111111 1111111111111111111111111111111111111111111111111111111111111 US008795991B2 c12) United States Patent (10) Patent No.: US 8, 795,991 B2 San et al. (45) Date of Patent: Aug. 5, 2014

(54) INCREASING BACTERIAL SUCCINATE FOREIGN PATENT DOCUMENTS PRODUCTIVITY WO PCT/US20111035001 9/2011 (75) Inventors: Ka-Yiu San, Houston, TX (US); George OTHER PUBLICATIONS Bennett, Houston, TX (US); Grant Balzer, Houston, TX (US); Jiangfeng Jantama et a!., Combining Metabolic Engineering and Metabolic Zhu, Houston, TX (US); Chandresh Evolution to Develop Nonrecombinant Strains of Escherichia coliC Thakker, Houston, TX (US); Ailen That Produce Succinate and Malate. Biotechnology and Sanchez, Foster City, CA (US) Bioengineering. vol. 99, No.5, 1140-1154. Apr. 1, 2008.* McKinlay et a!., Prospects for a bio-based succinate industry. Appl. (73) Assignee: William Marsh Rice University, Microbiol. Biotechnol. 76:727-740, 2007.* Houston, TX (US) Sanchez, A.M., Bennett, G.N., San, K.-Y., 2005. Novel pathway engineering design of the anaerobic central in ( *) Notice: Subject to any disclaimer, the term of this to increase succinate yield and productivity. Metab. patent is extended or adjusted under 35 Eng. 7, 229-239. Berrios-Rivera, S.J., Bennett, G.N., San, K.-Y., 2002a. Metabolic U.S.C. 154(b) by 0 days. engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+- dependent formate dehydrogenase. (21) Appl. No.: 13/696,268 Metab. Eng. 4, 217-229. Berrios-Rivera, S.J., Bennett, G.N., San, K.-Y., 2002b. The effect of (22) PCTFiled: May 3, 2011 increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab. Eng. 4, 230- (86) PCTNo.: PCT/US2011!035001 237. Berrios-Rivera, S.J., Sanchez, A.M., Bennett, G.N., San, K.-Y., 2004 § 371 (c)(l), Effect of different levels ofNADH availability on metabolite distri­ (2), ( 4) Date: Jan. 14, 2013 bution in Escherichia coli in defined and complex media. App. Microbiol. Biotechnol. 65, 426-432. (87) PCT Pub. No.: W020111140088 Lin H., San, K.-Y., Bennett, G.N. 2005. Effect of Sorghum vulgare PCT Pub. Date: Nov.10, 2011 phosphoenolpyruvate carboxylase and Lactococcus lactis coexpression on succinate production in mutant strains of Escherichia coli. Appl. Microbiol. Biotechnol. 67, 515-523. (65) Prior Publication Data Sakai,Y., Murdanato,A.P., Konishi, T., Iwamatsu,A., Kato, N., 1997. US 2013/0203137 Al Aug. 8, 2013 Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic Candida boidinii and growth characteristics of Related U.S. Application Data an FDH1-disrupted strain on , methylamine, and choline. J. Bacteriol. 179, 4480-4485. (60) Provisional application No. 61/332,427, filed on May Vemuri, G.N., Eiteman, M.A., Altman, E., 2002. Effects of growth 7, 2010. mode and pyruvate carboxylase on succinic acid production by meta­ bolically engineered strains of Escherichia coli. Appl. Environ. (51) Int. Cl. Microbiol. 68, 1715-1727. C12N 15170 (2006.01) * cited by examiner (52) U.S. Cl. USPC ...... 435/145; 435/252.3 Primary Examiner- Tekchand Saidha (58) Field of Classification Search Assistant Examiner- Rama P Ramanujam CPC ...... C12N 9/18; C12N 9/2437; C12N 9/54 (74) Attorney, Agent, or Firm- Boulware & Valoir USPC ...... 435/135 See application file for complete search history. (57) ABSTRACT (56) References Cited Improved for making succinate and other 4 carbon dicarboxylates from the Krebs cycle have modifications to U.S. PATENT DOCUMENTS reduce acetate, lactate, EtOH and formate, as well as tum on the glyoxylate shunt, produce more NADH and overexpress 7,223,567 B2 5/2007 Ka-Yiu In one embodiment, the bacteria are lladhEllldhAlliclR!lack­ 2005/0042736 A1 2/2005 San et al. 2006/0046288 A1 3/2006 Ka-Yiu et al. pta plus pyc+ and NAD+-dependant FDH+. 2010/0086958 A1 4/2010 Davis et al. 2013/0029378 A1 * 112013 Davis et al ...... 435/90 17 Claims, 8 Drawing Sheets U.S. Patent Aug. 5, 2014 Sheet 1 of8 US 8, 795,991 B2

FIGURE 1 U.S. Patent Aug. 5, 2014 Sheet 2 of8 US 8, 795,991 B2

FIGURE2 U.S. Patent Aug. 5, 2014 Sheet 3 of8 US 8, 795,991 B2

FIGURE 3A

I

FIGURE 3B

I

LEGEND for FIG 3A-B. SBS550MG pHL413Kan 11adhEMdhA11iclRI1ack-pta plus PYC+ SBS550MG pHL413KF12 11adhE11ldhAI1iclRI1ack-pta plus PYC+, FDH+ U.S. Patent Aug. 5, 2014 Sheet 4 of8 US 8, 795,991 B2

FIGURE 4A

6

FIGURE 4B

(}

{)

LEGEND for FIG 4A-B. • f..adhEf..ldhAf..ic!Rf..ack-pta plus PYC+ o f..adhEf..ldhAf..ic!Rf..ack-pta plus PYC+, FDH+ (first replicate) f.. f..adhEMdhAf..ic!Rf..ack-pta plus PYC+, FDH+ (second replicate) o f..adhEMdhAf..ic!Rf..ack-pta plus PYC+, FDH+ plus 6 giL glucose in the aerobic phase U.S. Patent Aug. 5, 2014 Sheet 5 of8 US 8, 795,991 B2

FIGURE 5A

0

FIGURE 5B

20

16

~ E~ 12 ~ '; 8 :9= "'tl.l 0:: ..

