HK1211318B

HK1211318B - Materials and methods for efficient succinate and malate production

Info

Publication number: HK1211318B
Application number: HK15112083.6A
Authority: HK
Inventors: Kaemwich Jantama; Mark John Haupt; Xueli Zhang; Jonathan C. Moore; Keelnatham T. Shanmugam; Lonnie O'neal Ingram
Original assignee: University Of Florida Research Foundation, Inc.
Priority date: 2007-03-20
Filing date: 2015-12-08
Publication date: 2019-06-21

Description

Materials and methods for efficient production of succinic and malic acid

The present application is a divisional application of PCT application PCT/US2008/057439 entitled "material and method for efficient production of succinic acid and malic acid" filed 3/19/2008, which entered the chinese national phase at a date of 18/9/2009, with application number 200880008975.1.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. provisional application serial No.60/895,806, filed 3/20/2007, the disclosure of which is incorporated by reference in its entirety, including all figures, tables, and amino acid or nucleic acid sequences.

Government support

The present invention was made in accordance with government support granted by Department of Energy (Department of Energy) under grant number USDOE-DE FG02-96ER20222 and granted by Department of Energy in conjunction with the United States Department of Agriculture (United States Department of Agriculture) under grant number USDA & DOE Biomass RDI DE FG36-04GO 14019. The government has certain rights in the invention.

Background

With increasing petroleum prices, the fermentative production of succinic acid from renewable feedstocks will become increasingly competitive. Succinic acid can be used as a substrate for conversion to plastics, solvents and other chemicals currently made from petroleum (Lee et al, 2004; Lee et al, 2005; McKinlay et al, 2007; Wendisch et al, 2006; Zeikus et al, 1999). Many bacteria are described as having the natural ability to produce succinic acid as the primary fermentation product (U.S. Pat. No.5,723,322; Table 1). However, complex processes, complex media and long incubation times are often required.

Various genetic approaches have been previously used to engineer strains of E.coli (Escherichia coli) for succinic acid production with varying degrees of success (Table 1). In most studies, the titers achieved are low and complex media components such as yeast extract or corn steep liquor are required. Strain NZN111 produced 108mM succinic acid in molar yield of 0.98 moles succinic acid per mole glucose metabolized (Chatterjee et al, 2001; Millard et al, 1996; Stols and Donnelly, 1997). The strain is obtained by the following modification: both genes were inactivated (pflB encoding pyruvate-formate lyase and ldhA encoding lactate dehydrogenase) and two e.coli genes from a multicopy plasmid were overexpressed: phosphoenolpyruvate carboxylase (ppc) and malate dehydrogenase (mdh). The strain HL27659k is obtained by the following modification: mutant succinate dehydrogenase (sdhAB), phosphate acetyltransferase (pta), acetate kinase (ackA), pyruvate oxidase (poxB), glucose transporter (ptsG) and isocitrate lyase repressor (iclR). This strain produces less than 100mM succinic acid and requires oxygen-limited fermentation conditions (Cox et al, 2006; Lin et al, 2005a, 2005b, 2005 c; Yun et al, 2005). Gene knockouts were designed using metabolic analysis on computer chips to create a pathway in E.coli similar to the native succinate pathway in Mannheimia succiniciproducens (Lee et al, 2005 and 2006). The resulting strain, however, produced very little succinic acid. Andersson et al, (2007) reported the highest level of succinate production (339mM) in engineered E.coli containing only the native gene.

Other researchers have pursued alternative approaches to the expression of heterologous genes in E.coli. Rhizobium eteloti pyruvate carboxylase (pyc) is overexpressed from a multicopy plasmid, directing carbon flux to succinate (Gokarn et al, 2000; Vemuri et al, 2002a, 2002 b). Strain SBS550MG was constructed as follows: the isocitrate lyase repressor (iclR), adhE, ldhA and ackA were inactivated, and citZ (citrate synthase) and R.etli pyc of Bacillus subtilis (Sanchez et al, 2005a) were overexpressed from the multicopy plasmid. Using this strain, 160mM succinic acid was produced from glucose with a molar yield of 1.6.

More complex processes for succinic acid production were also investigated (table 1). Many of these processes involve an aerobic growth phase followed by an anaerobic production phase. The anaerobic phase is typically provided by carbon dioxide, hydrogen, or both (Andersson et al, 2007; Sanchez et al, 2005a and 2005 b; Sanchez et al, 2006; U.S. Pat. No.5,869,301; Vemuri et al, 2002a and 2002 b). In a recent study using natural succinic acid producers for production of anaerobiospirillum succiniciproducens, electrodialysis, CO, was combined₂Sparging, cell recycling and fed-batch (meynal-Salles et al, 2007).

The present invention provides various forms of microorganisms, such as E.coli strains, that produce succinic acid in high titer and yield during simple, pH-controlled, batch fermentation in mineral salt media without the need for heterologous genes or plasmids. During development, the intermediate strains were characterized by the production of malic acid as the predominant product.

Summary of The Invention

The present invention provides novel microorganisms, such as E.coli, suitable for the production of lactic acid. Thus, the materials and methods of the present invention can be used to produce succinic acid and malic acid suitable for use in a variety of applications.

In certain embodiments, derivatives of E.coli (also referred to herein as E.coli) can be used to construct strains that produce succinic acid, malic acid, and alanine. In various embodiments, E.coli C (e.g., ATCC 8739) can be used as any other strain of E.coli, which can be obtained from various depositories or commercial sources. In some embodiments, the engineered microorganisms of the present invention also contain only native genes (i.e., do not contain genetic material from other organisms). Other advantages of the present invention will be readily apparent from the following description.

Brief description of the drawings

FIGS. 1A-1B fermentation of glucose to succinic acid. FIG. 1A shows a standard pathway for fermentation of glucose by E.coli. This pathway has been redrawn by Unden and Kleefeld (2004). Bold arrows indicate central fermentation pathways. Crosses represent gene deletions performed in this study to engineer KJ012 (ldhA, adhE, ackA). Genes and enzymes: ldhA, lactate dehydrogenase; pflB, pyruvate-formate lyase; focA, formate transporter; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB and fumC, fumarase isomerase; frdABCD, fumarate reductase; fdh, formate dehydrogenase; icd, isocitrate dehydrogenase; acs, acetyl-CoA synthetase; mgsA, methylglyoxal synthase; poxB, pyruvate oxidase; aldA, aldehyde dehydrogenase; and aldB, aldehyde dehydrogenase. FIG. 1B shows the coupling of ATP production and growth to succinate and malate production in engineered E.coli strains. The solid arrow connects the NADH pool. Dotted arrow connecting NAD⁺And (4) a pool. During glycolysis under anaerobic conditions, growth is coupled exclusively with the production of ATP and the oxidation of NADH.

FIGS. 2A-2D, a possible carboxylation pathway for succinic acid production by E.coli. The gene encoding the critical carboxylase is shown in bold. FIG. 2A shows PEP carboxylase. Phosphoenolpyruvate (PEP) does not produce ATP. This is considered to be the major pathway for succinate production by E.coli during glucose fermentation.

FIG. 2B shows malic enzyme (NADH). Energy is conserved during the production of ATP by pyruvate kinase (pykA or pykF) from ADP and PEP. Malic enzyme (sfcA) catalyzes NADH-related reductive carboxylation, thereby producing malic acid. Fig. 2C shows malic enzyme (NADPH). Energy is conserved during the production of ATP by pyruvate kinase (pykA or pykF) from ADP and PEP. Malic enzyme (maeB) catalyzes NADPH-related reductive carboxylation, thereby producing malic acid. FIG. 2D shows PEP carboxykinase (carboxykinase). Oxaloacetate is produced by conserving energy through the production of ATP during the carboxylation of PEP.

