase (Jin and Sonenshein 1994; Jin and Sonenshein 1996). Expression of citZ in KO11 stimulated growth and ethanol production by almost two-fold, substantially reducing the need to supply high levels of complex nutrients The pattern of carbon flow in KO11 is summarized in Figure 2-8. Expression of high levels of Z. mobilis pdc and adhB redirect py ruvate away from native fermentation pathways (py ruvate formate-ly ase, lactate dehy drogenase) and into ethanol, even in the presence of competing native enzy mes (Ohta et al. 1991). Since the K m of py ruvate decarboxylase f or pyruvate is approximately one-tenth that of the compe ting enzyme, pyruvate f ormate-lya se, production of acety l-CoA would also be limited. Integration of the ethanol-production genes into the chromosomal pfl gene ma y f urther c ontribute to this problem b y r educing the leve l of py ruvate f ormatelya se activ ity. Althoug h py ruvate

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42 dehydrog enase has a K m for pyruva te that is equal to that of pyruva te decarboxylase pyr uvate de hyd rogen ase is expr essed at low level s during fermen tation an d is allosterically inhibited by the high levels of NADH present during fermentation (de Graef et al. 1999; Graham et al. 1989). I n CSL+X medium, a portion of cellular py ruvate was converted to acety l-CoA by KO11 during the first 24 h as evidenced by the accumulation of acetate as a fermentation produc t. These acetate levels are pre sumed to be in excess of biosynthetic needs. The addition of either py ruvate or acetaldehy de dramatically stimulated the growth of KO11 in CSL+X medium. At lea st three sites of allosteric regulation may contribute to the increase in growth. Both py ruvate dehy drogenase (de Grae f et al. 1999; Graham et al. 1989) and citrate sy nthase (Faloona and Srere 1969; Weitzman 1981) are allosterically inhibited by NADH. Oxidation of NADH from gly colysis during the reducti on of ace taldehy de from a dded py ruvate o r added a cetalde hyd e would te nd to decrease the NADH/NAD + ratio. Th is should r educe th e alloste ric inhibi tion of py ruvate dehy droge nase and citrate syn thase by NADH. Ad ditional a cety l-CoA fro m py ruvate dehy droge nase wou ld supplem ent that p roduced by p yr uvate fo rmate-l ya se and inc rease the pool of acety l-CoA a substrate for citrate sy nthase and an allosteric antagonist of NADH inhi bition of th e native citrate syn thase (We itzman 1981 ). Toge ther, the se regulatory circuits would feed-forward to promote the flow of additional carbon skeletons into 2-ke togluta rate an d subsequ ent produ cts of bio syn thesis.

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Table 2-1. Effects of additives on the composition of fermentation products (24 h) in 1% CSL +X medium (9% xylose). M edium or strain Additive % of add itive remaining Fe r me nt at i on Pr od uc t s ( mM) Fum arate Succ inate Lacta te a For mate Ace tate Ethan ol CSL+X medium – <0.01 <0 .3 9.6 <1 .5 <1 .0 <1 .0 E. co li B, parent None – <0.01 53.4 27.7 95.8 91.4 71 E. co li KO11 None – 0.03 0.5 12.2 20.1 24.0 150 Sodi um py ruv ate (2 mg ml -1 ) < 5 0.29 0.9 12.1 13.2 27.4 208 Acet ald ehy de (2 x 0. 25 mg ml -1 ) < 5 0.21 0.5 11.1 25.6 26.6 161 Cit ri c aci d (2 mg ml -1 ) a < 5 1.65 0.6 11.8 11.9 27.0 154 Iso cit ri c aci d (2 mg ml -1 ) 78 <0.01 0.5 11.4 23.5 23.0 140 Sodi um 2ket ogl ut ara te (2 mg ml -1 ) < 5 0.28 0.9 12.5 37.8 32.1 193 Oxal oace ti c aci d (2 mg ml -1 ) 10 0.02 0.8 10.8 17.9 20.4 100 Sodi um mal ate (2 mg ml -1 ) 6 1.45 0.3 10.9 14.7 17.3 125 Sodi um f umar ate (2 mg ml -1 ) 16 1.22 0.4 10.1 19.7 18.8 110 Sodi um s ucci nat e (2 mg ml -1 ) 100 0.07 13.0 11.3 20.0 22.7 144

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Table 2-2. Effects of additives on growth and etha nol production by KO11 in 1% CSL +X medium (9% xylose). Additive No. Concent rat ion Cell Y ield Volumet ric Product ion of Et hanol Maximum Eth anol Yie ld b (% ) mg ml -1 mM Average % of Contr ol g li ter -1 h -1 % of Contr ol mg ml -1 % of Contr ol No ne (co ntrol) 26 – -0.94 0. 15 100 0.38 0. 08 100 31.12 4. 64 100 61 Sod ium p yruvate 3 0.5 4.6 1.32 0. 32 140 0.55 0. 17 144 37.60 5. 91 121 74 Sod ium p yruvate 3 1.0 9.1 1.49 0. 30 158 0.66 0. 17 173 40.09 5. 53 129 79 Sod ium p yruvate 15 2.0 18.2 1.99 0. 20 212 0.81 0. 14 213 44.22 2. 69 142 87 Sod ium p yruvate 3 4.4 36.4 2.08 0. 08 221 0.83 0. 01 218 43.23 1. 50 139 85 Ace taldeh yde (h alf initially + half af ter 12 h) 4 0.5 (to tal) 11.4 1.55 0. 07 165 0.60 0. 35 158 44.05 1. 89 142 86 citric acid 2 2.0 10.4 0.88, 0.75 86 0.35, 0.33 89 30.00, 27.59 93 56 isocitric a cid 2 2.0 6.6 1.11, 0.88 105 0.51, 0.36 113 36.45, 29.59 106 65 sod ium 2 -ketog lutarate 5 2.0 11.9 1.89 0. 18 201 0.84 0. 08 221 41.54 1. 24 133 81 oxa loace tic acid 2 2.0 15.1 0.75, 0.75 80 0.28, 0.28 74 23.58, 23.64 76 46 sod ium m alate 2 2.0 11.2 0.76, 0.84 85 0.27, 0.27 71 23.89, 24.97 79 48 sod ium fum arate 2 2.0 12.5 0.80, 0.96 91 0.37, 0.32 92 27.15, 29.84 92 56 sod ium suc cinate 3 2.0 7.4 1.00 0. 12 106 0.41 0. 05 108 35.00 1. 31 112 69 pota ssium g lutama te 3 2.0 10.8 1.66 0. 17 177 0.66 0. 13 174 43.14 0. 13 139 85 aspa rtic acid 2 2.0 15.0 0.85, 0.82 88 0.33, 0.32 84 28.40, 27.40 90 55 a Volumetric Productivity was ca lculated as the average hour ly rate of e thanol production between 24 h and 48 h after inoculation. When less than 3 replicates are presented, the value s of each replicate are shown. b Yield is expressed as a percentage of the theoretical y ield (100% = 0.51 g ethanol per g xy lose).

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45 Figure 2-1. Compa rison of ma ximal cell d ensities a chieved during a erobic a nd anae robic growth in 1% CSL mineral s alts mediu m contain ing eith er xy lose or g lucose. T hin lines rep resentin g the sta ndard er ror of th e mean a re shown for ave rages with three or more replicates.

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46 Figure 22. Co mp ar is on of gr ow th an d e th an ol pr od uc ti on fr om gl uc os e a nd xyl os e b y E. coli KO11 dur ing the f ermenta tion of 9% sugar i n 1% CSL mineral s alts mediu m. A. Growth. B. Ethanol. Thin lines representing the standa rd error of the mean are shown for averag es with thr ee or mor e replic ates.

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47 Figure 2-3. Effects of added py ruvate and acetaldehy de on growth and ethanol production by E. coli KO11 in CS L+X medium. A. Ce ll growth wit h added pyruvate. B. Ethanol production with added pyr uvate. C. Cell growth with added acetaldehy de. D. Ethanol production with added acetaldehy de. Thin lines represent the standard error of the mean.

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48 Figure 2-4. Initial effects of a dded TCA pathway intermediates on growth and ethanol pr od uc ti on by E. coli KO11 (24 h). A. Growth. B. Ethanol. Thin lines represent the standard error of the mean.

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49 Figure 2-5. Effect of metabolites on whole-cell fluoresc ence. A. Effects of aceta ldehyde on the xylose-dependent incre ase in fluorescence (time course) B. Effects of metabolites on the xylose-dependent incr ease in fluorescence. Values in B are expressed as a percentage of the xylose-dependent incre ase in the fluorescence of whole cells observed in the presence of both xy lose and the indicated additive. Note that a decre ase in the xylos e-depe ndent flu oresce nce is inte rprete d as a decrease in NAD(P)H and the NAD (P)H/NAD(P)+ ratio.

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50 Figure 2-6. B. subtilis c itZ increa ses the g rowth an d ethanol producti on of KO1 1 in CSL+X medium. A. Growth. B. Ethanol. Thin lines repre sent the standard error of the mean.

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51 Figure 2-7. Rela tionship be tween c ell y ield and f ermenta tion perf ormance In th is plot, results w ere com bined fr om ferme ntations w ith CSL+ X medium a lone and w ith suppleme nts. A comp uter-g enerat ed poly nomial wa s used to a pproximate cell yie lds. Result s from a li near re gressi on analy sis are sh own for v olumetric producti vity Dotted li nes repr esent the the 95% c onfidenc e interv als.

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52 Figure 2-8. Fermentation and TCA pathway Unless noted otherwise, enzyme s listed are native to E. coli Key to enzy mes: 1. pyruvate kinase ( pykA, pykF ); 2. py ruvate formate-lya se ( pflB ); 3. pyruvate de hydrog enase ( aceEF,lpd ); 4. phosphotransacety lase ( pta ); 5. acetate kinase ( ackA ); 6. alcohol/aldehyde dehydrog enase ( adhE ); 7. Z. mobilis pyruvate de carboxylase ( pdc ); 8. Z. mobilis alc oho l d ehyd rog ena se II ( adhB ); 9. lactate dehy drogenase ( ldhA ); 10. phosphoenolpyruvate c arboxylase ( ppc ); 11. citrate synthase ( gltA ); 12. ac onitase ( acn ); 13. isocitrate dehydr ogenase ( icd ); 14. glutamate dehy drogenase ( gdhA ); 15. malate dehydr ogenase ( mdh ); 16. fumarase ( fumB ); 17. fum arate r eductas e ( frdABC ); 18. aspartate transaminase ( aspA ); 19. aspartase ( aspC ). Arrows beneath citrate syn thase ind icate inh ibition of a ctivity by N ADH and a ntagon ism of NADH inhibition by ace tyl-CoA.

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53 CHAPTER 3 GEN ETIC CHA NGE S T O OP TIMIZ E C ARB ON P ART ITION ING IN ETHANOL OGENI C Escheric hia coli KO11 Introduction Citrate sy nthase, a key enzy me in the p artitioni ng of c arbon int o biosy nthesis (Walsh and Koshland, Jr. 1985), was shown to be growth limiting for KO11 (Chapter 2). Native citrate sy nthase is allosterically inhibited by high levels of NADH ty pical of fermen tation (We itzman 1981 ). Growt h and etha nol produc tion were substantia lly improved in KO11 by expression of an NA DH-insensitive citrate sy nthase ( citZ ) from Bacillus s ubtilis. A similar stimulation of growth and ethanol production was observed during low aeration (oxidation of NADH) and with the addition of py ruvate, 2-ketoglutarate, and aceta ldehyde. An alter native a pproach to enhanc e citrat e sy nthase a ctivity in KO11 is t o increa se available substrate pools (oxaloacetate and acety l-CoA). In vitro acetyl-CoA ha s been shown to se rve as a n alloster ic activa tor of pho sphoenolp yr uvate ca rboxy lase (I zui et al. 1981) for the production of oxaloacetic acid and to relieve the allosteric inhibition of citrate synthase by NADH (Weitzman 1981). I n this chapter, I demonstrate that physiological a nd genetic approaches which inc rease the availability of acety l-CoA for biosy nthesis sti mulate ce ll growt h and etha nol produc tion. Thes e results were us ed to engineer a second g eneration biocataly st, strain SU102, in which a small additional

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54 portion of substrate carbon was redirected f rom fermentation products to cellular biosynthesis. Materials and Methods Microor ganisms and Media Strains a nd plasmid s used in th is study are liste d in Table 3-1. KO1 1 and its derivat ives (SU1 02 and SU1 04) are prototro phic. Work ing cult ures of e thanolog enic strains were transferred daily on solid medium (1.5% agar) containing minera l salts, 2% xylose, and 1% CSL (Chapter 2). Stock cultures were stored frozen at 75C. Luria-ag ar plates were used for the maintenance of other strains. Ampicillin (50 : g/ml ), kanam yc in (50 : g/ml ) and tetracy cline (5 or 10 : g/ml ) were added as appropriate. Fermentation Seed cult ures and fermen tations (3 5C and 150 rpm) wer e grow n in minera l salts medium containing 1% CSL and 9% xy lose (CSL+X medium; Chapter 2). Fer mentations were maintained at pH 6.5 by automatic addition of 2N KOH (Moniruzzaman and Ing ram 1998). Sup plements w ere filt er steri lized as co ncentra tes and a dded dire ctly to fermentation broth. Samples were removed during fer mentation for the measurement of cell mass ethanol orga nic acid s and sug ars. Analytical Methods Cell mass was estimated from the optical density at 550 nm using a Bausch & Lomb Spectronic 70 spectrophotometer ( 1 OD 550 = 0.33 mg ml -1 dry cell we ight). Ethanol and acetaldehy de were measured by gas chromatography (Varian 3400CX) (Mo ni ruz za man and Ingr am 1 998 ). O rga ni c ac id s an d su gar s we re a nal yzed by HP LC (Hewlett Packard 1090 series I I chromatogr aph equipped with refractive index and UV 210

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55 detecto rs) with a BioRad A minex HPX-8 7H ion exclu sion colum n. Maximum vo lumetric productivity in mmol liter -1 h -1 was estimated as the first derivative of ethanol production using PSI -Plot softw are (Pol y Sof tware I nternat ional, Sal t Lak e City Utah). Specific productivity was estimated by dividing volumetric productivity by cell mass; units are mmol (gr am cell dr y w eight) -1 hour -1 Genetic Methods Standard methods were used for plasmid construction, DNA amplification (PCR), transformation, electroporation and P1 phage tra nsduction (Miller 1992; Sambrook and Russell 2001). Primers (ORFmers) for the amplification of the E. coli ackA and adhE coding region s were p urchase d from the Sigma Ge nosy s (The Wood lands, TX ). These primers included Sap I sites at both ends of the amplified product. Chromosomal DNA from E. coli W3110 (ATC C 27325) se rved as t he templa te for a mplifica tion. This s train was also used as an intermediate during the c onstruction of adhE deletion in KO11. Chromosoma l insertio n of dele ted gen es ( adhE and ackA ) was facilitated by inserting a tet ge ne fl an ke d b y FRT sites for removal of the antibiotic marker by the chlorotetracy cline-inducible FLP rec ombinase (pFT-A) in the final construct (Martinez-Morales et al. 1999; Posfai et al. 1997). I ntegration of linearized DNA was facilita ted by using pK D46 (temp eratur e conditi onal) co ntaining an arab inose-in ducible red recombinase (Datsenko and Wanner 2000) Putative deletion mutants were selected for tetracy cline resistance (5 mg liter -1 ) and screened for appropria te antibiotic resistance markers. At each step, mutants were verif ied by analy ses of PCR and fermentation products.

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56 Construct ion of pL OI20 65 Contain ing a Re movable T etracy cline Res istance Cassette To facil itate ant ibiotic re moval af ter chr omosomal in tegra tion, a re usable c assette was constructed from the tet gene of pKNOCK-Tc (Alexey ev 1999) and the FRT sites in pSG76-A and pSG76-K (Posfai et al. 1997). Both FRT sites wer e orient ed in the s ame directio n to allow e fficien t in vivo excision of the tet gene by the flp -encoded recombinase (Martinez-Morales et al. 1999). This casse tte was inserted into a modified pUC18 to produce pLOI 2065 (Fig. 3-2). Plasmid pLOI 2065 contains two Eco RI sites and two Sma I sites oriented to allow the isolation of the FRT-tet-FRT cassette as a Sma I to Eco RI fr agment for dire ctional in sertion, as a blunt fragm ent ( Sma I) and as a sticky -ended fragm ent ( Eco RI). Nucleotide Sequence Accession Number The sequence for plasmid pL OI2065 has been deposited in Ge nBank under acquisition number AF521666. Construction of SU102 Containing an Insertion Mutation in ackA Strain SU102 was made by introducing the ackA mutation directly into KO11. The PCR-amplified coding region of ackA was cloned into pCR2.1-TOPO. After digestion with Eco RI, the 1.2 kbp frag ment containing the ackA coding region was liga ted into the unique Eco RI site of pL OI2302. A recombinant plasmid was sele cted in which the direction of transcription of lac and ackA genes were opposite. The ackA gene was disrupted by dige stion with Eco RV (1 site) and the insertion of a 1.7 kbp Sma I frag ment from pLOI 2065 containing a tet gene flanked by two FRT sites for FLP recombinase. A 2.8 kbp Asc I fragment conta inin g ackA ’FRT tet FRT ‘ackA was isola ted from t his plasmid and ligated into the Asc I site of pL OI2224 containing a c onditional R6K replicon.

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57 The resu lting pla smid, pL OI23 75 (Fig 3-2), w as used a s a templa te for PCR amplification of the 2.8 kbp Asc I frag ment with ackA primers. After purification by phenol extraction, amplified DNA was used for electropora tion of E. coli KO11(pKD46) expressin g phag e lambda red rec ombinase (Datsen ko and Wan ner 2000 ). Recomb inants were selected for tetracy cline resistance. Plasmid pKD46 was eliminated by growth at 40C. The integrated tet gene was deleted using pF T-A expressing the flp recombi nase (Martinez-Morales et al. 1999; Posfai et al. 1997). After removal of this plasmid by growth at 40C, the resulting strain containing a mutation in ackA (insertion of 98 bases including stop codons in all three reading fra mes) was designated SU102. Construction of SU104 Containing a Deletion in adhE A mutation in adhE was initially constructed in W3110 prior to P1 transduction into KO11. The PCR-amplified coding region of adhE (2.7 kbp) was clon ed into pCR2.1-TOPO. A recombinant plasmid was selected in which the transcription of lac and adhE were oriented in the same direction. The ce ntral region of the adhE gene (1.1 kbp) was deleted by digestion with Hin CII (2 sites) and replaced with a 1.6 kbp Sma I frag ment from pLOI 2065 containing the FRT tet FRT casset te (1. 7 kbp) to pro duce p LOI2803 (Fi g. 3-2). After digestion of pL OI2803 with both Pvu I and Sca I, thi s plasmid se rved as a templ ate to ampli fy the 3.2 kbp re gion co ntain ing adhE’-FRT-tet-FRT-‘adhE usin g adhE primers. This amplified DNA was used for electropora tion. Recombinants were selected for tetracy cline resistance. Plasmid pKD46 was eliminated by growth at 42C. P1 transduction was used to transfer the adhE mutation in W3110 to KO11. To circumvent differences in restriction sy stems, the adhE::te t mutation w as trans duced int o a restriction-negative (modification-positive) der ivative of E. coli B (strain WA837) prior

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58 to transduction into KO11. The tetracy cline resistance gene was de leted from the KO11 derivative using pFT-A expressing the flp recombinase (Martinez-Morales et al. 1999; Posfai et al. 1997) After r emoval of this plasmi d by growth at 40C, th e resulti ng stra in containing an internal deletion in adhE was designated SU104. Results and Discussion Acetate Addition Sti mulates G rowth an d Ethanol P roductio n by Reducing Net Ace tate Productio n During Sugar M etabolism Du ri ng th e a er ob ic me ta bo li sm of su ga rs by E. coli acetate production has been asso ciat ed wi th a d ecre ase i n gr owth r ate. Consi dera ble e ffor t has b een m ade t o mini mize acetate production as a means of incre asing cell density and the production of recombinant proteins (Aristidou et al. 1995; Bauer et al. 1990; Chang et al. 1999; Contiero et al. 2000; Yang et al. 1999a; Yang e t al. 1999b ). The addition of as little as 2 g liter -1 sodium acetate (24 mM) has been shown to decrea se growth rate during oxidative sugar metabolism (Luli and Strohl 1990). During xylose fermentation by KO11, however, the addit ion of ac etate sti mulated g rowth an d ethanol producti on (Fig 3-3A an d B; Tabl e 3-2). A portion of the added acetate wa s initially consumed, in contrast to control fermen tations wh ere ac etate wa s continuo usly produce d (Fig 3-4A). Rates of a cetate production declined during subsequent incubation in both control and acetate-supplemented fermentations. Although a lmost twice as much sugar was metabolized by aceta te-supplemented fermentations than by control fermentations (no additions), net acetate production in the acetate-suppleme nted culture (7.0 mmol liter -1 ) was less than half that of the control (18.6 mmoles liter -1 ) after 72 h.

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59 Previous s tudies ha ve shown t hat the r eversib le phospho transac ety lase-a cetate kinase pathway can serve as a route for entry of added acetate into the intracellular pool of acety l-CoA (Brown et al. 1977; Higgins and Johnson 1970). Additional acetate uptake activity may be provid ed by the induc ible ace tylCoA sy nthetase althoug h this ge ne is typically repressed under fermentative conditions (Kumar i et al. 1995). Thus, the stimulation of growth and ethanol production by added acetate is presumed to result from the increased availability of acety l-CoA. Under anaerobic conditions, the primary role of the TCA pathway is to supply carbon skeletons for biosy nthesis. Increasing the availability of ace tyl-CoA would promote biosy nthesis by relieving the NADH-mediated alloster ic inhibiti on of citr ate sy nthase ( Weitzman 198 1) and by serving as an all osteric activator of phosphoenolpy ruvate carboxyla se (Izui et al. 1981). Stimulation of Growt h and Etha nol Produc tion by Added Py ruvate Ca n Be Prima rily Attributed to Increased A cetate Production. The stimula tion of g rowth an d ethanol producti on by pyr uvate re ported pr eviously (Chapter 2) appeared quite similar to the effe cts of added acetate (Fig 3-3A and B). Analysis of products during fermentation provided further evidence of a related mechanism of action for acetate and py ruvate (Fig. 3-4 A-E). With the exception of formate (Fig. 3-4B), pr ofiles of organic acids were similar for acetate and pyruvatesupplemented cultures. Both were distinctive from the control lacking supplements. Control fermentations produced lower levels of lactate than pyruvatesupplemented and acetate-supplemented fer mentations during the initial 72 h (Fig. 3-4C). Addition of py ruvate stimulated the production of acetate to levels equivalent to that of acetate-supplemented fermentations (F ig. 3-4A). In both py ruvate and

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60 acetat e-supple mented f ermenta tions, ac etate c oncentr ations we re appr oximately 2-fold higher than in the control after 36 h. Ace tate concentrations in all fermentations remained relatively consta nt during further incubation. Most of the suppleme ntal py ruvate ( 22 mM) was metabolize d during the initia l 3 h of incubation (Fig. 3-5) although the be nefits for growth and ethanol production persisted throughout fermentation. During the initial 3 h, the larg est change was an increase in acetate (Fig 3-6A). Smaller pyr uvate-dependent increases wer e observed for ethanol, formate lactate and ace taldehy de. Bios ynt hetic ne eds wer e estimat ed to be sm all (increase of approximately 0.06 mg dry c ell weight liter -1 ) and did n ot repre sent a significant sink for the added py ruvate (2 g liter -1 ). The partitioning of py ruvate between these different fermentation products (and biosy nthesis) in KO11 is generally regarded as the resu lt of 5 comp eting r eaction s: py ruvate d ecarbo xyla se (PDC), pyr uvate formate-lya se (PFL), py ruvate dehy drogenase (PDH), lactate de hydrog enase (LDH) and phosphoenolpyruvate c arboxylase (PPC). On a triose basis, re lative activities can be estimated from the distribution of fermentation products (Fig. 3-1; de G raef et al. 1999). The larg e py ruvatedepende nt incre ase in ac etate a fter 3 h ( Fig. 36A) ref lects an increa se in acetyl-CoA produc tion (PFL and PDH activities). I n the absence of formate hy drogen lyase induction (B ock and Sawers 1996), formate production provides an inde pendent measure of PFL activity and exhibit ed a mode st increa se compa red to ac etate. T hese results indicate that PDH activity (e stimated as acetate minus formate) serves as the prima ry source o f addi tion al acet yl-CoA du ring th e meta boli sm of a dded p yruvate (F ig. 3-6B). Productio n of etha nol also in crease d immediat ely after th e additio n of py ruvate due to an i ncreas e in the pr oduction o f aceta ldehy de by PDC. I ncreas ed py ruvate

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61 oxidation by PDH is presumed to provide the additional NADH re quired to reduce acetaldehy de produced from added py ruvate. The increase in L DH activity (estimated a s lactate production) can be attributed to substrate ac tivation (Tarmy and Ka plan 1968). Elevated extracellular levels of acetate in py ruvate-supplemented fermentations may serve to increase intracellular ac etyl-CoA pools, extending the per iod of growth and thereby incr easing the volumetric rate of ethanol produc tion. The cha nneling of py ruvate t o acety l-CoA and acetat e by the addit ion of py ruvate can be readily explained based on known allosteric controls (Fig. 3-1). Py ruvate is both a substrate for acety l-CoA production and a strong allosteric activator of phosphotr ansace tyla se (Suzuki 1 969). Add ition of py ruvate h as also be en shown t o increase acetaldehy de and decrease the level of NA DH (Chapter 2), an allosteric inhibitor of phosphotransacety lase (Suzuki 1969) and PDH (de Graef et al. 1999; Hansen and Henning 1966). Th ese act ions would also tend to incre ase the p artitioni ng of c arbon int o acetate. Higher levels of succinate and fuma rate (3-fold to over 10-fold, respectively ) were produce d by acetat eand py ruvatesuppleme nted fer mentation s (Fig. 3-4C and D ). PPC (Izui et al. 1981) and citrate sy nthase (Weitzman 1981) are both activated by acetyl-CoA and link the supply of this important intermediate to fer mentation and biosynthesis. Under anaerobic conditions, the reductive portion of the TCA pathway is used to produce succinate. Due to the deletion of fumarate re ductase ( frd ) in KO11, little succinate was produced and a small amount of fumarate acc umulated. The increases in succinate and fumarate levels in acetate and py ruvate-supplemented fermentations may result from an excess of citrate. Excess citrate can be cleave d into acetate and oxaloacetate by an

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62 inducible citrate lya se (Lutgens a nd Gottschalk 1980). Additional succinate can be produced from isocitrate by isocitrate lyase ( Weitzman 1981). Pyruvate and fre e CoA a re co-s ubst rates for for mate p roduc tion by PFL (Fig. 3-1). Formate levels increased during the initial 12 h of incubation in all fermentations and declined thereafter (F ig. 3-4B). The decline in formate can be attributed to the formate -inducib le forma te hy droge n ly ase (B ock and Sa wers 199 6). Supple menting with acetate and py ruvate had opposite effects on formate produc tion (Fig. 3-4B), higher concen trations i n py ruvatesuppleme nted fer mentation s and lowe r levels i n acetat e-supple mented f ermenta tions in co mparison to those of the contr ol. Both differe nces ar e in gen eral ag reemen t with the c entral r ole of ac ety l-CoA in me tabolism (Ch ang et a l. 199 9b; Co nt ier o et al. 200 0; Kir kpa tri ck e t al 20 01) In acetate-supplemented fermentations, formate production by PFL may be limited by a lack of free CoA. Conversely higher formate levels produced by pyruvatesupplemented fermentations may r esult from an increase in free CoA due to the a llosteric activation of phosphotransacety lase by py ruvate (Suzuki 1969). Stimulation of Growt h and Etha nol Produc tion by Acetald ehy de Can B e Attribu ted to Increased A cetyl-CoA. Growth and ethanol production were also stimulated by acetaldehy de (Chapter 2; Fig 3-3A and B). At conce ntrations above 5.6 mM, acetaldehy de strongly inhibited growth It w as empiri cally determin ed that sti mulation e quivalen t to that of pyr uvate could be a chieved by t he additi on of 11.2 mM aceta ldehy de, 5.6 mM i nitially and 5.6 mM after 12 h of fermentation (Chapter 2). Previous studies also demonstra ted that the

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63 addition of acetaldehy de caused a rapid decrea se in the intracellular concentration of NADH (Chapter 2). The in itia l port ion o f added of acet aldeh yde was met aboli zed wi thin 3 h (Fi g. 3-5A). During this time, ethanol increased by an amount equal to 70% of the added acetaldehy de (Fig. 3-6A). I ncreased py ruvate flux through PDH appears to provide the additional NADH required for acetalde hyde reduc tion (Fig. 3-6B). The second acetaldehy de addition was metabolized within 1 h (Fig. 3-5A) although bene fits persisted throughout fermentation (Fig. 3-3A a nd B). Following the second addition, production of acetate and ethanol was increa sed while formate production was reduced. The persisting benefit of acet aldehy de additi ons for g rowth an d ethanol producti on are pr esumed to result from an increase in the intracellular a cetyl-CoA pool as a c onsequence of higher extracellular levels of acetate. High le vels of NADH and global reg ulation by ArcA a nd FNR (de Graef e t al. 1999) may also limit PD H functio n in the ab sence of suppleme nts. Increased pr oduction of acety l-CoA by PDH (and per haps increased sy nthesis of PDH) would be expected in response to NADH oxidation. The production of formate by PFL may be limited by competition with PDH for free CoA. Patterns of organic ac id production in acetaldehy de-supplemented cultures provide f urther s upport fo r a mech anism of a ction simila r to that f or py ruvate a nd aceta te (Fig. 3A-E). Acetate le vels were higher in all three supplemented c ultures than in the unsupplemented control. Each supplemented fermentation also produce d higher levels of succina te, lacta te and fu marate t han the c ontrol.