0 () 12 18 24

LEGEND for FIG SA-B. • 11adhE!1ldhA!1iclRI1ack-pta plus PYC+ o 11adhEMdhA!1iclR11ack-pta plus PYC+, FDH+ (first replicate) 11 11adhEI1ldhA!1iclRI1ack-pta plus PYC+, FDH+ (second replicate) o 11adhE!1ldhA!1iclRI1ack-pta plus PYC+, FDH+ plus 6 giL glucose in the aerobic phase U.S. Patent Aug. 5, 2014 Sheet 6 of8 US 8, 795,991 B2

FIGURE 6A

1 I 1 1

FIGURE 6B

10

0

LEGEND for FIG 6A-B. SBS550MG pHL413Kan 11adhEMdhA!1iclRI1ack-pta plus PYC+ SBS550MG pHL413KF12 11adhE!1ldhA!1iclRI1ack-pta plus PYC+, FDH+ U.S. Patent Aug. 5, 2014 Sheet 7 of8 US 8, 795,991 B2

FIGURE 7

1 MKIVLVLYDA GKHAADEEKL YGCTENKLGI ANWLKDQGHE LITTSDKEGG NSVLDQHIPD 61 ADIIITTPFH PAYITKERID KAKKLKLVVV AGVGSDHIDL DYINQTGKKI SVLEVTGSNV 121 VSVAEHVVMT MLVLVRNFVP AHEQIINHDW EVAAIAKDAY DIEGKTIATI GAGRIGYRVL 181 ERLVPFNPKE LLYYDYQALP KDAEEKVGAR RVENIEELVA QADIVTVNAP LHAGTKGLIN 241 KELLSKFKKG AWLVNTARGA ICVAEDVAAA LESGQLRGYG GDVWFPQPAP KDHPWRDMRN 301 KYGAGNAMTP HYSGTTLDAQ TRYAQGTKNI LESFFTGKFD YRPQDIILLN GEYVTKAYGK 361 HDKK

SEQ ID NO 1: formate from Candida boidinii 1 GenBank Accession number CAA09466. Other sequences that can be used include XP 506003.1 [ Yarrowia 70-72% ] ; XP 462381.1 hansenii CBS767 65-66%]; and 071148.1 [Coccidioides C735 delta 64%], among others. U.S. Patent Aug. 5, 2014 Sheet 8 of8 US 8, 795,991 B2

GPRHASINVK DVRKYYAPFE 641 IQNELTEEDV YARGNELNFP

PGDHEVISYI MYPQVFLDYQ KMQREFGAVT LLDTPTFLHG MRLNEKIEVQ IEKGKTLSIR LDEIGEPDLA GNRVLFFNLN GQRREVVIND RKAETGNPNQ IGATMPGSVL EILVKAGDKV KKGQALMVTE AMKMETTIES PFDGEVIALH DLLIEID