KJ012 growth during metabolic evolution, resulting in KJ017, KJ032 and KJ 060. The strain KJ012 was continuously transferred into NBS medium containing 5% (w/v) (FIG. 3A) and 10% (w/v) (FIG. 3B) glucose to yield KJ017, respectively. After deletion of focA and pflB, the resulting strain (KJ032) was initially subcultured in medium supplemented with acetic acid (FIG. 3C). Acetic acid levels are reduced and subsequently eliminated on further transport to produce KJ 060. The broken line represents fermentation of KJ017 without acetic acid, added for comparison. Symbol: OD_550nmOptical density ●.

FIGS. 4A-4F. overview of fermentation products during metabolic evolution of strains used to produce succinic acid and malic acid the culture was supplemented with sodium acetate as shown black arrows represent transitions between fermentation conditions as shown in the text No formic acid was detected and only small amounts of lactic acid were detected during metabolic evolution of KJ032 No formic acid and lactic acid were detected during metabolic evolution of KJ070 and KJ072 FIGS. 4A (5% w/v glucose) and 4B (10% w/v glucose), KJ012 to KJ017, FIGS. 4C (5% w/v glucose) and 4D (10% w/v glucose), KJ032 to KJ060, FIGS. 4E, 10% glucose, KJ070 to KJ071, FIGS. 4F, 10% glucose, KJ072 to KJ073. symbols of all the figures: ■, succinic acid, □, formic acid, Δ, acetic acid, ▲, ◆, lactic acid and lactic acidPyruvic acid.

FIG. 5 is a simplified diagram summarizing the steps in the genetic engineering and metabolic evolution of E.coli C as a biocatalyst for succinic and malic acid production. This process represents 261 serial passages, which provides over 2000 generations of growth-based selection. Clones were isolated from the final culture of each protocol and assigned strain names, shown in parentheses in table 3.

FIG. 6 fermentation of glucose and related pathways. Central metabolism refers to genes that are deleted in constructs engineered for succinic acid production. Solid arrows represent central fermentation pathways. The dashed arrows indicate the microaerophilic pathway for oxidation of pyruvate to acetate (poxB). The dotted arrows show the pathways that normally function during aerobic metabolism, pyruvate dehydrogenase (pdh) and the glyoxylate shunt (aceAB). Boxed crosses represent the deletion of three original genes (ldhA, adhE, ackA) used to construct KJ012 and KJ 017. A simple cross marks an additional gene that is deleted during the construction of the KJ017 derivative: KJ032(ldhA, adhE, ackA, focA, pflB), and KJ070(ldhA, adhE, ackA, focA, pflB, mgsA), and KJ072(ldhA, adhE, ackA, focA, pflB, mgsA, poxB). Genes and enzymes: ldhA, lactate dehydrogenase; focA, formate transporter; pflB, pyruvate-formate lyase; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB and fumC, fumarase isomerase; frdABCD, fumarate reductase; fdh, formate dehydrogenase; mgsA, methylglyoxal synthase; gloAB, glyoxalase I and II; poxB, pyruvate oxidase; aceA, isocitrate lyase; aceB, malate synthase; acnAB, aconitase; and acs, acetyl-CoA synthetase.

FIGS. 7A-7C succinic and malic acid production by derivatives of E.coli C in mineral salts medium (10% glucose). Fig. 7A shows succinic acid production from KJ060 in AM1 medium. Fig. 7B shows succinic acid production by KJ073 in AM1 medium. FIG. 7C shows the production of malic acid by KJ071 in NBS medium. Fermentation with 33mg DCW l^-1All figures are numbered ○, glucose, ●, succinic acid, ■, malic acid,. DELTA.cell mass.

FIG. 8 construction of pLOI 4162. The short solid arrows associated with pEL04 and pLOI4152 represent the primers used for DNA amplification.

FIG. 9. succinic acid production pathway in KJ 073. The pck gene encoding the phosphoenolpyruvate carboxykinase, the major carboxylase involved in succinic acid production in this study, is shown in inverted form. The solid arrows indicate the reactions expected to be functional during anaerobic fermentation of glucose. The solid line crosses indicate the missing gene. The boxed crosses represent the key deletion (ldhA, adhE, ackA) for the construction of the succinic acid producing initial strain KJ 017. The dashed line indicates that PoxB oxidizes pyruvate to acetate, a process that is normally functional only under microaerophilic conditions. The dotted line indicates the reactions mainly associated with aerobic metabolism. Genes and enzymes: ldhA, lactate dehydrogenase; pflB, pyruvate-formate lyase; focA, formate transporter; pta, phosphate acetyltransferase; ackA, acetate kinase; adhE, alcohol dehydrogenase; ppc, phosphoenolpyruvate carboxylase; pdh, pyruvate dehydrogenase complex; gltA, citrate synthase; mdh, malate dehydrogenase; fumA, fumB, and fumC, fumarase isomerase; frdABCD, fumarate reductase; fdh, formate dehydrogenase; icd, isocitrate dehydrogenase; acs, acetyl-CoA synthetase; mgsA, methylglyoxal synthase; poxB, pyruvate oxidase; aldA, aldehyde dehydrogenase; and aldB, aldehyde dehydrogenase. the tdcE gene (pyruvate formate-lyase, homologous to pflB) and the tcdD gene (propionate kinase, homologous to ackA) are shown in parentheses and are typically expressed during threonine degradation.

FIG. 10. extended portion of metabolism illustrates the pathway of other genes (solid crosses) being deleted. Succinic acid and acetic acid are the main products (boxed) from the KJ073 fermentation. Genes and enzymes: citDEF, citrate lyase; gltA, citrate synthase; aspC, aspartate aminotransferase; pck, phosphoenolpyruvate carboxykinase; sfcA, NAD + -related malic enzyme; fumA & fumB, fumarase; frdABCD, fumarate reductase; pykA & pykF, pyruvate kinase; tdcE, pyruvate formate-lyase (homolog of pflB); pta, phosphate acetyltransferase; tcdD, acetate kinase (homolog of ackA).

Detailed Description

The present invention provides materials and methods in which unique and advantageous combinations of genetic mutations are used to direct carbon flux to desired products, such as succinic acid and/or malic acid. The techniques of the present invention can be used to obtain products from both natural and recombinant routes. Advantageously, the present invention provides a versatile platform for producing these products using only mineral salts and sugars as nutrients.