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64 Stimulation of Growt h and Etha nol Produc tion by Ina ctivation of Non-b iosy nthetic Pathways Which Consume Acety l-CoA. Acetyl-CoA ser ves as the single most important intermediate for cellular biosy nthesis, p roviding over hal f of the c ellular c arbon du ring sug ar metab olism (Neidhardt et al. 1990). Previous studies have shown that cell gr owth is limited by the availability of car bon skeletons during the fermentation of xy lose (Chapter 2), a limitation which was relieved (Fig. 3-1A and B) by supplements which increase the extracellular levels of acetate (acetate, py ruvte, acetaldehy de). During fermentation (Fig 3-1), two pathway s drain a cety l-CoA fro m the intra cellular pool but pr ovide limit ed bene fit to biosynthesis. Acety l-CoA can be reduced to acetaldehy de and ethanol by alcohol dehydrog enase E ( adhE ) as an alternative route for NADH oxidation in KO11 (Fig 3-1). Acetyl-CoA c an also be converted to acetate by phosphotransacety lase ( pta ) and ac etate kinase ( ackA ), increasing the production of ATP. Mutations in these pathway s were investig ated as a means of sparing acety l-CoA for biosy nthetic n eeds. Inactivation of ackA rather than pta was chos en to minimi ze potentia l problems associated with global regulation. Ace tyl-P is proposed to serve as a n important global regulator in E. coli (Bouche et al. 1998; Kirkpatrick et al. 2001; McCleary et al. 1993), affec ting ge ne expres sion and f undament al proce sses such as the tur nover of RpoS. Du ri ng ox id at iv e m et ab ol is m, in ac ti va ti on of th e a ce ta te pa th wa y ( pta, ackA ) is detrimental to growth (Chang et al. 1999b; Contiero et al. 2000; Kirkpatr ick et al. 2001). Although not fully under stood, this detrimental effect has been attributed to depletion of free CoA due to low rates of acety l-CoA turnover (Chang et al. 1999b). I n contrast to that found in previous studies concerning oxidative metabolism, inactivation of ackA (SU102)

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65 stimulated growth and ethanol production during the fer mentation of xylose (Fig. 33C and D). An adhE mutation in strain KO 11 (SU104) was of no benefit during xy lose fermen tation. To gether these r esults sug gest tha t ADH con tributes l ittle to met abolism in KO11. The ben efici al effe ct of i nacti vatin g ackA is presume d to resul t from an i ncreas e in the availability of ac etyl-CoA for biosy nthesis, the genetic equivalent of adding a cetate, pyruvate, or acetaldehy de. Strains SU104 ( adhE mutant) and SU102 ( ackA mutant) w ere als o tested in fermentations with supplements that had been shown to increase the g rowth and ethanol producti on in KO11 (Table 3 -2). Add ition of ac etate, p yr uvate an d aceta ldehy de to SU104 increased growth and ethanol produc tion indicating that the native alcohol dehydrog enase ( adhE ) was not essential for this response. Growth and ethanol production by SU102 ( ackA ) without s upplemen ts were e quivalen t to that of KO11 with supplements. The addition of pyruva te, acetate, 2-ketoglutarate or acetaldehy de to SU102 provided little further improvement in growth or ethanol production. HPLC a naly sis of org anic ac ids reve aled simil arities i n the patt erns of f umarate (Fig. 3-3J) and succinate (Fig 3-3I) production betwee n SU102 ( ackA mutant) and KO11 supplemented with acetate, py ruvate or acetaldehy de (Fig. 3-4E). The ackA mutation in SU102 also increa sed lact ate prod uction (F ig. 3-4I ) and del ay ed the pr oduction o f format e (Fig. 3-4G) a nd aceta te (Fig 3-4F) The del ay in format e produc tion in SU10 2 could res ul t fr om in cre ase d ac etyl -Co A, r edu cin g th e po ol of f ree Co A (c o-s ubs tra te f or P FL) analogous to acetate-supplemented KO11 ( Fig. 3-4B). Both ace tate addition and mutations in the acetate pathway have been shown to cause a similar repre ssion of 37 genes (Kirkpatrick et al. 2001), attributed to a n increase in the acety l-CoA pool.

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66 Ina ctivation of acet ate kina se (SU102 ) cause d an initia l delay in aceta te producti on but did no t block la ter sy nthesis. T he pathw ay responsi ble for a cetate production during the latter stages of fe rmentation remains unknown but may be the result of spontane ous depho sphory lation of a cety l-P as pre viously proposed (Brown et al. 1977) or from induction of cry ptic enzyme(s). Despite the potential benefit of increased ATP pr oduct ion b y acetate k inase the i ncreas ed drai n of ace tyl-CoA to acet ate th rough this pathway appea rs to be more detrimental for growth and etha nol production by KO11 than the reduction in ATP. With the exception of acetate (Fig. 3-4F ), the production of fermentation products by the adhE mutant (strain SU104) was essentially the same as for the parent strain, KO11 (Fig. 3-4A) Acetate production by SU104 continued throughout fermentation and reached highe r final concentrations than KO11. Conclusions Increasing the availability of ac etyl-CoA stimulated gr owth and ethanol production from xylose by prolonging the growth phase of ethanologenic E. coli The resulting increa se in bioc ataly st rather than an in crease in cellula r activit y w as respo nsible for the increased rate of etha nol production (Table 3-2). Similar benefits were obtained by minimizing the loss of acetyl-CoA as ac etate ( ackA mutation) and by incre asing intracellular levels of acetate (supplementing with acetate, py ruvate, or acetaldehy de). Inactivation of the native E. coli alcohol/aldehyde dehydrog enase ( adhE ) had little effect indicating that this pathway has limited function in ethanologenic KO11. ATP production during xylose fer mentation does not appear to limit growth or cell y ield in KO 11. I ncluding the ener gy require d for xy lose upta ke and a ctivation less than 1 ATP (net) is produced from the metabolism of each xy lose converted to ethanol

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67 (Tao et al. 2001; Chapter 2). During the initial 12 hours of g rowth, up to 31% of the ATP (net) produ ced by KO11 is pro vided by the acet ate pat hway (calcu lated by assumi ng 1 ATP per acetate from acetate kinase and 0.4 ATP per py ruvate from gly colysis). Di sr up ti on of th is pa th wa y ( ackA ) in SU102 increased cell y ield by 2-fold (Ta ble 3-2). Thus, the partition ing of c arbon ske letons ra ther tha n the prod uction of ATP appe ars to limit the growth of ethanologenic E. coli during xylose fer mentation. The mechanism for the stimulation of growth in ethanologenic E. coli KO11 is consistent with established patterns of allosteric regulation althoug h further controls of gene e xpression ( FNR, Arc A) may also cont ribute to t he obser ved eff ects. Mor e than ha lf the am ino ac ids p roduc ed in t he cell are der ived f rom th e TCA p athwa y. Flux t hrough this pathway is controlled by PPC and citrate synthase (L ee et al. 1994; Walsh and Koshland, Jr. 1985), activities which can be stimulated by acetyl-CoA ( Weitzman 1981). The individual addition of pyruvate, a cetate, and acetaldehy de increased the extracellular levels of acetate which can in turn serve to elevate intracellular pools of acety l-CoA by reversible reactions. Additional benefits of supplements include a re duction in the level of NADH (a dded py ruvate a nd aceta ldehy de), an a llosteric inhibitor o f citrat e sy nthase which is a ntagon ized by high lev els of ac ety l-CoA. B ased on th ese res ults with mut ants and with supplements, we conclude that the regulation of a cetyl-CoA produc tion and consumption can be used to make small changes in the partitioning of carbon between bi os ynt he si s a nd fe rm en ta ti on du ri ng th e e th an ol pr od uc ti on by E. coli KO11.

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68 Table 3-1. Strains and plasmids used in Chapter 3. Strain o r Plas mid Relevant Character ist ics Reference or source Strains KO11 frd cat p fl + pfl:: ( Z. m obilis pdc + adhB + ) Ohta et al. 1991 SU102 KO11 ackA This work SU104 KO11 adhE This work W3110 wild t ype ATCC 27325 WA837 r Bm B+ gal met Wood 1966 Plasmi ds pKD46 ( $ exo repA 101 pSC101 repl icon ts (red recombin ase) Datse nko and Wanner 2000 pFT -A bla flp pSC101 repl icon ts (FLP recombinase) Posfa i et al. 1997 pCR2. 1TOPO bla kan Col E1 Invit rogen pKNO CKTc tet R6K ( pir dependent replicon) Alexe yev 1999 pSG 76-K kan FRT R6K ( pir dependent replicon) Posfa i et al. 1997 pSG 76-A bla FRT R6K ( pir dependent replicon) Posfa i et al. 1997 pLOI2065 bla FRT tet FRT Col E1 This work pLOI2224 kan R6K ( pir dependent replicon) Marti nezMorales 1999 pLOI2302 bla ColE1 ( Eco RI flank ed b y Asc I sites) Zhou an d Ingr am 1999 pLOI2375 ack A:: FRTte tFRT kan R6K ( pir dependent replicon) This work pLOI2803 adhE: :FRT -t et -FRT kan Col E1 This work

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Table 3-2. Eff ects of mu tations an d additive s on cell y ield and e thanol pr oductivity Cell M ass Ethan ol Strain and additive Concent rat ion ( mM) N Maximum (g li ter -1 ) Time (h) : a (h -1 ) Maximum ( mM) Max VP b (mmol li ter -1 h -1 ) Sp. Prod. c (mm ol g -1 h -1 ) Theoretical Yie ld d ( %) KO11 none 44 0.95 0. 13 72 0.63 641 93 8.3 10.1 63 + pyru vate 21 18 2.00 0. 18 72 0.76 955 55 18.3 11.5 94 + ace tate 24 2 1.92 0. 01 48 0.75 930 11 18.7 12.0 92 + ace tate 19 6 1.61 0. 30 72 ND f 901 118 ND ND 89 + acet aldeh yde e 11 6 1.51 0. 15 96 0.47 909 98 18.2 11.3 90 + 2-ke toglutar ate 12 6 1.84 0. 20 72 ND 907 ND ND 90 SU102 none 8 1.94 0. 14 48 0.66 946 20 17 13.7 93 + pyru vate 21 4 1.93 0. 13 48 0.44 926 19 17.4 10.0 92 + ace tate 19 2 2.24 0. 17 48 ND 933 26 ND ND 92 + acet aldeh yde 11 2 2.02 0. 17 48 ND 952 6 ND ND 94 + 2-ke toglutar ate 12 2 1.83 0. 01 96 ND 901 1 ND ND 89 SU104 none 8 1.02 0. 08 96 0.71 550 89 8.9 13.7 55 + pyru vate 21 2 1.96 0. 06 48 ND 885 24 ND ND 87 + ace tate 19 2 1.68 0. 02 48 ND 889 10 ND ND 88 + acet aldeh yde 11 2 1.57 0. 31 48 ND 856 100 ND ND 84 + 2-ke toglutar ate 12 2 1.97 0. 15 48 ND 971 20 ND ND 96 a Specific growth rat e at 2 h. b VP, maximum volumetric productivit y, mM ethanol produced per liter per hour. c Specific productivit y at 12 h, mmol ethanol produced per gram cell dry weight per hour. d Theoretical yiel d from 91g li ter -1 xylose (1.667 mmol ethanol/mmol xylose). e Half added initial ly, hal f added after 12 h. f ND not de termin ed. E stimates o f specific a nd vo lumetr ic pro duc tivity were n ot calc ulated due to the limited numb er of sa mplin g times.

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70 Figure 3-1. Allosteric control of central metabolism. Unless noted otherwise, enzy mes listed ar e native to E. coli Enzymes: 1. py ruvate kinase ( pykA, pykF ); 2. py ruvate formate-lya se ( pflB ); 3. pyruvate de hydrog enase ( aceEF,lpd ); 4. phosphotransacety lase ( pta ); 5. acetate kinase ( ackA ); 6. alcohol/aldehyde dehydrog enase ( adhE ); 7. Z. mobilis pyruvate de carboxylase ( pdc ); 8. Z. mobilis alc oho l d ehyd rog ena se II ( adhB ); 9. lactate dehy drogenase ( ldhA ); 10. phosphoenolpyruvate c arboxylase ( ppc ); 11. citrate synthase ( gltA ); 12. ac onitase ( acn ); 13. isocitrate dehydr ogenase ( icd ); 14. glutamate dehy drogenase ( gdhA ); 15. glutamine syntheta se ( glnA ); 16. malate dehydr ogenase ( mdh ); 17. fum arase ( fumB ); 18. fumarate reductase ( frdABCD ); 19. aspartate transaminase ( aspA ); 20. aspartase ( aspC ). r indicates allosteric activation, s indicates allosteric inhibition.

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71 Figure 3-2. Plasmids used to construct mutations in KO11. FRT sites allow in vivo excision of the tet gene after integr ation using FLP recombinase ( flp ). A. Plas mid pLOI 2065 containing a tet ge ne fl an ke d b y FRT sites. B. Plasmid pLOI 2375 containing an interrupted ackA gene.

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72 Figure 3-3. Effect of media additions and mutations on growth (A, C) and e thanol producti on (B, D ). Sy mbols for A and B:  KO11 no a dditive; ) KO11+ pyr uvate;  KO11 + acetate; and ” KO11+ acetaldehy de. Symbols for C and D:  KO11 no a dditive; ” SU102 ( ackA ) no addit ive; ) SU104 ( adhE ) no additive; and  SU102 + pyruvate.

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73 Figure 3-4. Eff ect of me dia addit ions and mu tations on organ ic acid p roductio n: aceta te (A/F), formate (B/G), lactate (C/H), fu marate ( D/I) and succ inate (E /J). Sy mbols for A-E : KO11 no a dditive; ) KO11+ p yr uvate; KO11 + acetate; and KO11+ a cetalde hyd e. Sy mbols for F -J: KO11 no a dditive; SU102 no additive ; ) SU104 no additive; and SU102 + pyruvate.

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74 Figure 3-4. continued.

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75 Figure 3-5. Metabolism of added acetaldehy de and pyruva te during fermentation. Pyr uvate ad dition is ind icated b y th e larg e arrow Aceta ldehy de additi ons (5.6 mM each) are indicated by the large arrow (initial addition) and the small arr ow (second addition at 12 h). A. Utilization of added py ruvate and acetaldehy de. Symb ols: ) py ruvate u tilization by KO11; py ruvate u tilization by SU102; acetal dehy de utilizat ion by KO11; acetaldehy de in KO11 broth with no additions; and pyruvate in KO11 br oth with no additions. B. Effect of second acetal dehy de additi on on prod uction of fermen tation pro ducts by KO11. Sy mbols: cell mas s; ethanol ; formate ; lactate ; ) aceta te; t succinate; and fumarate.

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76 Figure 3-6. Partitioning of carbon among competing pathways during the initial 3 h of fermentation. A. Fermentation products after 3 h. B Relative activity of primar y enzymes that partition 3-car bon intermediates carbon (py ruvate and phosphoenolpyruvate) through competing pathway s. Relative activities were estimate d using f ermenta tion produ cts, expre ssed as : mol of pro duct per ml during t he initial 3 h of incu bation. En dogeno us produc tion of ac etate in acetate-supplemented fermentations was a ssumed to be equal to that for the control fermentation without additives. Pyruva te decarboxylase activity was assumed to be equal to ethanol production, except for the acetaldehy de-supplemented fermentations where it could not be ca lculated. Pyruvate dehy drogenase was calculate d as the difference between a cetate and formate production. Lac tate dehydrog enase, pyr uvate formately ase, and phosphoenolpyruvate c arboxylase activities wer e assumed to equal to the producti on of lac tate, fo rmate, a nd succin ate, re spective ly. Abbrevi ations: PPC, phosphoenolpyruvate c arboxylase; PPS, phosphoenolpy ruvate syntheta se; LDH, lactate dehy droge nase; PF L, py ruvate f ormately ase; PDH, pyr uvate dehydrog enase; PDC, pyruvate decarboxylase.

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77 CHAPTER 4 A DEFICI T IN PROTECTI VE OSMOLYTES I S RESPONSIBLE F OR THE DECREASED GROWTH AN D ETHANO L PROD UCTI ON DURI NG XYL OSE FERMENTATION Introduction Maintaini ng inexpe nsive sour ces of f uels and c ommodity chemica ls for the U. S. is a matter of national security Increasing the production of fuel ethanol offers a potential solution to this problem. The conversion of lignocellulose to fuel ethanol and other chemicals typica lly derived from pe troleum would decrease the U. S. dependance on imported oil (Artzen and Dale 1999). Enteric bacteria a re noted for their broad rang e of growth substrates, including all the sugar s present in the polymers of lignocellulose. Escheric hia coli a microbial platform for the commercial production of amino ac ids and recombi nant prot eins (Chot ani et al. 2000; Ake sson et al 2001), w as previ ously engineered for the produc tion ethanol by integr ating a sy nthetic operon containing the ethanol p athway from Z. mobilis ( pdc and adhB ) into the chromosome (Ohta et al. 1991). The resultant strain, designated KO11, fer mented all the sugar constituents of lignoce llulose to e thanol wit h yi elds appr oaching 100% (Oh ta et al. 1 991; Mart inez et al. 1999; Ingram et a l. 1999). During batch fermentations with strain KO11, the volumetric ra te of ethanol production was directly related to the growth of the biocataly st (Martinez et al. 1999; Chapter 2 ; Chapter 3). Cell y ield for b oth the et hanolog enic stra in and its p arent ( E. coli B) was dependent upon the availability of nutrients in a variety of media, despite the

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78 absence of any specific auxotrophic requirements. During the batch f ermentation of xylose (90 g liter -1 ) to ethanol by strain KO11, c ell growth appeared to be limited by the availab ility of carb on skelet ons deriv ed from th e citrat e arm of the anae robic TCA pathway (Cha pter 2; Chapter 3) (Fig. 4-1). During fermentative metabolism, the TCA pathway is interrupted by the repression of 2-ketoglutarate dehy drogenase (I uchi and Lin 1988). The ultimate product of this pa thway 2-ketog lutarate is a subst rate fo r gluta mate biosy nthesis. D uring g rowth in minimal media, glutamate is the most abundant free amino acid in the cy toplasm of E. coli (Cay ley et al. 199 1). I n addition to its role s in metabo lism and pr otein sy nthesis, glutamate biosynthe sis is part of the primary r esponse to osmotic stress (Csonka 1989; Csonka and Hanson 1991). The high osmolarity of the medium used for ethanol production (0.6 M xylose) would be expected to incre ase the requirement for glutamate a s a protective osmolyte. Po ta ss iu m i on s a re ra pi dl y ac cu mu la te d b y E. coli in respon se to osmoti c stress. This is rapidly followed by the accumulation of glutamate (McL aggan et al. 1994), a negatively charged amino acid and protec tive osmolyte, to balance the positive c harge of the acc umulated potassium. In the closely related organ ism Salmonella typhimurium cells tha t are re stricted in their a bility to sy nthesize g lutamate have be en demons trated to grow poorly during osmotic stress (Csonka 1988; Yan et al. 1996). Mutations preventing glutamate production were associated w ith the inability to balance the cha rge of intracellular potassium. The resulting decrea se in steady-state potassium levels has been proposed to limit cell growth (Yan et al. 1996).

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79 Alternate protective osmoly tes such as gly cine betaine (hereafter r eferred to as betaine), proline, taurine, dimethy lsulfoniopropionate and many other s can be accumulated from the environment. A hierarc hy for these osmoprote ctants was empirically deter mined for salt stress (Randall et al. 1995). Although there have been conflicting reports concerning this hierarchy f or sugar-mediated osmotic stress (Glaasker et al. 1998), betaine is generally regarded as the most effe ctive protective osmolyte f or E. coli. The eff ectiven ess of be taine fo r restor ation of g rowth ha s been sho wn to car y w ith the sugar used for osmotic stress (Dulaney et al. 1968).. In this study NMR was used to examine changes in the intracellular pools of osmoly tes in res ponse to g enetic c hange s and nutr ient suppl ements tha t stimulate d cell growth and ethanol production. L ow cell yield and low e thanol production in the absence of supplements appears to result from a deficit in intracellular g lutamate or alternative protect ive osmoly tes. Materials and Methods Microorganisms and Media. The ethanologenic E. coli strains KO11 and SU102 (KO11 ) ackA ; Chapter 3) are prototrophic. Working cultures were transfer red daily on solid medium (1.5% ag ar) containing mineral salts, 2% xylose, a nd 1% corn steep liquor (CSL+X medium; Chapter 2) alternating between 40 mg liter -1 and 600 mg liter -1 chloramphenicol. Stock cultures were sto red fro zen at -70 C in 40% g lyc erol. Fermentation. Seed cultures (35C and 120 rpm) and fermentations (35C and 100 rpm) we re grown i n either mineral s alts mediu m contain ing 1% c orn stee p liquor a nd 9% xy lose

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80 (Chapter 3) or Luria br oth (10 g liter -1 tryptone, 5 g liter -1 yeast extract, 5 g liter -1 NaCl ) with 9% xylose. For ferme ntations, sufficient cell mass to achieve and initial concentration of 33 mg liter -1 were harvested by centrifugation (5000 x g; 5 min) and suspended in appropriate fresh medium. Fermenta tions were maintained at pH 6.5 by automatic addition of 2N KOH (Moniruzzaman and Ing ram 1998). Stock solutions (100 mM) of betaine (Sigma, St. Louis, MO) and dimethy lsulfoniopropionate (TCI America, Portland, OR) were dissolved in deionized water and filter sterilized directly into the fermentation vessel. Glutamate and acetate w ere added as described previously (Chapter 2; Chapter 3). 13 C NMR. Intracellular osmoly tes were analy zed by NMR (Park et al. 1997). Af ter a 24 h incubation in the fermentation chamber, 700 mL culture was harvested by centrifugation, washed twice in mineral salt solution containing NaCl (0.6 M) and resuspende d in 3 volumes of ethanol (95%). Suspensions were rocked g ently 16-24 hr at 4C. Cellular debris was removed by centrifugation (4 / C, 10,000 x g, 30 min). Extracts were dried under vacuum, disloved in deionized water, dried under vacuum, re suspended in 33% D 2 O and filtered (0.2 : m) Acetone (10 : L) was used as an inter nal reference. Data wa s obtained using a modified Nicolet NT300 spectrometer ope rating in the Fourier transform mode as f ollows: 75. 46 MHz; excit ation puls e width, 2 5 us; pulse repetiti on delay 40s; spectra l width 18 kH z and broa dband (b i-level) decoupli ng of pr otons. Fo r cell extr acts, at least 1000 scans were obtained.

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81 Analy tical Met hods. Cell mass was determined from the optical density at 550 nm using a Bausch & Lomb Spectronic 70 spectrophotometer ( 1 OD 550 = 0.33 g liter -1 dry cell we ight), and ethanol was measured by gas chromatography (Varian 3400CX) (Moniruzzaman. and Ingram 1998). F or the quantitation of the compounds detected by NMR, a standard curve was ge nerate d. The av erage of all the chemica l shift val ues are reporte d normalize d to cell mass Results and Discussion Citrate Synthase Flux L imits the Biosynthesis of Glutamate, a Primary Intracellular Osmolyte. E. coli typically maintains large cy toplasmic pools of potassium, glutamate and trehalose during growth in media conta ining high concentrations of suga rs or salt (Cayley et al. 1991; Lewis et al. 1990). Previous studies with the ethanolog enic E. coli strain KO11 demo nstrated that the f lux throug h citrate syn thase, th e first st ep in glu tamate biosynthesis, limited growth and etha nol production during the fermentation of 90 g liter -1 xylose (0.6 M) to ethanol (Chapter 2; Chapter 3) This limitation was proposed to be due to the dra in of py ruvate t o ethanol by t he reco mbinant pa thway (py ruvate decarboxylasealcohol dehydrog enase) which has a higher a ffinity for py ruvate than pyruvate f ormate-lya se or pyruva te dehydrog enase, competing pathway s for acey l-CoA biosy nthesis (C hapter 2 ; Chapter 3). Supple menting the CSL +X medium with ace tate increa sed the a vailabili ty o f acety l-CoA (Cha pter 3), an activ ator of c itrate sy nthase (Weitzman 1966). During fermentations with strain KO11, supplementing the CSL +X

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82 medium with glutamate increased the final cell y ield and ethanol productivity (Chapte r 2) by by passing the growth-limiting citrate sy nthase. The intracellular osmoly te pools were compared between conditions of lower growth (CSL+ X medium w ithout add itives) a nd highe r grow th (supple mented wi th glutama te or ac etate) t o investig ate whe ther the inability of strain KO11 to ac cumulate glutamate resulted from restricted citra te synthase flux (Fig 4-2; Table 4-1). Fermentations without additives accumulated only proline, while those supplemented with acetate (activating citrate sy nthase) or glutamate (by passing citrate sy nthase) resulted in approxima tely 2-fold hi gher g rowth an d ethanol producti vity Cells fr om these fermentations accumulated approximately the same level of intracellular glutamate, supportin g the hy pothesis th at a def icit in the accumul ation of th is protec tive osmoly te limited growth. The intracellular proline, a known osmoprotectant (Csonka 1989;Csonka and Hanson 1991), was likely a ccumulated from the CSL provided as a source of complex nutri ents. Though some o rganis ms synth esiz e prol ine in respo nse to osmo tic ch allen ge (Kawahara et al. 1989), E. coli can only ac cumulate this protective osmolyte by active transport (Smith et al. 1984). However, mutants have been isolated that are less sensitive to the feedback-inhibition of the proline biosy nthetic pathway ( Smith 1985). Strains expressing these genes accumulate d high levels of intracellular proline (derived f rom glutamate) and were more r esistant to high osmotic environments (Csonka 1981; Csonka et al. 198 8). Presu mably the intra cellular proline in strain KO 11 may have re sulted fr om a similar spontaneous mutation. Accordingly such a mutation would also reduce the glutamate pool.

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83 Glutamate was accumulated in fermentations with increa sed growth y ield, and the intracellular concentration of proline decr eased (Table 4-1). This further supports the hyp othesis tha t the intra cellular proline w as taken up from th e medium a nd was not a result of biosynthesis. I f the drain of intracellular glutamate f or proline production had starved the cells for glutamate, supplementing the medium with proline should have a sparing effect on the consumption of g lutamate. However, proline addition only increased the intra cellular proline p ool without affec ting eith er gro wth or eth anol prod uction (T able 4-1). Glutamate is a product of proline degrada tion (McFall and Newman 1996), but the deg radatio n of proli ne has be en shown t o be inhibi ted in med ia of hig h osmotic streng th (Csonka 1988). Sup plementin g the me dium with a n excess of proline ( 17 mM) should provide excess proline for glutamate production. However the absence of increased growth and intrace llular glutamate in these fermentations confirms the previously observe d inhibition of proline degradation during osmotic stress (Csonka 1988). The acc umulation o f proline was pre viously shown not t o affec t glutama te pools (Cay ley et al. 199 2). Durin g experi ments in a g lucosemineral s alts mediu m buffer ed with MO PS an d h ig h N aC l, th e p ri ma ry o sm ol yte s a cc um ul at ed by E. coli were K + glutamate, MOPS and trehalose (Cayle y et al. 1991; Cay ley et al. 1992; L ewis et al. 1990). Cultures in this medium supplemented with proline accumulated this protective osmolyte and reduced the biosy nthesis of trehalose (Cay ley et al. 1992). How ever, the intracellular glutamate concentration was not significa ntly altered by the accumulation of proline.