SEQ ID NO: 2: pyruvate Enterococcus .1 faecal is ] ; Enterococcus US 8,795,991 B2 1 2 INCREASING BACTERIAL SUCCINATE Metabolic engineering has the potential to considerably PRODUCTIVITY improve bacterial productivity by manipulating the through­ put of metabolic pathways. Specifically, manipulating PRIOR RELATED APPLICATIONS levels through the amplification, addition, or deletion of a particular pathway can result in high yields of a desired This invention is a National Phase filing under 35 U.S.C. product. Several examples of increasing succinate levels §3 71 oflnternational Application PCT/US 11/3 5001, filed on through metabolic engineering are known, including several May 3, 2011 which claims priority to U.S. 61/332,427, filed patented examples from our own group. However, there is on May 7, 2010. Both applications are incorporated by refer- always room for continued improvement. ence in their entirety herein. 10 What is needed in the art is an improved bacterial strain that produces higher levels of succinate and other carboxy lie acids FEDERALLY SPONSORED RESEARCH than heretofor provided. STATEMENT SUMMARY OF THE INVENTION This invention was made with government support under 15 Grant No: BES 0000303 awarded by the NSF. The govern­ The present invention establishes an improved in vivo ment has certain rights in the invention. method for production of succinic acid that increases the yield, and the production rate of succinate and other compo­ REFERENCE TO MICROFICHE APPENDIX nents of the Krebs cycle, and reduces the byproduct formate. 20 Generally speaking, the invention includes recombinant Not applicable. bacteria engineered to produce fewer by products (acetate, lactate, EtOH), balancing the carbon flux through the fermen­ FIELD OF THE INVENTION tative pathway and the glyoxylate cycle (which has less NADH requirement), as well as driving the Krebs cycle Engineered bacteria that produce higher levels of 4-carbon 25 through increased expression of PYC and supplying dicarboxylic acids from the Krebs Cycle, especially succi­ increased NADH through overexpression ofFDH, which also nate, and methods and uses for same. has the effect of reducing the byproduct formate. Thus, genes encoding proteins essential for the production BACKGROUND OF THE INVENTION oflactate, acetate and ethanol are disrupted and the glyoxy- 30 late cycle is turned on. The glyoxylate cycle (aka glyoxylate Succinate has many industrial uses. As a specialty chemi­ shunt or bypass) like the , begins with the cal, it is a flavor and formulating ingredient in food process­ condensation of acetyl CoA and oxaloacetate to form citrate, ing, a pharmaceutical ingredient, and a surfactant. Succi­ which is then isomerized to isocitrate. Instead ofbeing decar­ nate's greatest market potential, though, would be its use as boxylated, isocitrate is cleaved by isocitrate into succi- an intermediary commodity chemical feedstock for produc­ 35 nate and glyoxylate. The subsequent steps regenerate oxalo­ ing bulk chemicals, stronger-than-steel plastics, ethylene acetate from glyoxylate. Acetyl CoA condenses with diamine disuccinate (a biodegradable chelator), and diethyl glyoxylate to form malate in a reaction catalyzed by malate succinate (a green solvent for replacement of methylene chlo­ synthase, which resembles . Finally, malate is ride). Along with succinic acid, other 4-carbon dicarboxylic oxidized to oxaloacetate, as in the citric acid cycle. The sum acids from the Krebs Cycle, such as malic acid and fumaric 40 of these reactions of the glyoxylate-TCA pathway is: acid, also have feedstock potential. More than 17,000 tons of succinate are sold per year. It is 2Acetyl CoA+NAD.2H2 0~succinate+2CoA+ currently sold in the U.S. for $2.70-4.00/lb, depending on its NADH+2H+ purity. Succinate is currently produced petrochemically from Thus, using the glyoxylate cycle, allows conversion of butane through maleic anhydride. It can also be made by 45 acetate to succinate and reduces the requirements for NADH. fermentation from glucose at a production cost of about Additional NADH is produced with the added FDH, which $1.00/lb, but for succinate to be competitive with maleic also reduces formate byproduct. anhydride as a commodity chemical, its overall production Thus, such bacteria have disrupted alcohol dehydrogenase cost should be lowered to approximately 15 cents/lb. (thus producing less EtOH), disrupted lactate dehydrogenase The production of succinate, malate, and fumarate from 50 (thus producing less lactate), disrupted acetate kinase or glucose, xylose, sorbitol, and other "green" renewable feed­ phosphotransacetylase or both ack-pta (thus producing less stocks (in this case through fermentation processes) is a way acetate), a disruption in iclR (thus allowing expression of to supplant the more energy intensive methods of deriving aceBAK and operation of the glyoxylate shunt to utilize such acids from nonrenewable sources. acetate to make succinate) and overexpression of NAD+ Succinate is an intermediate for anaerobic 55 dependent FD H (thus producing needed NAD H and reducing by propionate producing bacteria (e.g., Actinobacillus succi­ formate) and overexpressed PYC (further driving the Kreb's nogenes), but those processes result in low yields and con­ cycle). centrations and these bacteria are generally not cost effective In a preferred embodiment, the invention is E. coli com­ to use. prising a disruption in adhE, a disruption in ldh, a disruption It has long been known that mixtures of acids are produced 60 in ack-pta, a disruption in iclR and overexpression of both from E. coli fermentation. However, for each mole of glucose FDHandPYC. fermented, only 1.2 moles of , 0.1-0.2 moles of The invention also includes methods employing such bac­ lactic acid, and 0.3-0.4 moles of succinic acid are produced. teria, including methods of making succinate and other com­ As such, efforts to produce carboxylic acids fermentatively ponents of the TCA cycle, such as citrate, alpha-ketoglut­ have resulted in relatively large amounts of growth substrates, 65 arate, fumarate, malate and oxaloacetate. such as glucose, not being converted to desired product, and In more detail, one embodiment of the invention is a bac­ this greatly reduces the cost effectiveness of the method. teria comprising reduced activity of alcohol hydrogenase US 8,795,991 B2 3 4 (ADH), reduced activity of lactate dehydrogenase (LDH), alignment (in bits) 30 for blastn, 15 for other programs; Z reduced activity of acetate kinase or phosphotransacetylase final X dropoff value for gapped aligrillent (in bits) 50 for or both, reduced activity of the aceBAK operon repressor blastn, 25 for other programs. This program is available (ICLR) and overexpression of NAD+-dependent formate online at NCBI™ (ncbi.nlm.nih.gov/BLAST/). dehydrogenase (FDH) and overexpression of pyruvate car- 5 As used herein, "ADH" means a protein having alcohol boxy lase (PYC). More preferred is E. coli bacteria compris­ dehydrogenase activity. Many such proteins are available in ing lladhE, llldh, !lack-pta, lliclR and FDH+ and pyc+. GenBank. The E. coli gene encoding this protein is adhE, but The above bacteria can be used to make any four carbon the gene may have other names in other species. dicarboxylic acid from the Krebs cycle simply by culturing As used herein, "PYC" means pyruvate carboxylase. Many the bacteria in a medium, and isolating a four carbon dicar- 10 such proteins are available in GenBank. In a preferred boxy lie acid from said bacteria or medium or both. In pre­ embodiment, the pyruvate carboxylase (PYC) is fromLacto­ ferred embodiments, the four carbon dicarboxylic acid is coccus lactisis (SEQ ID NO: 2 ofFIG. 8), but other sequences succinate, and the medium is supplemented with 25-250 mM formate. having 60-75% identity are available in a variety of other species. The gene encoding PYC in Lactococcus is called Succinic acid and succinate are used interchangeably 15 herein, as are other acid/base nomenclature for organic acids. pycA, but the gene may have other names in other species. As used herein, "FDH" means a protein having NAD+­ We have exemplified the invention herein using E. coli, but dependent formate dehydrogenase activity. Many such pro­ it can easily be performed in other bacteria, including Bacil­ teins are available in GenBank and exemplary proteins are lus, Lactobacillus, Lactococcus, Clostridia and the like, pro- described in U.S. Pat. No. 7,256,016, incorporated herein in 20 vided the bacteria have the same or equivalent pathways to its entirety by reference. The gene encoding FHD in Candida those diagrammed in FIG. 1 such that the same modifications is fdhl, but the gene may have other names in other species. can be made therein. Since the Krebs cycle and the glyoxylate In the exemplified embodiment the FDH is from Candida bypass are common in bacteria and plants, the invention is boidinii, but obviously any functional FDH can be used from believed to be generally applicable. any source since by definition FDH is an NAD+formate dehy- 25 As used herein, "LDH" means a protein having lactate drogenase and will catalyze the same reaction. Thus, the FDH dehydrogenase activity. Many such proteins are available in can selected from the group consisting of Candida boidinii GenBank. The E. coli gene encoding this protein is ldhA, but FDH, Candida methylica FDH, Pseodomonas sp 101 FDH, it may have other names in other species. Arabidopsis thaliana FDH, Staphylococcus aureus FDH, As used herein, "ADH" means a protein having alcohol Saccharomyces bayanus FDH, Saccharomyces exiguus FDH, 30 dehyrogenase activity. Many such proteins are available in Saccharomyces servazzii FDH, Zygosaccharomyces rouxil GenBank. The E. coli gene encoding this protein is adhE, but FDH, Saccharomyces kluyveri FDH, Kluyveromyces thermo­ it may have other names in other species. tolerans FDH, Kluyveromyces lactis FDH, Kluyveromyces As used herein the aceBAK