The microorganism of the present invention can be obtained by modifying one or more target genes in a bacterium such as a bacterium belonging to the genus Escherichia (Escherichia). In some embodiments, the modified bacteria may be e.coli, or a specific strain thereof, e.g. e.coli B, e.coli C, e.coli W or the like. In some other embodiments of the invention, bacteria that may be modified according to the invention include, but are not limited to, Gluconobacter oxydans (Gluconobacter oxydans), Gluconobacter shallot (Gluconobacter asaii), Achromobacter delavayi (Achromobacter delmarmorale), Achromobacter viscosus (Achromobacter viscosus), Achromobacter lactis (Achromobacter lacticum), Agrobacterium tumefaciens (Agrobacterium tumefaciens), Agrobacterium radiobacter (Agrobacterium tumefaciens), Alcaligenes faecalis (Alcaligenes faecalis), Arthrobacter citrobacter (Arthrobacter), Arthrobacter tumefaciens (Arthrobacter tumefaciens), Arthrobacter paraffineus (Arthrobacter paraffineus, Arthrobacter albobacter, Arthrobacter carthamiae, Arthrobacter nigripes, Arthrobacter oxydans, Arthrobacter roseum (Brevibacterium), Brevibacterium flavum, Brevibacterium fusobacter (Brevibacterium) and Brevibacterium flavum, Brevibacterium fusobacter acidum, Brevibacterium fusobacter, Brevibacterium immariophilium, Brevibacterium (Brevibacterium strain), Brevibacterium proteorum, Corynebacterium prototheciae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum (Corynebacterium glutamicum), Enterobacter aerogenes (Enterobacter calamus), Corynebacterium amyloliquefaciens (Corynebacterium acetoacidophilum), Corynebacterium acetoglutamicum (Corynebacterium acetobacter), Enterobacter aerogenes (Enterobacter aerogenes), Erwinia amyloliquefaciens (Erwinia amylovora), Erwinia carotovora (Erwinia carotovora), Erwinia herbicola (Erwinia carotovora), Erwinia carotovora (Erwinia rhodobacter), Pseudomonas aeruginosa (Corynebacterium glutamicum), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Corynebacterium, Pseudomonas aeruginosa), Pseudomonas aeruginosa (Corynebacterium strain (Corynebacterium, Pseudomonas aeruginosa), Pseudomonas aeruginosa (Corynebacterium strain (Corynebacterium glutamicum), Pseudomonas aeruginosa), Pseudomonas strain (Corynebacterium, Pseudomonas), Pseudomonas aeruginosa (Corynebacterium strain (Corynebacterium), Pseudomonas aeruginosa (Corynebacterium strain (Corynebacterium), Pseudomonas strain (Corynebacterium strain (Pseudomonas), Pseudomonas aeruginosa, Pseudomonas), Pseudomonas strain (Corynebacterium strain (Pseudomonas), Pseudomonas strain (Corynebacterium strain (Pseudomonas), Pseudomonas strain (Corynebacterium strain, Pseudomonas ovorans (Pseudomonas ovilis), Pseudomonas stutzeri (Pseudomonas stutzeri), Pseudomonas pseudomonads (Pseudomonas mucosae), Pseudomonas fragi (Pseudomonas aeruginosa), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Rhodococcus erythropolis (Rhodococcus rhodochrous), Rhodococcus rhodochrous (ATCC 15592), Rhodococcus species ATCC 19070, Bacillus urealyticus (Sporosarcina ureus), Staphylococcus aureus (Staphylococcus aureus), Streptomyces metschnikovii, Clostridium typrogens, Nocardia madura (Aspergillus oliv), Streptomyces actinovaeae, Streptomyces clavuligerus (Streptomyces viridis), Streptomyces viridans (Streptomyces viridis), Streptomyces viridis (Streptomyces viridis), Streptomyces viridis (Streptomyces viridis ), Streptomyces viridis, Streptomyces viridis, Streptomyces viridis, streptomyces viridochromogenes, Aeromonas salmonicida (Aeromonas salmonicida), Bacillus pumilus (Bacillus pumilus), Bacillus circulans (Bacillus circulans), Bacillus thiaminolyticus (Bacillus thiaminolyticus), Escherichia coli (Escherichia freundii), Microbacterium ammoniaphilum, Serratia marcescens (Serratia marcocens), Salmonella typhimurium (Salmonella typhimurium), Salmonella scheotmulleri, Xanthomonas citri (Xanthomonas citri) and the like.

In certain embodiments, the invention provides strains (e.g., e.coli) lacking plasmids, antibiotic resistance genes, and/or material from other organisms, which are suitable for the production of succinic acid or malic acid. Unlike other microbial systems, the microorganisms of the present invention can be used in a single step production process using sugars as substrates, with high product productivity, high yield, simple nutritional requirements (e.g., mineral salt media), and robust metabolism that allows for the bioconversion of hexoses, pentoses, and many disaccharides.

Thus, a microorganism produced according to the present disclosure may have one or more target genes inactivated by various methods known in the art. For example, the target gene may be inactivated by introducing an insert, deletion, or random mutation into the target gene. Thus, certain aspects of the invention provide for the insertion of at least one stop codon (e.g., one to ten or more stop codons) into a target gene. Some aspects of the invention provide for insertion or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more bases to introduce a frame shift mutation in a target gene. Other aspects of the invention provide for the insertion or deletion of 1, 2, 4, 5,7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or more bases to introduce a frame shift mutation in a target gene. Other embodiments of the present application also provide for the introduction of one or more point mutations (e.g., 1 to 30 or more) in a target gene, while other aspects of the invention provide for the partial, total, or complete deletion of a target gene from a microorganism of the invention. In each of these aspects of the invention, the metabolic pathway is inactivated by inactivating the enzymatic activity of the polypeptide encoded by the target gene.

The term "target gene" as used herein refers to a gene encoding acetate kinase, alcohol dehydrogenase, aspartate aminotransferase, citrate lyase, formate transporter, lactate dehydrogenase, methylglyoxal synthase, pyruvate-formate lyase, pyruvate oxidase, phosphate acetyltransferase, malic enzyme, and/or propionate kinase/α -ketobutyrate formate lyase (α -ketobutyrate transporter) in certain preferred embodiments the gene is ackA (acetate kinase), adhE (alcohol dehydrogenase), aspC (aspartate aminotransferase), citCDEF (citrate lyase), focA (formate transporter), ldhA (lactate dehydrogenase), mgsA (methylglyoxal synthase), pflB (pyruvate-formate lyase), poxB (pyruvate oxidase), pta (phosphate acetyltransferase), sfcA (malic enzyme) and/or cde (propionate kinase/α -ketobutyrate lyase) in certain aspects of the invention, it is therefore understood that in certain aspects of the invention, in strains containing such microorganisms (bacteria), the genes are found in any strain known as "native microorganism" and that the genes are naturally evolved "or" are obtained from "any other than the native microorganism.

Various non-limiting embodiments of the invention include:

1. a genetically modified bacterial strain comprising genetic modifications to one or more of the following target genes: a) acetate kinase, b) lactate dehydrogenase, c) alcohol dehydrogenase, d) pyruvate formate lyase, e) methylglyoxal synthase, f) pyruvate oxidase, and/or g) citrate lyase, said genetic modification inactivating the enzymatic activity of the polypeptide produced by said target gene;

2. the genetically modified bacterial strain according to embodiment 1, wherein the genetically modified bacterial strain is Escherichia coli, Gluconobacter oxydans, Gluconobacter shallii, Achromobacter demarkii, Achromobacter viscosus, Achromobacter lactis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citrobacter, Arthrobacter tumefaciens, Arthrobacter hydrogluceracum, Arthrobacter oxydans, Brevibacterium bovis, Azotobacter indicum, Brevibacterium ionogenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium Brevibacterium luteum, Brevibacterium divaricatum, Brevibacterium ketoglutarate, Brevibacterium helcola, Brevibacterium pusillus, Brevibacterium tristearum, Brevibacterium roseum, Brevibacterium immarilicium, Brevibacterium expansum, Brevibacterium (Brevibacterium), Brevibacterium glutamicum, Corynebacterium acetobacter aceticum, Corynebacterium glutamicum, Acetobacter aceticum, Acetobacter xylinum, Acetobacter asiaticum, Brevibacterium glutamicum, Corynebacterium glutamicum, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantium, Flavobacterium reinhardtii, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus species CCM825, Proteus morganii, Nocardia opacea, Nocardia crassa, Planococcus euticus, Proteus raperii, Propionibacterium schermans, Pseudomonas flavus, Pseudomonas fluorescens, Pseudomonas ovoid, Pseudomonas stutzeri, Pseudomonas aeruginosa, Rhodococcus rhodochrous species ATCC 15592, Rhodococcus species ATCC 19070, Rhodococcus carotovora, Staphylococcus aureus, Vibrio bacteriovorans, Streptomyces violaceus, Streptomyces viridans, Streptomyces coelicolor, Mycospora, Streptomyces, streptomyces violaceorum, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacao, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia coli, Microbacterium ammoniaphilus, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, or Xanthomonas citri;