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84 Thus, the intracellular accumulation of proline (from the CSL ) by strain KO11 should not have af fected the glut amate re quireme nt. Strain KO 11 faile d to sy nthesize d etecta ble leve ls of the o smoprote ctant tre halose (<10 mM) under these conditions. While the presence of proline would have decreased the synthesis of trehalose (Cayley et al. 1992), significant trehalose should have bee n detected. This may be a result of growth on xy lose, a pentose. Perhaps the relatively low ATP y ield from xylos e catab olism (0.4 A TP/py ruvate) restric ts the gl uconeog enic producti on of glu cose and uridine d iphosphat e-gluc ose, subst rates fo r treha lose biosynthesis. Genetic Change s to Optimize Carbon Pa rtitioning Inc reased the Gluta mate Pool. The functional expression of citZ by s train KO 11 (pL OI25 14) was p reviousl y shown to in crease growth and etha nol produc tion. To co nfirm tha t the expre ssion of th is enzyme aides in g lutamate accumulation, the intracellular osmoly te pool during fermentations in the CSL+X medium were a nalyzed (Fig 4-2; Table 4-1). Similar to the fermentations supplemented with glutamate or aceta te, cells from these fermentations had an increased glutamate pool. The intrac ellular accumulation of proline was similar to that of other experiments with increased growth y ields. Thus, the expression of citZ provided more citr ate, ultim ately increa sing g lutamate biosy nthesis a nd the g lutamate pool. A m ut at io n i n t he pr im ar y ac et at e p ro du ct io n p at hw ay ( ) pta ) was pre viously shown to increase glutamate biosy nthesis (Chang et al. 1999b). This likely resulted from the accumulation of acety l-CoA, an activator of citrate sy nthase (Weitzman 1966). Acetyl-CoA is also an a ctivator of phosphoenolpyr uvate carboxylase ( ppc ) (I zui et al. 1981), the controlli ng step i n the biosy nthesis of oxaloace tate and co-subst rate fo r citrat e

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85 synthase. Thus, the biosy nthesis of citrate is regulated, in part, by the availability of acetyl-CoA. I n an analogous study presented here, blocking ace tate production ( ) ack ) increased the intracellular glutama te pool level (Fig. 4-2, Table 4-1). Glutamate Accumulation Functions in Osmoprotection. Three different osmoprotectants were tested for their ability to re store growth and ethanol production, replacing the additional glutama te requirement.(Fig. 4-3). The addition o f 1.0 mM be taine or dimethy lsulfoniop ropionat e (DMSP) in crease d the cel l yield and ethanol produc tion similar to experiments where glutamate production had been increased. Taurine, a weak osmoprote ctant for E. coli (McLagg an and Epstein 1991), failed to increa se grow th or etha nol produc tion. Neit her beta ine nor pr esumably DMSP should provide a source of glutamate. Thus, supply ing osmoprotectants to the medium replaced the need for the ac cumulation of intracellular glutamate. To determine the optimal concentration required to restore growth, betaine and DMSP were added fr om 0.1-2.0 mM and 0.11.0 mM, re spective ly ( Fig. 44). Grow th and ethanol were increased in a dose -dependant manner in each instance. The maximum stimulation of growth and ethanol production by betaine was at the highest level of betaine tested, 2.0 mM. However, only 0.25 mM DMSP was required for maximum benefit. Surprisingly during the fermentation of xy lose to ethanol, DMSP is 10-fold more effective in restoring gr owth and ethanol production than betaine. Though this is contrary to previo us repor ts that bet aine is the most effe ctive pr otective osmoly te (Randa ll et al. 1995), the se studie s were d one using high lev els NaCl f or osmotic challen ge. The ability of betai ne to res tore gr owth has b een rep orted to v ary during o smotic cha llenge with

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86 different sugars (Dulaney et al. 1968). Thus, a specific protective osmoly te may be more effective during challeng e by diffe rent osmolytes (sug ars and salts). Replace ment of Gl utamate b y O ther Osmo protect ants. The intra cellular osmoly te pools of cells in f ermenta tions suppl emented with betaine and DMSP we re examine d by NMR. Cells f rom ferm entation s suppleme nted with betaine were fo und to con tain only detecta ble leve ls of beta ine (Fig 4-2), c onsistent w ith previous studies (Cayley et al. 1992). This was likely a result of the properties of the osmotically activated tra nsport pathways. While the K m of ProP and ProU for proline are 0.3 mM and 2 : M, respectively, ProU has a 1.3 : M K m for betaine (Luc ht and Bremer 1994). The high level of betaine in the medium (2.0mM) coupled with the low Km of the primary betaine transpor t pathway (ProU) e xplains the e xclusive a ccumula tion of this protect ive osmoly te. Additi onally the ProP sy stem has a peripla smic, hig h-affin ity betaine binding protein (K D 1 : M) which aids in the accumulation of this preferred substrate. Cells from the fermentations supplemented with 0.25 mM DMSP accumulated both proline and DMSP. There are three possible explanations for the contempora neous accumulation of proline and DMSP under these conditions. I t is possible that there is an independent transporter for DMSP. However, DMSP is structurally similar to betaine (Fig. 4-5), and it is likely transported by the sa me mechanism. Possibly, the accumulation of both proline and DMSP results from the K m for proline and DMSP being more similar to each other. Alternatively the low level of DMSP in the medium (0.25 mM) compared to the concentration of betaine used (2.0 mM) may have allowed for the proline (equal

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87 concentrations in both experiements) to more effectively compete for transport by ProP and/or ProU. Neither the beta ine nor D MSP suppleme nted fer mentation s contain ed detec table levels of glutamate (>10 mM; Fig 4-2) confirming that the high glutamate pool wa s not needed for biosy nthesis, p er se. Th us, the re quireme nt for ad ditional g lutamate is presume d to be ass ociated with adap tation to th e highe r sugar environm ent. This i s consistent with previous reports using strains of E. coli or S. typhimurium which were deficie nt in glut amate bio syn thesis (Mc Lag gan et a l. 1994; Cso nka et al 1994; Ya n et al. 1996). While the growth of these strains was poor in high osmotic environments, normal growth was observed in more optimal osmotic environments. Thus, the observed deficie ncy in gluta mate biosy nthesis in s train KO 11 was at tributed t o the inab ility to accumulate large quantities of intrac ellular glutamate for osmoadaptation but not necessa rily for mac romolec ular bios ynt hesis. Betaine from Dif co Yeas t Extract Re stores Gr owth in L uria Br oth Ferm entation s. Dulaney a nd coworkers (1968) demonstrated that Difco y east extract a component of Luria broth (10 g liter -1 tryptone, 5 g liter -1 yeast extract and 5 g liter -1 ), contains betaine by e xtracting and fractionating Difco y east extract. Xylose f ermentations with these nutrients yie lded hig h biocata lys t concen trations a nd high e thanol pr oductivity (Table 4-1). Cells harvested from these fer mentations at 12 h (when the sugar concentration would be similar to that of the CSL fer mentations at 24h) accumulated proline a nd betain e were accumul ated by strain KO 11 (Fig 4-2). Th e ratio o f proline to betaine was much higher in L uria broth than in CSL+X medium supplemented with 2.0 mM betaine, thus allowing proline to more effectively compete with betaine for transport

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88 by t he osmotic ally active tr anporte rs, ProP an d ProU. Th e lower g rowth y ield in betaine-supplemented fermentations indicated that although be taine aided in restoring growth it was not as effe ctive as the rich Difco nu trients. So me other nutrient i n the L uria broth may be nec essary f or even higher growth y ields (>2 g liter -1 ). The hig h availa bility of carbon skeletons, essential vitamins and minerals in the L uria broth would also have a sparing effect on all biosy nthetic pathways. The higher growth y ield and ethanol producti vity observe d in ferme ntations w ith these n utrients m ay have re sulted fr om this gener al sparin g effe ct. Conclusions Growth a nd ethano l product ion in the CSL +X medium was rest ricted b y a deficit in the ac cumulatio n of prote ctive osmo lyte s. Glutama te, ty pically accumul ated in response to osmotic s tress, fa iled to ac cumulate due to re stricted citrate biosy nthesis. Supplementing the medium with potassium glutamate (2 g liter -1 ) bypassed this limitation and rest ored the intrace llular g lutamate pool. Alte rnately the addi tion of sod ium aceta te (2 g liter -1 ), an ac tivator of citrate syn thase an d precur sor of g lutamate biosy nthesis, restored the intracellular glutamate pool and inc reased growth and ethanol produc tion. Together, these results sugg ested that a deficit in the production of glutamate restric ted growth. Betaine and DMSP increased the gr owth and ethanol production in a dose-dependent manner when added to the medium. NMR ana lysis of the intracellular osmolytes during these fermentations demonstrated the accumulation of these protec tive osmolytes. While the addition of DMSP to the CSL+X medium resulted in the accumulation of both proline and DMSP, cells from betaine supplemented cultures

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89 accumulated betaine exclusively Although growth and ethanol production were stimulated by betaine and DMSP, the g lutamate pool was not restored. Thus, the accumulation of protective osmoly tes (glutamate, betaine or DMSP) was require d for adaptat ion to the h igh sug ar envir onment ra ther tha n macrom olecula r sy nthesis. Fermen tations in r ich media (Lur ia broth) achieve compara tively higher cell densities (3.5 g liter -1 cell dry we ight) than those in the CSL+X medium supplemented with betaine (1.6 g liter -1 cell dry we ight) or DMSP (1.8 g liter -1 cell dry we ight). Betaine (from Di fco y east extra ct) and p roline we re acc umulated in ferme ntations w ith Lu ria broth (90 g liter -1 xylos e), cons istent with a need f or osmopr otectan ts. While supplementing the CSL+X medium with protective osmoly tes (glutamate, betaine or DMSP) restored growth, the maximum biocataly st concentration was still less than that of fermentations with Luria broth. Ba sed on these results, a deficit in the accumulation of protective osmolytes appe ars to be the primary factor that restricts cell growth and e thanol producti on in the CSL +X medium

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90 Table 4-1. Intracellular a ccumulation of protective osmolyte s by KO11. Fermentaion conditions a Fermentation Parameters Intr acell ular Osmoly tes b M ax. C ell conc. c Avg Vol Pro duc tivity d Proline Gluta mate Betaine DM SP Total No A dditive 0.95 0. 13 0.33 0. 05 135 6 <10 e <10 <10 128 + P r ol i ne ( 17 mM) 0.98 0. 10 0.35 0. 05 158 <10 <10 <10 158 + G l ut am at e ( 11 mM) 1.78 0. 20 0.56 0. 03 88 31 <10 <10 119 + A ce t at e ( 24 mM) 1.77 0. 12 0.57 0. 05 82 28 <10 <10 111 KO11 (pLOI 2514) 1.72 0. 11 0.50 0. 04 91 34 <10 <10 125 SU102 ( ) ackA ) 1.94 0. 12 0.54 0. 02 84 38 <10 <10 122 + B et ai ne ( 2. 0 m M) 1.58 0. 25 0.49 0. 06 <10 <10 107 <10 107 + D MSP ( 0. 25 mM) 1.84 0. 08 0.58 0. 03 41 <10 <10 54 95 + T au r i ne ( 1. 0 m M) 0.96; 0.94 0.33; 0.31 133 <10 <10 <10 133 LB Xylose st rai n KO11 3.53 0. 34 0.88 0. 02 67 6 <10 81 0.3 <10 147 a All fermentations were in the CSL+X me dium unless otherwise indicated a Co nc en tr at io ns (m M) we re de te rm in ed by 13 C NMR and a ssumed 1 mg DCW = 1 mL intracellular volume. b Maximum cell mass (dry weight) g liter -1 Standar d deviati ons are g iven in expe riments with more than 3 replicates. Otherwise, individual replicates ar e given. c Average volumetric productivity (g ethanol liter -1 h -1 ) for the first 72 hours for CSL+X fermentation, 48 h for LB fermentation. d Limit o f detec tion for in tracell ular osmo lyte s was > 10 mM.

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91 Figure 41. Ca rb on fl ow du ri ng fe rm en ta ti on of xyl os e b y E. coli KO11. Unless noted otherwis e, enzy mes listed are nat ive to E. coli Enzy mes: 1. py ruvate k inase ( pykA, pykF ); 2. pyruvate f ormate-lya se ( pflB ); 3. phosphotransacety lase ( pta ); 4. acetate kinase ( ackA ); 5. alcohol/aldehyde dehydrog enase ( adhE ); 6. Z. mobilis pyruvate de carboxylase ( pdc ); 7. Z. mobilis alc oho l d ehyd rog ena se II ( adhB ); 8. phosphoenolpyruvate c arboxylase ( ppc ); 9. citrate synthase ( gltA ); 10. ac onitase ( acn ); 11. isocitrate dehydr ogenase ( icd ); 12. glutamate dehy drogenase ( gdhA ); 13. malate dehydr ogenase ( mdh ); 14. fumarase ( fumB ); 15. fum arate r eductas e ( frdABC ); r indicates allosteric activation, s indicates allosteric inhibition.

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92 Figure 4-2. Major intracellular osmoly tes accumulated by ethanologenic E. coli during fermentation of 90 g liter -1 xylose to ethanol. 13 C NMR spectra (1000 scans) were obtained as described in the materials and methods. (A) CSL +X medium with out additives, strain KO11. (B) CSL+X me dium supplemented with 11 mM (2 g liter -1 ) potassiu m glutama te, strai n KO11. ( C) CSL+ X medium su pplement ed with 24 mM (2 g liter -1 ) sodium acetate, strain KO11. (D) CSL +X medium without additives, strain SU102 ( ) ackA ). (E) CSL+X medium with 17 mM (2 g liter -1 ) proline (F) Luria br oth, strain KO11. (G) CSL+X medium supplemented with 2 mM betaine strain K O11. (H) CSL+X medium sup plemente d with 0.25 mM DMSP, stra in KO11. Sig nals are labeled as follow s: aceto ne, A; pr oline, P; glutamate, G; betaine, B; DMSP, D.

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93 Figure 4-3. Effect of osmoprotectants (1.0 mM) on maximum cell concentration. Fermentations were supplemented with the indicated supplements. Small bars indicate the standard deviation.

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94 Figure 4-4. Effects of Betaine and DMSP on gr owth and ethanol production. Dose-dependent increase in biocataly st and ethanol with the addition of betaine (A and B) o r DMSP (C an d D):  no additi on; • 0.1 mM; Ž 0.25 mM; ) 0.5 mM;  1.0 mM; and ” 2.0 mM.

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95 Figure 4-5. The chemical structure of betaine a nd DMSP. Both osmoprotectants are zwitterionic and proposed to be transported by either the ProP or ProU transport sys tems due to structur al similar ity o f the com pounds.

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96 CHAPTER 5 GENERAL DISCU SSION AND CONCL USIO NS This study investigated the ba sis for the high nutrient requirement of ethanologenic E. coli strain KO11 during fermentation of xy lose to ethanol, with the ultimate goal of minimizing the requirement for nutritional supplements. As a starting point, the fermentation of 9% xylose to ethanol in a 1% CSL medium was evaluated. Despite the absence of any specific auxotrophic requirement, the growth of strain KO 11 was limited by the availability of complex nutrients. Cell growth was limited due to an imbalance in carbon partitioning between biosy nthesis (glutamate) and product formation (ethan ol), l ikely res ulti ng from the me tabol ic engi neerin g of the strai n. The high expression of the low K m pdc from Z. mobilis out-competed PFL for py ruvate, restricting citrate synthase activity resulting in a glutamate deficiency Several approaches were evaluated that significantly increased the growth and ethanol pr oduction by strain KO11 in this medium. Ultimately, genetic and phy siological approaches decre ased the nutrient requirement 5-fold. Increased A cetyl-CoA Ava ilability Stimulated Growth. Aerobic growth experiments were the first indica tion that an imbalance in carbon partitioning restricted growth (Chapter 2) Under these conditions, the oxidative conversion of pyr uvate to acety l-CoA (PDH) is active due to decreased fe edback inhibition by NADH (de Gra ef et al. 1999; Hansen and Henning 1966) whic h are known to be hig her duri ng fer mentative or anae robic g rowth (de Graef e t al. 1999; Snoep et a l. 1990). The PDH K m for pyruva te (0.52 mM; Nemeria et al. 2001) is more similar to that

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97 of the to th e recombinant PDC (Km 0. 4 mM; Raj e t al. 2002) than the ferm entative P FL (2.0 mM; Knappe and Sawers 1990). Thus, PDH is more competitive for essential ca rbon skeleton s than the fermen tative pa thway (PFL ). Supplem enting t he CSL +X medium with acetate reduced its production to less than half that of fe rmentations without additives (Chapter 3), increasing the ava ilability of acety l-CoA for biosynthetic pathwa ys. Added pyr uvate wa s consume d within the first thr ee hours resultin g in incr eased a cetate producti on. I ncreas ed PDH ac tivity accoun ted for th e additio nal ace tylCoA biosy nthesis required for acetate production. This also re sulted in a transient increase in the availability of ace tyl-CoA during the f irst 3 h of incubation. However, the beneficial effects on growth were not r ealized until after more than 9 h of incubation. The final acetate concentrations in these ferme ntations were similar to the acetate supplemented culture s. Thus, th e longterm ben efit res ulting f rom py ruvate s upplemen tation wa s due to the accumulation of acetate in the medium, increasing the availability of ac etyl-CoA. Acetaldehy de supplemented experiments were shown to have decre ased relative intracellular NADH concentration (dec reased NADH/NAD + ratio), i ncreas ing gr owth yield and ethanol produc tivity (Chapter 2). Thoug h the initial 0.25 g liter -1 were consumed in the first three hours of incubation (Chapter 3), the ef fects of this addition were not realized, in terms of increased g rowth, until after 12 hours. The second addition of acetaldehy de at this time increased PDH flux and produced moderately more acetate. This is indic ative of increa sed ace tylCoA produ ction dur ing a cr itical time of grow th where fermentations without additives ceased to g row.

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98 Some TCA Intermediates I ncrease Growth. Addition of intermediates from the succinate production pathway (oxaloacetate, malate or fumarate) did not increase g rowth yields or ethanol pr oductivity. Fermenta tions suppleme nted with s uccinat e did not no ticeably affec t the per formanc e of stra in KO11 in this medium. Supplementing fermentations with malate or fumarate resulted in eleva ted fumartate levels compared to those without additives (Chapter 2). As the pr oduction of succinate by this pathwa y is blocked in strain KO11 ( ) frdABCD ), the elevated levels of fumarate were expected. Howeve r, fermentations with added oxaloacetate had lower biocata lys t yie lds and de crease d ethanol producti vity While thes e cultur es consum ed all of the ad ded oxaloa cetate within the first 24 h, the car bon could not be ac counted f or in the fermentation broth as succinate or fumara te (Table 2-1). This might be explained as the induction of a futile cy cle for ATP generated by the contemporaneous expression of phosphoenolpyruvate c arboxylase ( ppc ) and the glucon eogen ic phospho enolpy ruvate carboxykinase ( pck ). The c reation of this fut ile cy cle has b een pre viously shown to restrict growth (Chao et al. 1993; Chao and L iao 1994; Gokarn et al. 2000). While citr ate and i socitrat e did not in crease growth of strain KO11, the ir derivatives 2-ketoglutarate and g lutamate were beneficial (Chapter 2) The addition of citrate resulted in higher levels of fumar ate and acetate, while formate produc tion was lower tha n ferme ntations w ithout supp lements. T his is cons istent with citrate lya se induction (Lutgens and Gottscha lk 1980), which cleaves citrate into acetate ( without formate production) and oxaloacetate (leading to fumarate). Most of the isocitrate added to the med ium remai ned at 24 h ours. How ever, 2ketoglu tarate a nd its amino acid derivative, glutamate, stimulated growth and etha nol production.

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99 Citrate Sy nthase–A Unify ing Hy pothesis. Based o n these r esults, c itrate sy nthase a ctivity was prop osed to limi t the gr owth of strain KO11 in the CSL+X medium. Supplementing the medium with glutama te or 2-ketoglutarate by passed the need for additional citrate biosy nthesis. Acetyl-CoA, increased with the addition of acetate, py ruvate or acetaldehy de, is both an activator and co-substrate for E. coli citrate synthase (Weitzman 1969). The biosynthetic pathway for the other co-substrate, oxaloacetate, is also activated by acetyl-CoA ( Izui et al. 1981), linking the availability of the two substra tes. The decreased NADH/ NAD+ ratio in th e cell resulting from supplementing the medium with py ruvate, acetaldehy de or 2-ketoglutarate, decreased the NADH-mediated inhibition of citrate synthase. Thus, all of the be neficial suppleme nts can be linked by a common a ctivity citrate biosy nthesis. The biosynthesis of citrate is regulated by the allosteric controls of this enzyme. By var ying the le vels of citrate sy nthase expression in E. coli from a plasmid, Walsh and Koshland, Jr. (1985) demonstrated that the over-expression of citrate sy nthase did not increase flux through this pathway The carbon flow through this pathway is linked to the regeneration of NAD + As the electron transport chain becomes more r educed, NADH accumulates in the cell (de Graef e t al. 1999). Under these conditions, the further production of NADH is not favored. This is indicated by the inhibition of the oxidative cleavage of py ruvate to acety l-CoA (PDH) which produces NADH (de Gra ef et al. 1999; Snoep et a l. 1990). F urthermo re, the o xidation of a cety l-CoA by the TCA cy cle (produc ing NAD H) is dec reased by t he NADH -mediate d inhibition of citra te sy nthase (Weitzman 1966), the first committed step of the TCA cycle

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100 In strain KO11, the normal patterns of carbon flow were altered by the expression of the Z. mobilis pdc which co mpetes fo r py ruvate. During fermen tation, a cety l-CoA is produced by PF L which has a highe r K m for pyruva te (2.0 mM; Knappe and Sawers 1990) tha n the rec ombinant PD C (0.4 mM; Ra j et al. 20 02). Thus most of the pyr uvate produce d from g lyc oly sis is dire cted towa rd ethan ol, resul ting in hi gh etha nol y ields. However, this decreased the production of a cetyl-CoA, an e ssential substrate and allosteric regulator of many biosynthetic pathway s including citrate sy nthase (Weitzman 1966). Thus, carbon partitioning between biosy nthesis and cofactor regene ration was disrupte d, result ing in de crease d biocata lys t yie ld in the nu trient-p oor mediu m. Two genetic solutions to increase citrate biosy nthesis were tested. The functional expression of citZ from B. subtilis increa sed gro wth and e thanol pr oduction i n strain KO11 (Cha pter 2). This could be due to t wo facto rs, neith er of wh ich are mutually exclusive Ty pical of the citra te sy nthases f rom Gram -positive or eukar yot ic orga nisms, this enzy me is insen sitive to N ADH (Jin an d Sonenshe in 1996). A dditionall y, t his enzyme has a sig nificantly lower K m for ace tylCo A th an t he n ati ve c it rat e syn th ase In light of the evidence presented in these studies, either of these factors may have contributed to the increased growth of strain KO 11. Expressing citrate sy nthases with a range K m values between that of the E. coli and B. subtilis may provide fur ther evidence as to the exact cause of the increased g rowth during the expression of the heterologous citrate synthase Eliminating the non-biosynthetic dra in of the acety l-CoA through the primary ac et at e p ro du ct io n p at hw ay ( ) ackA ) also increased growth and etha nol production (Chapter 3). Though the ATP resulting from the a cetate pathway may have c aused the

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101 slight growth lag initially observed with this strain, the maximum growth rate was not affected. The specific produc tivity was also unchang ed by this mutation. Thus, the elimination of competing pathway s for acety l-CoA increased the availability of acetyl-CoA, pa rtitioning more carbon into biosy nthesis. Similar effects have been achieved in other E. coli strains e nginee red for the produ ction of o ptically -pure la ctic ac id (Zhou et al. 2002; Zhou et al. 2003). A Defic it in Protec tive Osmol yte s Limit ed Growt h Intr acellula r osmoly te pools a re reg ulated by cells for adaptat ion to the o smotic streng th of the me dium (Csonk a 1989; Cso nka and H anson 199 1) Durin g gro wth in minimal media of high osmotic strength, E. coli and other related organ isms will accumulate large quantities of K + glutamate and trehalose (Cay ley et al. 1991; L ewis et al. 1990). The biosynthesis of g lutamate requires 2-ketoglutarate, a product of citrate. However, citrate biosy nthesis has been demonstrated to limit growth under the conditions of this study (Chapter 2; Chapter 3). I n the CSL+X medium (0.6M xy lose), strain KO11 accumulated only the protective osmolyte proline. When this medium was supplemente d with additives that increased growth and ethanol produc tion, glutamate accumulated. Thus, the accumulation of the protective osmoly te glutamate was necessary for extended growth. Trehalose likely f ailed to accumulate to detectable levels (<10 mM) be cause of the carbon source used for g rowth. Glucose-6-phosphate and uridine diphosphate gluc ose, both derived from glucose, are substrate s for trehalose biosy nthesis. During growth on xylos e, fruc tose-6phosphate and gly cerald ehy de-3-ph osphate e nter gl yc oly sis at thei r respective steps of gly colysis. Trehalose biosy nthesis would require 2 molecules of

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102 fructos e-6-pho sphate be convert ed to the s ubstrate s for tre halose bi osy nthesis. T his would result in an expenditure of ATP. However, the ATP y ield during growth on xy lose (0.4 ATP per pyruva te produced) is low compared to that of glucose (1 ATP per pyr uvate). Additionally, ATP can only be made by substra te-level phosphoryla tion during fermentation. Thus, the energetic dema nds for the biosynthesis would have f urther restricted growth. The accumulation of proline from the medium should not have affec ted the steady-state g lutamate pool (Cayley et al. 1992). The accumulation of intracellular glutama te in fer mentation s with hig her gr owth y ields (sup plements o r gene tic manipulation) indicated that additional protective osmolyte s were necessary for continued growth under the se condit ions. Thou gh proli ne can b e degr aded to g lutamate this pathway wa s inhibited by high osmotic environment. Thus, eve n an excess of proline (added as a supplement) did not result in increased g rowth or accumulation of intracellular glutamate. Supplementing the medium with betaine or DMSP increased growth and e thanol producti on. Analy sis of the i ntrace llular osm oly tes acc umulated by c ells from these fermen tations did not detec t increa sed glut amate po ols. Thus, the rate of gluta mate biosynthesis is sufficient for g rowth of strain KO11 in the CSL+X medium, but not for adapting to the high osmolarity of the growth medium. This is consistent with previous reports with stra ins restr icted in th eir abili ty to make gl utamate. By deleting the glut amate pathway with a hig h affinity for a mmonium (glutamine synthaseglutamate sy nthase) and growing these strains in media with low ammonium and high osmolarity cells continued to grow at a lower rate (Csonka e t al. 1994; Yan et al. 1996), indicating that the

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103 accumulation of glutamate for adaptation to the osmotic environment ( and not for biosynthesis) limited growth. While DMSP supplemented fermentations (0.25 mM) accumulated both proline and DMSP, c ells from the beta ine supple mented f ermenta tions (2.0 mM) conta ined only betaine (Chapter 4). This supports previous observations that betaine is the pref erred substrate for the osmotically a ctivated transport mechanisms ProP and ProU (Randall et al 1995). However, significantly less DMSP (0.25 mM) than betaine (2.0 mM) was required to restore the growth of stra in KO11, in contrast to previous reports establishing betaine as the mos t effec tive osmop rotecta nt (Randa ll et al. 19 95). I t is importa nt to note that studies of osmoprotectants commonly use Na Cl for increasing the osmolarity of the medium, while the experiments presented here used xy lose as both a growth substrate and most predominant osmolyte in the medium. There has be en at least one report of betaine not being as effective in restoring growth when different suga rs were the challenging osmolyte and gr owth substrate (Dulaney et al. 1968). Thus, during fermentation in the CSL+X medium, DMSP seems to be the more effec tive protective osmolyte. Difco yea st extract has been reported to have betaine (Dula ney et al. 1968), despite th e absenc e of a bio syn thetic pa thway for beta ine in y east. Fe rmentati ons with Difco nutrients had higher growth y ields (>3 g liter -1 ) and ethanol productivity tha n that of fermentations in CSL+X medium. I n fermentations with Difco nutrients, both proline and beta ine were accumul ated by the cells This sug gests th at the ra tio of beta ine to proline in the mediu m was much lower, th us allowin g prolin e to more effec tively compete with betaine for the transport mechanisms. While supplementing the CSL +X medium with betaine or DMSP restored growth (~2 g liter -1 ), cell y ields wer e still

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104 somewhat lower than those during fermentations with Difco nutrie nts. Further studies are necessa ry to deter mine the b asis for t his appar ent requ irement f or additio nal nutrie nts. Beet mol asses an d cane mo lasses a re natur al sourc es of bet aine and DMSP, respectively While these byproducts of the sug ar processing industry are more expensive than CSL they are ine xpensive so urces of protect ive osmoly tes. Thei r potentia l to increase ethanol productivity and replace the high levels of complex nutrients require d by strain KO11 should be investigated. Futur e Pros pects for Met aboli c Engin eering. With recent advances in DNA technology it is now possible to engineer microorganisms to produce a wide variety of products by fe rmentation. Researchers have examined the possibility of using a variety of host strains for the production of ethanol (Ing ram et al 1999; Gon g et al, 1999), su ccinate (Donnel ly e t al. 1998a ; Donnelly et al. 1998b; Vemuri et al. 2002), opticallypure lactic acid (Zhou et al. 2002; Z hou et al. 2003; Dien et a l. 2001; Ky la-Nikki la et al. 2 000; Bia nchi et a l. 2001; Cha ng et al 1999), a dipic acid (Niu et al. 2002), 1,3-propandiol (Nakamura e t al. 2000; Tong et al. 1991; Diaz-Torres et al. 2000) and many other compounds (Chotani et al. 2000). The success of these efforts is reflected by the recent commercialization of lactic acid production for poly -lactide syn thesis, a biodeg radable thermopla stic, by Cargill Dow L LC. Du Pont is examining the feasibility of using genetically engineered E. coli for the production of 1,3-prop andiol, a copoly mer in the ir latest poly ester ma terial, So rona. Ar cher Da niels Midland Company is currently using genetically engineered y east for the production of etha nol fr om co rn fi ber h yd roly sate Seve ral o ther comp anie s are try ing t o com merc ializ e the pr oduct ion o f ethan ol fro m bio mass u sing a v ariet y of biocat alysts, inclu ding E. coli

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105 KO11. The economic viability of these and other fermentation processes for value-added compounds is reliant upon the development of the most efficient biocataly st for each specific process. The studies presented here have provided insight into the phy siological effec ts of meta bolic eng ineerin g and of fered p hys ical and geneti c solution s to allevi ate th e d el et er io us ef fe ct s o f a lt er in g c ar bo n f lo w o n g ro wt h a nd pr od uc ti vi ty.