operon repressor is ICLR and marxianus FDH, Pichi a an gusta FDH, Debaryomyces hans­ the gene encoding same is iclR, but it may have other names enii FDH, Pichia sorbitophila FDH, Candida tropicalis 35 in other species. FDH, and Yarrowia lipolytica FDH, among others. The disruptions inADH, LDH, ACK-PTA and ICLR can be In a preferred embodiment, the FDH has SEQ ID NO. 1 derived as described in U.S. Pat. No. 7,223,567, incorporated (FIG. 7), but variations having 65-75% identity are found in herein in its entirety by reference. other species. Understanding the inherent degeneracy of the genetic code 40 BRIEF DESCRIPTION OF THE DRAWINGS allows one of ordinary skill in the art to design multiple nucleotides that encode the same sequence. FIG. 1 is a diagram showing the genetically engineered NCBI™ provides codon usage databases for optimizing central anaerobic metabolic pathway of E. coli strain DNA sequences for protein expression in various species. SBS550MG (lladhEllldhAlliclR!lack-pta) harboring Using such databases, a gene or eDNA may be "optimized" 45 pHL413KF1 (PYC+, FDW). for expression in E. coli, or other bacterial species using the Shaded X-boxes in FIG. 1 represent inactivated pathways codon bias for the species in which the gene will be expressed. in E. coli strain SBS550MG (lladhEllldhAlliclR!lack-pta) In calculating"% identity" the unaligned terminal portions (Sanchez et a!., 2005): lactate (LDH), acetate (PTA, ACK), of the query sequence are not included in the calculation. The ethanol (ADH), and glyoxylate bypass operon (aceBAK) identity is calculated over the entire length of the reference 50 repressor gene (iclR). The asterisks indicate the overex­ sequence, thus short local aliguments with a query sequence pressed heterologous pyruvate carboxylase (PYC) are not relevant (e.g., % identity=number of aligned residues from Lactococcus lactis (Lin eta!., 2004) and the NAD+­ in the query sequence/length of reference sequence). Align­ dependent formate dehydrogenase (FDH) from Candida boi­ ments are performed using BLAST homology alignment as dinii. The bold arrow in the dotted box represents the newly described by Tatusova T A & Madden T L (1999) FEMS 55 introduced NADH regenerating formate dehydrogenase Microbial. Lett. 174:247-250. The default parameters were (FDH) pathway demonstrated herein. The glyoxylate shunt, used, except the filters wereturnedOFF.As ofJan.l, 2001 the turned on by deleting iclR, is shown crossing the Kreb' s cycle default parameters were as follows: BLASTN or BLASTP as (see straight arrow). appropriate; Matrix=none for BLASTN, BLOSUM62 for FIG. 2 Schematic diagram showing the construction of BLASTP; G Cost to open gap default=5 for nucleotides, 1 1 60 pHL413KF1 ((PYC+, FDW). The 1.1 Kb fdhl (Candida for proteins; E Cost to extend gap [Integer] default=2 for biodinii) fragment was PCR amplified from pFDHl (Sakai et nucleotides, 1 for proteins; q Penalty for nucleotide mismatch a!. 1997), restriction digested with Psti, and ligated to the 7.8 [Integer] default=-3; r reward for nucleotide match [Integer] Kb Pstl restriction digested pHL413Kan (PYC+). The newly default=!; e expect value [Real] default=lO; W word size constructed 8.8 Kb pHL413KF1 (PYC+, FDH+) co-expresses [Integer] default=!! fornucleotides, 3 for proteins; y Dropoff 65 the heterologous pyruvate carboxylase gene (pycA) from (X) for blast extensions in bits (default if zero) default=20 for Lactococcus lactis and the NAD+-dependent formate dehy­ blastn, 7 for other programs; X dropoff value for gapped drogenase gene (fdhl) from Candida boidinii. US 8,795,991 B2 5 6 Abbreviations in FIG. 2: Ptrc, trc promoter; PpycA, native As used herein "recombinant" is relating to, derived from, promoter of the Lactococcus lactis pyruvate carboxylase or containing genetically engineered material. In other words, (pycA) gene; Km, kanamycin resistance gene; pBR322 ori, the genome was intentionally manipulated in some way. origin of replication; laclq: lac operon repressor; rbs: ribo­ "Reduced activity" or "inactivation" is defined herein to be some ; restriction enzyme sites: Neal, EcoRI, at least a 7 5% reduction in protein activity, as compared with Sacl, Kpnl, Smal, Hindiii, Psti, Bg!I, Ndel, Apal, Narl. an appropriate control species. Preferably, at least 80, 85, 90, FIG. 3 is metabolite data from anaerobic shake flask fer­ 9 5% reduction in activity is attained, and in the most preferred mentation experiments showing the relative succinate yield embodiment, the activity is eliminated (100%). Proteins can with the control strain normalized to 100% (A) and the 10 be inactivated with inhibitors, by mutation, or by suppression residual formate (mM) (B) using the genetically engineered of expression or translation, and the like. E. coli strain SBS550MG (lladhEllldhAlliclR!lack-pta) har­ Overexpression" or "overexpressed" is defined herein to be boring either pHL413Kan (PYC+ overexpressing control at least 150% of protein activity as compared with an appro­ vector) or vector pHL413KF1 (PYC+FDH+). The reported priate control species. Overexpression can be achieved by values represent averages of HPLC data from at least three 15 mutating the protein to produce a more active form or a form independent experiments and error bars indicate the standard that is resistant to inhibition, by removing inhibitors, or add­ error of the means. ing activators, and the like. Overexpression can also be FIG. 4 is metabolite data from the anaerobic phase of achieved by removing repressors, adding multiple copies of bioreactor experiments showing the glucose consumption the gene to the cell, or upregulating the endogenous gene, and (mM) (A) and the succinate production (mM) (B) over time 20 the like. An overexpressed gene can be represented by the + by the same two mutants. symbol, e.g., pyc+. FIG. 5 is metabolite data from the anaerobic phase of bioreactor experiments showing the succinate production rate The terms "disruption" as used herein, refer to cell strains (giL/h) (A) and the residual formate (mM) (B) over time by in which the native gene or promoter is mutated, deleted, the same two mutants. 25 interrupted, or down regulated in such a way as to decrease FIG. 6 is metabolite data from anaerobic shake flask fer­ the activity of the protein at least 90% over the wild type mentation experiments showing the relative succinate yield un-disrupted protein. A gene or protein can be completely with the control strain at 0 mM formate normalized to 100% (100%) reduced by knockout or removal of the entire (A) and the residual formate concentration (mM) with and genomic DNA sequence. A knockout mutant can be repre- without 50 mM sodium formate supplementation (B). The 30 sented by the ll symbol. reported values represent averages of HPLC data from two Use of a frame shift mutation, early stop codon, point independent experiments using the same pair of mutants. mutations of critical residues, or deletions or insertions, and FIG. 7. Exemplary FDH sequence SEQ ID NO.1. the like, can completely inactivate (100%) gene product by FIG. 8. Exemplary PYC sequence SEQ ID NO. 2. completely preventing transcription and/or translation of 35 active protein. DESCRIPTION OF EMBODIMENTS OF THE INVENTION Generally speaking we have referenced protein names herein, but it is understood that a change in protein activity As used herein the specification, "a" or "an" may mean one can of course be effected by changing the gene. This provides or more. As used herein in the claim(s), when used in con­ 40 clarity since the gene nomenclature can be widely divergent junction with the word "comprising", the words "a" or "an" in bacteria, but the proteins are defined by their activities and may mean one or more than one. As used herein "another" thus names. may mean at least a second or more. The following abbreviations, plasmids and strains are used The term "about" means the stated value plus or minus the herein: margin of error of measurement or plus or minus 10% if no 45 method of measurement is indicated. The use of the term "or" in the claims is used to mean ABBREVIATION FULL NAME "and/or" unless explicitly indicated to refer to alternatives aceBAK operon Encodes genes required for the glyoxylate only or if the alternatives are mutually exclusive. bypass and is essential for growth on The terms "comprise", "have", "include" and "contain" 50 acetate or fatty acids. Isocitrate lyase and (and their variants) are open-ended linking verbs and allow malate synthase are encoded by aceA and the addition of other elements when used in a claim. aceB, respectively, while (IDH) kinase/phosphatase is encoded by aceK. As used herein, the expressions "cell", "cell line" and "cell ACK acetate kinase culture" are used interchangeably and all such designations ackA E. coli gene encoding ACK include progeny. Thus, the words "cells" and similar desig­ 55 ADH Alcohol dehydrogenase nations include the primary subject cell and cultures derived adhE E. coli gene encoding ADH CmR chloramphenicol resistance gene therefrom without regard for the number of transfers. It is also EtOH ethanol understood that all progeny may not be precisely identical in FDH Formate dehydrogenase, dependant DNA content, due to deliberate or inadvertent mutations that iclR E. coli gene encoding ICLR aka aceBAK operon repressor 60 arise after genetic engineering is concluded. Mutant progeny ICLR the aceBAK operon repressor that have the same function or biological activity as screened LDH lactate dehydrogenase for in the originally transformed cell are included. Where ldhA E. coli gene encoding LDH, NAD•-dependent distinct designations are intended, it will be clear from the PTA Phosphotransacetylase context. pta E. coli gene encoding PTA PYC Pyruvate carboxylase The terms "operably associated" or "operably linked," as 65 pycA Gene encoding PYC from Lactococcus lactis used herein, refer to functionally coupled nucleic acid ------sequences. US 8,795,991 B2 7 8