3. the genetically modified bacterial strain according to embodiment 1 or 2, wherein the modified bacterial strain is escherichia coli B;

4. the genetically modified bacterial strain of embodiments 1, 2 or 3, wherein the following target genes are inactivated: a) acetate kinase, b) lactate dehydrogenase, c) alcohol dehydrogenase, d) pyruvate formate lyase, and e) pyruvate oxidase;

5. the genetically modified bacterial strain of embodiment 4, wherein the bacterial strain further comprises an inactivated methylglyoxal synthase gene;

6. the genetically modified bacterial strain of embodiment 4 or 5, wherein the bacterial strain further comprises an inactivated citrate lyase gene;

7. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4 or 5, wherein said genetically modified bacterial strain is metabolically evolved;

8. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5, 6 or 7, wherein the gene or a portion thereof is deleted;

9. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5, 6 or 7, wherein the gene is inactivated by a frameshift mutation, a point mutation, insertion of a stop codon or a combination thereof;

10. the genetically modified bacterial strain according to embodiment 2, 3, 4, 5, 6, 7, 8 or 9, wherein the genetically modified bacterial strain is an escherichia coli strain and does not contain a foreign gene or a fragment thereof (or contains only a native escherichia coli gene);

11. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, with the proviso that: 1) the genetically modified bacterial strain has one or more of the following genes not inactivated: a) fumarate reductase; b) an ATP synthase; c) 2-oxoglutarate dehydrogenase (sucAB); d) succinate dehydrogenase (e.g., sdhAB), phosphate acetyltransferase (e.g., pta); e) glucose transporters (e.g., ptsG); f) isocitrate lyase repressors (e.g., iclR); and/or 2) the genetically modified strain does not contain plasmids or multicopy plasmids encoding and/or overexpressing genes such as malate dehydrogenase (mdh) and phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc) and/or citrate synthase (e.g., citZ of bacillus subtilis);

12. the genetically modified bacterial strain according to embodiments 1-11, wherein the genetically modified bacterial strain is KJ012, KJ017, KJ032, KJ044, KJ059, KJ060, KJ070, KJ071, KJ072 or KJ 073;

13. a method of culturing or breeding a genetically modified bacterial strain, comprising inoculating a culture medium with one or more genetically modified bacterial strains according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and culturing or breeding the genetically modified bacterial strain;

14. a method of producing succinic acid or malic acid comprising culturing one or more genetically modified bacterial strains according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 under conditions that allow for the production of succinic acid or malic acid;

15. the method according to embodiment 14, wherein the one or more genetically modified bacterial strains is KJ012, KJ034, KJ044, KJ059, KJ060, KJ070, KJ071, KJ072 or KJ 073;

16. the method according to any one of embodiments 13, 14 or 15, wherein the genetically modified bacterial strain is cultured in a mineral salt medium;

17. the method according to embodiment 16, wherein the mineral salts medium comprises between 2% and 20% (w/v) carbohydrate;

18. the method according to embodiment 17, wherein the mineral salts medium contains 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% (w/v) of a sugar;

19. the method according to claim 17 or 18, wherein the carbohydrate is glucose, fructose, xylose, arabinose, galactose, mannose, rhamnose, sucrose, cellobiose, hemicellulose or a combination thereof;

20. the method according to embodiment 14, 15, 16, 17, 18 or 19, wherein succinic acid or malic acid is produced at a concentration of at least 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.55M, 0.60M, 0.65M or 0.70M;

21. the method according to embodiment 14, 15, 16, 17, 18, 19 or 20, wherein the culture medium is NBS mineral salts medium or AM1 medium (see table 4);

22. the method according to embodiment 14, 15, 16, 17, 18, 19, 20 or 21, wherein the yield of succinic acid or malic acid is at least or greater than (or greater than or equal to) 90%;

23. the method according to embodiment 22, wherein the yield is at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99%;

24. the method according to any one of claims 13-16 or 20-23, wherein the growth medium comprises glycerol as substrate for the production of succinic acid, malic acid or fumaric acid;

25. the method according to claims 17-19, wherein the culture medium further comprises glycerol as substrate for the production of succinic acid, malic acid or fumaric acid; or

26. A composition comprising one or more genetically modified bacterial strains according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and a culture medium.

The present application also provides other embodiments of the following:

1. a genetically modified bacterial strain comprising a genetic modification to a target gene encoding a) acetate kinase, b) lactate dehydrogenase, c) alcohol dehydrogenase, d) pyruvate formate lyase, e) methylglyoxal synthase, f) pyruvate oxidase, g) citrate lyase, h) aspartate aminotransferase, i) formate transporter, j) phosphate acetyltransferase, k) malic enzyme, and l) propionate kinase/α -ketobutyrate formate lyase, said genetic modification inactivating the enzymatic activity of a polypeptide produced by said target gene;

3. the genetically modified bacterial strain according to embodiment 2, wherein the genetically modified bacterial strain is escherichia coli;

4. a genetically modified bacterial strain comprising:

(a) genetic modification of a citrate lyase gene and one or more target genes encoding a) an acetate kinase, b) a lactate dehydrogenase, c) an alcohol dehydrogenase, d) a pyruvate formate lyase, e) a methylglyoxal synthase, f) a pyruvate oxidase, g) an aspartate aminotransferase, h) a formate transporter, i) a phosphate acetyltransferase, j) a malic enzyme, and/or k) a propionate kinase/α -ketobutyrate formate lyase, or

(b) Genetic modification of a citrate lyase gene, a lactate dehydrogenase gene, an alcohol dehydrogenase gene, an acetate kinase gene, a formate transporter gene, a pyruvate formate lyase gene, a methylglyoxal synthase gene, a pyruvate oxidase gene and one or more target genes encoding a) an aspartate aminotransferase, b) a phosphate acetyltransferase, c) a malic enzyme, and/or d) a propionate kinase/α -ketobutyrate formate lyase;

the genetic modification inactivates the enzymatic activity of the polypeptide produced by the target gene;

5. the genetically modified bacterial strain according to embodiment 4, wherein the genetically modified bacterial strain is Escherichia coli, Gluconobacter oxydans, Gluconobacter shallii, Achromobacter malorum, Achromobacter viscosus, Achromobacter lactis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citrobacter, Arthrobacter tumefaciens, Arthrobacter hydrogluceramicus, Arthrobacter oxydans, Brevibacterium longissimum, Azotobacter indicum, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium lactoglobosum, Brevibacterium melanobacter, Brevibacterium ketoglutarate, Brevibacterium helcola, Brevibacterium lactococcus, Brevibacterium lactofermentum, Brevibacterium tristicum, Brevibacterium roseum, Brevibacterium immaribacter, Brevibacterium expansum, Brevibacterium oxyphenicum, Brevibacterium proucens, Corynebacterium protolyticum, Corynebacterium acetobacter aceticum, Corynebacterium glutamicum, Bacillus caldaricola, Bacillus caldovelox, Bacillus cereus, Bacillus cerealopecurobacter asiaticum, Bacillus cerealopecorubidus, Bacillus cere, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucidum fucatum, Flavobacterium aurantium, Flavobacterium reinhardtii, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningoticum, Micrococcus species 825, Proteus morganii, Nocardiaa, Nocardia crassa, Planococcus eutica, Proteus rapae, Propionibacterium klebsiella schermans, Pseudomonas azotoformis, Pseudomonas fluorescens, Pseudomonas ovoid, Pseudomonas schutzfeldti, Pseudomonas aeruginosa, Streptomyces erythropolis, Rhodococcus rhodochrous, Rhodococcus rhodococcus species ATCC 15592, Rhodococcus species ATCC 19070, Sporococcus sarcina, Staphylococcus aureus, Vibrio bacteriovorans, Streptomyces Victorius, Streptomyces violaceus, Streptomyces aureofaciens, Streptomyces aureocauliflora, Streptomyces aureofaciens, Streptomyces coelicolor strain, Streptomyces aureocauliflora, Streptomyces aureoviridans, Streptomyces aureophycus, Streptomyces aur, Streptomyces theobromae, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia coli, Microbacterium ammoniaphilus, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, or Xanthomonas citri;