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106 REFERENCES Akesson, M., P. Hag ander, a nd J. P. Axelsso n. 2001. A voiding a cetate accume lation in Esherich ia coli culture s using f eedbac k control of gluc ose fee ding. B iotechno l. Bioeng. 73: 223-230. Alexeyev, M.F. 1999. The pKNOCK series of broad host-range mobilizable suicide vectors for gene knockout and tar geted DNA insertion into the chromosome of gram-negative bac teria. BioTechniques. 26: 824-828. Alterthu m, F., an d L.O Ing ram. 1989 Effici ent etha nol produc tion from g lucose, l actose and xy lose by recombi nant Escheric hia coli Appl. Env iron. Mic robiol. 55:1943-1948. Aristidou, A. A., K. San, and G. N. Bennett. 1995. Metabolic eng ineering of Escheric hia coli to enhance recombinant protein production through a cetate reduction. Biotechnol. Prog. 11:475-478. Aristidou A., and M Penttila. 2000. Meta bolic eng ineerin g applic ations to r enewab le resource utilization. Curr. Opin. Biotechnol. 11:187-198. Arntzen, C. E., and B. E. Dale (co-chairs). 1999. B iobased industrial products, priorities for res earch a nd commer cializatio n. Nation al Acad emy Press, Was hington, D.C. Asghari, A, R. J. Bothast, J. B. Doran, and L. O Ingram. 1996. Ethanol pr oduction from hemicellulose hydroly sates of agricultural residues using g enetically e ngineered Escheric hia coli strain KO11. J. Ind. Microbiol. 16:42-47. Barbosa, M. de F. S., and L O. Ingra m. 1994. Expression of the Zymomona s mobilis alc oho l d ehyd rog ena se II ( adhB ) and pyruva te decarboxylase ( pdc ) gene s in Bacillus Cur. Microbiol. 28:279-282. Barbosa, M. F. S., M. J. Beck, J. E. Fein, D. Potts, and L. O. I ngram. 1992. Efficient fermentation of Pinus sp. acid hydroly sates by an e thanologenic strain of Escheric hia coli Appl. Environ. Microbiol. 58:1382-1384. Bauer, K. A., A. Ben-B assat, M. Dawson, V. T. de la Puente, and J. O. Neway 1990. Improved expression of human interleukin-2 in hig h-cell-density f ermentor

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110 Dinnbier, U., E. Limpinsel, R. Schmidt, and E. P. Bakker. 1988. Tra nsient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escheric hia coli K-12 to elevated sodium chloride concentrations. Arch. Microbiol. 150:348-357. Donnelly, M. I ., C. Sanville-Millard, and R. Chatterjee. 1998a. Method for construction of bacterial strains with increased succinic a cid production. U. S. Patent No. 6159738. Donnelly, M. I ., C. S. Millard, D. P. Clark, M. J. Chen, and J. W. Rathke. 1998b. A novel fermentation pathway in an Escheric hia coli mutant producing succinic acid, acetic acid and ethanol. Appl. Biochem. B iotechnol. 70-72:187-198. Dulaney, E. L ., D. D. Dulaney and E. L. Rickes. 1968. Fac tors in yeast extract which relieve growth inhibition of bacteria in defined medium of hig h osmolarity. Dev. Indust. Microbiol. 9:260-269. Evans, C. T., B. Sumeg, P. A. Srere, A. D. Sherry and C. R. Malloy. 1993. [C-13]propi onate oxida tion in wild -ty pe and c itrate sy nthase mu tant Escheric hia coli evidence for multiple pathway s of propionate utilization. Biochem. J. 291: 927-932. Faloona, G. R., and P. A. Srere. 1969. Escheric hia coli citrate synthase Purification and the effect of potassium on some properties. Biochemistry 8:4497-4503. Furlong, C. E. 1987. Osmotic-shock-sensitive transport sy stems, p. 768-796. In F. C. Neidhardt, J. L. I ngraham, K. B. L ow, B. Magasanik, M. Schaechter and H. E. Umbarger (ed.). Escheric hia coli and Salmonella typhimurium : Cellular and molecula r biolog y, v ol 1. Amer ican Soci ety for Micr obiology Washing ton, D.C. Gennis, R. B., and V. Stewart. 1996. Respiration, pp. 217-261. In Neidhar dt, F. C, R. Curtiss, I II J. L. I ngrah am, E. C. C. Lin, K B. L ow, B Ma gasan ik, W. S. Reznikoff, M. Riley, M. Schaechter, a nd H. E. Umbarger. (ed.) Escheric hia coli and Salmonella typhimurium : Cellular and molecular biology 2nd edition. America n Society for Micr obiology Washing ton, D.C. Giaever, H. M., O. B. Sty rvold, I. Kaasen, and A R. Strm. 1988. Biochemical and genetic character ization of osmoregulatory trehalose synthesis in Escheric hia coli J. Bacteriol. 170:2841-2849. Glaasker, E., F. S. B. Tjan, P. F. ter Steeg W. N. Konings, and B. Poolman. 1998. Physiological re sponse of Lactobacillus plantarum to salt and nonelec troly te stress. J. Bacteriol. 180:4718-4723.

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118 Yan, D. L ., T. P. I keda, A. E. Shaug er, and S Kustu. 19 96. Gluta mate is re quired to maintain the steady-state potassium pool in Salmonella typhimurium Proc. Na tl. Acad. Sci. USA 93:6527-6531. Yang, Y., A. A. Aristidou, K. San, and G. N. Benne tt. 1999a. Metabolic flux analysis of Escheric hia coli deficient in the acetate production pathway and expressing the Bacillus s ubtilis acetolactate sy nthase. Metab. Eng. 1:26-34. Yang, Y ., K. San, and G. N. Bennet t. 1999b. Re distributi on of meta bolic fluxe s in Escheric hia coli with fermentative lactate dehy drogenase overexpression and deletion. Metab. Eng. 1:141-152. Yang, Y ., G. N. B ennett, a nd K. San. 2001. The effec ts of fee d and intr acellula r py ruvate levels on the redistribution of metabolic fluxes in Escheric hia coli Meta b. Eng. 3:115-123. York, S. W., and L. O. I ngram. 1996a. Soy -based medium for ethanol production by Escheric hia coli KO11. J. Ind. Microbiol. 16:374-376. York S. W., a nd L. O Ing ram. 1996 b. Ethano l product ion by recombi nant Escheric hia coli KO11 using crude y east aut oly sate as a nutrient s upplemen t. Biotec hnol. L ett. 18:683-688. Zaldivar, J., J. Neilsen, and L. Olsson. 2001. Fuel ethanol produc tion from lignocellulose: a challa nge fo r metabo lic eng ineerin g and pr ocess int egrat ion. Appl. Microbio l. Biotechnol. 56:17-34. Zhou, S. and L. O. I ngram. 1999. Engineering endoglucanase-secre ting strains of ethanologenic Klebsiella oxytoca P2. J. Ind. Microbiol. Biotechnol. 22: 600-607. Zhou, S., T. B. Causey A. Hasona, K. T., Shanmugam, and L O. Ingra m. 2002. Production of optically pure Dlactic acid in mineral salts medium by metabolically eng ineered Escheric hia coli W3110. Appl Environ Microbi ol. 69:399-407. Zhou, S. D., K. T. Shanmugam, and L O. Ingra m. 2003. Functional replacement of the Escheric hia coli D-(-)lactate dehy droge nase g ene (ldh A) with the L-( +)-lac tate dehy droge nase g ene (ldh L) f rom Pediococcus acidilactici Appl. Environ. Microbiol. 69:2237-2244

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119 BIO GRAPHI CAL SK ETCH Stuart was born in Clanton, Alabama, in 1975. After a short stay in Texas, in 1979 his fa mily move d to th e Pen saco la, F lorid a, ar ea wh ere h e att ende d Gulf Bre eze Elementary a nd Middle School. After moving to Destin, Florida, he attended For t Walton Beach High Sch ool for tw o ye ars. See king a b etter ed ucation, Stuart en rolled in D eerfie ld Academy in Deerfield, Massachusetts. I n addition to several Advanced Placement courses, he was on the varsity water polo, swimming and golf teams. Following gradu ation, he began his pursui t of a Ba chelor o f Scienc e degr ee in micr obiology and cell science from the U niversity of Flori da in the f all of 199 4. Follow ing gr aduation in the fa ll of 1997, he worked for a small start-up biotechnology company. Afte r the company collapse d, he move d to Resea rch Tria ngle Pa rk, North Carolina where he worke d with Novartis Biotechnology and then Duke University Seeking further education and a r eturn to microbiology re search, he came back to the Univer sity of Florida for his doctoral degree in the spring of 1999. Unde r the guidance of Dr. L onnie O. Ingra m, Stuart studied the me tabol ism an d physio logy of the ethan ol pro ducin g E. coli strain KO 11. While attendin g gra duate sc hool, he me t and mar ried his w ife Bev erly a Ph. D. c andidate in plant molecular biology Together, they are looking forward to building a fa mily and long caree rs in scie ntific pur suits.


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Physical Description: Mixed Material
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Publication Date: 2003
Copyright Date: 2003

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IMPROVING THE PERFORMANCE OF Escherichia coli KO 11 DURING THE
FERMENTATION OF XYLOSE TO ETHANOL
















By

STUART A. UNDERWOOD


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

UNIVERSITY OF FLORIDA


2003































Copyright 2003

by

Stuart A. Underwood



























This work is dedicated to my wife, Beverly, and my family. The years of their endless
love and support made this work possible.














ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Lonnie O. Ingram, for his insight and

guidance in my academic development. His inspiration and general enthusiasm for

science will have lasting effects on the development of my career.

My deepest appreciation is extended to the other members of my graduate

committee: Dr. K. T. Shanmugam, Dr. Julie A. Maupin-Furlow, Dr. Gregory W. Luli and

Dr. Jon D. Stewart. Without their guidance and sagacity, this work would not have been

possible.

I am grateful to Lorraine Yamano, Dr. Shengde Zhou and Dr. Fernando

Martinez-Morales for their help in learning the intricacies of molecular biology. Many

thanks to Sean York and Alfredo Martinez for their help in learning our fermentation

processes. I would also like to thank Dr. Marian L. Buszko for his guidance with NMR

experiments.

My warmest thanks to the other members of Dr. Ingram's laboratory and the

biomass research group for the many insightful discussions about scientific matters.

The text and figures in Chapter 2 and Chapter 3, in part or in full, are reprints of

the material as it appears in Applied and Environmental Microbiology (vol. 68, pp.

1071-1081 and pp. 6263-6272, respectively).














TABLE OF CONTENTS



ACKNOWLEDGMENTS ................. .......................... iv

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

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

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

CHAPTER

1 INTRODUCTION ............ ......................... ...... 1

Lignocellulose as a Carbohydrate Source ............................ 4
Adaptation to High Sugar Environments ............................. .. 5
Xylose versus Glucose Metabolism ................ ................. 8
Pyruvate Dissimilation ............... .......................... 10
Engineering E. coli for Ethanol Production ............................. 15
Deleterious Effects of Metabolic Engineering ........................... 16
Project Goals .......... .. ...................... ............ 18

2 FLUX THROUGH CITRATE SYNTHASE LIMITS THE GROWTH OF
ETHANOLOGENIC Escherichia coli KO 11 DURING XYLOSE
FERMENTATION ................................... ............ 26

Introduction ........... ........................................26
Materials and Methods ..........................................27
Microorganisms and Media ................ ................... 27
Fermentation Conditions .................................... 28
Aerobic Growth Studies ........................................29
Analytical M ethods ........................................... 29
G enetic M ethods .............................. ..... ........ 30
NAD(P)H/NAD(P)+ ratio .......................................31
Enzyme Assays .............. .................... ......... 31
Results ................... ...................... ............. 32
Macro-nutrient Limitation. ......... ...... ................... 32









Energy Limitation ............. ...... .......................... 33
Metabolic Imbalance Relieved by Addition of Pyruvate or Acetaldehyde. 33
Pyruvate as a Source of Carbon Skeletons for Biosynthesis ............. 35
Whole-cell Fluorescence ....................................... 37
Citrate Synthase, a Link Between NADH and 2-Ketoglutarate. .......... 39
Discussion ........... .........................................40

3 GENETIC CHANGES TO OPTIMIZE CARBON PARTITIONING IN
ETHANOLOGENIC Escherichia coli KO11 ........................... 53

Introduction ....................................................53
M materials and M ethods ............................................ 54
Microorganisms and Media ................ ................... 54
Ferm entation ............ ........................ ........ 54
Analytical M ethods ........................................... 54
Genetic Methods .............. ................................ 55
Construction of pLOI2065 Containing a Removable Tetracycline Resistance
Cassette .............. .................... .............56
Nucleotide Sequence Accession Number ......................... 56
Construction of SU102 Containing an Insertion Mutation in ackA ........ 56
Construction of SU104 Containing a Deletion in adhE ................. 57
Results and Discussion ............................................ 58
Acetate Addition Stimulates Growth and Ethanol Production by Reducing Net
Acetate Production During Sugar Metabolism .................... 58
Stimulation of Growth and Ethanol Production by Added Pyruvate Can Be
Primarily Attributed to Increased Acetate Production. .............. 59
Stimulation of Growth and Ethanol Production by Acetaldehyde Can Be
Attributed to Increased Acetyl-CoA. .......................... 62
Stimulation of Growth and Ethanol Production by Inactivation of Non-
biosynthetic Pathways Which Consume Acetyl-CoA ............... 64
Conclusions ........... ........................................66

4 A DEFICIT IN PROTECTIVE OSMOLYTES IS RESPONSIBLE FOR THE
DECREASED GROWTH AND ETHANOL PRODUCTION DURING XYLOSE
FERMENTATION .............. .............................77

Introduction ............... .............................. 77
M materials and M ethods ............................................ 79
Microorganisms and Media. ................. ................ 79
Fermentation. ........... ................... ............ 79
13C NMR. ........... .................... ............ 80
Analytical Methods .................. ............ ........... 81
Results and Discussion .......... ............. ....... 81









Citrate Synthase Flux Limits the Biosynthesis of Glutamate, a Primary
Intracellular Osmolyte .................. ...................... 81
Genetic Changes to Optimize Carbon Partitioning Increased the Glutamate
Pool ............................................... 84
Glutamate Accumulation Functions in Osmoprotection ................ 85
Replacement of Glutamate by Other Osmoprotectants ................. 86
Betaine from Difco Yeast Extract Restores Growth in Luria Broth
Fermentations. ...........................................87
Conclusions ............... ...............................88

5 GENERAL DISCUSSION AND CONCLUSIONS ...................... 96

Increased Acetyl-CoA Availability Stimulated Growth ................... 96
Some TCA Intermediates Increase Growth. ............................ 98
Citrate Synthase-A Unifying Hypothesis. ........................... 99
Future Prospects for Metabolic Engineering ........................... 104

REFERENCES ............. .......................................... 106

BIOGRAPHICAL SKETCH ............................................ 119














LIST OF TABLES


Table pa e

2-1. Effects of additives on the composition of fermentation products ............. 43

2-2. Effects of additives on growth and ethanol production by KO 11 .............. 44

3-1. Strains and plasmids used in Chapter 3 .................................. 68

3-2. Effects of mutations and additives on cell yield and ethanol productivity ........ 69

4-1. Intracellular accumulation of protective osmolytes by KO 11 ................. 90














LIST OF FIGURES


Figure pge

1-1. Glucose transport by the phosphotransferase system. ....................... 20

1-2. Glycolysis. ....................................................... .21

1-3. Xylose transport in E. coli ................ ......................... 22

1-4. Xylose metabolism .......... ................... ............23

1-5. Reactions of the pyruvate dehydrogenase complex ....................... 24

1-6. Fermentation pathways ofE. coli ................ ................... 25

2-1. Comparison of maximal cell densities ............... ................ 45

2-2. Comparison of growth and ethanol production from glucose and xylose ........ 46

2-3. Effects of added pyruvate and acetaldehyde .......................... 47

2-4. Initial effects of added TCA pathway intermediates ..................... .. 48

2-5. Effect ofmetabolites on whole-cell fluorescence ....................... 49

2-6. B. subtilis citZ increases the growth and ethanol production .................. 50

2-7. Relationship between cell yield and fermentation performance ................ 51

2-8. Fermentation and TCA pathway ....................................... 52

3-1. Allosteric control of central metabolism ................................. 70

3-2. Plasmids used to construct mutations ................................... 71

3-3. Effect of media additions and mutations ................................. 72

3-4. Effect of media additions and mutations on organic acid production ........... 73









3-5. Metabolism of added acetaldehyde and pyruvate during fermentation .......... 75

3-6. Partitioning of carbon among competing pathways ......................... 76

4-1. Carbon flow during fermentation of xylose by E. coli KOl l.................. 91

4-2. Major intracellular osmolytes accumulated by ethanologenic E. coli during
fermentation .................................... .. ...........92

4-3. Effect of osmoprotectants (1.0 mM) on maximum cell concentration ........... 93

4-4. Effects of Betaine and DMSP on growth and ethanol production .............. 94

4-5. The chemical structure ofbetaine and DMSP ............................. 95














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

IMPROVING THE PERFORMANCE OF Escherichia coli KO 11 DURING THE
FERMENTATION OF XYLOSE TO ETHANOL

By

Stuart A. Underwood

August 2003

Chair: Lonnie O. Ingram
Co-Chair: Keelnatham T. Shanmugam
Major Department: Microbiology and Cell Science

The large-scale conversion of lignocellulose to fuel ethanol would greatly reduce

the U.S. dependence on imported oil. To facilitate this need, Escherichia coli has been

genetically engineered for the homofermentative production of ethanol from all

constituent sugars of lignocellulose. However, high levels of complex nutrients are

required for rapid fermentation of xylose, the second most abundant sugar in

lignocellulose. With low levels of complex nutrients, the rate of xylose fermentation was

limited by the growth of the biocatalyst. In a mineral salts medium containing 1% corn

steep liquor as a nutrient source (90 g liter1 xylose), growth was limited by an imbalance

in the partitioning of carbon between ethanol production and biosynthetic pathways.

Citrate synthase was shown to catalyze the specific growth-limiting reaction. The

allosteric controls of citrate synthase regulate carbon flow through the oxidizing arm of

the TCA pathway, ultimately producing 2-ketoglutarate and glutamate. Functionally









expressing citrate synthase II (citZ) from Bacillus subtilis stimulated growth due to its

different allosteric and kinetic properties. Acetyl-CoA server as an antagonist to the

NADH-mediated allosteric inhibition of the E. coli citrate synthase. Supplementing the

medium with pyruvate, acetate, acetaldehyde, 2-ketoglutarate or glutamate increased

growth and ethanol production by activating, relieving or bypassing the allosteric

regulation of the E. coli citrate synthase. Conservation of acetyl-CoA by mutating acetate

kinase (AackA) also increased growth and ethanol production, presumably by increasing

the availability of acetyl-CoA (activating citrate synthase). In addition to biosynthetic

needs, large intracellular pools of glutamate (>20 mM) function as a protective osmolyte.

During growth in the high osmotic environment of the corn steep liquor medium

containing 0.6 M xylose, intracellular glutamate was low (< 10 mM) and cells grew

poorly, consistent with a glutamate deficiency. The addition of glutamate to the medium

and all approaches that stimulated citrate synthase increased the high intracellular pool of

glutamate during growth in this medium. Supplementing with other protective osmolytes,

such as betaine and dimethylsulfoniopropionate, restored growth without affecting the

intracellular pool of glutamate and appear to act directly as alternative osmolytes. These

results indicate that the poor growth and ethanol production in 1% cor steep liquor

medium (0.6 M xylose), the apparent requirement for high levels of nutrients without a

specific auxotrophic requirement and the beneficial effects of increased intracellular

glutamate all result from the requirement for high levels of protective osmolytes. Under

these conditions, the growth of the biocatalyst (E. coli) and ethanol production are limited

by insufficient levels of intracellular osmoprotectants rather than the synthesis of

glutamate, per se.














CHAPTER 1
INTRODUCTION

The production of fuel ethanol from renewable feedstocks could potentially

decrease the U.S. dependance on imported oil as well as decrease the release of fossilized

carbon into the atmosphere as carbon dioxide (CO2), a greenhouse gas. Blends of 95%

ethanol with gasoline are effective motor fuels, as demonstrated by Brazil's use of such

blends for more than 20 years prior to securing inexpensive sources of fossil fuels. In the

year 2002, approximately 140 billion gallons of gasoline were consumed in the United

States, most of which was derived from foreign oil. Approximately 2.9 billion gallons of

ethanol are produced annually in the U.S., slightly more than 2% of the gasoline

consumed. While the volume of ethanol produced increases each year, demands for

ethanol and energy also increase. For example, the phasing out of the gasoline oxygenate

methyl tertiary-butyl ether (MTBE) over the next several years will further increase the

demand for fuel ethanol, an alternate oxygenate. A substantial increase in ethanol

production must be achieved to replace MTBE with 10% ethanol.

Today, most of the ethanol derived from fermentation uses cornstarch as the

feedstock with yeast as the biocatalyst. Competing demands for cornstarch and variable

crop yields cause price volatility. Feedstock is the major contributor to the cost of current

ethanol processes. The cost of ethanol production must remain low in order for it to be an

economically competitive automobile fuel. The necessity for a less expensive, lower

demand feedstock is obvious. Agricultural wastes (corn stover, sugarcane bagasse, wheat








2

straw, etc.) are relatively inexpensive sources of carbohydrates that can be converted to

ethanol (Amtzen and Dale 1999; Ingram and Doran 1995; Ingram et al. 1999; Zaldivar et

al. 2002). More than 200 billion gallons of ethanol could be produced using these

lignocellulosic materials, sufficient to replace all of the gasoline burned by automobiles in

the United States (Arntzen and Dale 1999). As these agricultural wastes have little or no

competing uses, they offer long-term solutions to the necessity for inexpensive

carbohydrate sources.

However, there is no known organism in nature capable of fermenting all of the

various hexose and pentose components ofbiomass to ethanol. This difficulty is further

compounded by the complex, polymeric and somewhat variable structure of the

lignocellulosic biopolymers (Clarke 1997). Harsh treatments are required to breakdown

these sugar polymers into suitable substrates for fermentation. During these processes,

furfural, hydroxymethylfurfural, acetate, and many other cytotoxic byproducts are

released into the resulting solutions. An organism must tolerate the environmental

conditions created by these treatments to be an effective biocatalyst. With advances in

molecular biology, genetically engineering a desirable microorganism to produce ethanol

should be possible.

There are essentially two approaches to engineering an organism for the

production of ethanol from lignocellulosic residues. Either an ethanol producing

microorganism could be engineered to use all of the various sugars or a microorganism

already capable of fermenting all of these sugars could be engineered to produce

exclusively ethanol. The former approach has been pursued by many groups through the

engineering ofSaccharomyces cerevisiae or Zymomonas mobilis (deficient in pentose










metabolism) to utilize these carbohydrates by expressing heterologous transport and

metabolic pathways (Aristidou and Penttila 2000; Chotani et al. 2000; Gong et al. 1999).

While high productivities have been reported for both organisms in optimal conditions,

yeasts capable of fermenting both xylose and arabinose have not been reported in the

literature. Z. mobilis, a very fastidious organism, is not environmentally hardy, and the

harsh conditions resulting from the pretreatment of the lignocellulose severely hinders its

productivity.

One of the most studied and characterized organisms, Escherichia coli is an

excellent candidate for genetic engineering. The complete genetic sequence has been

published (Blattner et al. 1997), and much is known about its physiology (Neidhardt et al.

1990). The utility of this organism in industrial processes is second only to yeast. Typical

of enteric organsism, E. coli is capable of fermenting both the pentoses and hexoses

present in lignocellulose. However, E. coli is a mixed acid fermenter, producing lactate,

acetate, ethanol, format and succinate as its major fermentation products. Previous work

in our laboratory engineered the metabolism ofE. coli to produce exclusively ethanol

(Ohta et al. 1991).

The sugars of hemicellulose hydrolysates, containing mostly xylose, were

fermented by the engineered E. coli strain, with yields approaching 100% (0.51 g ethanol

/ g sugar) (Asghari et al. 1996; Lawford and Rouseau 1996; Martinez et al. 1999; York

and Ingram 1996a; York and Ingram 1996b). However complex, expensive nutrients

(Luria broth) are required to obtain these high yields. High levels of inexpensive nutrients

are required to replace these rich nutrients (Asghari et al. 1996; Lawford and Rouseau

1996; Martinez et al. 1999; York and Ingram 1996a; York and Ingram 1996b), but this










creates waste management problems and increases cost. Fermentations which use low

levels of complex nutrients or no nutritional supplements would be most desirable for

industrial fermentations.

Corn steep liquor (CSL), a by-product from the wet milling of corn, is an

inexpensive nutrient source with demonstrated utility in industrial processes.

Fermentations of hemicellulose hydrolysate with CSL as the nutrient source exhibited

dose-dependent change in ethanol productivities (Martinez et al. 1999). To equal the

ethanol productivity achieved with Difco nutrients (5 g liter' yeast extract and 10 g liter'

tryptone), 50 g liter-' CSL (wet weight; 50% solids) were required. The goal of this

present study is to understand the basis of the need for complex nutrients and develop

physiological and genetic solutions to circumvent this requirement.

Lignocellulose as a Carbohydrate Source

Most of the dry weight biomass is lignocellulose, composed of cellulose,

hemicellulose, pectin and lignin (Clarke 1997). Cellulose, the most abundant polymer on

the planet, is a homopolymer of cellobiose (P-1,4-glucose) and represents 20-50% of the

dry weight of plant matter. Lignin is a polymer of aromatic alcohols, comprising 10-20%

of the dry weight of plant biomass. Representing only 1-10% of the dry weight, pectin is a

methylated homopolymer of galacturonic acid. Hemicellulose is a complex, branched

polymer ofhexoses (glucose, galactose, mannose, rhamnose, and fucose) and pentoses

xylosee and arabinose). This polymer represents 20-40% of the plant dry weight and is the

most easily solubilized component of lignocellulose.

The sugars ofhemicellulose are released as monomers through a variety of

hydrolysis procedures, but dilute acid hydrolysis is currently the preferred method










(Ingram et al. 1999). This procedure uses moderate heat and low pH to release the sugars

of hemicellulose into solution as monomers (Grohmann et al. 1985). The exact ratio of

sugars in these hydrolysates can vary considerably depending on the feedstock, but xylose

is the most prevalent sugar in hydrolysates of hard woods and grasses (sugarcane, wheat

straw, etc.). Generating a concentrated sugar solution during hydrolysis is a formidable

challenge, but a goal of 100 g liter-' total sugar monomers in hemicellulose hydrolysates

should be achievable. Most of the studies presented here have used 90 g liter-' xylose as

the fermentation substrate.

Adaptation to High Sugar Environments

Many industrial fermentation processes operate as either batch fermentations

(with all required nutrients and substrates supplied initially) or fed-batch fermentations

(multiple additions of nutrients; requires concentrated feed solutions). As the hydrolysis

ofhemicellulose produces sugar streams up to 100 g liter-' (Ingram et al. 1999), their

fermentation to ethanol favors a batch fermentation process to avoid the additional cost of

concentrating these sugar streams and potentially concentrating growth inhibitory

compounds. However, this relatively high sugar concentration requires E. coli to adapt to

this higher osmolarity.

The rapid accumulation of potassium is the first response ofE. coli and related

organisms to an increase in the osmotic strength of the medium. Within a minute after an

increase in osmotic pressure, glutamate (a negatively-charged amino acid) synthesis is

increased to provide charge balance for the accumulated potassium (McLaggan et al.

1994). The short time between the accumulation of potassium and the biosynthesis of

glutamate, suggests that the onset of glutamate biosynthesis is a result of allosteric










regulation (<5 min) rather than genetic induction (10-20 min). Additionally, the

accumulation of glutamate in response to osmotic stress was found to be dependent on

the presence of K in the medium (McLaggan et al. 1994).

Escherichia coli has two biosynthetic pathways for glutamate. Under a nitrogen

limitation (0.1 mM ammonium), glutamate synthase-glutamine synthetase has been

shown to be the predominant glutamate biosynthetic pathway (Pahel et al. 1978). During

growth in excess nitrogen, glutamate dehydrogenase (GDH), a pathway that does not

consume ATP, is the primary glutamate biosynthetic pathway (Helling 1994).

Additionally, GDH is activated by K (Measures 1975). This allosteric regulation of GDH

has been proposed to be responsible for osmotically activated glutamate biosynthesis

(Helling 1994).

The intracellular concentration of K can be as high as 800 mM in E. coli during

growth in media of high osmolarity (Cayley et al. 1991; Cayley et al. 1992). Cells

deficient in glutamate accumulation have demonstrated growth defects during osmotic

challenge (Csonka 1988; McLaggan et al. 1991; Yan et al 1996) due to an inability to

maintain sufficient K (Yan et al. 1996). The large increases in intracellular potassium

and glutamate are transient, and their levels begin to decrease to 20-50 mM as trehalose

or other protective osmolytes accumulate in the cytoplasm (Dinnbier et al. 1988; Giaever

et al. 1988). However, glutamate pools remain elevated during growth the higher osmotic

conditions (Yan et al. 1996).

For the long-term adaptation to media of high osmolarity, E. coli synthesizes

trehalose (Boos et al. 1990; Dinnbier et al. 1988; Giaever et al. 1988) or accumulates

other charge-neutral (zwitterionic) compatible solutes (betaine, proline ectoine,










dimethylsulfonioproprionate, etc.) (Csonka and Hanson 1991). E. coli has a limited

capacity for biosynthesis of these compounds. Although E. coli is incapable of de novo

betaine biosynthesis, choline can be oxidized to betaine. However, this process is

restricted to aerobic growth (Landfald and Strom 1986). Some organisms synthesize

proline for long-term osmoadaptation (Kawahara et al. 1989). However, the y-glutamyl

kinase step in proline biosynthesis is subject to strong feedback inhibition in E. coli,

preventing the biosynthesis of this protective osmolyte (Csonka 1988; Smith 1985; Smith

et al. 1984). Thus, many of the protective osmolytes accumulated by E. coli must be taken

from their environment.