nase converts formate to C02 and H2 with no cofactor PLASMID AND STRAINS involvement. The new system retains the reducing power of formate resulting in a more robust strain with a much faster pHL413Kan- plasmid containing only PYC pHL413KF1 -plasmid containing PYC and FDH (see FIG. 2) average succinate production rate (15 mM!h) and reduced SBS550MG- L\.adhELI.ldhAL\.iclR.L\.ack-pta::CmR average byproduct formate concentration (3 mM), thereby SBS550MG pHL413KF1-L\.adhELI.ldhALI.iclR.L\.ack-pta: :CmR leading to opportunities for reduced costs associated with plus overexpressed PYC and FDH downstream processing, purification, and waste disposal. The functionality of the new succinate production system The following examples are illustrative only, and are not that regenerates NADH leading to an increase in succinate intended to unduly limit the scope of the invention. 10 production rate and decrease in the byproduct formate, was successfully demonstrated in anaerobic shake flask fermen­ EXAMPLE 1 tations (FIG. 3, 6) and anaerobic bioreactor experiments (FIGS. 4-5). Methods An additional feature of the new system was that this 15 NAD+-dependent FDH resulted in an increase in the succi­ Disrupted bacteria were prepared according to known nate production yield. As previously mentioned, the new techniques, essentially as described in U.S. Pat. No. 7,223, pathway converts 1 mol of formate into 1 mol ofNADH and 567. Overexpressed PYC and FDH was achieved by combin­ (Berrios-Rivera eta!., 2002a, b, 2004). This ing these two genes on one plasmid as shown in FIG. 2, but the conversion can be exploited by supplementing fermentations with external formate thereby providing an additional genes can also be added to the bacterial chromosome or added 20 by any other vector or even via separate vectors. increase in the NADH availability. A demonstration of the Anaerobic shake flasks experiments were performed at 3 7° increased succinate yield using externally supplemented for­ mate was performed using shake flask fermentations (FIG. 6). C. with shaking at 250 rpm for 20-24 hours using C02 purged flasks containing 200 OD units of cells resuspended in 10 ml The percentage increase in molar yield is 2-5%. In summary, the advantages of using the new improved LB broth supplemented with 20 giL glucose, 1 g/L NaHC03 , 25 system include: 50 g/L MgC03 , 50 f.tg/ml kanamycin (FIG. 3), and 50 mM sodium formate (FIG. 6). 1. Increased rate of succinate production because of an Bioreactor experiments were performed using aerobically increase in NADH availability. grown biomass using modified dual-phase medium with 4 2. Reduced levels of byproduct formate due to overexpres­ g/L glucose unless noted otherwise followed by the anaerobic sion of an NAD+ -dependent formate dehydrogenase that con- succinate production phase using 0.2 Llmin C0 , 20 g/L 30 verts 1 mole of formate into 1 moll of NADH and carbon 2 dioxide. glucose, 6.4 g/L MgC03 , 50 flg/ml kanamycin, and 14.3 M NH 0H to maintain the pH at 7.0 (FIGS. 4 and 5). 3. Increased succinate yields using externally supple­ 4 mented formate because the NAD+-dependent formate dehy­ EXAMPLE2 drogenase provides additional NADH availability. The following references are incorporated by reference 35 Results herein in their entirety. Berrios-Rivera, S. J., Bennett, G. N., San, K.-Y., 2002a. Meta­ bolic engineering of Escherichia coli: increase ofNADH To demonstrate the utility and advantages of the invention, availability by overexpressing an NAD+-dependent for­ we used the previously engineered high succinate producing E. coli strain having lladhEllldhA!lic!Rllack-pta plus pyc+ mate dehydrogenase. Metab. Eng. 4, 217-229. 40 Berrios-Rivera, S. J., Bennett, G. N., San, K.-Y., 2002b. The [strain SBS550MG pHL413Kan]. This strain was further enhanced by adding a NAD+ -dependent formate dehydroge­ effect of increasing NADH availability on the redistribu­ tion of metabolic fluxes in Escherichia coli chemostat cul­ nase (FDH+) to regenerate NADH in vivo and manipulate tures. Metab. Eng. 4, 230-237. intracellular NADH availability (see dotted box in FIG. 1). Berrios-Rivera, S. J., Sanchez, A. M., Bennett, G. N., San, The performance of the two strains were then compared. In previous studies, it was demonstrated that this NAD+- 45 K.-Y., 2004 Effect of different levels ofNADH availability dependent FDH pathway converts 1 mol offormate into 1 mol on metabolite distribution in Escherichia coli fermentation ofNADH and carbon dioxide (Berrios-Rivera eta!., 2002a, b, in defined and complex media. App. Microbial. Biotech­ 2004). Implementation of the NADH regeneration system no!. 65, 426-432. thus doubled the maximum yield ofNADH using glucose as Lin H., San, K.-Y., Bennett, G. N. 2005. Effect of Sorghum a substrate from 2 to 4 mol NADH/mol of substrate consumed 50 vulgare phosphoenolpyruvate carboxylase and Lactococ­ in complex medium. cus lactis pyruvate carboxylase coexpression on succinate In our experiments, this increase in NADH availability production in mutant strains of Escherichia coli. Appl. significantly changed the final metabolite concentration pat­ Microbial. Biotechnol. 67, 515-523. tern under anaerobic conditions. The parent strain used in this Sakai, Y., Murdanato, A. P., Konishi, T., Iwamatsu, A., Kato, demonstration was capable of producing succinate from glu- 55 N., 1997. Regulation of the formate dehydrogenase gene, case to a yield of about 1.6 mol/mol with an average anaero­ FDH1, in the methylotrophic yeast Candida boidinii and bic productivity rate of 10 mM/h (Sanchez et a!., 2005), but growth characteristics of an FDH1-disrupted strain on adding in an NAD+-dependant FDH resulted in much faster methanol, methylamine, and choline. J. Bacterial. 179, average succinate production rate (15 mM!h). 4480-4485. Along with succinate as a major product, the parent strain Sanchez, A. M., Bennett, G. N., San, K.-Y., 2005. Novel also produced 12.7 mM of formate during the anaerobic 60 production phase. The overexpressed biologically active pathway engineering design of the anaerobic central meta­ NAD+-dependent formate dehydrogenase (FDH) from Can­ bolic pathway in Escherichia coli to increase succinate dida boidinii (FIG. 7) also served to reduce production of yield and productivity. Metab. Eng. 7, 229-239. formate. Vemuri, G. N., Eiteman, M.A., Altman, E., 2002. Effects of The newly introduced NADH regenerating formate dehy- 65 growth mode and pyruvate carboxylase on succinic acid drogenase pathway will provide one mole ofNADH from one production by metabolically engineered strains ofEscheri­ mole of formate. In contrast, the native formate dehydroge- chia coli.Appl. Environ. Microbial. 68, 1715-1727. US 8,795,991 B2 9 10