6. the genetically modified bacterial strain according to embodiment 5, wherein the genetically modified bacterial strain is escherichia coli;

7. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5 or 6, wherein said genetically modified bacterial strain is metabolically evolved;

8. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5 or 6, wherein the target gene or part thereof, or target gene or part thereof is inactivated by deletion, frameshift mutation, point mutation, insertion of a stop codon or a combination thereof;

9. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5 or 6, wherein said genetically modified bacterial strain does not contain any foreign genes or fragments thereof, or contains only native genes;

10. the genetically modified bacterial strain according to embodiment 1, 2, 3, 4, 5 or 6, with the proviso that: 1) the genetically modified bacterial strain has not been inactivated by one or more of the following enzymes: a) fumarate reductase; b) an ATP synthase; c) 2-ketoglutarate dehydrogenase; d) a succinate dehydrogenase; e) a glucose transporter; f) a repressor of isocitrate lyase; and/or 2) the genetically modified strain does not contain a plasmid or multicopy plasmid encoding and/or overexpressing malate dehydrogenase, phosphoenolpyruvate carboxylase, pyruvate carboxylase and/or citrate synthase;

11. the genetically modified bacterial strain according to embodiment 7, 8, 9, 10 or 11, wherein said genetically modified bacterial strain is metabolically evolved.

12. The genetically modified bacterial strain according to any of embodiments 1-10, wherein the genetically modified bacterial strain produces:

a) succinic acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM or 700 mM;

b) fumaric acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM or 700 mM; or

c) Malic acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM;

13. a genetically modified bacterial strain, wherein the genetically modified bacterial strain is KJ012, KJ017, KJ032, KJ044, KJ059, KJ060, KJ070, KJ071, KJ072, KJ073, KJ076, KJ079, KJ091, KJ098, KJ104, KJ110, KJ119, KJ122, or KJ 134;

14. a method of culturing or growing a genetically modified bacterial strain comprising inoculating a culture medium with one or more genetically modified bacterial strains of any one of embodiments 1-13 and culturing or growing the genetically modified bacterial strain;

15. a method of producing succinic acid, fumaric acid or malic acid comprising culturing one or more genetically modified bacterial strains according to any one of embodiments 1-13 under conditions that allow for the production of succinic acid or malic acid or fumaric acid;

16. the method according to embodiment 15, wherein the one or more genetically modified bacterial strains is KJ012, KJ017, KJ032, KJ044, KJ059, KJ060, KJ070, KJ071, KJ072, KJ073, KJ076, KJ079, KJ091, KJ098, KJ104, KJ110, KJ119, KJ122, or KJ 134;

17. the method according to any one of embodiments 14-16, wherein the genetically modified bacterial strain is cultured in a mineral salt medium;

18. the method according to embodiment 17, wherein the mineral salts medium comprises between 2% and 20% (w/v) carbohydrate;

19. the method of claim 18, wherein the mineral salt medium contains 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% (w/v) of a sugar;

20. the method according to embodiment 18 or 19, wherein the carbohydrate is glucose, fructose, xylose, arabinose, galactose, mannose, rhamnose, sucrose, cellobiose, hemicellulose or a combination thereof;

21. the method according to any one of embodiments 15-20, wherein the yield of succinic acid or malic acid is greater than or equal to 90%;

22. the method according to embodiment 21, wherein the yield is at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99%;

23. the method according to any one of embodiments 15-22, wherein the genetically modified bacterial strain produces succinic acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, or 700 mM;

24. the method according to any one of embodiments 15-22, wherein the genetically modified bacterial strain produces malic acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM;

25. the method according to any one of embodiments 15-22, wherein the genetically modified bacterial strain produces fumaric acid at a concentration of at least 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, 500mM, 550mM, 600mM, 650mM, or 700 mM;

26. the method according to any one of embodiments 14-17 or 21-25, wherein the growth medium comprises glycerol as a substrate for the production of succinic acid, malic acid or fumaric acid;

27. the method according to any one of embodiments 18-20, wherein the culture medium further comprises glycerol as a substrate for the production of succinic acid, malic acid or fumaric acid; or

28. A composition comprising one or more genetically modified bacterial strains according to any one of embodiments 1-13 and a culture medium.

Microorganisms were deposited at the american agricultural research Culture Collection (agricutural research Service Culture Collection), 1815n. university Street, Peoria, Illinois, 61604 u.s.a., as shown in the examples. According to 37CFR 1.14 and 35USC 122, the deposit is made under conditions that ensure that the patent and trademark office chief (Commission of patents and Trademarks) determines that anyone to whom the culture is entitled will be able to obtain it during the pendency of this patent application. Deposits are available as required by foreign patent laws in countries where copies of the present application or their progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the invention in the detriment of patent rights granted by governmental action.

In addition, the subject culture deposits should be deposited and made publicly available, i.e., they should be deposited and used with all the necessary care to survive for a period of at least five years after the last requirement to provide a deposited sample and not be contaminated, and in any event, for a period of at least 30 (thirty) years after the date of deposition or the actionable life of any patent that issued the culture, in accordance with the provisions of the budapest treaty on the deposit of microorganisms. The depositor has the responsibility of changing the deposit when the depository cannot provide the sample on demand due to the condition of the deposit. All restrictions on the public availability of the subject culture deposits will be irrevocably removed upon grant of the patent disclosing the culture deposit.

The following examples illustrate the steps for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise indicated.

Example 1

The microorganisms were deposited at the ARS culture Collection as follows:

culture of microorganisms	Strain nomenclature	Date of storage
			KJ012	B-50022	2007, 3, 15 days
KJ017	B-50023	2007, 3, 15 days
			KJ032	B-50024	2007, 3, 15 days
KJ060	B-50025	2007, 3, 15 days
			KJ070	B-50026	2007, 3, 15 days
KJ071	B-50027	2007, 3, 15 days
			KJ072	B-50028	2007, 3, 15 days
KJ073	B-50029	2007, 3, 15 days

Materials and methods

Strains, media and culture conditions

The strains used in this study are summarized in table 2. Through a combination of unique gene deletions and selection for increased productivity, derivatives of escherichia coli C (ATCC 8739) were developed for succinic acid production. Only during the strain construction, cultures were grown in modified Luria-Bertani (LB) liquid medium (per liter: 10g Difco tryptone, 5g Difco yeast extract, 5g sodium chloride) (Miller, 1992) at 37 ℃. Antibiotics are included where appropriate.

In most studies, 100mM KHCO supplementation was used₃NBS mineral salt medium (Caus) with 1mM betaine HCl and sugar (2% to 10%)ey, 2004) as fermentation broth and for maintaining the strain. A new low salt medium AM1(4.2g l) was developed in the late stages of the study^-1Total salt; martinez et al, 2007) and used in the fermentation of KJ060 and KJ 073. With 100mM KHCO₃And the indicated sugar supplements the medium and contains 1mM betaine when the initial sugar concentration is 5% or higher. In addition to intermediates during construction, no genes encoding antibiotic resistance, plasmids or other foreign genes were present in the strains developed for succinic acid production.