E. coli and related organisms have two primary transport systems for protective

osmolytes during osmotic stress, ProP and ProU (Randall et al. 1995). The ProP system

uses the proton gradient maintained by the cell to drive the uptake ofosmoprotectants.

This low affinity system (Km for proline is 0.3 mM) also transports many other

osmoprotectants (Lucht and Bremer 1994). The ProU transport system consists of a

periplasmic binding protein with a high affinity for betaine (Km 1.3 [IM), a

membrane-spanning component and a membrane bound enzyme which hydrolyzes ATP

for the active transport of betaine (Lucht and Bremer 1994).

A hierarchy for osmoprotectants has been empirically established for E. coli,

primarily for salt-mediated osmotic stress (Randall et al. 1995). Although there have been

conflicting reports concerning the validity of this hierarchy for sugar-mediated osmotic

stress (Glaasker et al. 1998), betaine is generally regarded as the most effective protective

osmolyte for E. coli. In at least one report, the ability of betaine to restore growth during

osmotic challenge with different carbon sources was dependent on the particular sugar










(Dulaney et al. 1968). Thus, the sugar-mediated osmotic stress anticipated for

fermentations of hemicellulose hydrolysates (100 g liter-' sugar) may require the

accumulation of different osmolytes.

Xylose versus Glucose Metabolism

The reactions involved in the transport and metabolism of glucose are well

understood and outlined in Figures 1-1 and 1-2. Transport of glucose into the E. coli

cytoplasm is mediated by a phospho-transferase system (PTS). The energy and phosphate

required for translocation and phosphorylation of PTS sugars comes from

phosphoenolpyruvate (PEP). An additional ATP is required for the phosphorylation of

fructose-6-phosphate to fructose-1,6-bisphosphate. Thus, to metabolize glucose, an initial

investment of 2 ATP equivalents (1 ATP and 1 PEP) is required.

Fructose-1,6-bisphosphate is cleaved into dihydroxyacetone-phosphate and

glyceraldehyde-3-phosphate. These two molecules are interconverted via triose-phosphate

isomerase. For the production of pyruvate, the terminal product of glycolysis,

glyceraldehyde-3-phosphate is oxidized and phosphorylated to form

1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. During this step,

nicotinamide adenine dinucleotide (NAD ) is reduced to NADH. The high group-transfer

potential of the phosphate bond on carbon 1 is used in the production of ATP from ADP

in the proceeding reaction catalyzed by phosphoglycerate kinase. The reactions leading to

the formation of phosphoenolpyruvate do not result in any further energy yield or

reducing equivalents.

In converting glyceraldehyde-3-phosphate to PEP, 1 ATP and 1 NADH are

produced. The conversion of PEP to pyruvate is either carried out via the










phosphotransferase system or through an ATP yielding reaction catalyzed by a pyruvate

kinase. The net reaction ofglycolysis can be written as follows:

glucose + 2NAD+ + 2ADP + 2 Pi 2 pyruvate + 2NADH + 2H' + 2 ATP

The production of pyruvate from 1/2 molecule glucose yields a net of 1 ATP and 1

reducing equivalent (NADH). Reducing equivalents are often considered as pools of both

their reduced and oxidized forms, and their ratio is indicative of the metabolic state of the

cell (respiration or fermentation) (de Graef et al. 1999; Snoep et al. 1990). Though the

ratio of the reduced to oxidized form (NADH/NAD+ ratio) varies widely with different

growth conditions, the absolute concentration of the two forms remains relatively

constant (de Graef et al. 1999).

In contrast to glucose, xylose is transported into the cell by either a proton

symport pathway (xylE) or an ATP dependant transporter (xylFGH) (Song and Park 1997;

Tao et al. 2001; Fig. 1-3). During fermentation, the cellular proton gradient is maintained

presumably by energy-consuming reactions (F1/F0 ATPase, for example). Thus, a proton

symport pathway is fueled indirectly by the hydrolysis of ATP. The transport of each

xylose is energized by the hydrolysis of 1 ATP. Once inside the cell, xylose is converted

into xylulose by xylose isomerase. Xylulose is then phosphorylated by xylulokinase,

utilizing the hydrolysis of a second ATP. Regardless of the pathway, xylose uptake and

activation phosphorylationn) require energy derived from the hydrolysis of 2 ATP

molecules. In contrast, glucose transport uses a single ATP equivalent (PEP) for both

transport and activation.

Intracellular xylulose-5-phosphate is metabolized by the pentose-phosphate

pathway (Fig 1-4). Through a series of reactions catalyzed by transketolase and








10

transaldolase, xylose is converted into intermediates of glycolysis (fructose-6-phosphate

and glyceraldehyde-3-phosphate). For every 6 xyloses consumed (30 carbon atoms), 4

fructose-6-phosphates and 2 glyceraldehyde-3-phosphates are produced. These molecules

are further metabolized by glycolysis to ultimately yield 10 molecules of pyruvate. Thus,

all 30 carbon atoms which began in xylose are converted into pyruvate.

The energy (ATP) and reducing equivalents (NADH) produced from the reactions

common to xylose and glucose metabolism are the same regardless of the original

substrate. However, since xylose is a pentose and requires separate energy for transport

and activation, growth on xylose results in a relatively low ATP yield. The transport and

activation of 6 xylose molecules (30 carbons) requires 12 ATPs, assuming 1 ATP is

required for transport regardless of the pathway. In the conversion of this

xylulose-5-phosphate to 10 molecules of glyceraldehyde-3-phosphate, an additional 4

ATPs are consumed. The conversion of these 10 molecules of

glyceraldehyde-3-phosphate to pyruvate yields 20 ATPs. The net gain of energy in the

conversion of 6 molecules of xylose to 10 pyruvate is 4 ATPs. An equal amount of

glucose on the basis of moles of carbon (5 molecules; 30 carbon atoms) produces a net of

10 ATPs during conversion to 10 molecules of pyruvate. The net energy gain for glucose

catabolism is 1 ATP per pyruvate, 2.5-fold more ATP than from xylose catabolism.

While there is a considerable difference in ATP yield between xylose and glucose, only

one NADH is produced per pyruvate from either glucose and xylose.

Pyruvate Dissimilation

A facultative anaerobe, E. coli accomplishes redox balance either by respiration

(Gennis and Stewart 1996) or fermentation (Bock and Sawers 1996). During respiration,










reducing equivalents are oxidized when their electrons are donated to the primary

oxido-reductases of the electron transport system. These electrons are passed between the

various proteins of the electron transport chain and ultimately used to reduce a terminal

electron acceptor (oxygen during aerobic respiration, for example). The energy from these

reducing equivalents is preserved in the form of a proton gradient established by the

concomitant translocation of H' from the cytosol to the periplasm. This proton gradient is

used to produce ATP via the F1/F0 ATPase.

The pyruvate dehydrogenase complex (PDH) catalyzes the oxidative metabolism

of pyruvate to acetyl-Coenzyme A (acetyl-CoA) and CO2, with the formation of 1

reducing equivalent (NADH). This complex consists of three activities: pyruvate

decarboxylase (aceE), acetyltransferase (aceF), and lipoate dehydrogenase (Ipd) (Fig.

1-5). The decarboxylation of pyruvate to an enzyme-bound acetyl moiety by the pyruvate

decarboxylase of this complex requires a thiamine pyrophosphate (TPP) cofactor, a

carrier of the "active" acetaldehyde. The acyl moiety is then transferred to an acyl-carrier

through the reduction of a disulfide bond. The resulting thioester has a high group transfer

potential and is transferred to Coenzyme A, an acetyltransferase reaction. The disulfide

which accepts the acetaldehyde from the TPP must be regenerated through an

oxidation/reduction reaction. NAD+ is reduced to NADH as the sulfhydryl group is

oxidized, forming the required disulfide bond. This reaction is subject to strong feedback

inhibition by NADH (Hansen and Henning 1966).

During aerobic growth, the tricarboxylic acid (TCA) cycle is responsible for the

total oxidation of acetyl-CoA to CO2 (Cronan, Jr. and LaPort 1996). The first step in this

cycle, citrate synthase, is also the rate controlling step (Lee et al. 1994; Walsh and










Koshland, Jr. 1985). This enzyme catalyzes the condensation of acetyl-CoA and

oxaloacetate to form citrate (Weitzman 1981). Citrate synthase is primarily regulated by

allosteric controls, activated by acetyl-CoA and inhibited either by NADH and 2-

ketoglutarate (Gram-negative) or ATP (Gram-positive, archea and eukaryotes) (Weitzman

1981). This provides a link between the energetic needs of the cell and the generation of

reducing equivalents (and ultimately ATP) through the TCA cycle.

The TCA cycle is also a source of carbon skeletons for biosynthesis. More than

half of the amino acids made by the cell are derived from intermediates of the TCA cycle

(Neidhardt et al. 1990). Oxaloacetate must be regenerated for the continued cyclic action

as intermediates are drawn into biosynthesis. This anapleurotic reaction is catalyzed by

phosphoenolpyruvate carboxylase in E. coli. The biosynthetic needs and metabolic state

of the cell dictate the activity of this reaction through allosteric control. Acetyl-CoA,

fructose-1,6-bisphosphate and GTP are activators of this enzyme (Izui et al. 1981), while

malate and aspartate (products of oxaloacetate utilizing reactions) are inhibitors (Izui et

al. 1981). Acetyl-CoA and oxaloacetate are co-substrates for citrate synthase. Thus, the

allosteric activation of phosphoenolpyruvate carboxylase and citrate synthase by

acetyl-CoA links the availability of the two co-substrates for citrate synthase.

During fermentation, no external terminal electron acceptors are available for

respiration, resulting in the accumulation of NADH to higher levels than during

respiration (de Graef et al. 1999). For redox balance to be maintained, intracellular

metabolites serve as electron acceptors. As NAD+ regeneration becomes difficult, NADH

generation is not favored. The formation of acetyl-CoA from the pyruvate dehydrogenase

reaction is inhibited by the high NADH/NAD+ ratio (Hansen and Henning 1966),










necessitating an alternate, non-oxidative route to acetyl-CoA generation. The

non-oxidative cleavage of pyruvate to acetyl-CoA and format is catalyzed by pymvate

formate-lyase (PFL; Knappe and Sawers 1990).

PFL activity is relative to the metabolic state of the cell, similar to PDH and

citrate synthase. However, PFL activity is regulated by post-translational modification

enzymes which are allosterically controlled. The protein is translated in an inactive form.

An oxygen-labile free radical is placed on a glycine residue by a PFL-activase enzyme

(pflA) forming the active PFL enzyme (Conradt et al. 1984). To protect the enzyme from

irreversible inactivation by oxygen, the multi functional alcohol dehydrogenase (adhE)

also has a PFL-deactivase activity to remove the oxygen-labile free radical (Kessler et al.

1991). The PFL-deactivase activity of adhE is inhibited by NADH (Kessler et al. 1992),

linking the activation state of PFL to the metabolic state of the cell as described by the

NADH/NAD+ ratio. When oxygen supply is limited, NADH accumulates and inhibits

PDH and the PFL deactivase activity. This causes a shift in flux to acetyl-CoA from

oxidative pyruvate cleavage (PDH) to the non-oxidative cleavage (PFL).

In contrast to respiration, acetyl-CoA is an electron acceptor during fermentation.

The two-step reduction of acetyl-CoA to ethanol is catalyzed by alcohol dehydrogenase

(adhE; Fig. 1-6), regenerating 2 NAD Glycolysis produces only one NADH per

pyruvate. Thus, the native alcohol production pathway results in an NADH deficit. This is

overcome by converting one of the acetyl-CoA to acetate, producing an additional ATP

by substrate-level phosphorylation. In E. coli grown under anaerobic fermentation

conditions with glucose as the carbon and energy source, equal amounts of acetate and

ethanol are produced.








14

Lactic acid is often produced by E. coli during fermentation in addition to acetate

and ethanol, primarily as active growth slows and stationary growth. Pyruvate is reduced

in a single-step reaction catalyzed by lactate dehydrogenase (LDH, IdhA gene product;

Bunch et al. 1997), resulting in the re-oxidation of 1 NADH per lactate produced. The

pathway for lactate production in E. coli is controlled by allosteric regulation, activated

by pyruvate (Tarmy and Kaplan 1968). In conditions of surplus supply of pyruvate, the

lactate pathway is activated. There is an associated energetic loss as a result of lactate

production compared to the co-production of acetate and ethanol, as no ATP is made in

the reduction ofpyruvate to lactate.

In contrast to respiration, the TCA cycle is interrupted at 2-ketoglutarate

dehydrogenase due to transcriptional regulation during fermentation (Iuchi and Lin 1988).

The resulting pathway has two sides, the reductive (leading to succinate production) and

the oxidative (stopping at 2-ketoglutarate). For succinate production during fermentation,

the anapleurotic pathway for oxaloacetate production (PPC) is the first step. As described

previously, there are multiple allosteric effectors of this enzyme which control its

physiological activity. Oxaloacetate is converted to malic acid through the reverse activity

of the malate dehydrogenase, regenerating 1 NAD+ (Bock and Sawers 1996). Fumarase

catalyzes the conversion ofmalate to fumarate. Fumarate is reduced to succinate by a

fermentation specific fumarate reductase (frdBACD gene products) with the oxidation of

a reduced menaquinone (Cronan, Jr. and LaPort 1996). The oxidative side of the TCA

pathway provides carbon skeletons for biosynthesis (Neidhardt et al. 1990).










Engineering E. coli for Ethanol Production

The enteric bacterium E. coli can use all of the sugar constituents of

lignocellulose, while the natural ethanol producing Saccharomyces cerevisiae and

Zymomonas mobilis are limited to growth on hexoses,. Wild-type E. coli produces

ethanol from the two step reduction of acetyl-CoA oxidizing two NADH to NAD As a

result, acetate (no further reduction required) is made in approximately equal amounts to

ethanol. However, Z. mobilis and S. cerevisiae produce ethanol from pyruvate through a

pathway which only re-oxidizes one NADH. The irreversible, non-oxidative cleavage of

pyruvate into acetaldehyde and carbon dioxide is catalyzed by pyruvate decarboxylase

(PDC). Acetaldehyde is reduced to ethanol, oxidizing 1 NADH. Thus, for each pyruvate

that is converted to ethanol via this pathway, one NADH is re-oxidized. With this

stoichiometry, all of the pyruvate generated by glycolysis can be converted to ethanol

without the necessity of other oxidized products to maintain redox balance.

Previous studies demonstrated that the Z. mobilis genes involved in ethanol

production are expressed well in E. coli (Ingram and Conway 1988). These genes (pdc

and adhB) were used to construct a synthetic operon which was integrated into the

chromosome for increased genetic stability of the recombinant strain (Ohta et al. 1991). A

deletion was introduced in fumarate reductase to decrease succinate production,

problematic in xylose fermentation. The resultant strain, designated K 11, fermented

both pentoses and hexoses to ethanol with yields approaching 100% of total sugars

present (0.51 g ethanol/g sugar = 100% theoretical yield) during fermentation in

laboratory media containing excess complex nutrients. In addition to the alterations to the

fermentation profile, there were some notable effects on growth physiology. In broth










cultures, comparatively high cell yields were achieved. On solid media, colonies

exhibited a raised morphology, similar to yeast.

Deleterious Effects of Metabolic Engineering

The engineering of metabolic pathways for the production of industrial chemicals

as an alternative to chemical synthesis has been performed for a variety of chemicals

(Chotani et al. 2000). Metabolic engineering for renewable chemicals such as ethanol

(Ingram et al. 1999), acetate (Causey et al, 2003), lactate (Bianchi et al. 2001; Chang et

al. 1999a; Dien et al 2001; Kyla-Nikkila et al. 2000; Zhou et al. 2002; Zhou et al. 2003),

propanediol (Nakamura et al. 2000; Tong et al. 1991), adipic acid (Niu et al. 2002) and

succinate (Donnelly et al 1998a; Donnelly et al. 1998b; Vemuri et al. 2002) have focused

primarily on product yields. The metabolic engineering of these new products has often

resulted in unexpected changes which increased the need for complex nutrients and

decreased potential utility (Bunch et al. 1997; Chang et al. 1999a; Chao and Liao 1994;

Chao et al. 1993; Martinez et al. 1999).

Undesirable changes such as reduced growth, decreased glycolytic flux and low

volumetric productivity are generally attributed to a lack ofATP (Gokarn et al. 2000; Xie

et al. 2001), creation of futile cycles (Chao and Liao 1994; Chao et al. 1993; Patnaik et al.

1992), changes in intracellular metabolite pools or a metabolic imbalance (Aristidou et al.

1992; Bunch et al. 1997; Chang et al. 1999b; Contiero et al. 2000; Liao et al. 1996; Yang

et al. 1999a; Yang et al. 1999b; Zhou et al. 2002; Zhou et al. 2003). Often, these

detrimental effects are masked by abundant complex nutrients in laboratory media and are

only apparent in mineral salts or low-nutrient media (Bunch et al. 1997; Chao and Liao

1994; Chao et al. 1993; Martinez et al. 1999).










Salmonella typhimurium was engineered for succinate production by increasing

the expression ofpyc encoding pyruvate carboxylase (Xie et al. 2001). Although

succinate production increased, growth rate declined by 18% and glycolytic flux

decreased by 40%. Similar results were reported for an analogous construction in E. coli

(Gokarn et al. 2000). Donnelly and coworkers (1998a and 1998b) isolated E. coli mutants

which produced 5-times more succinate than the parent strain, and again growth rate was

impaired. Growth rate and cell yield were also decreased by engineering E. coli for the

production of 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) by over-expression

ofpps (phosphoenolpyruvate synthase) (Patnaik and Liao 1994; Patnaik et al. 1992). This

inhibition of growth was more pronounced in minimal medium (Chao and Liao 1994).

Acetate production during the aerobic growth ofE. coli on sugars has been

correlated with a decline in metabolic activity and reduced expression of heterologous

genes (Aristidou et al. 1995; Bauer et al. 1990; Chang et al. 1999b; Luli and Strohl 1990).

Many approaches have been employed to decrease acetate production and increase

recombinant products (Aristidou et al. 1995; Barbosa and Ingram 1994; Bauer et al. 1990;

Chang et al. 1999b; Contiero et al. 2000; Yang et al. 1999a). Mutations in the primary

acetate pathway (pta, phosphotransacetylase; ackA, acetate kinase) increased the yield of

recombinant proteins, but usually reduced cell growth. The detrimental effect on growth

was attributed to the accumulation of metabolic intermediates such as acetyl-CoA or

acetyl-phosphate. An alternative approach, channeling pyruvate away from acetate by

expressing the Bacillus subtilis alsA gene encoding acetolactate synthase, also reduced

acetate production by 80% and increased product yields, but again reduced cell growth

(Yang et al. 1999a). Other attempts to decrease acetate production by increased










expression of IdhA (lactate dehydrogenase) were ineffective in rich medium (Yang et al.

1999b). In mineral salts medium, over-expression of IdhA was accompanied by a severe

growth limitation (Bunch et al. 1997).

Lactate dehydrogenase (IdhA) has been expressed to divert carbon away from

acetate accumulation. Despite the relatively high Km for pyruvate, lactate production

increased by 50% (Yang et al. 1999b). Interestingly, the amount of acetate produced in

these fermentations was not altered. However, these studies were conducted in a rich

laboratory medium. In a mineral salts medium (M9), expression of LDH resulted in

severe growth defects (Bunch et al. 1997). These growth defects were attributed to a

decrease in pyruvate availability necessary for growth.

Project Goals

Though strain KO 11 is prototrophic, high levels of complex nutritional

supplements are required for the rapid fermentation of sugars to ethanol (Martinez et al.

1999). For example, during the fermentation of 90 g liter-' xylose to ethanol, the addition

of CSL as a nutritional supplement (0-50 g liter') had a dose dependent effect on final

cell concentration. With the increase in biocatalyst concentration, there was a

proportional increase in fermentation rate and decrease in required fermentation time.

Although the addition of 50 g liter' CSL is not cost-prohibitive, the handling of this much

material on the scale of an industrial fermentation could be problematic and generate

excessive cost for waste disposal.

These studies will examine the basis for the high nutrient requirement for KO 11

for the rapid conversion of sugar to ethanol. As a starting point, growth and ethanol

production will be evaluated in a medium containing 1% CSL, 90 g liter' xylose, and








19

mineral salts. Physiological and genetic approaches will be used to characterize the

growth limitation. Solutions for solving this limitation will be presented. Further work

will demonstrate the basis for a specific biosynthetic pathway under the conditions tested.

Knowledge gained in these studies will have applications in the further development of

the commercial production of ethanol from plant biomass by metabolically engineered E.

coli and will significantly contribute to the field of metabolic engineering by emphasizing

the importance of metabolic intermediates down-stream of the product forming node.










n-i vmil v.cZISCZ


Glucose 6-
Cytoplasm G
II A-
Periplasm P-HPr nzyme PEP
cBrr pH ptS i
EnzymeA
G Enzyme HPr nzyme I- Pyruvate

Glucose
Figure 1-1. Glucose transport by the phosphotransferase system. The phosphate from PEP
passes through a cascade of enzymes and ultimately to intracellular glucose. The
hydrolysis of 1 ATP equivalent (PEP) is used to energize transport and activate
glucose.


1


I --











Glucose-6-P

1

Fructose-6-P
ATP
ADP
Fructose-1 j-P

4
Dihydroxyacetone-P Glyceraldehyde-3-P
NAD* ,Pi
NADH

1 3-bisphosphoglycerate

ADP -

ATP

3-phosphoglycerate

7


2-phosphoglycerate


8


phosphoenolpyruvate

ADP 9,

ATP
pyruvate


Figure 1-2. Glycolysis. The enzymes and genes that catalyze the conversion of glucose to
pyruvate are as follows: (1) phosphoglucose isomerase,pgi; (2)
phosphofructokinase,pflkA; (3) fructose-6-phosphate aldolase,fba; (4) triose
phosphate isomerase, tpi; (5) glyceraldehyde-3-phosphate dehydrogenase, gapA;
(6) phosphoglycerate kinase,pgk; (7) phosphoglycerate mutase, gpmA orpgml;
(8) enolase, eno; (9) phosphotransferase system; (10) pyruvate kinase, pykA or
pykF.










H+
H+ H+ H
H+
Xylose H+
H+ H+
\ xyl Outside

Inside

Xylose Xyl ATP H+ ADP


nH+ xyIB
nH+ Xylulose Xylulose-SP

Xylose ATP ADP +P,

ATP

ADP

Figure 1-3. Xylose transport in E. coli. In contrast to glucose, the transport and activation
of xylose is not coupled, each requiring energy.









Ribulose-5-P


rpA9


Idehyde-3-P


Xylulose-5-P


Glyceraldehyde-3-P Fructose--P


Figure 1-4. Xylose metabolism. Xylose is metabolized to intermediates of glycolysis (in
bold) by the pentose-phosphate pathway. For the sake of carbon balance, 6 xylose
are converted to 4 fructose-6-phosphate and 2 glyceraldehyde-3-phosphate. Note
that neither ATP nor reducing equivalents are produced or consumed in this
pathway.













SDecarboxyati
- O


0 II
C -C -CH3
0 P
Pyruvate H


0
II
Enzyme I-TPP -c -CH,


S
Enzyme 2- R- I


II
oEny Enzyme 1-TPP


Enzm 2 -C-CH3
Enzyme 2 R.


Transa cety- tn Coe nzym e A-SH

SH II
Coenzyme A-S~ C -CH-
Acetyl-CoA


Enzyme 3- FAD


NAD+


NADH + H+


Figure 1-5. Reactions of the pyruvate dehydrogenase complex. Enzyme I (aceE) catalyzes
the oxidative decarboxylation of pyruvate through a pyruvate dehydrogenase
activity. The thiamine pyrophosphate bound activated acetaldehyde is passed to
the lipoate transacetylase (Enzyme 2; aceF), and ultimately to Coenzyme A. To
regenerate the reduced lipoate, the FAD bound to Enzyme 3 (dihydrolipoate
dehydrogenase; Ipd) is reduced. NAD+ is reduced to NADH by this FADH,,
allowing for another cycle.


Redox


Enzyme 3 FADH2


Reactants: Products:
Pyruvate CO2
NAD+ NADH
Coenzyme A Acetyl-CoA


co


Enzyme 1-TPP









GLYCOLYSIS


Co2


Oxaloacetate


Form;


Malate


8

Fumarate


ccinat

Succinate


- PEP
ATP
1
ADP
NADH
Pyruvate NAI

j 2 Lactate


Acetyl-CoA NADH
-_ NAD+


Ace P [Acetalhyde]
Acetyl-P [Acetaldehyde]


5 NADH


Eth NADn
Ethanol


Acetate


Figure 1-6. Fermentation pathways of E. coli. (1) pyruvate kinase, pykA orpykF or PTS
sugar transport; (2) pyruvate formate-lyase, pflB; (3) phosphotransacetylase, pta;
(4) acetate kinase, ackA; (5) PFL-deactivase / alcohol dehydrogenase /
acetaldehyde dehydrogenase, adhE; (6) PEP carboxylase, ppc; (7) malate
dehydrogenase, mdh; (8) fumarase,fumB; (9) fumarate reductase,frdABCD; (10)
lactate dehydrogenase, IdhA.














CHAPTER 2
FLUX THROUGH CITRATE SYNTHASE LIMITS THE GROWTH OF
ETHANOLOGENIC Escherichia coli KO 11 DURING XYLOSE FERMENTATION

Introduction

Our laboratory has previously engineered E. coli strain B for the production of

ethanol from pentose-rich, hemicellulose syrups by expressing high levels of Zymomonas

mobilis pdc (pyruvate decarboxylase) and adhB (alcohol dehydrogenase) (Ingram et al.

1999; Ohta et al. 1991). This strain was chosen for metabolic engineering because of its

hardiness, wide substrate range, and ability to grow well in mineral salts medium without

organic nutrients (Alterthum et al. 1989; Luli and Strohl 1990). During xylose

fermentation, ATP yield in E. coli is low (-0.67 ATP per xylose) due to separate energy

requirements for uptake and phosphorylation (Tao et al. 2001). Unlike most genetically

engineered strains of E. coli, KO 11 grew to higher densities than the parent in both

mineral salts and complex media (Martinez et al. 1999). Initial studies with Luria broth

demonstrated rapid and efficient conversion of sugars to ethanol by KO 11, with yields

approaching 95% of the theoretical maximum. However, volumetric productivity and

ethanol yields were considerably lower in mineral salts medium without complex

nutrients (Lawford and Rouseau 1996; Martinez et al. 1999; Moniruzzaman and Ingram

1998; York and Ingram 1996a; York and Ingram 1996b).

Supplementing mineral salts medium with complex nutrients significantly

increased ethanol production. The least expensive complex nutrient, corn steep liquor,

supported growth rates and ethanol productivities near those for Luria broth but only

26










when provided at high concentrations (5% w/v). Although not prohibitively expensive,

the addition of high levels of complex nutrients adds to the cost of ethanol production and

increases the requirements for waste treatment.

The lower rate of ethanol production volumetricc productivity) in minimal media

(compared to Luria broth) resulted from low cell densities and reduced expression of

recombinantpdc and adhB (lower metabolic activity). Inorganic components did not

appear to be limiting and no specific auxotrophic requirements could be identified

(Martinez et al. 1999). Reduced expression of heterologous genes was attributed to

"biosynthetic burden", the competitive reduction in synthesis of heterologous products

due to de-repression of native genes for biosynthetic enzymes (Martinez et al. 1999). In

this study, I have used a mineral salts medium containing 1% CSL and investigated the

basis of the requirement for higher levels of nutrients during xylose fermentation. Four

hypotheses were examined as the basis for the decreased growth in the CSL medium: 1)

availability of macro-nutrients; 2) loss of a biosynthetic pathway due to metabolic

engineering; 3) insufficient ATP during xylose fermentation; and 4) an imbalance in

central metabolism.

Materials and Methods

Microorganisms and Media

E. coli B (ATCC 11303) and an ethanologenic derivative, strain KO11 (Ohta et al.

1991), were used in all fermentation experiments. KO11 contains a deletion in thefrd

region (anaerobic fumarate reductase) which eliminates succinate production. Genes

encoding the Zymomonas mobilis ethanol pathway (pdc, adhB) and chloramphenicol

acetyltransferase (cat) were integrated into thepfl gene (chromosome) by a single








28

cross-over event resulting in a functional, full length pfl gene downstream. Both E. coli B

and K 11 are prototrophic. Stock cultures were stored in glycerol at -750C. Working

cultures were transferred daily on solid medium containing mineral salts and 1% CSL.

Xylose (2%) and chloramphenicol (alternating between 40 and 600 mg liter-') were

included in solid media for K011.

A citrate synthase mutant, E. coli W620 (glnV44, gltA6, galK30, pyrD36,

spdL129, thi-1), was obtained from the E. coli Genetic Stock Center (CGSC # 4278) and

used to test expression of the B. subtilis citZ gene (citrate synthase). This strain contains a

gltA6 mutation (citrate synthase) that prevents growth on M9 medium containing thymine

and glucose (Herbert and Guest 1968).