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS, 2

<210> SEQ ID NO 1 <211> LENGTH, 364 <212> TYPE, PRT <213> ORGANISM, Candida boidinii <300> PUBLICATION INFORMATION, <308> DATABASE ACCESSION NUMBER, CAA0946 <309> DATABASE ENTRY DATE, 2005-04-15 <313> RELEVANT RESIDUES IN SEQ ID NO, (1) .. (364)

<400> SEQUENCE, 1

Met Lys Ile Val Leu Val Leu Tyr Asp Ala Gly Lys His Ala Ala Asp 1 5 10 15

Glu Glu Lys Leu Tyr Gly Cys Thr Glu Asn Lys Leu Gly Ile Ala Asn 20 25 30

Trp Leu Lys Asp Gln Gly His Glu Leu Ile Thr Thr Ser Asp Lys Glu 35 40 45

Gly Gly Asn Ser Val Leu Asp Gln His Ile Pro Asp Ala Asp Ile Ile 50 55 60

Ile Thr Thr Pro Phe His Pro Ala Tyr Ile Thr Lys Glu Arg Ile Asp 65 70 75 80

Lys Ala Lys Lys Leu Lys Leu Val Val Val Ala Gly Val Gly Ser Asp 85 90 95

His Ile Asp Leu Asp Tyr Ile Asn Gln Thr Gly Lys Lys Ile Ser Val 100 105 110

Leu Glu Val Thr Gly Ser Asn Val Val Ser Val Ala Glu His Val Val 115 120 125

Met Thr Met Leu Val Leu Val Arg Asn Phe Val Pro Ala His Glu Gln 130 135 140

Ile Ile Asn His Asp Trp Glu Val Ala Ala Ile Ala Lys Asp Ala Tyr 145 150 155 160

Asp Ile Glu Gly Lys Thr Ile Ala Thr Ile Gly Ala Gly Arg Ile Gly 165 170 175

Tyr Arg Val Leu Glu Arg Leu Val Pro Phe Asn Pro Lys Glu Leu Leu 180 185 190

Tyr Tyr Asp Tyr Gln Ala Leu Pro Lys Asp Ala Glu Glu Lys Val Gly 195 200 205

Ala Arg Arg Val Glu Asn Ile Glu Glu Leu Val Ala Gln Ala Asp Ile 210 215 220

Val Thr Val Asn Ala Pro Leu His Ala Gly Thr Lys Gly Leu Ile Asn 225 230 235 240

Lys Glu Leu Leu Ser Lys Phe Lys Lys Gly Ala Trp Leu Val Asn Thr 245 250 255

Ala Arg Gly Ala Ile Cys Val Ala Glu Asp Val Ala Ala Ala Leu Glu 260 265 270

Ser Gly Gln Leu Arg Gly Tyr Gly Gly Asp Val Trp Phe Pro Gln Pro 275 280 285

Ala Pro Lys Asp His Pro Trp Arg Asp Met Arg Asn Lys Tyr Gly Ala 290 295 300

Gly Asn Ala Met Thr Pro His Tyr Ser Gly Thr Thr Leu Asp Ala Gln 305 310 315 320

Thr Arg Tyr Ala Gln Gly Thr Lys Asn Ile Leu Glu Ser Phe Phe Thr 325 330 335

Gly Lys Phe Asp Tyr Arg Pro Gln Asp Ile Ile Leu Leu Asn Gly Glu 340 345 350 US 8,795,991 B2 11 12 -continued