Genetic method

The plasmids and primers used in this study are summarized in table 2. Methods for chromosomal deletions, integration and antibiotic resistance gene removal have been previously described (Datsenko and Wanner, 2000; Grabar et al, 2006; Posfai et al, 1997; Zhou et al, 2006). The sense primer contains a sequence corresponding to the N-terminus of each target gene (bold), followed by 20bp (underlined) corresponding to the FRT-kan-FRT cassette. The antisense primers contain a sequence corresponding to the C-terminus of each target gene (bold), followed by 20bp corresponding to the cassette (underlined). The amplified DNA fragment was electroporated into a strain of E.coli harboring the Red recombinase (pKD 46). In the resultant recombinants, the FRT-kan-FRT cassette replaced the deleted target gene region by homologous recombination (double crossover event). The resistance gene (FRT-kan-FRT) was then excised from the chromosome using plasmid pFT-A using FLP recombinase, leaving a scar region containing one FRT site. Chromosomal deletions and integrations were verified by tests against antibiotic markers, PCR analysis and fermentation product analysis. Transduction using the ubiquitous P1 phage (Miller, 1992), Δ focA-pflB: : the FRT-kan-FRT mutation was transferred from strain SZ204 into strain KJ017, yielding KJ 032.

Deletion of mgsA and poxB genes

An improved method was developed for deletion of E.coli chromosomal genes using a two-step homologous recombination process (Thomason et al, 2005). When this method is used, there is no antibiotic gene or scar sequence remaining on the chromosome after the gene deletion. In the first recombination, part of the target gene was replaced with a DNA cassette containing a chloramphenicol resistance gene (cat) and a levan sucrase gene (sacB). In the second recombination, the cat-sacB cassette is replaced with the native sequence omitting the deleted region. Cells containing the sacB gene accumulate fructan and are killed during incubation with sucrose. Surviving recombinants were highly enriched for loss of the cat-sacB cassette.

A cassette was constructed to simplify gene deletion. The cat-sacB region was amplified by PCR from pEL04(Lee et al, 2001; Thomason et al, 2005) using the JMcatacSacB primer set (Table 2), digested with NheI, and ligated into the corresponding site of pLOI3421, resulting in pLOI 4151. The cat-sacB cassette was amplified by PCR using pLOI4151 (template) and the cat-up2/sacB-down2 primer set (each primer contains an EcoRV site), digested with EcoRV, and used in subsequent ligations.

The mgsA gene and the adjacent 500bp region (yccT '-mgsA-help', 1435bp) were amplified using the primer set mgsA-up/down and cloned into the pCR2.1-TOPO vector (Invitrogen) to generate plasmid pLOI 4228. This 1000-fold diluted preparation of plasmid DNA functions as a template, and inside-out (inside-out) amplification was performed using the mgsA-1/2 primer set (both primers are within the mgsA gene and face outward). The resulting replicon-containing 4958bp fragment was ligated with the cat-sacB cassette amplified from pLOI4151 and digested with EcoRV, resulting in pLOI 4229. This 4958bp fragment was also used to construct a second plasmid, pLOI4230 (phosphorylated and self-ligated). In pLOI4230, the central region of mgsA was deleted (yccT ' -mgsA ' -mgsA "-helD ').

After digestion of pLOI4229 and pLOI4230 with XmnI (in vector), they were amplified as templates using the mgsA-up/down primer set to generate linear DNA fragments for integration of step I (yccT '-mgsA' -cat-sacB-mgsA "-helD ') and step II (ycC T' -mgsA '-mgsA" -helD'), respectively. After electroporation of the step I fragment into KJ060 containing pKD46(Red recombinase) and incubation at 30 ℃ for 2 hours to allow expression and isolation, chloramphenicol (40mg l) was targeted on plates (30 ℃, 18 hours)^-1) And ampicillin (20mg l)^-1) Resistant selection recombinants. Three clones were selected containing ampicillin and5% w/v arabinose in Luria broth and ready for electroporation. After electroporation with the step II fragment, cells were incubated at 37 ℃ for 4 hours and transferred to 250-ml flasks containing 100ml of modified LB containing 10% sucrose (100 mM MOPS buffer was added and NaCl was omitted). After overnight incubation (37 ℃), clones were selected on modified LB plates (without NaCl; with 100mM MOPS addition) containing 6% sucrose (39 ℃, 16 h). The resulting clones were tested for loss of ampicillin and chloramphenicol resistance. The constructs were further verified by PCR analysis. A clone lacking the mgsA gene was selected and designated KJ 070.

In a manner similar to that used for deletion of mgsA gene, poxB gene was deleted from KJ 071. Additional primer sets (poxB-up/down and poxB-1/2) for the construction of poxB deletions were included in Table 2 along with the corresponding plasmids (pLOI4274, pLOI4275 and pLOI 4276). The resulting strain was named KJ 072.

Enzyme assay

Cells were cultured in NBS medium containing 5% or 10% glucose, harvested by centrifugation (8,000 g for 5 min at 4 ℃) during mid-log phase, washed with cold 100mM Tris-HCl (pH 7.0) buffer, and resuspended in the same buffer (5 ml). The cells were disrupted by Bead-treatment (MP Biomedicals; Solon, Ohio) with glass beads and then centrifuged at 13,000g for 15 minutes to give a crude extract. The protein was measured by the BCA method using bovine serum albumin as a standard (Pierce BCA protein assay kit).

PEP carboxylase activity was measured as described previously (Canovas and Kornberg, 1969). The reaction mixture contained 100mM Tris-HCl buffer (pH8.0), 10mM MgCl₂、1mMDTT、25mM NaHCO₃0.2mM NADH, 20U malate dehydrogenase and 10mM PEP, the reaction was initiated by addition of the crude extract. PEP carboxykinase activity was measured as previously described (Vander Werf et al, 1997). The reaction mixture contained 100mM MES buffer (pH6.6), 10mM MgCl₂、75mM NaHCO₃、5mM MnCl₂50mM ADP, 1mM DTT, 0.2mM NADH, 20U malate dehydrogenase, and 10mM PEP. By addingCrude extract initiated the reaction.

Measuring NAD in both directions as described previously⁺Dependent malic enzyme activity (Stols and Donnelly, 1997). For the direction of carboxylation, the reaction mixture contained 100mM Tris-HCl buffer (pH7.5), 25mM NaHCO₃、1mM MnCl₂1mM DTT, 0.2mM NADH and 25mM pyruvate. The reaction was initiated by the addition of the crude extract. However, this assay method cannot measure the malate activity in wild-type E.coli C because of the presence of lactate dehydrogenase. For decarboxylation, the reaction mixture contained 100mM Tris-HCl buffer (pH7.5), 2.5mM NAD⁺、1mM DTT、10mM MgCl₂20mM KCl and 20mM L-malic acid. The reaction was initiated by the addition of the crude extract.

By reaction with NADP⁺NADP was measured in the same manner as dependent malic enzyme⁺Dependent malic enzyme Activity with the exception of NADP (H)⁺In place of NAD (H)⁺. One activity unit is defined as the amount of enzyme that oxidizes or reduces 1nmol of substrate per minute.