Corn steep liquor medium (CSL+X) contained (per liter in distilled water): 10 g of

corn steep liquor (-50% solids), 1 g of KH2PO4, 0.5 g of K2HPO4, 3.1 g of (NH4)2SO4, 0.4

g of MgCL2,6H20, and 20 mg of FeCl3*6H20. A one-liter stock solution of CSL was

prepared by dilution of 200 g with distilled water, adjustment to pH 7.2 with 50% NaOH

and steam sterilization. Before use, the sterile stock solution of CSL was aseptically

clarified by centrifugation (10,000 x g, 5 minutes). Mineral solutions were prepared as

described previously (Martinez et al. 1999). Broth cultures and fermentations contained

9% (w/v) xylose medium, unless indicated otherwise. In some experiments, Luria broth

containing xylose was included for comparison.

Fermentation Conditions

Seed cultures (100 ml in 250 ml-flask) were grown 14-16 hours at 350C with

agitation (120 rpm). Cells were harvested by centrifugation (5,000 x g, 5 min) and used as

an inoculum to provide an initial concentration of 33 [ig ml-' dry weight (0.1 OD550nm).








29

Fermentation vessels contained a total volume of 350 ml ( 350C, 100 rpm). Cultures were

maintained at pH 6.5 by the automatic addition of 2N KOH (Moniruzzaman and Ingram

1998). For strain B, 6 N KOH was used to maintain pH after the initial 24 h.

Supplements were added with distilled water as necessary (10 ml total volume).

Organic acids and amino acids were neutralized with NaOH, sterilized by filtration and

added at a final concentration of 2 mg ml-'. Acetaldehyde was added at a final

concentration of 0.25 mg ml-' or 0.5 mg ml-'. Cell mass, ethanol, organic acids and sugars

were monitored at 24 h intervals.

Aerobic Growth Studies

Cells were grown with aeration in 250 ml, baffled flask (350C, 220 rpm)

containing 50 ml of CSL+X medium. A range of sugar concentrations was tested (0.5% -

5%) to determine maximal cell density under conditions of sugar excess. Media were

inoculated directly using cells grown on solid media (18-24 h). Ethanol and cell mass

were measured after 16 h. For comparison, Luria broth containing 5% (w/v) xylose was

also included.

Analytical Methods

Cell mass was estimated as OD550nm using a Baush & Lomb Spectronic 70 (1

OD550 = 0.33 mg ml-' dry cell weight). Ethanol was measured by gas chromatography

using a Varian model 3400 CX as described previously (Moniruzzaman and Ingram

1998). Organic acids and sugars were measured by HPLC using a HP 1090 Series II

chromatograph equipped with a BioRad Aminex HPX-87H ion exclusion column (450C,

4 mM H2SO4, 0.5 ml minf', 10 pl injection) and dual detectors (refractive index monitor

and UV detector at 210 nm).










Fermentation products were also analyzed by NMR to confirm the identities of

HPLC peaks. Broth samples were centrifuged to remove cells. Supernatants (0.9 ml) were

mixed with deuterium oxide (0.1 ml) and sodium 3-(trimethylsilyl)propionate (10 mM

internal standard) in 5 mm sample tubes. Proton spectra were obtained using a modified

Nicolet NT300 spectrometer in the Fourier transform mode (Buszko et al. 1998) as

follows: frequency, 300.065 MHz; excitation pulse width, 5 ps; pulse repetition delay, 3

s; spectral width, 3.6 KHz. A minimum of 100 acquisitions were obtained for each

sample.

Genetic Methods

The citZ gene encoding B. subtilis citrate synthase II has been previously

described (Jin and Sonenshein 1994). This gene was amplified by PCR (forward primer,

5'-TGTGCTCTTCCATGTTTTTACAACACTGTTAAAG-3'; reverse primer,

5'-TTGCTCTTCGTTAGGCTCTTTCTTCAATCG-3') using genomic DNA from B.

subtilis strain YB886 as the template (Barbosa and Ingram 1994). Primers were added to

the Taq PCR Master mix (Qiagen) as recommended by the manufacturer. Conditions of

thermal cycling were as follows: 1) two initial cycles with denaturation at 940C (60 s),

annealing at 500C (60 s) and elongation at 680C (90 s); 2) twenty-eight cycles with

denaturation at 940C (10 s), annealing at 700C (60 s), and elongation at 680C (90 s); and

3) a final elongation step at 720C (10 min). The PCR product (1.5 kbp) was cloned into

pCR2.1-TOPO (Invitrogen) using ampicillin (50 [ig ml1) for selection. Colonies were

screened for size and ability to complement the gltA mutation ofE. coli W620 on

glucose-minimal medium (Herbert and Guest 1968). The citZ gene was also confirmed by

DNA sequencing using a LI-COR model 4000L sequencer (Middendorf et al. 1992).










NAD(P)H/NAD(P) ratio

Whole cell fluorescence was used as a relative measure of reduced nucleotides in

situ (Tartakosvsky et al. 1996; Trivedi and Ju 1994). Since only the reduced form of

NAD(P)H fluoresces at 460 nm, an immediate decrease in the fluorescence of fermenting

cells is interpreted as a decline in the level of NAD(P)H and the NAD(P)H/NAD(P)+

ratio. Cells were grown for 12 h in CSL+X medium, harvested by centrifugation (5,000 x

g, 5 min) and washed 3 times in mineral salts. The pellet was then suspended in mineral

salts solution at a concentration of 1.0 OD550. Emission at 460 nm (excitation at 340 nm)

was recorded at 5 s intervals using an Aminco-Bowman Series 2 Luminescence

Spectrometer. Cells were energized by the addition of 1% xylose resulting in an

immediate increase in fluorescence, primarily due to the increase in NADH/NAD+ ratio.

Test compounds were added at a final concentration of 2 mg ml-' (organic acids, amino

acids) or 0.25 mg ml-' acetaldehydee) using distilled water as a control. Results for each

test compound were expressed relative to the xylose-dependent increase in fluorescence.

Control experiments confirmed that quenching of cellular fluorescence did not occur

when additives were mixed with energy-deficient cells (without xylose).

Enzyme Assays

Citrate synthase was assayed using a modification of the method described

previously (Evans et al. 1993, Faloona and Srere 1969). Cultures were grown in one-liter

flasks (250 ml Luria broth) for 16 h at 350C (150 rpm). Cells were harvested by

centrifugation, washed 3 times in buffer containing 50 mM Tris-Cl (pH 8.0) and 20%

glycerol and suspended in 2 volumes of same buffer. Cell-free preparations were made by

two passages through a French Pressure cell (20,000 psi) followed by treatment with








32

-100 ag ml' deoxyribonuclease I. Cell debris was removed by centrifugation (15,000 x g,

Ih, 40C). The supernatant was dialyzed against 20 mM Tris-Cl and 20% glycerol. Each

assay (1 ml) contained 20 mM Tris-Cl (pH 8.0), 10 mM KC1, 1 mM

5',5'-dithio-bis-(2-nitrobenzoic acid), 10 mM oxaloacetate and 0.5 mM acetyl-CoA.

Reactions were initiated by the addition of cell lysate and monitored for 300 s at 412 nm.

Specific activity was reported as pmol of reduced coenzyme A produced per min per mg

protein.

Results

Macro-nutrient Limitation.

Previous studies have shown that up to 5% CSL is needed to support anaerobic

growth and ethanol production at a rate near that of Luria broth (Martinez et al. 1999).

Based on a comparison of E. coli elemental composition (Taylor 1946), the

macro-nutrient salts in CSL+X medium should provide sufficient nitrogen and

phosphorus to support the growth of at least 5 mg ml-' dry weight. During anaerobic

growth in pH-stats with CSL+X (Fig. 2-1), the maximal cell density for KO11 was only

about 1 mg ml-', 33% lower than the parent E. coli B (1.5 mg ml-') in CSL+X medium

and only 25% of that reached by KO 11 (4 mg ml-') in Luria broth plus sugar (Martinez et

al. 1999, York and Ingram 1996b, York and Ingram 1996b). During aerobic growth with

the same nutrients, however, KO 11 grew to a maximum density of 2.7 mg ml-'. This is

almost two-fold higher than E. coli B under the same conditions and 2.7-fold higher than

KO 11 during anaerobic growth. Together, these results indicate that the anaerobic growth

of KO 11 in 1% CSL with 9% xylose is not limited by the availability of macro-nutrients

(ie. N, P, etc.) or by the inactivation of a biosynthetic pathway due to genetic










manipulation. However, metabolic engineering of the ethanol pathway does appear to

contribute to the reduced growth of KO 11 in this medium under anaerobic conditions.

Energy Limitation.

The separate energy requirement for uptake (ATP-dependent transporter or proton

symport) and phosphorylation (xylulokinase) results in a low net yield of ATP from

xylose fermentation (0.67 ATP per xylose), 33% of the yield from glucose (2 ATP per

glucose) (Tao et al. 2001). An additional ATP can also be produced from pyruvate by the

acetate pathway. Since KOl1 produced less acetate than strain B (Table 2-1), the growth

ofKO 11 could be limited by the availability of ATP. To test this hypothesis, I compared

the growth of KO11 in 1% CSL containing 9% xylose with growth in 1% CSL containing

9% glucose (Fig. 2-2A and Fig. 2-2B). Glucose was fermented to ethanol at a higher rate

than xylose. However, the cell yield of KO 11 was identical for both sugars, despite the

3-fold difference in net ATP production. Although cell densities were low, cells remained

metabolically active for at least 96 h and produced most of the ethanol after growth had

ceased.

Metabolic Imbalance Relieved by Addition of Pyruvate or Acetaldehyde.

Pyruvate serves a dual role during fermentation, as a source of carbon skeletons

for biosynthesis and as a source of electron acceptors acetaldehydee) to allow continued

ATP production by glycolysis. During sugar fermentation to ethanol, one NADH is

produced per pyruvate. Each NADH must be oxidized by reducing an electron acceptor

such as acetaldehyde or by biosynthetic reactions (Mat-Jan et al. 1989). In a growing wild

type E. coli, partitioning of pyruvate between biosynthesis and redox needs is presumed

to be balanced for optimal growth. Metabolic engineering of the ethanol pathway










contributed to the reduced growth of KO 11 only under fermentative conditions,

consistent with a metabolic imbalance resulting from uncontrolled utilization of pyruvate

for ethanol production. This possibility was confirmed by the addition of pyruvate to

CSL+X medium. Pyruvate addition resulted in a dose-dependent increase in cell growth

and ethanol production that was particularly evident after 24 h (Fig. 2-3A and Fig. 2-3B).

With 2 mg ml-' of added pymvate, growth and ethanol production were twice that of the

control without pyruvate addition (Table 2-2). Supplementing with pyruvate did not cause

a buildup of TCA intermediates or acidic fermentation products (Table 2-1). Note that

format was produced in all fermentations, confirming the that the pfl gene encoding

pyruvate formate-lyase remains functional in KO 11.

The addition ofpyruvate to media has been shown to increase the intracellular

pyruvate pool in E. coli (Yang et al. 2001), increasing the ratio of potential electron

acceptors for the oxidation of NADH (from glycolysis). When added at a level of 2 mg

ml-', pyruvate was metabolized concurrently with cell growth during the first 24 h after

inoculation (Table 2-1). The pyruvate-dependent increase in cell mass (-1 mg ml-') was

roughly equivalent to half of the added pyruvate (Table 2-2, Fig. 2-3A). Remaining

pyruvate is presumed to be metabolized to acetaldehyde by recombinant Z. mobilis

pyruvate decarboxylase. Since acetaldehyde has been previously shown to stimulate

growth and ethanol production by yeasts (Walker-Caprioglio et al. 1985) and Z. mobilis

(Stanley et al. 1997), it seemed possible that the stimulation of cell growth by pyruvate

could be mediated in part by an increase in acetaldehyde from pyruvate (Table 2-2, Fig.

2-3C and Fig. 2-3D). Concentrations of acetaldehyde above 0.50 mg ml' were toxic.

With lower concentrations of acetaldehyde (0.25 and 0.50 mg ml-'), cell growth and










ethanol production were increased. Like pyruvate, added acetaldehyde was fully

metabolized during the initial 24 h after inoculation (Table 2-1). A near optimal level of

acetaldehyde was provided by 2 additions of 0.25 mg ml' each to CSL+X medium

(initially and after 12 h). This was almost as effective as pyruvate (2 mg ml-') in

stimulating ethanol production and also caused a 65% increase in cell mass. The basis for

the increase in cell growth is not readily explained by the limited routes for acetaldehyde

metabolism in E. coli as compared to those for pyruvate, a key central metabolite. These

results provide evidence that the beneficial effect of added pyruvate results primarily

from an increase in electron acceptors.

Pyruvate as a Source of Carbon Skeletons for Biosynthesis.

The pyruvate-stimulated increase in cell growth reflects a two-fold increase in the

flow of carbon into biosynthesis. Pyruvate and upstream metabolites in glycolysis are

used for the biosynthesis of approximately half of cellular constituents. Pools for these

upstream intermediates may increase when pyruvate is added, increasing availability for

biosynthesis. Pyruvate (and phosphoenolpyruvate) is also converted to a series of

biosynthetic intermediates by the TCA pathway and linking reactions. The TCA pathway

provides half of the carbon skeletons for cell protein. None of the TCA intermediates can

be produced readily from acetaldehyde by biosynthetic reactions. Note that the TCA

pathway is not cyclic during fermentation. This pathway is interrupted between

2-ketoglutarate and succinate by ArcAB-mediated repression of genes (sucAB) encoding

2-ketoglutarate dehydrogenase (Iuchi and Lin 1988). One side of the TCA pathway

produces 2-ketoglutarate, the precursor for the glutamic acid family of amino acids,

polyamines, among others. Precursors such as oxaloacetate on the other side of the TCA








36

pathway are derived from phosphoenolpyruvate. Oxaloacetate is used for synthesis of the

aspartic acid family of amino acids, etc. The addition of pyruvate could potentially

increase the flow of carbon into both sides.

TCA pathway intermediates were tested as additives to CSL+X medium.

Utilization of these additives was investigated using HPLC and NMR (Table 2-2, Fig.

2-4). All except two, succinate (100% remaining) and isocitrate (78% remaining), were

metabolized efficiently during the initial 24 h of fermentation (Table 2-1). Additions of

malate and fumarate resulted in a similar small increase in fumarate, but did not stimulate

growth or ethanol production. Despite the potential interconversion of these

intermediates, fumarate did not accumulate when oxaloacetate was added. Addition of

aspartate, the transamination product of oxaloacetate, was similarly ineffective. Indeed,

addition of oxaloacetate, malate, fumarate and aspartic acid reduced growth and ethanol

production. In contrast, 2-ketoglutarate was almost as effective as pyruvate in stimulating

growth and ethanol production by KO 11. A similar stimulation was also observed for

glutamate, the transamination product of 2-ketoglutarate.

TCA intermediates that are immediate precursors of 2-ketoglutarate were not

beneficial. Isocitrate was not readily metabolized. Citrate was metabolized but had no

effect on growth and ethanol production. Growth with added citrate was accompanied by

an accumulation of fumarate and a high acetate/formate ratio similar to that with pyruvate

(Table 2-1). Note that this ratio is near unity for other fermentations with TCA

intermediates, providing a clue to the ineffectiveness of citrate. The addition of citrate

may induce citrate lyase (Furlong 1987; Lutgens and Gottschalk 1980; Schneider et al.

2000), an enzyme that cleaves citrate into an equimolar mixture of oxaloacetate and








37

acetate. Oxaloacetate is readily metabolized to furmarate. Both fumarate and acetate were

higher in fermentations with added citrate than with 2-ketoglutarate and other TCA

intermediates, consistent with the induction of citrate lyase. Induction of this enzyme is

presumed to block the beneficial effects of this TCA intermediate for biosynthesis.

When considered together, studies with added TCA intermediates provide

evidence that the beneficial effect ofpyruvate for growth and ethanol production by

KO 11 in CSL+X medium results in large part from an increase in the flow of carbon

skeletons into 2-ketoglutarate and subsequent products of biosynthesis. However,

investigations with added pyruvate and acetaldehyde provided evidence that an increase

in electron acceptors was arguably of primary importance for the beneficial effect of

pyruvate. For both to be possible, both must be mediated by a common mechanism.

Whole-cell Fluorescence.

The ratio of NAD(P)H/NAD(P)+ has been shown to alter cellular patterns of

metabolic flux (de Graef et al. 1999). NAD(P)H is an allosteric inhibitor of many

enzymes including pyruvate dehydrogenase (Graham et al. 1989), phosphotransacetylase

(Suzuki 1969), malate dehydrogenase (Sanwal 1969) and citrate synthase (Faloona and

Srere 1969; Weitzman 1966). In KO11, the addition of acetaldehyde or pyruvate

(metabolized to acetaldehyde by recombinant pyruvate decarboxylase) would be expected

to decrease the level of NAD(P)H and the NAD(P)H/NAD(P)+ ratio by increasing the

pool of acetaldehyde available for reduction to ethanol. This has been investigated in

non-growing cells by examining the effects of these additives on whole-cell fluorescence.

(Fig. 2-5A and Fig. 2-5B).










Fluorescence changes in responses to additives were immediate and stable as

shown for acetaldehyde (Fig. 2-5A). Relative fluorescence increased when fermentation

was initiated by the addition of xylose, and decreased immediately upon the addition of

acetaldehyde (alcohol dehydrogenase) and pyruvate (pyruvate decarboxylase plus alcohol

dehydrogenase), consistent with expected changes in the oxidation of NADH. The

fluorescence of energized cells also decreased immediately upon the addition of

2-ketoglutarate and oxaloacetate (Fig. 2-5B). The apparent decline in NAD(P)H in

response to these two TCA pathway intermediates may be due to reductive amination

oxaloacetatee and 2-ketoglutarate). Addition of the respective amino acid products,

glutamic acid and aspartic acid, did not cause a similar change. With added oxaloacetate,

malate dehydrogenase provides an additional opportunity for NADH oxidation.

Additions of malate, fumarate, succinate, citrate and isocitrate did not

significantly alter whole cell fluorescence. Together, these data demonstrate that three

compounds which increased the growth and fermentation of KO 11 in CSL+X medium

acetaldehydee, pyruvate and 2-ketoglutarate) also decreased the NAD(P)H/NAD(P)+ ratio

in cells. Compounds which did not decrease this ratio were not beneficial. Oxaloacetate

was an exception. Although this compound decreased the NAD(P)H/NAD(P)+, growth

and fermentation were retarded. The negative effects of added oxaloacetate may be

attributed to the induction of pyruvate carboxykinase. Together with

phosphoenolpyruvate carboxylase, this enzyme creates a futile cycle for ATP (Chao et al.

1994; Chotani et al. 2000 ). ATP yields are low for xylose and ATP wasted by this futile

cycle may offset any potential benefits from increased oxidation of NADH.










Citrate Synthase, a Link Between NADH and 2-Ketoglutarate.

In E. coli (gltA) as in most Gram-negative bacteria, citrate synthase is

allosterically inhibited by NADH and activated by acetyl-CoA (Weitzman 1981). The

activity of this enzyme serves to regulate the flow of carbon into the 2-ketoglurate side of

the TCA pathway, linking the cellular abundance of NADH and acetyl-CoA to the

production of 2-ketoglutarate for biosynthesis (Faloona and Srere 1969; Lee et al. 1994;

Walsh and Koshland, Jr. 1985). This enzyme integrates both beneficial effects of added

pyruvate, increased electron acceptors acetaldehydee) and increased carbon skeletons in

the 2-ketoglutarate arm of the TCA pathway (2-ketoglutarate). The allosteric control of

this enzyme by NADH could restrict the flow of carbon into the biosynthesis of

2-ketoglutarate and other products. This hypothesis can be readily tested by expressing an

NADH-insensitive recombinant citrate synthase gene in K 11.

The primary citrate synthase in Gram-positive bacteria is allosterically regulated

by ATP and relatively insensitive to NADH (Jin and Sonenshein 1996). Since an

over-abundance of ATP is not anticipated during xylose fermentation (Tao et al. 2001),

expression ofB. subtilis citZ in KO11 would be expected to increase carbon flow into the

oxidizing arm of the TCA pathway. Primers were used to clone the citZ gene (including

ribosomal binding site) into pCR2.1-TOPO to produce pLOI2514. Plasmid pLOI2514

was found to complement a gltA mutation in E. coli W620 on plates containing M9

minimal media supplemented with glucose and thymine. Citrate synthase activity (0.08 U

mg protein-') was also confirmed in strain W620(pLOI2514) and absent in strain W620

lacking citZ.








40

Expression of citZ in KO 11 (pLOI2514) increased growth and ethanol production

by approximately 75% (Fig. 2-6) in comparison to the control with vector alone,

KO 1 (pCR2.1-TOPO). The low level of NADH-insensitive citrate synthase produced

from pLOI2514 was almost as effective as pyruvate, acetaldehyde and 2-ketoglutarate

additions in stimulating growth. Thus the allosteric regulation of the native citrate

synthase by high NADH appears to limit the flow of carbon skeletons into biosynthesis in

CSL+X medium.

Discussion

The rate of ethanol production and ethanol yield are important factors in

determining the cost of large-scale fermentation processes. For KO 11, both of these are

directly related to the extent of growth of the biocatalyst (Fig. 2-7). In CSL+X medium,

more than half of the ethanol was produced after cells entered stationary phase (Fig. 2-1).

By increasing cell densities, fermentation times can be reduced without sacrificing

ethanol yield. However, previous studies with KO 11 have shown that high levels of

complex nutrients were needed for cell growth and rapid ethanol production (Martinez et

al. 1999; York and Ingram 1996b; York and Ingram 1996b). This apparent requirement

for high levels of complex nutrients now appears to reflect a regulatory error in the

partitioning of pyruvate skeletons between competing requirements for the oxidation of

NADH and biosynthesis. Our study demonstrates that the growth of KO 11 was not

limited by nutrients, a lack of biosynthetic enzymes, or insufficient ATP from xylose

metabolism (0.67 ATP per xylose). During the fermentation of 9% xylose, growth was

limited by a lack of carbon skeletons for the biosynthesis of products derived from










2-ketoglutarate. Growth and ethanol production were increased by the addition of

pyruvate or 2-ketoglutarate, but not by the addition of oxaloacetate, malate or fumarate.

The apparent starvation for carbon skeletons to produce 2-ketoglutarate was also

alleviated by the addition of acetaldehyde, consistent with an involvement of NADH or

NADH/NAD+ ratios. The ratio ofNADH/NAD+ is typically higher during fermentation

than during oxidative metabolism (de Graef et al. 1999). High levels of NADH serve as

an allosteric inhibitor of citrate synthase, the first committed step for the production of

2-ketoglutarate and a likely bottleneck for the biosynthesis of many amino acids (Walsh

and Koshland, Jr. 1985). Addition of acetaldehyde decreased the NADH/NAD+ ratio by

increasing the pool of electron acceptors, potentially increasing the function of the native

citrate synthase in vivo. This hypothesis was confirmed, in part, using the B. subtilis citZ

gene encoding an NADH-insensitive citrate synthase (Jin and Sonenshein 1994; Jin and

Sonenshein 1996). Expression of citZ in KO 11 stimulated growth and ethanol production

by almost two-fold, substantially reducing the need to supply high levels of complex

nutrients.

The pattern of carbon flow in KO 11 is summarized in Figure 2-8. Expression of

high levels of Z. mobilis pdc and adhB redirect pyruvate away from native fermentation

pathways (pyruvate formate-lyase, lactate dehydrogenase) and into ethanol, even in the

presence of competing native enzymes (Ohta et al. 1991). Since the Km of pyruvate

decarboxylase for pyruvate is approximately one-tenth that of the competing enzyme,

pyruvate formate-lyase, production of acetyl-CoA would also be limited. Integration of

the ethanol-production genes into the chromosomalpfl gene may further contribute to this

problem by reducing the level of pyruvate formate-lyase activity. Although pyruvate










dehydrogenase has a Km for pyruvate that is equal to that of pyruvate decarboxylase,

pymvate dehydrogenase is expressed at low levels during fermentation and is

allosterically inhibited by the high levels of NADH present during fermentation (de Graef

et al. 1999; Graham et al. 1989). In CSL+X medium, a portion of cellular pyruvate was

converted to acetyl-CoA by KO 11 during the first 24 h as evidenced by the accumulation

of acetate as a fermentation product. These acetate levels are presumed to be in excess of

biosynthetic needs.

The addition of either pyruvate or acetaldehyde dramatically stimulated the

growth of KO 11 in CSL+X medium. At least three sites of allosteric regulation may

contribute to the increase in growth. Both pyruvate dehydrogenase (de Graef et al. 1999;

Graham et al. 1989) and citrate synthase (Faloona and Srere 1969; Weitzman 1981) are

allosterically inhibited by NADH. Oxidation of NADH from glycolysis during the

reduction of acetaldehyde from added pyruvate or added acetaldehyde would tend to

decrease the NADH/NAD+ ratio. This should reduce the allosteric inhibition ofpyruvate

dehydrogenase and citrate synthase by NADH. Additional acetyl-CoA from pyruvate

dehydrogenase would supplement that produced by pyruvate formate-lyase and increase

the pool of acetyl-CoA a substrate for citrate synthase and an allosteric antagonist of

NADH inhibition of the native citrate synthase (Weitzman 1981). Together, these

regulatory circuits would feed-forward to promote the flow of additional carbon skeletons

into 2-ketoglutarate and subsequent products of biosynthesis.















Table 2-1. Effects of additives on the composition of fermentation products (24 h) in 1% CSL+X medium (9% xylose).
% of additive Fermentation Products (mM)
Medium or strain Additive
remaining Fumarate Succinate Lactatea Formate Acetate Ethanol
CSL+X medium <0.01 <0.3 9.6 <1.5 <1.0 <1.0
E. coli B, parent None <0.01 53.4 27.7 95.8 91.4 71


None
Sodium pyruvate (2 mg ml'1)
Acetaldehyde (2 x 0.25 mg ml'1)
Citric acid (2 mg ml ')a
Isocitric acid (2 mg ml'1)
Sodium 2-ketoglutarate (2 mg ml'1)
Oxaloacetic acid (2 mg ml'1)
Sodium malate (2 mg ml'1)
Sodium fumarate (2 mg ml'1)
Sodium succinate (2 mg ml'1)


0.03
0.29
0.21
1.65
<0.01
0.28
0.02
1.45
1.22
0.07


E. coli KO 11














Table 2-2. Effects of additives on growth and ethanol production by KO 11 in 1% CSL+X medium (9% xylose).
Volumetric Production
SConcentration Cell Yieldc o ton Maximum Ethanol Yieb
Additive No. of Ethanol Yieldb (%)
mg ml1- mM Average % of Control g liter' h-1 % of Control mg ml1- % of Control

None (control) 26 -- 0.94 0.15 100 0.38 0.08 100 31.12 4.64 100 61
Sodium pyruvate 3 0.5 4.6 1.32 0.32 140 0.55 0.17 144 37.60 5.91 121 74
Sodium pyruvate 3 1.0 9.1 1.49 0.30 158 0.66 0.17 173 40.09 5.53 129 79
Sodium pyruvate 15 2.0 18.2 1.99 0.20 212 0.81 0.14 213 44.22 2.69 142 87
Sodium pyruvate 3 4.4 36.4 2.08 0.08 221 0.83 0.01 218 43.23 1.50 139 85
Acetaldehyde (half initially + 4 0.5 (total) 11.4 1.55 0.07 165 0.60 0.35 158 44.05 1.89 142 86
half after 12 h)
citric acid 2 2.0 10.4 0.88, 0.75 86 0.35, 0.33 89 30.00, 27.59 93 56
isocitric acid 2 2.0 6.6 1.11, 0.88 105 0.51, 0.36 113 36.45, 29.59 106 65
sodium 2-ketoglutarate 5 2.0 11.9 1.89 0.18 201 0.84 0.08 221 41.54 1.24 133 81
oxaloacetic acid 2 2.0 15.1 0.75, 0.75 80 0.28, 0.28 74 23.58, 23.64 76 46
sodium malate 2 2.0 11.2 0.76, 0.84 85 0.27, 0.27 71 23.89, 24.97 79 48
sodium fumarate 2 2.0 12.5 0.80, 0.96 91 0.37, 0.32 92 27.15, 29.84 92 56
sodium succinate 3 2.0 7.4 1.00 0.12 106 0.41 0.05 108 35.00 1.31 112 69
potassium glutamate 3 2.0 10.8 1.66 0.17 177 0.66 0.13 174 43.14 0.13 139 85
aspartic acid 2 2.0 15.0 0.85, 0.82 88 0.33, 0.32 84 28.40, 27.40 90 55
a Volumetric Productivity was calculated as the average hourly rate of ethanol production between 24 h and 48 h after inoculation.
When less than 3 replicates are presented, the values of each replicate are shown.
b Yield is expressed as a percentage of the theoretical yield (100% = 0.51 g ethanol per g xylose).












5 25 I Anaerobic
2.5
2.0-
1.5-
1.0-
0.5
0.0
: ^ H5: 5Aerob c I









Figure 2-1. Comparison of maximal cell densities achieved during aerobic and anaerobic
growth in 1% CSL mineral salts medium containing either xylose or glucose. Thin
lines representing the standard error of the mean are shown for averages with
three or more replicates.