Tyr Val Thr Lys Ala Tyr Gly Lys His Asp Lys Lys 355 360

<210> SEQ ID NO 2 <211> LENGTH, 1137 <212> TYPE, PRT <213> ORGANISM: Lactococcus lactis subsp. lactis <300> PUBLICATION INFORMATION, <308> DATABASE ACCESSION NUMBER, AF068759 <309> DATABASE ENTRY DATE, 2000-03-01 <313> RELEVANT RESIDUES IN SEQ ID NO, (1) .. (1137)

<400> SEQUENCE, 2

Met Lys Lys Leu Leu Val Ala Asn Arg Gly Glu Ile Ala Val Arg Val 1 5 10 15

Phe Arg Ala Cys Asn Glu Leu Gly Leu Ser Thr Val Ala Val Tyr Ala 20 25 30

Arg Glu Asp Glu Tyr Ser Val His Arg Phe Lys Ala Asp Glu Ser Tyr 35 40 45

Leu Ile Gly Gln Gly Lys Lys Pro Ile Asp Ala Tyr Leu Asp Ile Asp 50 55 60

Asp Ile Ile Arg Val Ala Leu Glu Ser Gly Ala Asp Ala Ile His Pro 65 70 75 80

Gly Tyr Gly Leu Leu Ser Glu Asn Leu Glu Phe Ala Thr Lys Val Arg 85 90 95

Ala Ala Gly Leu Val Phe Val Gly Pro Glu Leu His His Leu Asp Ile 100 105 110

Phe Gly Asp Lys Ile Lys Ala Lys Ala Ala Ala Asp Glu Ala Gln Val 115 120 125

Pro Gly Ile Pro Gly Thr Asn Gly Ala Val Asp Ile Asp Gly Ala Leu 130 135 140

Glu Phe Ala Gln Thr Tyr Gly Tyr Pro Val Met Ile Lys Ala Ala Leu 145 150 155 160

Gly Gly Gly Gly Arg Gly Met Arg Val Ala Arg Asn Asp Ala Glu Met 165 170 175

His Asp Gly Tyr Ala Arg Ala Lys Ser Glu Ala Ile Gly Ala Phe Gly 180 185 190

Ser Gly Glu Ile Tyr Val Glu Lys Tyr Ile Glu Asn Pro Lys His Ile 195 200 205

Glu Val Gln Ile Leu Gly Asp Ser His Gly Asn Ile Val His Leu His 210 215 220

Glu Arg Asp Cys Ser Val Gln Arg Arg Asn Gln Lys Val Ile Glu Ile 225 230 235 240

Ala Pro Ala Val Gly Leu Ser Pro Glu Phe Arg Asn Glu Ile Cys Glu 245 250 255

Ala Ala Val Lys Leu Cys Lys Asn Val Gly Tyr Val Asn Ala Gly Thr 260 265 270

Val Glu Phe Leu Val Lys Asp Asp Lys Phe Tyr Phe Ile Glu Val Asn 275 280 285

Pro Arg Val Gln Val Glu His Thr Ile Thr Glu Leu Ile Thr Gly Val 290 295 300

Asp Ile Val Gln Ala Gln Ile Leu Ile Ala Gln Gly Lys Asp Leu His 305 310 315 320

Thr Glu Ile Gly Ile Pro Ala Gln Ala Glu Ile Pro Leu Leu Gly Ser 325 330 335 US 8,795,991 B2 13 14 -continued

Ala Ile Gln Cys Arg Ile Thr Thr Glu Asp Pro Gln Asn Gly Phe Leu 340 345 350

Pro Asp Thr Gly Lys Ile Asp Thr Tyr Arg Ser Pro Gly Gly Phe Gly 355 360 365

Ile Arg Leu Asp Val Gly Asn Ala Tyr Ala Gly Tyr Glu Val Thr Pro 370 375 380

Tyr Phe Asp Ser Leu Leu Val Lys Val Cys Thr Phe Ala Asn Glu Phe 385 390 395 400

Ser Asp Ser Val Arg Lys Met Asp Arg Val Leu His Glu Phe Arg Ile 405 410 415

Arg Gly Val Lys Thr Asn Ile Pro Phe Leu Ile Asn Val Ile Ala Asn 420 425 430

Glu Asn Phe Thr Ser Gly Gln Ala Thr Thr Thr Phe Ile Asp Asn Thr 435 440 445

Pro Ser Leu Phe Asn Phe Pro Arg Leu Arg Asp Arg Gly Thr Lys Thr 450 455 460

Leu His Tyr Leu Ser Met Ile Thr Val Asn Gly Phe Pro Gly Ile Glu 465 470 475 480

Asn Thr Glu Lys Arg His Phe Glu Glu Pro Arg Gln Pro Leu Leu Asn 485 490 495

Ile Glu Lys Lys Lys Thr Ala Lys Asn Ile Leu Asp Glu Gln Gly Ala 500 505 510

Asp Ala Val Val Glu Tyr Val Lys Asn Thr Lys Glu Val Leu Leu Thr 515 520 525

Asp Thr Thr Leu Arg Asp Ala His Gln Ser Leu Leu Ala Thr Arg Leu 530 535 540

Arg Leu Gln Asp Met Lys Gly Ile Ala Gln Ala Ile Asp Gln Gly Leu 545 550 555 560

Pro Glu Leu Phe Ser Ala Glu Met Trp Gly Gly Ala Thr Phe Asp Val 565 570 575

Ala Tyr Arg Phe Leu Asn Glu Ser Pro Trp Tyr Arg Leu Arg Lys Leu 580 585 590

Arg Lys Leu Met Pro Asn Thr Met Phe Gln Met Leu Phe Arg Gly Ser 595 600 605

Asn Ala Val Gly Tyr Gln Asn Tyr Pro Asp Asn Val Ile Glu Glu Phe 610 615 620

Ile His Val Ala Ala His Glu Gly Ile Asp Val Phe Arg Ile Phe Asp 625 630 635 640

Ser Leu Asn Trp Leu Pro Gln Met Glu Lys Ser Ile Gln Ala Val Arg 645 650 655

Asp Asn Gly Lys Ile Ala Glu Ala Thr Ile Cys Tyr Thr Gly Asp Ile 660 665 670

Leu Asp Pro Ser Arg Pro Lys Tyr Asn Ile Gln Tyr Tyr Lys Asp Leu 675 680 685

Ala Lys Glu Leu Glu Ala Thr Gly Ala His Ile Leu Ala Val Lys Asp 690 695 700

Met Ala Gly Leu Leu Lys Pro Gln Ala Ala Tyr Arg Leu Ile Ser Glu 705 710 715 720

Leu Lys Asp Thr Val Asp Leu Pro Ile His Leu His Thr His Asp Thr 725 730 735

Ser Gly Asn Gly Ile Ile Thr Tyr Ser Gly Ala Thr Gln Ala Gly Val 740 745 750

Asp Ile Ile Asp Val Ala Thr Ala Ser Leu Ala Gly Gly Thr Ser Gln US 8,795,991 B2 15 16 -continued