Real-time RT-PCR analysis

Messenger RNA levels were measured using real-time RT-PCR as previously described (Jarboe et al, 2008). Cells were cultured in NBS medium containing 5% or 10% glucose, harvested during mid-log phase of growth by shaking in a dry ice/ethanol bath followed by centrifugation, and stored in RNALater (Qiagen, Valencia CA) at-80 ℃ until purification. RNA purification was performed using RNeasy Mini columns (Qiagen) followed by digestion with DNaseI (Invitrogen). Reverse transcription was performed using Superscript II (Invitrogen, Carlsbad CA) using 50ng of total RNA as template. Real-time PCR was performed in a Bio-Rad iCycler using SYBRGreen RT-PCR mixture (Bio-Rad, Hercules CA). The RNA was tested for genomic DNA contamination by RT-PCR in the absence of reverse transcription. Transcript abundance was assessed using genomic DNA as a standard and expression levels were normalized by the transcription repressor birA gene (Jarboe et al, 2008). RT-PCR primers for pck and birA are listed in Table 2.

Sequencing of the pck region

To know if any mutations occurred in the pck gene of KJ073, the coding region and the promoter region of the pck gene (about 800bp before the coding region) were amplified in both KJ012 and KJ073 by Pfuultra high fidelity DNA polymerase (Stratagene; Wilmington, DE). The coding region was amplified by transcription terminator using primer set pck-F/R. The promoter region was amplified using the primer set pck-2. DNA sequencing was provided by the University of Florida Biotechnology Research Center (University of Florida Interdisciplicity Center for Biotechnology Research) (using an applied biosystems automated sequencer).

Fermentation of

In the presence of 100mM KHCO containing glucose₃And 1mM betaine HCl in NBS or AM1 mineral salt medium at 37 ℃, 100rpm incubation inoculation culture and fermentation. These were maintained at ph7.0 during the initial experiment by automatic addition of KOH. Followed by the addition of 3MK₂CO₃And a 1: 1 mixture of 6N KOH maintains the pH. The fermentation was carried out in a small fermentor with a working volume of 350 ml. As indicated, at 0.01(3.3mg CDW l)^-1) Or 0.1(33.3mg CDW l)^-1) Initial OD of₅₅₀And (5) inoculating and fermenting. Antibiotic resistance genes were not present in the strains tested. The fermenter was sealed except for a 16 gauge needle (gauge needle) which served as a sampling port. Fast achievement of anaerobic life during growth with added bicarbonate that acts to ensure atmospheric CO₂The function of (1).

Analysis of

By Bausch&The cell mass was evaluated on the basis of the absorbance at 550nm using a Lomb Spectronic 70 spectrophotometer (OD 1.0: 333mg dry cell weight l)^-1). Organic acids and sugars were determined by using high performance liquid chromatography (Grabar et al, 2006).

Results and discussion

Construction of KJ012 for succinic acid production: deletions of ldhA, adhE, and ackA

Most of the scientific knowledge on E.coli to date has been from studies in complex media (e.g., Luria broth) rather than mineral salt media using low concentrations of sugar substrate (typically 0.2% w/v; 11mM) rather than the 5% (w/v) glucose (278mM) and 10% w/v (555mM) used in the studies reported herein. Large amounts of sugar are required to produce commercially significant levels of product. Previous researchers described the construction of a number of E.coli derivatives for succinic acid production in complex media (Table 1). Rational design based on the basic pathway has been reasonably successful in the academic demonstration of metabolic engineering when complex media are used. However, the use of complex nutrients for the production of bacterial fermentation products increases material costs, purification costs, and costs associated with waste disposal. The use of mineral salts media without complex media components should be more cost effective.

Coli C grew well in NBS mineral salt medium with glucose and produced a mixture of lactic, acetic, ethanol and succinic acid as the fermentation product (FIG. 1A; Table 3). In contrast to other studies using e.coli (table 1), the studies reported herein focused on the development of strains capable of converting high levels of sugars to succinic acid, using mineral salt media to minimize the cost of materials, succinic acid purification, and waste disposal. By examining fig. 1, which illustrates the generally accepted standard fermentation pathway for e.coli, a rational design for metabolic engineering of succinic acid producing strains was invented, wherein among all the alternative products: deletions were made in the genes encoding the terminal steps of lactate (ldhA), ethanol (adhE) and acetate (ackA). The results obtained from this metabolic engineering by rational design are completely unexpected. The resulting strain (KJ012) grew very poorly in mineral salt medium containing 5% glucose (278mM) under anaerobic conditions and produced acetic acid instead of succinic acid as the main fermentation product. In contrast to the expectation from rational design, succinic acid remains as a secondary product. These mutations resulted in no change in the molar yield of succinate based on metabolized glucose, 0.2 moles succinate per mole glucose for the parent and KJ012 during fermentation in NBS mineral salts medium with 5% glucose. We demonstrate that NBS mineral salt medium contains all the mineral nutrients required for KJ012 growth when incubated under aerobic conditions (aerobic shake flask; 5% glucose). In aerobic shake flasks, the cell yield of KJ012 was 5-fold higher than during anaerobic growth and 75% of that of e.coli C (parent) during anaerobic growth. These results also confirm that all central biosynthetic pathways remain functional in KJ 012.

In the presence of complex nutrients (Luria broth), the fermentative succinic acid production of KJ012 was increased 20-fold and the succinic acid molar yield increased 3.5-fold compared to KJ012 in mineral salts medium. Clearly, rational design based on the main approach is better suited for academic demonstration or for designing processes aimed at using complex nutrients.

The principles of poor growth, poor succinic acid production and increased acetic acid production during anaerobic metabolism of KJ012(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT) in mineral salt media are unknown. These are unexpected consequences of using rationally designed metabolic engineering based on standard pathway tables. In minimal media, rational design for metabolic engineering is clearly unpredictable. The resulting strain KJ012 was inferior in growth to the parent and almost equal in succinic acid production to the parent.

Development of KJ017 for succinic acid production by growth-based KJ012 selection

KJ012(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT) grew poorly, showed lower succinic acid productivity, and provided almost equal molar yields compared to the parent E.coli C (Table 3). Despite these results, based on the following theory, serial passage using this strain was attempted as a method for co-selecting improved production and succinic acid growth. The main route of glucose fermentation to succinate (FIGS. 1A and 2A) is generally thought to be the carboxylation step using phosphoenolpyruvate carboxylase (ppc) (Unden and Kleefeld, 2004; Fraenkel 1996; Keseler et al, 2005; Millard et al, 1996; Gottschalk, 1985; Karp et al, 2007). The carboxylase does not preserve the high-energy phosphate in phosphoenolpyruvate and reduces the pure ATP available for growth. Alternative pathways for succinate production can be postulated using known E.coli gene repertoires that increase ATP yield and thus promote growth (FIG. 1A; FIGS. 2B, 2C and 2D). However, none of these alternative pathways have been shown to play a role in succinic acid production during fermentation with a native strain of E.coli. Key enzymes in these alternative pathways are inhibited by glucose and are generally active during gluconeogenesis. In general, the levels of these gluconeogenic enzymes vary inversely with the availability of glucose and other metabolites (Goldie and Sanwal, 1980 a; Wright and Sanwal, 1969; Sanwal and Smando, 1969a) and function in the opposite direction-decarboxylation (Keseler et al, 2005; Oh et al, 2002; Kao et al, 2005; Stols and Donnelly, 1997; Samuelov et al, 1991; Sanwal, 1970 a; Delbaere et al, 2004; Goldie and Sanwal, 1980 b; Sanwal and Smando, 1969 b; Sanwal1970 b).