2.5
A
2.0


1.5


3 1.0
ID~ ---------------]

0.5- --9% Xylo se
S-A- 9% Glucose
0.0-
0 24 48 72 96
Time (h)
40
B

30-


20


10-
-m- 9% Xyl ose
-- 9% Glucose
0-
0 24 48 72 96
Time (h)

Figure 2-2. Comparison of growth and ethanol production from glucose and xylose by E.
coli KO11 during the fermentation of 9% sugar in 1% CSL mineral salts medium.
A. Growth. B. Ethanol. Thin lines representing the standard error of the mean are
shown for averages with three or more replicates.



























0 24 48
Time (h)


72 96


S -u-No addition
-&-0.25 g Acetaldehyde
---0.50 g Acetaldehyde
--o-2x 0.25 g Acetaldelh
0 24 48 72
Time (h)


30


20


10


0




50


40


30


20


10


0


48
Time (h)


---No addition
S0.25 g Acetaldehyde
--0.50 g Acetaldehyde
S--2x 0.25 g Acetaldelyc
0 24 48 72
Time (h)


Figure 2-3. Effects of added pyruvate and acetaldehyde on growth and ethanol production
by E. coli KO11 in CSL+X medium. A. Cell growth with added pyruvate. B.
Ethanol production with added pyruvate. C. Cell growth with added acetaldehyde.
D. Ethanol production with added acetaldehyde. Thin lines represent the standard
error of the mean.












3



02








0


S30


20

M1


Lv


Figure 2-4. Initial effects of added TCA pathway intermediates on growth and ethanol
production by E. coli KO11 (24 h). A. Growth. B. Ethanol. Thin lines represent
the standard error of the mean.


eee
g"~;b~~











5.0
A 1% Xylose 0.25 g/L Acetaldehyde

S4.5
0
S4.0


~3.5


3.0 ,
0 60 120 180 240 300
Time (s)


125

O 100 B

S75

8 50-

25

0




Figure 2-5. Effect of metabolites on whole-cell fluorescence. A. Effects of acetaldehyde
on the xylose-dependent increase in fluorescence (time course). B. Effects of
metabolites on the xylose-dependent increase in fluorescence. Values in B are
expressed as a percentage of the xylose-dependent increase in the fluorescence of
whole cells observed in the presence of both xylose and the indicated additive.
Note that a decrease in the xylose-dependent fluorescence is interpreted as a
decrease in NAD(P)H and the NAD(P)H/NAD(P)+ ratio.











2.5
A
2.0


I1.5


1.0
^-




0.5
-- K011 (pCR2.1-TOPO)
0.0 -T-KOll (pLOI2514)
II' I I
0 24 48 72 96
Time (h)
50
B
40


30-


S20



-eKO 11 (pCR2.1-TOPO)
0A --KO 11 (pLOI2514)
0-
0 24 48 72 96
Time (h)


Figure 2-6. B. subtilis citZ increases the growth and ethanol production of KO 1l in
CSL+X medium. A. Growth. B. Ethanol. Thin lines represent the standard error of
the mean.











50 1.0
<
40- ,-0.8
A-'A-
30- 0.6
a CD
CO -'
20- 0.4


10- -0.2
m Ethanol Yield
A Volumetric Productivity
0 0.0
0.0 0.5 1.0 1.5 2.0
Cell Mass (g/L)

Figure 2-7. Relationship between cell yield and fermentation performance. In this plot,
results were combined from fermentations with CSL+X medium alone and with
supplements. A computer-generated polynomial was used to approximate cell
yields. Results from a linear regression analysis are shown for volumetric
productivity. Dotted lines represent the the 95% confidence intervals.








52
glyceraldehyde-3-P -------------------------- XYLOSE 52
SNAD+
l NADH


phosphoenolpyruvate NAD+

NADH

1 ^


lactate

NADH


NAD+


ETHANOL


NAD+


acetyl-P acetate
citrate syntmse
;NADH t; acetyl-CoAl t


NADH
NAD+ 1
nalate aspartate

6+ I9d
fiumarate 4-
NADH (e
NAD 17 (deleted)
NAD+ *^


succnate


gutamate 7-

NADP+


citrate
12

isocitrate
A NADP+
C02 -'r1 NADPH

3& 2-ketoglutarate


NADPH
+NH4


Figure 2-8. Fermentation and TCA pathway. Unless noted otherwise, enzymes listed are
native to E. coli. Key to enzymes: 1. pyruvate kinase (pykA, pykF); 2. pyruvate
formate-lyase (pflB); 3. pyruvate dehydrogenase (aceEF,lpd); 4.
phosphotransacetylase (pta); 5. acetate kinase (ackA); 6. alcohol/aldehyde
dehydrogenase (adhE); 7. Z. mobilis pyruvate decarboxylase (pdc); 8. Z. mobilis
alcohol dehydrogenase II (adhB); 9. lactate dehydrogenase (IdhA); 10.
phosphoenolpyruvate carboxylase (ppc); 11. citrate synthase (gltA); 12. aconitase
(acn); 13. isocitrate dehydrogenase (icd); 14. glutamate dehydrogenase (gdhA);
15. malate dehydrogenase (mdh); 16. fumarase (fumB); 17. fumarate reductase
(frdABC); 18. aspartate transaminase (aspA); 19. aspartase (aspC). Arrows
beneath citrate synthase indicate inhibition of activity by NADH and antagonism
of NADH inhibition by acetyl-CoA.


CO2














CHAPTER 3
GENETIC CHANGES TO OPTIMIZE CARBON PARTITIONING IN
ETHANOLOGENIC Escherichia coli KO 11

Introduction

Citrate synthase, a key enzyme in the partitioning of carbon into biosynthesis

(Walsh and Koshland, Jr. 1985), was shown to be growth limiting for KO11 (Chapter 2).

Native citrate synthase is allosterically inhibited by high levels of NADH typical of

fermentation (Weitzman 1981). Growth and ethanol production were substantially

improved in KO 11 by expression of an NADH-insensitive citrate synthase (citZ) from

Bacillus subtilis. A similar stimulation of growth and ethanol production was observed

during low aeration (oxidation of NADH) and with the addition ofpyruvate,

2-ketoglutarate, and acetaldehyde.

An alternative approach to enhance citrate synthase activity in KO 11 is to increase

available substrate pools oxaloacetatee and acetyl-CoA). In vitro, acetyl-CoA has been

shown to serve as an allosteric activator ofphosphoenolpyruvate carboxylase (Izui et al.

1981) for the production of oxaloacetic acid and to relieve the allosteric inhibition of

citrate synthase by NADH (Weitzman 1981). In this chapter, I demonstrate that

physiological and genetic approaches which increase the availability of acetyl-CoA for

biosynthesis stimulate cell growth and ethanol production. These results were used to

engineer a second generation biocatalyst, strain SU102, in which a small additional










portion of substrate carbon was redirected from fermentation products to cellular

biosynthesis.

Materials and Methods

Microorganisms and Media

Strains and plasmids used in this study are listed in Table 3-1. KOl and its

derivatives (SU102 and SU104) are prototrophic. Working cultures of ethanologenic

strains were transferred daily on solid medium (1.5% agar) containing mineral salts, 2%

xylose, and 1% CSL (Chapter 2). Stock cultures were stored frozen at -750C. Luria-agar

plates were used for the maintenance of other strains. Ampicillin (50 [ig/ml), kanamycin

(50 lig/ml) and tetracycline (5 or 10 [ig/ml) were added as appropriate.

Fermentation

Seed cultures and fermentations (350C and 150 rpm) were grown in mineral salts

medium containing 1% CSL and 9% xylose (CSL+X medium; Chapter 2). Fermentations

were maintained at pH 6.5 by automatic addition of 2N KOH (Moniruzzaman and Ingram

1998). Supplements were filter sterilized as concentrates and added directly to

fermentation broth. Samples were removed during fermentation for the measurement of

cell mass, ethanol, organic acids and sugars.

Analytical Methods

Cell mass was estimated from the optical density at 550 nm using a Bausch &

Lomb Spectronic 70 spectrophotometer (1 OD50 = 0.33 mg ml-' dry cell weight). Ethanol

and acetaldehyde were measured by gas chromatography (Varian 3400CX)

(Moniruzzaman and Ingram 1998). Organic acids and sugars were analyzed by HPLC

(Hewlett Packard 1090 series II chromatograph equipped with refractive index and UV210








55

detectors) with a BioRad Aminex HPX-87H ion exclusion column. Maximum volumetric

productivity in mmol liter' h-' was estimated as the first derivative of ethanol production

using PSI-Plot software (Poly Software International, Salt Lake City, Utah). Specific

productivity was estimated by dividing volumetric productivity by cell mass; units are

mmol (gram cell dry weight)-' hour'.

Genetic Methods

Standard methods were used for plasmid construction, DNA amplification (PCR),

transformation, electroporation and P1 phage transduction (Miller 1992; Sambrook and

Russell 2001). Primers (ORFmers) for the amplification of the E. coli ackA and adhE

coding regions were purchased from the Sigma Genosys (The Woodlands, TX). These

primers included SapI sites at both ends of the amplified product. Chromosomal DNA

from E. coli W3110 (ATCC 27325) served as the template for amplification. This strain

was also used as an intermediate during the construction of adhE deletion in KO 11.

Chromosomal insertion of deleted genes (adhE and ackA) was facilitated by

inserting a tet gene flanked by FRT sites for removal of the antibiotic marker by the

chlorotetracycline-inducible FLP recombinase (pFT-A) in the final construct

(Martinez-Morales et al. 1999; Posfai et al. 1997). Integration of linearized DNA was

facilitated by using pKD46 (temperature conditional) containing an arabinose-inducible

red recombinase (Datsenko and Wanner 2000). Putative deletion mutants were selected

for tetracycline resistance (5 mg liter-') and screened for appropriate antibiotic resistance

markers. At each step, mutants were verified by analyses of PCR and fermentation

products.










Construction of pLOI2065 Containing a Removable Tetracycline Resistance Cassette

To facilitate antibiotic removal after chromosomal integration, a reusable cassette

was constructed from the tet gene of pKNOCK-Tc (Alexeyev 1999) and the FRT sites in

pSG76-A and pSG76-K (Posfai et al. 1997). Both FRT sites were oriented in the same

direction to allow efficient in vivo excision of the tet gene by theflp-encoded

recombinase (Martinez-Morales et al. 1999). This cassette was inserted into a modified

pUC18 to produce pLOI2065 (Fig. 3-2). Plasmid pLOI2065 contains two EcoRl sites and

two Smal sites oriented to allow the isolation of the FRT-tet-FRT cassette as a Smal to

EcoRl fragment for directional insertion, as a blunt fragment (Smal) and as a sticky-ended

fragment (EcoRI).

Nucleotide Sequence Accession Number

The sequence for plasmid pLOI2065 has been deposited in GenBank under

acquisition number AF521666.

Construction of SU102 Containing an Insertion Mutation in ackA

Strain SU102 was made by introducing the ackA mutation directly into KO11.

The PCR-amplified coding region of ackA was cloned into pCR2.1-TOPO. After

digestion with EcoRl, the 1.2 kbp fragment containing the ackA coding region was ligated

into the unique EcoRl site of pLOI2302. A recombinant plasmid was selected in which

the direction of transcription of lac and ackA genes were opposite. The ackA gene was

disrupted by digestion with EcoRV (1 site) and the insertion of a 1.7 kbp Smal fragment

from pLOI2065 containing a tet gene flanked by two FRT sites for FLP recombinase. A

2.8 kbp AscI fragment containing ackA'-FRT-tet-FRT- 'ackA was isolated from this

plasmid and ligated into the AscI site ofpLOI2224 containing a conditional R6K replicon.










The resulting plasmid, pLOI2375 (Fig. 3-2), was used as a template for PCR

amplification of the 2.8 kbp AscI fragment with ackA primers. After purification by

phenol extraction, amplified DNA was used for electroporation ofE. coli KO 1 (pKD46)

expressing phage lambda red recombinase (Datsenko and Wanner 2000). Recombinants

were selected for tetracycline resistance. Plasmid pKD46 was eliminated by growth at

400C. The integrated tet gene was deleted using pFT-A expressing theflp recombinase

(Martinez-Morales et al. 1999; Posfai et al. 1997). After removal of this plasmid by

growth at 400C, the resulting strain containing a mutation in ackA (insertion of 98 bases

including stop codons in all three reading frames) was designated SU102.

Construction of SU104 Containing a Deletion in adhE

A mutation in adhE was initially constructed in W3110 prior to P1 transduction

into KO 11. The PCR-amplified coding region of adhE (2.7 kbp) was cloned into

pCR2.1-TOPO. A recombinant plasmid was selected in which the transcription of lac and

adhE were oriented in the same direction. The central region of the adhE gene (1.1 kbp)

was deleted by digestion with HinCII (2 sites) and replaced with a 1.6 kbp Smal fragment

from pLOI2065 containing the FRT-tet-FRT cassette (1.7 kbp) to produce pLOI2803 (Fig.

3-2). After digestion of pLOI2803 with both PvuI and Scal, this plasmid served as a

template to amplify the 3.2 kbp region containing adhE'-FRT-tet-FRT- 'adhE using adhE

primers. This amplified DNA was used for electroporation. Recombinants were selected

for tetracycline resistance. Plasmid pKD46 was eliminated by growth at 420C.

P1 transduction was used to transfer the adhE mutation in W3110 to KO 11. To

circumvent differences in restriction systems, the adhE::tet mutation was transduced into

a restriction-negative (modification-positive) derivative ofE. coli B (strain WA837) prior










to transduction into K 11. The tetracycline resistance gene was deleted from the K 11

derivative using pFT-A expressing theflp recombinase (Martinez-Morales et al. 1999;

Posfai et al. 1997). After removal of this plasmid by growth at 400C, the resulting strain

containing an internal deletion in adhE was designated SU104.


Results and Discussion

Acetate Addition Stimulates Growth and Ethanol Production by Reducing Net Acetate
Production During Sugar Metabolism.

During the aerobic metabolism of sugars by E. coli, acetate production has been

associated with a decrease in growth rate. Considerable effort has been made to minimize

acetate production as a means of increasing cell density and the production of

recombinant proteins (Aristidou et al. 1995; Bauer et al. 1990; Chang et al. 1999;

Contiero et al. 2000; Yang et al. 1999a; Yang et al. 1999b). The addition of as little as 2 g

liter-' sodium acetate (24 mM) has been shown to decrease growth rate during oxidative

sugar metabolism (Luli and Strohl 1990). During xylose fermentation by KO 11, however,

the addition of acetate stimulated growth and ethanol production (Fig. 3-3A and B; Table

3-2). A portion of the added acetate was initially consumed, in contrast to control

fermentations where acetate was continuously produced (Fig. 3-4A). Rates of acetate

production declined during subsequent incubation in both control and

acetate-supplemented fermentations. Although almost twice as much sugar was

metabolized by acetate-supplemented fermentations than by control fermentations (no

additions), net acetate production in the acetate-supplemented culture (7.0 mmol liter'1)

was less than half that of the control (18.6 mmoles liter-') after 72 h.










Previous studies have shown that the reversible phosphotransacetylase-acetate

kinase pathway can serve as a route for entry of added acetate into the intracellular pool

of acetyl-CoA (Brown et al. 1977; Higgins and Johnson 1970). Additional acetate uptake

activity may be provided by the inducible acetyl-CoA synthetase, although this gene is

typically repressed under fermentative conditions (Kumari et al. 1995). Thus, the

stimulation of growth and ethanol production by added acetate is presumed to result from

the increased availability of acetyl-CoA. Under anaerobic conditions, the primary role of

the TCA pathway is to supply carbon skeletons for biosynthesis. Increasing the

availability of acetyl-CoA would promote biosynthesis by relieving the NADH-mediated

allosteric inhibition of citrate synthase (Weitzman 1981) and by serving as an allosteric

activator of phosphoenolpyruvate carboxylase (Izui et al. 1981).

Stimulation of Growth and Ethanol Production by Added Pyruvate Can Be Primarily
Attributed to Increased Acetate Production.

The stimulation of growth and ethanol production by pyruvate reported previously

(Chapter 2) appeared quite similar to the effects of added acetate (Fig. 3-3A and B).

Analysis of products during fermentation provided further evidence of a related

mechanism of action for acetate and pyruvate (Fig. 3-4 A-E). With the exception of

format (Fig. 3-4B), profiles of organic acids were similar for acetate and

pyruvate-supplemented cultures. Both were distinctive from the control lacking

supplements. Control fermentations produced lower levels of lactate than

pyruvate-supplemented and acetate-supplemented fermentations during the initial 72 h

(Fig. 3-4C). Addition of pyruvate stimulated the production of acetate to levels equivalent

to that of acetate-supplemented fermentations (Fig. 3-4A). In both pyruvate and










acetate-supplemented fermentations, acetate concentrations were approximately 2-fold

higher than in the control after 36 h. Acetate concentrations in all fermentations remained

relatively constant during further incubation.

Most of the supplemental pyruvate (22 mM) was metabolized during the initial 3

h of incubation (Fig. 3-5) although the benefits for growth and ethanol production

persisted throughout fermentation. During the initial 3 h, the largest change was an

increase in acetate (Fig. 3-6A). Smaller pyruvate-dependent increases were observed for

ethanol, format, lactate and acetaldehyde. Biosynthetic needs were estimated to be small

(increase of approximately 0.06 mg dry cell weight liter') and did not represent a

significant sink for the added pyruvate (2 g liter'). The partitioning of pyruvate between

these different fermentation products (and biosynthesis) in KO 11 is generally regarded as

the result of 5 competing reactions: pyruvate decarboxylase (PDC), pyruvate

formate-lyase (PFL), pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH) and

phosphoenolpyruvate carboxylase (PPC). On a triose basis, relative activities can be

estimated from the distribution of fermentation products (Fig. 3-1; de Graef et al. 1999).

The large pyruvate-dependent increase in acetate after 3 h (Fig. 3-6A) reflects an increase

in acetyl-CoA production (PFL and PDH activities). In the absence of format hydrogen

lyase induction (Bock and Sawers 1996), format production provides an independent

measure of PFL activity and exhibited a modest increase compared to acetate. These

results indicate that PDH activity (estimated as acetate minus format) serves as the

primary source of additional acetyl-CoA during the metabolism of added pyruvate (Fig.

3-6B). Production of ethanol also increased immediately after the addition of pyruvate

due to an increase in the production of acetaldehyde by PDC. Increased pyruvate










oxidation by PDH is presumed to provide the additional NADH required to reduce

acetaldehyde produced from added pyruvate. The increase in LDH activity (estimated as

lactate production) can be attributed to substrate activation (Tarmy and Kaplan 1968).

Elevated extracellular levels of acetate in pyruvate-supplemented fermentations may

serve to increase intracellular acetyl-CoA pools, extending the period of growth and

thereby increasing the volumetric rate of ethanol production.

The channeling of pyruvate to acetyl-CoA and acetate by the addition of pyruvate

can be readily explained based on known allosteric controls (Fig. 3-1). Pyruvate is both a

substrate for acetyl-CoA production and a strong allosteric activator of

phosphotransacetylase (Suzuki 1969). Addition of pyruvate has also been shown to

increase acetaldehyde and decrease the level of NADH (Chapter 2), an allosteric inhibitor

of phosphotransacetylase (Suzuki 1969) and PDH (de Graef et al. 1999; Hansen and

Henning 1966). These actions would also tend to increase the partitioning of carbon into

acetate.

Higher levels of succinate and fumarate (3-fold to over 10-fold, respectively) were

produced by acetate- and pyruvate-supplemented fermentations (Fig. 3-4C and D). PPC

(Izui et al. 1981) and citrate synthase (Weitzman 1981) are both activated by acetyl-CoA

and link the supply of this important intermediate to fermentation and biosynthesis. Under

anaerobic conditions, the reductive portion of the TCA pathway is used to produce

succinate. Due to the deletion of fumarate reductase (frd) in KO 11, little succinate was

produced and a small amount of fumarate accumulated. The increases in succinate and

fumarate levels in acetate and pyruvate-supplemented fermentations may result from an

excess of citrate. Excess citrate can be cleaved into acetate and oxaloacetate by an










inducible citrate lyase (Lutgens and Gottschalk 1980). Additional succinate can be

produced from isocitrate by isocitrate lyase (Weitzman 1981).

Pyruvate and free CoA are co-substrates for format production by PFL (Fig.

3-1). Formate levels increased during the initial 12 h of incubation in all fermentations

and declined thereafter (Fig. 3-4B). The decline in format can be attributed to the

formate-inducible format hydrogen lyase (Bock and Sawers 1996). Supplementing with

acetate and pyruvate had opposite effects on format production (Fig. 3-4B), higher

concentrations in pyruvate-supplemented fermentations and lower levels in

acetate-supplemented fermentations in comparison to those of the control. Both

differences are in general agreement with the central role of acetyl-CoA in metabolism

(Chang et al. 1999b; Contiero et al. 2000; Kirkpatrick et al. 2001). In

acetate-supplemented fermentations, format production by PFL may be limited by a lack

of free CoA. Conversely, higher format levels produced by pyruvate-supplemented

fermentations may result from an increase in free CoA due to the allosteric activation of

phosphotransacetylase by pyruvate (Suzuki 1969).

Stimulation of Growth and Ethanol Production by Acetaldehyde Can Be Attributed to
Increased Acetyl-CoA.

Growth and ethanol production were also stimulated by acetaldehyde (Chapter 2;

Fig 3-3A and B). At concentrations above 5.6 mM, acetaldehyde strongly inhibited

growth. It was empirically determined that stimulation equivalent to that of pyruvate

could be achieved by the addition of 11.2 mM acetaldehyde, 5.6 mM initially and 5.6 mM

after 12 h of fermentation (Chapter 2). Previous studies also demonstrated that the










addition of acetaldehyde caused a rapid decrease in the intracellular concentration of

NADH (Chapter 2).

The initial portion of added of acetaldehyde was metabolized within 3 h (Fig.

3-5A). During this time, ethanol increased by an amount equal to 70% of the added

acetaldehyde (Fig. 3-6A). Increased pyruvate flux through PDH appears to provide the

additional NADH required for acetaldehyde reduction (Fig. 3-6B). The second

acetaldehyde addition was metabolized within 1 h (Fig. 3-5A) although benefits persisted

throughout fermentation (Fig. 3-3A and B). Following the second addition, production of

acetate and ethanol was increased while format production was reduced. The persisting

benefit of acetaldehyde additions for growth and ethanol production are presumed to

result from an increase in the intracellular acetyl-CoA pool as a consequence of higher

extracellular levels of acetate. High levels of NADH and global regulation by ArcA and

FNR (de Graef et al. 1999) may also limit PDH function in the absence of supplements.

Increased production of acetyl-CoA by PDH (and perhaps increased synthesis of PDH)

would be expected in response to NADH oxidation.

The production of format by PFL may be limited by competition with PDH for

free CoA. Patterns of organic acid production in acetaldehyde-supplemented cultures

provide further support for a mechanism of action similar to that for pyruvate and acetate

(Fig. 3A-E). Acetate levels were higher in all three supplemented cultures than in the

unsupplemented control. Each supplemented fermentation also produced higher levels of

succinate, lactate and fumarate than the control.










Stimulation of Growth and Ethanol Production by Inactivation of Non-biosynthetic
Pathways Which Consume Acetyl-CoA.

Acetyl-CoA serves as the single most important intermediate for cellular

biosynthesis, providing over half of the cellular carbon during sugar metabolism

(Neidhardt et al. 1990). Previous studies have shown that cell growth is limited by the

availability of carbon skeletons during the fermentation of xylose (Chapter 2), a limitation

which was relieved (Fig. 3-1A and B) by supplements which increase the extracellular

levels of acetate (acetate, pyruvte, acetaldehyde). During fermentation (Fig. 3-1), two

pathways drain acetyl-CoA from the intracellular pool but provide limited benefit to

biosynthesis. Acetyl-CoA can be reduced to acetaldehyde and ethanol by alcohol

dehydrogenase E (adhE) as an alternative route for NADH oxidation in KO 11 (Fig. 3-1).

Acetyl-CoA can also be converted to acetate by phosphotransacetylase (pta) and acetate

kinase (ackA), increasing the production of ATP. Mutations in these pathways were

investigated as a means of sparing acetyl-CoA for biosynthetic needs.

Inactivation ofackA rather thanpta was chosen to minimize potential problems

associated with global regulation. Acetyl-P is proposed to serve as an important global

regulator in E. coli (Bouche et al. 1998; Kirkpatrick et al. 2001; McCleary et al. 1993),

affecting gene expression and fundamental processes such as the turnover of RpoS.

During oxidative metabolism, inactivation of the acetate pathway (pta, ackA) is

detrimental to growth (Chang et al. 1999b; Contiero et al. 2000; Kirkpatrick et al. 2001).

Although not fully understood, this detrimental effect has been attributed to depletion of

free CoA due to low rates of acetyl-CoA turnover (Chang et al. 1999b). In contrast to that

found in previous studies concerning oxidative metabolism, inactivation of ackA (SU102)










stimulated growth and ethanol production during the fermentation of xylose (Fig. 3-3C

and D). An adhE mutation in strain KO11 (SU104) was of no benefit during xylose

fermentation. Together, these results suggest that ADH contributes little to metabolism in

KO11. The beneficial effect of inactivating ackA is presumed to result from an increase in

the availability of acetyl-CoA for biosynthesis, the genetic equivalent of adding acetate,

pyruvate, or acetaldehyde.

Strains SU104 (adhE mutant) and SU102 (ackA mutant) were also tested in

fermentations with supplements that had been shown to increase the growth and ethanol

production in KO 11 (Table 3-2). Addition of acetate, pyruvate and acetaldehyde to

SU104 increased growth and ethanol production indicating that the native alcohol

dehydrogenase (adhE) was not essential for this response. Growth and ethanol production

by SU102 (ackA) without supplements were equivalent to that of KO11 with

supplements. The addition of pyruvate, acetate, 2-ketoglutarate, or acetaldehyde to SU102

provided little further improvement in growth or ethanol production.

HPLC analysis of organic acids revealed similarities in the patterns of fumarate

(Fig. 3-3J) and succinate (Fig. 3-31) production between SU102 (ackA mutant) and KO11

supplemented with acetate, pyruvate or acetaldehyde (Fig. 3-4E). The ackA mutation in

SU102 also increased lactate production (Fig. 3-41) and delayed the production of format

(Fig. 3-4G) and acetate (Fig. 3-4F). The delay in format production in SU102 could

result from increased acetyl-CoA, reducing the pool of free CoA (co-substrate for PFL)

analogous to acetate-supplemented KO 11 (Fig. 3-4B). Both acetate addition and

mutations in the acetate pathway have been shown to cause a similar repression of 37

genes (Kirkpatrick et al. 2001), attributed to an increase in the acetyl-CoA pool.










Inactivation of acetate kinase (SU102) caused an initial delay in acetate

production but did not block later synthesis. The pathway responsible for acetate

production during the latter stages of fermentation remains unknown but may be the

result of spontaneous dephosphorylation of acetyl-P as previously proposed (Brown et al.

1977) or from induction of cryptic enzyme(s). Despite the potential benefit of increased

ATP production by acetate kinase, the increased drain of acetyl-CoA to acetate through

this pathway appears to be more detrimental for growth and ethanol production by KO 11

than the reduction in ATP. With the exception of acetate (Fig. 3-4F), the production of

fermentation products by the adhE mutant (strain SU104) was essentially the same as for

the parent strain, K 11 (Fig. 3-4A). Acetate production by SU104 continued throughout

fermentation and reached higher final concentrations than K 11.

Conclusions

Increasing the availability of acetyl-CoA stimulated growth and ethanol

production from xylose by prolonging the growth phase of ethanologenic E. coli. The

resulting increase in biocatalyst rather than an increase in cellular activity was responsible

for the increased rate of ethanol production (Table 3-2). Similar benefits were obtained by

minimizing the loss of acetyl-CoA as acetate (ackA mutation) and by increasing

intracellular levels of acetate (supplementing with acetate, pyruvate, or acetaldehyde).

Inactivation of the native E. coli alcohol/aldehyde dehydrogenase (adhE) had little effect

indicating that this pathway has limited function in ethanologenic KO 11.

ATP production during xylose fermentation does not appear to limit growth or

cell yield in KO11. Including the energy required for xylose uptake and activation, less

than 1 ATP (net) is produced from the metabolism of each xylose converted to ethanol








67

(Tao et al. 2001; Chapter 2). During the initial 12 hours of growth, up to 31% of the ATP

(net) produced by KO11 is provided by the acetate pathway (calculated by assuming 1

ATP per acetate from acetate kinase and 0.4 ATP per pyruvate from glycolysis).

Disruption of this pathway (ackA) in SU102 increased cell yield by 2-fold (Table 3-2).

Thus, the partitioning of carbon skeletons rather than the production of ATP appears to

limit the growth of ethanologenic E. coli during xylose fermentation.

The mechanism for the stimulation of growth in ethanologenic E. coli KO 11 is

consistent with established patterns of allosteric regulation although further controls of

gene expression (FNR, ArcA) may also contribute to the observed effects. More than half

the amino acids produced in the cell are derived from the TCA pathway. Flux through

this pathway is controlled by PPC and citrate synthase (Lee et al. 1994; Walsh and

Koshland, Jr. 1985), activities which can be stimulated by acetyl-CoA (Weitzman 1981).

The individual addition of pyruvate, acetate, and acetaldehyde increased the extracellular

levels of acetate which can in turn serve to elevate intracellular pools of acetyl-CoA by

reversible reactions. Additional benefits of supplements include a reduction in the level of

NADH (added pyruvate and acetaldehyde), an allosteric inhibitor of citrate synthase

which is antagonized by high levels of acetyl-CoA. Based on these results with mutants

and with supplements, we conclude that the regulation of acetyl-CoA production and

consumption can be used to make small changes in the partitioning of carbon between

biosynthesis and fermentation during the ethanol production by E. coli KO11.