755 760 765

Pro Ser Met Gln Ser Ile Tyr Tyr Ala Leu Glu His Gly Pro Arg His 770 775 780

Ala Ser Ile Asn Val Lys Asn Ala Glu Gln Ile Asp His Tyr Trp Glu 785 790 795 800

Asp Val Arg Lys Tyr Tyr Ala Pro Phe Glu Ala Gly Ile Thr Ser Pro 805 810 815

Gln Thr Glu Val Tyr Met His Glu Met Pro Gly Gly Gln Tyr Thr Asn 820 825 830

Leu Lys Ser Gln Ala Ala Ala Val Gly Leu Gly His Arg Phe Asp Glu 835 840 845

Ile Lys Gln Met Tyr Arg Lys Val Asn Met Met Phe Gly Asp Ile Ile 850 855 860

Lys Val Thr Pro Ser Ser Lys Val Val Gly Asp Met Ala Leu Phe Met 865 870 875 880

Ile Gln Asn Glu Leu Thr Glu Glu Asp Val Tyr Ala Arg Gly Asn Glu 885 890 895

Leu Asn Phe Pro Glu Ser Val Val Ser Phe Phe Arg Gly Asp Leu Gly 900 905 910

Gln Pro Val Gly Gly Phe Pro Glu Glu Leu Gln Lys Ile Ile Val Lys 915 920 925

Asp Lys Ser Val Ile Met Asp Arg Pro Gly Leu His Ala Glu Lys Val 930 935 940

Asp Phe Ala Thr Val Lys Ala Asp Leu Glu Gln Lys Ile Gly Tyr Glu 945 950 955 960

Pro Gly Asp His Glu Val Ile Ser Tyr Ile Met Tyr Pro Gln Val Phe 965 970 975

Leu Asp Tyr Gln Lys Met Gln Arg Glu Phe Gly Ala Val Thr Leu Leu 980 985 990

Asp Thr Pro Thr Phe Leu His Gly Met Arg Leu Asn Glu Lys Ile Glu 995 1000 1005

Val Gln Ile Glu Lys Gly Lys Thr Leu Ser Ile Arg Leu Asp Glu 1010 1015 1020

Ile Gly Glu Pro Asp Leu Ala Gly Asn Arg Val Leu Phe Phe Asn 1025 1030 1035

Leu Asn Gly Gln Arg Arg Glu Val Val Ile Asn Asp Gln Ser Val 1040 1045 1050

Gln Thr Gln Ile Val Ala Lys Arg Lys Ala Glu Thr Gly Asn Pro 1055 1060 1065

Asn Gln Ile Gly Ala Thr Met Pro Gly Ser Val Leu Glu Ile Leu 1070 1075 1080

Val Lys Ala Gly Asp Lys Val Lys Lys Gly Gln Ala Leu Met Val 1085 1090 1095

Thr Glu Ala Met Lys Met Glu Thr Thr Ile Glu Ser Pro Phe Asp 1100 1105 1110

Gly Glu Val Ile Ala Leu His Val Val Lys Gly Glu Ala Ile Gln 1115 1120 1125

Thr Gln Asp Leu Leu Ile Glu Ile Asp 1130 1135

What is claimed is: (ACK) or phosphotransacetylase (PTA) or both (ACK-PTA), 1. An engineered E. coli comprising reduced activity of 65 reduced activity of the aceBAK operon repressor (ICLR) and alcohol dehydrogenase (ADH), reduced activity of lactate overexpressed NAD+ -dependent formate dehydrogenase dehydrogenase (LDH), reduced activity of acetate kinase (FDH+) and overexpressed pyruvate carboxylase (PYC+). US 8,795,991 B2 17 18 2. The E. coli of claim 1, wherein said E. coli comprising 11. A method of making a four carbon dicarboxylic acid lladhE, llldh, !lack-pta, lliclR and FDH+ and pyc+. from the Krebs cycle, comprising culturing the E. coli of any 3. The E. coli of claim 1, wherein the FDH has at least 65% one of claim 1-10 in a medium, and isolating a four carbon identity to SEQ ID NO 1. dicarboxylic acid from said E. coli or medium or both. 4. The E. coli of claim 1, wherein the PYC has at least 63% 5 12. A method of making a succinate, comprising culturing identity to SEQ ID NO. 2. the E. coli of any one of claim 1-10 in a medium, and isolating 5. The E. coli of claim 1, wherein the FDH has SEQ ID NO succinate from said E. coli or medium or both. 1. 13. The method of claim 11, further comprising supple­ menting said medium with 25-250 mM formate. 6. TheE. coli of claim 1, wherein thePYChas SEQIDNO. 2. 10 14. The method of claim 12, further comprising supple­ 7. The E. coli of claim 2, wherein the FDH has at least 65% menting said medium with 25-250 mM formate. identity to SEQ ID NO 1. 15. The method of claim 12, wherein the yield of succinate 8. The E. coli of claim 2, wherein the PYC has at least 63% is > 1.6 moles/mole of glucose. identity to SEQ ID NO. 2. 16. The method of claim 14, wherein the yield of succinate 9. TheE. coli ofclaim2, wherein the FDHhas SEQ ID NO 15 is > 1.7 moles/mole of glucose. 1. 17. The method of claim 11, wherein theE. coli comprising lladhE, llldh, !lack-pta, lliclR and FDH+ and pyc+. 10. The E. coli of claim 2, wherein the PYC has SEQ ID N0.2. * * * * *