Table 3-1. Strains and plasmids used in Chapter 3.

Strain or Plasmid Relevant Characteristics


Strains

KO11

SU102

SU104

W3110

WA837


frd cat pfl' pfl::(Z. mobilis pdc' adhB')

KO11 ackA

KO11 adhE

wild type

rB m,+ gal met


Reference or source


Ohta etal. 1991

This work

This work

ATCC 27325

Wood 1966


Plasmids

pKD46

pFT-A

pCR2.1-TOPO

pKNOCK-Tc

pSG76-K

pSG76-A

pLOI2065

pLOI2224

pLOI2302

pLOI2375

pLOI2803


y P exo repAlOl pSC101 repliconts (red recombinase)

blaflp pSC101 repliconts (FLP recombinase)

bla kan ColEl

tet R6K (pir dependent replicon)

kan FRT R6K (pir dependent replicon)

bla FRT R6K (pir dependent replicon)

bla FRT-tet-FRT ColEl

kan R6K (pir dependent replicon)

bla ColEl (EcoRI flanked by AscI sites)

ackA::FRT-tet-FRTkan R6K (pir dependent replicon)

adhE::FRT-tet-FRTkan ColE 1


Datsenko and Wanner 2000

Posfai et al. 1997

Invitrogen

Alexeyev 1999

Posfai et al. 1997

Posfai et al. 1997

This work

Martinez-Morales 1999

Zhou and Ingram 1999

This work

This work
















Table 3-2. Effects of mutations and additives on cell yield and ethanol productivity.

Cell Mass Ethanol

Concentration Maximum Time l a Maximum Max VP b Sp. Prod.c Theoretical
Strain and additive (mM) N (g liter') (h) (h-1) (mM) (mmol liter' h-1) (mmol g-1 h-') Yield d (%)


KO11

+ pyruvate

+ acetate

+ acetate

+ acetaldehydee

+ 2-ketoglutarate


SU102

+ pyruvate

+ acetate

+ acetaldehyde

+ 2-ketoglutarate

SU104

+ pyruvate

+ acetate

+ acetaldehyde

+ 2-ketoglutarate
a Specific growth rate at 2 h.


none

21

24

19

11

12


0.95 0.13

2.00 0.18

1.92 0.01

1.61 0.30

1.51 0.15

1.84 0.20


1.94 0.14

1.93 0.13

2.24 0.17

2.02 0.17

1.83 0.01

1.02 0.08

1.96 0.06

1.68 0.02

1.57 0.31

1.97 0.15


none

21

19

11

12


none

21

19

11

12


0.63

0.76

0.75

NDf

0.47

ND


0.66

0.44

ND

ND

ND

0.71

ND

ND

ND

ND


641 93

955 55

930 11

901 118

909 98

907


946 20

926 19

933 26

952 6

901 1

550 89

885 24

889 10

856 100

971 20


8.3

18.3

18.7

ND

18.2

ND


10.1

11.5

12.0

ND

11.3

ND


63

94

92

89

90

90


b VP, maximum volumetric productivity, mM ethanol produced per liter per hour.
' Specific productivity at 12 h, mmol ethanol produced per gram cell dry weight per hour.
d Theoretical yield from 91g liter' xylose (1.667 mmol ethanol/mmol xylose).
e Half added initially, half added after 12 h.
fND, not determined. Estimates of specific and volumetric productivity were not calculated due to the limited number of sampling times.











glyceraede-3-P ------------------ XYLOSE
NAD'
NADH lactate
CO,
phosphDenalpyiUvate 9
ID -- NAD'

NAD 1 NADH NADH NAD'
SADH
@AcCoA Npywrate acetaBehyde ETHANOL

10, 3(DNADH RDA te 6,AD'
10AcCoA 2 : 6 NADH

CO, acetyl-CoA 4H I acetyl-P 5 acetale

W oaloacetate 0 NADH- cta
NADH Ad aoA n- citraa
NADH 1A

malate
SATP ADP+P 12

17 17 + NH4' I R\ NAAfl
fuuu~ate
RH1 ghltamaite 2-loetogtalate isocitbae

NADP' NADPH CO,
+ NH

Figure 3-1. Allosteric control of central metabolism. Unless noted otherwise, enzymes
listed are native to E. coli. Enzymes: 1. pyruvate kinase (pykA, pykF); 2. pyruvate
formate-lyase (pflB); 3. pyruvate dehydrogenase (aceEF,lpd); 4.
phosphotransacetylase (pta); 5. acetate kinase (ackA); 6. alcohol/aldehyde
dehydrogenase (adhE); 7. Z. mobilis pyruvate decarboxylase (pdc); 8. Z. mobilis
alcohol dehydrogenase II (adhB); 9. lactate dehydrogenase (IdhA); 10.
phosphoenolpyruvate carboxylase (ppc); 11. citrate synthase (gltA); 12. aconitase
(acn); 13. isocitrate dehydrogenase (icd); 14. glutamate dehydrogenase (gdhA);
15. glutamine synthetase (glnA); 16. malate dehydrogenase (mdh); 17. fumarase
(fumB); 18. fumarate reductase (frdABCD); 19. aspartate transaminase (aspA); 20.
aspartase (aspC). o indicates allosteric activation, o indicates allosteric inhibition.





























m '.kA EcoRVmISm
(inactive)

pLOI2375
5317 bps


AscI
Mack' FRT

EcoRVISnao
(irmtive)

Figure 3-2. Plasmids used to construct mutations in KO 11. FRT sites allow in vivo
excision of the tet gene after integration using FLP recombinase (flp). A. Plasmid
pLOI2065 containing a tet gene flanked by FRT sites. B. Plasmid pLOI2375
containing an interrupted ackA gene.



















l "JO lJ -__

I jSynk for A ad B -abols fa C rad D
U 0.5 ) K0No Adi 0.5a K11 NoAdd
-0-KOll I+ e SJ104
S-o- K11+ Acet~ ---- SUl ad
OD0 -Ko- K11O+ cAdehydte OJO-- -o-- -SU12 + Pynwrate
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96
Time (h) Time (h)




1000 10 D

800 :R 800 -

600- 600-
400- 400

200- 200 -
0-l- 0-
0 12 24 36 48 6 72 84 6 0 12 24 36 48 60 72 84 96
Tim e (i) Time (1I


Figure 3-3. Effect of media additions and mutations on growth (A, C) and ethanol
production (B, D). Symbols for A and B: 0, KO11 no additive; A, K1 1+
pyruvate; 0, KO11 + acetate; and O KO11+ acetaldehyde. Symbols for C and
D: 0, KO11 no additive; 0, SU102 (ackA) no additive; A, SU104 (adhE) no
additive; and 0, SU102 + pyruvate.

















20- 20-
-- _--~_OII_+_Ae____-l 0 UU 2 Pv___

m-KOll + ANo ta1 0 ; 31I2PM v
-ci-- KOll + AcetaHeyde -SUlU 1 Py iivate
0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84


20- B20



S10-
Z5. 5-

t 0-1
I0 12 24 36 48 60 72 84





0 12 24 48 72 84 0 1224 48 7284








Figure 3-4. Effect of media additions and mutations on organic acid production: acetate
(A/F), format (B/G), lactate (C/H), fumarate (D/I) and succinate (E/J). Symbols
for A-E: U, KO 1l no additive; A, KOl + pyruvate; 0, KO 1l + acetate; and O,
KOl 1+ acetaldehyde. Symbols for F-J: U, KOl 1 no additive; O, SU102 no
additive; A, SU14 no additive; and SU102 + pyruvate.
0- 0




Figure 3-4. Effect of media additions and mutations on organic acid production: acetate
(A/F), format (B/G), lactate (C/H), fumarate (D/I) and succinate (E/J). Symbols
for A-E: 0, KO11 no additive; A, KO11+ pyruvate; C, KO11 + acetate; and 7,
KO11+ acetaldehyde. Symbols for F-J: 0, KO11 no additive; 7, SU102 no
additive; A, SU104 no additive; and C, SU102 + pyruvate.


?6


6


























0 12 24 36 48 60 72 84 96


1.4 I I I I
,-1.2 E
1.0-
D 0.8-
S0.6
0.4
0.2
0.0 0


0 12 24 36 48 60 72 84 96
Time (h)


0 12 24 36 48 60 72 84 96


0 12 24 36 48 60 72 84 96
Time (h)


Figure 3-4. continued.


I I I











25

-20




10

A

0













/- -


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


- 200-

-i

E-
150-


M 100-


S50-
-I
ZSO-
:9


I I I I
6 12 18 24
Time (h)


>
-20

-15,

-10




-0




25


-20


-15


-10



-0


Figure 3-5. Metabolism of added acetaldehyde and pyruvate during fermentation.
Pyruvate addition is indicated by the large arrow. Acetaldehyde additions (5.6 mM
each) are indicated by the large arrow (initial addition) and the small arrow
(second addition at 12 h). A. Utilization of added pyruvate and acetaldehyde.
Symbols: A, pyruvate utilization by KO 1; 0, pyruvate utilization by SU102; O,
acetaldehyde utilization by KO 11; E, acetaldehyde in KO 11 broth with no
additions; and @, pyruvate in KO 11 broth with no additions. B. Effect of second
acetaldehyde addition on production of fermentation products by KO 11. Symbols:
U, cell mass; A, ethanol; @, format; 0, lactate; A, acetate; *, succinate; and 0,
fumarate.














15-




5-

0--







. 66-


4-


2-

0


a.) ( D a.) ( (D 6
e 4M 4 e 4M W1 0 -d






2 I lPyruva e B
N^I2 gLAcetate
FEIJT l Acetaldehyde


PPC/PPS LDH PFL PDH PDC


Figure 3-6. Partitioning of carbon among competing pathways during the initial 3 h of
fermentation. A. Fermentation products after 3 h. B. Relative activity of primary
enzymes that partition 3-carbon intermediates carbon (pyruvate and
phosphoenolpyruvate) through competing pathways. Relative activities were
estimated using fermentation products, expressed as [imol of product per ml
during the initial 3 h of incubation. Endogenous production of acetate in
acetate-supplemented fermentations was assumed to be equal to that for the
control fermentation without additives. Pyruvate decarboxylase activity was
assumed to be equal to ethanol production, except for the
acetaldehyde-supplemented fermentations where it could not be calculated.
Pyruvate dehydrogenase was calculated as the difference between acetate and
format production. Lactate dehydrogenase, pyruvate formatelyase, and
phosphoenolpyruvate carboxylase activities were assumed to equal to the
production of lactate, format, and succinate, respectively. Abbreviations: PPC,
phosphoenolpyruvate carboxylase; PPS, phosphoenolpyruvate synthetase; LDH,
lactate dehydrogenase; PFL, pyruvate formatelyase; PDH, pyruvate
dehydrogenase; PDC, pyruvate decarboxylase.














CHAPTER 4
A DEFICIT IN PROTECTIVE OSMOLYTES IS RESPONSIBLE FOR THE
DECREASED GROWTH AND ETHANOL PRODUCTION DURING XYLOSE
FERMENTATION

Introduction

Maintaining inexpensive sources of fuels and commodity chemicals for the U. S.

is a matter of national security. Increasing the production of fuel ethanol offers a potential

solution to this problem. The conversion of lignocellulose to fuel ethanol and other

chemicals typically derived from petroleum would decrease the U. S. dependance on

imported oil (Artzen and Dale 1999). Enteric bacteria are noted for their broad range of

growth substrates, including all the sugars present in the polymers of lignocellulose.

Escherichia coli, a microbial platform for the commercial production of amino acids and

recombinant proteins (Chotani et al. 2000; Akesson et al. 2001), was previously

engineered for the production ethanol by integrating a synthetic operon containing the

ethanol pathway from Z. mobilis (pdc and adhB) into the chromosome (Ohta et al. 1991).

The resultant strain, designated KO 11, fermented all the sugar constituents of

lignocellulose to ethanol with yields approaching 100% (Ohta et al. 1991; Martinez et al.

1999; Ingram et al. 1999).

During batch fermentations with strain KO 11, the volumetric rate of ethanol

production was directly related to the growth of the biocatalyst (Martinez et al. 1999;

Chapter 2; Chapter 3). Cell yield for both the ethanologenic strain and its parent (E. coli

B) was dependent upon the availability of nutrients in a variety of media, despite the










absence of any specific auxotrophic requirements. During the batch fermentation of

xylose (90 g liter1) to ethanol by strain KO 11, cell growth appeared to be limited by the

availability of carbon skeletons derived from the citrate arm of the anaerobic TCA

pathway (Chapter 2; Chapter 3) (Fig. 4-1).

During fermentative metabolism, the TCA pathway is interrupted by the

repression of 2-ketoglutarate dehydrogenase (Iuchi and Lin 1988). The ultimate product

of this pathway, 2-ketoglutarate, is a substrate for glutamate biosynthesis. During growth

in minimal media, glutamate is the most abundant free amino acid in the cytoplasm of E.

coli (Cayley et al. 1991). In addition to its roles in metabolism and protein synthesis,

glutamate biosynthesis is part of the primary response to osmotic stress (Csonka 1989;

Csonka and Hanson 1991). The high osmolarity of the medium used for ethanol

production (0.6 M xylose) would be expected to increase the requirement for glutamate as

a protective osmolyte.

Potassium ions are rapidly accumulated by E. coli in response to osmotic stress.

This is rapidly followed by the accumulation of glutamate (McLaggan et al. 1994), a

negatively charged amino acid and protective osmolyte, to balance the positive charge of

the accumulated potassium. In the closely related organism Salmonella typhimurium,

cells that are restricted in their ability to synthesize glutamate have been demonstrated to

grow poorly during osmotic stress (Csonka 1988; Yan et al. 1996). Mutations preventing

glutamate production were associated with the inability to balance the charge of

intracellular potassium. The resulting decrease in steady-state potassium levels has been

proposed to limit cell growth (Yan et al. 1996).










Alternate protective osmolytes such as glycine betaine (hereafter referred to as

betaine), proline, taurine, dimethylsulfoniopropionate and many others can be

accumulated from the environment. A hierarchy for these osmoprotectants was

empirically determined for salt stress (Randall et al. 1995). Although there have been

conflicting reports concerning this hierarchy for sugar-mediated osmotic stress (Glaasker

et al. 1998), betaine is generally regarded as the most effective protective osmolyte for E.

coli. The effectiveness of betaine for restoration of growth has been shown to cary with

the sugar used for osmotic stress (Dulaney et al. 1968)..

In this study, NMR was used to examine changes in the intracellular pools of

osmolytes in response to genetic changes and nutrient supplements that stimulated cell

growth and ethanol production. Low cell yield and low ethanol production in the absence

of supplements appears to result from a deficit in intracellular glutamate or alternative

protective osmolytes.

Materials and Methods

Microorganisms and Media.

The ethanologenic E. coli strains KO 11 and SU102 (KO 11 AackA; Chapter 3) are

prototrophic. Working cultures were transferred daily on solid medium (1.5% agar)

containing mineral salts, 2% xylose, and 1% corn steep liquor (CSL+X medium; Chapter

2) alternating between 40 mg liter' and 600 mg liter' chloramphenicol. Stock cultures

were stored frozen at -700C in 40% glycerol.

Fermentation.

Seed cultures (350C and 120 rpm) and fermentations (350C and 100 rpm) were

grown in either mineral salts medium containing 1% corn steep liquor and 9% xylose








80

(Chapter 3) or Luria broth (10 g liter -1 tryptone, 5 g liter-' yeast extract, 5 g liter-' NaC )

with 9% xylose. For fermentations, sufficient cell mass to achieve and initial

concentration of 33 mg liter' were harvested by centrifugation (5000 x g; 5 min) and

suspended in appropriate fresh medium. Fermentations were maintained at pH 6.5 by

automatic addition of 2N KOH (Moniruzzaman and Ingram 1998). Stock solutions (100

mM) ofbetaine (Sigma, St. Louis, MO) and dimethylsulfoniopropionate (TCI America,

Portland, OR) were dissolved in deionized water and filter sterilized directly into the

fermentation vessel. Glutamate and acetate were added as described previously (Chapter

2; Chapter 3).

WCNMR

Intracellular osmolytes were analyzed by NMR (Park et al. 1997). After a 24 h

incubation in the fermentation chamber, 700 mL culture was harvested by centrifugation,

washed twice in mineral salt solution containing NaCl (0.6 M) and resuspended in 3

volumes of ethanol (95%). Suspensions were rocked gently 16-24 hr at 40C. Cellular

debris was removed by centrifugation (4"C, 10,000 x g, 30 min). Extracts were dried

under vacuum, disloved in deionized water, dried under vacuum, resuspended in 33%

D20 and filtered (0.2[im) Acetone (10[iL) was used as an internal reference. Data was

obtained using a modified Nicolet NT300 spectrometer operating in the Fourier transform

mode as follows: 75.46 MHz; excitation pulse width, 25 us; pulse repetition delay, 40s;

spectral width 18 kHz and broadband (bi-level) decoupling of protons. For cell extracts,

at least 1000 scans were obtained.










Analytical Methods.

Cell mass was determined from the optical density at 550 nm using a Bausch &

Lomb Spectronic 70 spectrophotometer (1 OD550= 0.33 g liter' dry cell weight), and

ethanol was measured by gas chromatography (Varian 3400CX) (Moniruzzaman. and

Ingram 1998). For the quantitation of the compounds detected by NMR, a standard curve

was generated. The average of all the chemical shift values are reported normalized to

cell mass.

Results and Discussion

Citrate Synthase Flux Limits the Biosynthesis of Glutamate, a Primary Intracellular
Osmolyte.

E. coli typically maintains large cytoplasmic pools of potassium, glutamate and

trehalose during growth in media containing high concentrations of sugars or salt (Cayley

et al. 1991; Lewis et al. 1990). Previous studies with the ethanologenic E. coli strain

KO 11 demonstrated that the flux through citrate synthase, the first step in glutamate

biosynthesis, limited growth and ethanol production during the fermentation of 90 g liter'

xylose (0.6 M) to ethanol (Chapter 2; Chapter 3). This limitation was proposed to be due

to the drain ofpyruvate to ethanol by the recombinant pathway (pyruvate

decarboxylase-alcohol dehydrogenase) which has a higher affinity for pyruvate than

pyruvate formate-lyase or pyruvate dehydrogenase, competing pathways for aceyl-CoA

biosynthesis (Chapter 2; Chapter 3). Supplementing the CSL+X medium with acetate

increased the availability of acetyl-CoA (Chapter 3), an activator of citrate synthase

(Weitzman 1966). During fermentations with strain K 11, supplementing the CSL+X








82

medium with glutamate increased the final cell yield and ethanol productivity (Chapter 2)

by bypassing the growth-limiting citrate synthase.

The intracellular osmolyte pools were compared between conditions of lower

growth (CSL+X medium without additives) and higher growth (supplemented with

glutamate or acetate) to investigate whether the inability of strain KO 11 to accumulate

glutamate resulted from restricted citrate synthase flux (Fig. 4-2; Table 4-1).

Fermentations without additives accumulated only proline, while those supplemented

with acetate (activating citrate synthase) or glutamate (bypassing citrate synthase) resulted

in approximately 2-fold higher growth and ethanol productivity. Cells from these

fermentations accumulated approximately the same level of intracellular glutamate,

supporting the hypothesis that a deficit in the accumulation of this protective osmolyte

limited growth.

The intracellular proline, a known osmoprotectant (Csonka 1989;Csonka and

Hanson 1991), was likely accumulated from the CSL provided as a source of complex

nutrients. Though some organisms synthesize proline in response to osmotic challenge

(Kawahara et al. 1989), E. coli can only accumulate this protective osmolyte by active

transport (Smith et al. 1984). However, mutants have been isolated that are less sensitive

to the feedback-inhibition of the proline biosynthetic pathway (Smith 1985). Strains

expressing these genes accumulated high levels of intracellular proline (derived from

glutamate) and were more resistant to high osmotic environments (Csonka 1981; Csonka

et al. 1988). Presumably, the intracellular proline in strain KO11 may have resulted from

a similar spontaneous mutation. Accordingly, such a mutation would also reduce the

glutamate pool.








83

Glutamate was accumulated in fermentations with increased growth yield, and the

intracellular concentration of proline decreased (Table 4-1). This further supports the

hypothesis that the intracellular proline was taken up from the medium and was not a

result of biosynthesis. If the drain of intracellular glutamate for proline production had

starved the cells for glutamate, supplementing the medium with proline should have a

sparing effect on the consumption of glutamate. However, proline addition only increased

the intracellular proline pool without affecting either growth or ethanol production (Table

4-1).

Glutamate is a product of proline degradation (McFall and Newman 1996), but

the degradation of proline has been shown to be inhibited in media of high osmotic

strength (Csonka 1988). Supplementing the medium with an excess ofproline (17 mM)

should provide excess proline for glutamate production. However, the absence of

increased growth and intracellular glutamate in these fermentations confirms the

previously observed inhibition of proline degradation during osmotic stress (Csonka

1988).

The accumulation of proline was previously shown not to affect glutamate pools

(Cayley et al. 1992). During experiments in a glucose-mineral salts medium buffered with

MOPS and high NaC1, the primary osmolytes accumulated by E. coli were K glutamate,

MOPS and trehalose (Cayley et al. 1991; Cayley et al. 1992; Lewis et al. 1990). Cultures

in this medium supplemented with proline accumulated this protective osmolyte and

reduced the biosynthesis of trehalose (Cayley et al. 1992). However, the intracellular

glutamate concentration was not significantly altered by the accumulation of proline.








84

Thus, the intracellular accumulation of proline (from the CSL) by strain KO 11 should not

have affected the glutamate requirement.

Strain KO11 failed to synthesize detectable levels of the osmoprotectant trehalose

(<10 mM) under these conditions. While the presence of proline would have decreased

the synthesis of trehalose (Cayley et al. 1992), significant trehalose should have been

detected. This may be a result of growth on xylose, a pentose. Perhaps the relatively low

ATP yield from xylose catabolism (0.4 ATP/pyruvate) restricts the gluconeogenic

production of glucose and uridine diphosphate-glucose, substrates for trehalose

biosynthesis.

Genetic Changes to Optimize Carbon Partitioning Increased the Glutamate Pool.

The functional expression of citZ by strain KO 11 (pLOI2514) was previously

shown to increase growth and ethanol production. To confirm that the expression of this

enzyme aides in glutamate accumulation, the intracellular osmolyte pool during

fermentations in the CSL+X medium were analyzed (Fig 4-2; Table 4-1). Similar to the

fermentations supplemented with glutamate or acetate, cells from these fermentations had

an increased glutamate pool. The intracellular accumulation of proline was similar to that

of other experiments with increased growth yields. Thus, the expression of citZ provided

more citrate, ultimately increasing glutamate biosynthesis and the glutamate pool.

A mutation in the primary acetate production pathway (Apta) was previously

shown to increase glutamate biosynthesis (Chang et al. 1999b). This likely resulted from

the accumulation of acetyl-CoA, an activator of citrate synthase (Weitzman 1966).

Acetyl-CoA is also an activator ofphosphoenolpyruvate carboxylase (ppc) (Izui et al.

1981), the controlling step in the biosynthesis of oxaloacetate and co-substrate for citrate










synthase. Thus, the biosynthesis of citrate is regulated, in part, by the availability of

acetyl-CoA. In an analogous study presented here, blocking acetate production (Aack)

increased the intracellular glutamate pool level (Fig. 4-2, Table 4-1).

Glutamate Accumulation Functions in Osmoprotection.

Three different osmoprotectants were tested for their ability to restore growth and

ethanol production, replacing the additional glutamate requirement.(Fig. 4-3). The

addition of 1.0 mM betaine or dimethylsulfoniopropionate (DMSP) increased the cell

yield and ethanol production similar to experiments where glutamate production had been

increased. Taurine, a weak osmoprotectant for E. coli (McLaggan and Epstein 1991),

failed to increase growth or ethanol production. Neither betaine nor presumably DMSP

should provide a source of glutamate. Thus, supplying osmoprotectants to the medium

replaced the need for the accumulation of intracellular glutamate.

To determine the optimal concentration required to restore growth, betaine and

DMSP were added from 0.1-2.0 mM and 0.1-1.0 mM, respectively (Fig. 4-4). Growth

and ethanol were increased in a dose-dependant manner in each instance. The maximum

stimulation of growth and ethanol production by betaine was at the highest level of

betaine tested, 2.0 mM. However, only 0.25 mM DMSP was required for maximum

benefit. Surprisingly, during the fermentation of xylose to ethanol, DMSP is 10-fold more

effective in restoring growth and ethanol production than betaine. Though this is contrary

to previous reports that betaine is the most effective protective osmolyte (Randall et al.

1995), these studies were done using high levels NaCl for osmotic challenge. The ability

of betaine to restore growth has been reported to vary during osmotic challenge with








86

different sugars (Dulaney et al. 1968). Thus, a specific protective osmolyte may be more

effective during challenge by different osmolytes (sugars and salts).

Replacement of Glutamate by Other Osmoprotectants.

The intracellular osmolyte pools of cells in fermentations supplemented with

betaine and DMSP were examined by NMR. Cells from fermentations supplemented with

betaine were found to contain only detectable levels of betaine (Fig. 4-2), consistent with

previous studies (Cayley et al. 1992). This was likely a result of the properties of the

osmotically activated transport pathways. While the Km of ProP and ProU for proline are

0.3 mM and 2 tiM, respectively, ProU has a 1.3 [iM Km for betaine (Lucht and Bremer

1994). The high level of betaine in the medium (2.0mM) coupled with the low Km of the

primary betaine transport pathway (ProU) explains the exclusive accumulation of this

protective osmolyte. Additionally, the ProP system has a periplasmic, high-affinity

betaine binding protein (KD 1 tiM) which aids in the accumulation of this preferred

substrate.

Cells from the fermentations supplemented with 0.25 mM DMSP accumulated

both proline and DMSP. There are three possible explanations for the contemporaneous

accumulation of proline and DMSP under these conditions. It is possible that there is an

independent transporter for DMSP. However, DMSP is structurally similar to betaine

(Fig. 4-5), and it is likely transported by the same mechanism. Possibly, the accumulation

of both proline and DMSP results from the Km for proline and DMSP being more similar

to each other. Alternatively, the low level of DMSP in the medium (0.25 mM) compared

to the concentration of betaine used (2.0 mM) may have allowed for the proline (equal










concentrations in both experiments) to more effectively compete for transport by ProP

and/or ProU.

Neither the betaine nor DMSP supplemented fermentations contained detectable

levels of glutamate (>10 mM; Fig 4-2), confirming that the high glutamate pool was not

needed for biosynthesis, per se. Thus, the requirement for additional glutamate is

presumed to be associated with adaptation to the higher sugar environment. This is

consistent with previous reports using strains ofE. coli or S. typhimurium which were

deficient in glutamate biosynthesis (McLaggan et al. 1994; Csonka et al. 1994; Yan et al.

1996). While the growth of these strains was poor in high osmotic environments, normal

growth was observed in more optimal osmotic environments. Thus, the observed

deficiency in glutamate biosynthesis in strain KO11 was attributed to the inability to

accumulate large quantities of intracellular glutamate for osmoadaptation but not

necessarily for macromolecular biosynthesis.

Betaine from Difco Yeast Extract Restores Growth in Luria Broth Fermentations.

Dulaney and coworkers (1968) demonstrated that Difco yeast extract, a

component of Luria broth (10 g liter-' tryptone, 5 g liter-' yeast extract and 5 g liter-'),

contains betaine by extracting and fractionating Difco yeast extract. Xylose fermentations

with these nutrients yielded high biocatalyst concentrations and high ethanol productivity

(Table 4-1). Cells harvested from these fermentations at 12 h (when the sugar

concentration would be similar to that of the CSL fermentations at 24h) accumulated

proline and betaine were accumulated by strain KO11 (Fig 4-2). The ratio of proline to

betaine was much higher in Luria broth than in CSL+X medium supplemented with 2.0

mM betaine, thus allowing proline to more effectively compete with betaine for transport










by the osmotically active tranporters, ProP and ProU. The lower growth yield in

betaine-supplemented fermentations indicated that although betaine aided in restoring

growth, it was not as effective as the rich Difco nutrients. Some other nutrient in the Luria

broth may be necessary for even higher growth yields (>2 g liter'). The high availability

of carbon skeletons, essential vitamins and minerals in the Luria broth would also have a

sparing effect on all biosynthetic pathways. The higher growth yield and ethanol

productivity observed in fermentations with these nutrients may have resulted from this

general sparing effect.

Conclusions

Growth and ethanol production in the CSL+X medium was restricted by a deficit

in the accumulation of protective osmolytes. Glutamate, typically accumulated in

response to osmotic stress, failed to accumulate due to restricted citrate biosynthesis.

Supplementing the medium with potassium glutamate (2 g liter') bypassed this limitation

and restored the intracellular glutamate pool. Alternately, the addition of sodium acetate

(2 g liter'), an activator of citrate synthase and precursor of glutamate biosynthesis,

restored the intracellular glutamate pool and increased growth and ethanol production.

Together, these results suggested that a deficit in the production of glutamate restricted

growth.

Betaine and DMSP increased the growth and ethanol production in a

dose-dependent manner when added to the medium. NMR analysis of the intracellular

osmolytes during these fermentations demonstrated the accumulation of these protective

osmolytes. While the addition of DMSP to the CSL+X medium resulted in the

accumulation of both proline and DMSP, cells from betaine supplemented cultures