It has been demonstrated that one of the key enzymes of one of these pathways, NADH-linked malic enzyme (sfcA) (FIG. 2B), is capable of increasing succinate production in E.coli, but requires overexpression from plasmids (Stols and Donnelly, 1997). However, none of these alternative pathways is expected to function in either the native strain of escherichia coli or KJ012 during anaerobic growth using high levels of glucose. Serial passage of KJ012 and selection for improved production provided an opportunity to select for mutational activation of alternative pathways for succinic acid production (fig. 1B) that maintained redox balance and increased ATP yield.

KJ012 was serially passaged as quickly as possible as allowed by growth in NBS glucose medium under fermentation conditions (FIG. 3A; FIG. 4A; FIG. 5). Between the first 9 passages, growth continued slowly requiring incubation for 4-5 days, then increased appreciably, allowing passage at 24 hour intervals. This event was accompanied by an increase in acetic acid (fig. 4A) with little improvement in succinic acid production. After 27 passages (60 days), 3M K was added₂CO₃And a 1: 1 mixture of 6N KOH instead of KOH to provide additional carbon dioxide (to all NBS mineral salts medium initially supplemented with 100 mM). Serial passage resulted in improved succinic acid production. A total of 40 passages were performed in 5% glucose (227mM), followed by another 40 passages in 10% glucose (555 mM). During the passage in 10% glucose, the succinic acid yield remained at about 0.7mol per mol of metabolized glucose, with lactic acid, acetic acid and formic acid as byproducts (table 3). The yield was 3 times higher than E.coli C and KJ 012. The clone was isolated and designated KJ 017. The improvement in growth of KJ012 was selected, resulting in KJ017 co-selected for the improvement in succinic acid production (rate, titer and molar yield).

Physiological principles of enhanced succinic acid production of KJ017

The use of succinic acid produced by E.coli, which is generally considered to be the natural fermentation pathway (phosphoenolpyruvate carboxylase; ppc), wastes the energy of phosphoenolpyruvate by producing inorganic phosphate. By this route, one ATP is lost per succinate produced (FIG. 1; FIG. 6). Conservation of this energy as ATP by using alternative enzyme systems represents an opportunity to promote cell growth and co-select for increased succinate growth. Based on known genes in E.coli, three other enzymatic pathways for succinate production are envisioned, which preserve ATP and thus are capable of promoting growth (FIG. 1; FIG. 6). However, all carboxylation steps in these alternative pathways are thought to act in the opposite direction (decarboxylation), primarily for gluconeogenesis during growth on substrates such as organic acids (Keseler et al, 2005; Oh et al, 2002; Kao et al, 2005; Stols and Donnelly, 1997; Samuelov et al, 1991; Sanwal, 1970 a; Depalere et al, 2004; Goldie and Sanwal, 1980 b; Sanwal and ndSmao, 1969 b; Sanwal1970 b). To test the hypothesis that KJ017 actually activated one or more of these alternative pathways, developed based on growth selection, the specific activities of the key carboxylation steps were compared (table 4). Three of these were similar or lower in E.coli C, KJ012 and KJ 017. Energy-conserving phosphoenolpyruvate carboxykinase (pck) was increased 4-fold in KJ017 compared to KJ012, consistent with the following hypothesis: selection for improved growth will co-select for increased growth of succinate by increasing ATP yield, which results from increased expression of energy conserving pathways for succinate production.

Growth-based selection and additional gene deletion was also used to construct a number of additional strains with further improvements in growth and succinate production (fig. 3 and 4). Enzyme levels in KJ073, one of these strains, were also examined. In this strain, the in vitro activity of phosphoenolpyruvate carboxykinase was further increased 8-fold over KJ017 while the other carboxylases remained essentially unchanged (Table 4).

Pck and surrounding regions were cloned from KJ012 and KJ073 and sequenced. No changes were found in the coding region. Without post-translational modification, the catalytic properties of the enzyme should be unchanged. A single mutation was detected in the pck promoter region, changing G to A at a-64 bp site relative to the translation start. This mutation follows the transcription start site, which is a-139 bp site relative to the translation start site. Restoration of this sequence (a to G) to e.coli C in KJ073 did not affect cell growth, fermentation or succinate production, indicating that the mutation was not critical (data not shown). RT-PCR confirmed that the level of information in KJ073 was increased. These results are consistent with regulatory mutations underlying increased pck expression.

Previous researchers have noted that the kinetic parameters of phosphoenolpyruvate carboxylase (ppc) and phosphoenolpyruvate carboxykinase (pck) can have a significant impact on carboxylation and succinate production (Millard et al, 1996; Kim et al, 2004). For E.coli phosphoenolpyruvate carboxylase (ppc), Km towards bicarbonate was 0.15mM (Morikawa et al, 1980), 9-fold lower than for E.coli phosphoenolpyruvate carboxykinase (pck) (13mM) (Krebs and bridge 1980). Although overexpression of pck from E.coli in a multicopy plasmid increased phosphoenolpyruvate carboxykinase activity by a factor of 50, it was reported that this had no effect on succinate production (Millard et al, 1996). Succinate production was also not enhanced when phosphoenolpyruvate carboxykinase from Anaerobiospirillum succiniciproducens (anamobium succiniciproducens) was overexpressed in E.coli K12 (Kim et al, 2004). The enzyme also has a high Km for bicarbonate (30 mM; Laivenieks et al, 1997). However, succinate production was increased 6.5 fold when pck of anaerobiospirillum succinogenes was overexpressed in ppc mutant of E.coli K12 (Kim et al, 2004). In KJ017 and subsequent derivatives, phosphoenolpyruvate carboxykinase is undoubtedly the predominant carboxylation activity, even in the presence of a functional native phosphoenolpyruvate carboxylase.

The results of the enzyme measurements from E.coli C are very surprising. The enzyme that is generally considered to be the dominant carboxylation activity for succinate production in native E.coli during growth (phosphoenolpyruvate carboxylase; ppc) (Unden and Kleefeld, 2004; Fraenkel 1996; Keseler et al, 2005; Millard et al, 1996; Gottschalk 1985; Karp et al, 2007) is not the most active enzyme in vitro for E.coli C. Thus, the metabolic pathways generally accepted for E.coli (Unden and Kleefeld, 2004; Fraenkel 1996; Sanchez et al, 2006; Cox et al, 2006; Vemuri et al, 2002 a; Wang et al, 2006; Sanchez et al, 2005 ab; Gokarn et al, 2000; Karp et al, 2007) may not accurately reflect metabolism in all strains, and rational design of metabolic engineering and estimation of metabolic flux are generally based on such pathways. The phosphoenolpyruvate carboxykinase activity is most active under in vitro conditions of substrate saturation. In Escherichia coli K12, the activities of both phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase were reported to be equivalent in vitro (140nmmin^-1mg^-1A cellular protein; van der Werf et al, 1997), the former as the major pathway towards succinic acid.

Previous studies have shown that overexpression of the native ppc gene in E.coli leads to higher specific succinate production (Millard et al, 2000), higher specific growth rate and lower specific acetate production (Farmer and Liao, 1997) due to more PEP carboxylation supplementing the intermediates of the TCA cycle. However, since PEP is required for the glucose transport system, overexpression of ppc also reduced the glucose uptake rate by 15-40% compared to isogenic controls without significantly increasing the succinate yield (per glucose) (Chao and Liao, 1993; Gokarn et al, 2000). The failure of native phosphoenolpyruvate carboxylase to increase succinic acid yields has turned most of the research attention to new metabolic designs, overexpressing PYC (pyruvate carboxylase) from Lactobacillus lactis or Rhizobium phaseoli (Rhizobium etli) as the carboxylation step (Vemuri et al, 2002 ab; Gokarn et al, 2000; Lin et al, 2005abc) rather than pursuing further work with all the components of the E.coli gene that are native.

Rumen bacteria such as actinomycetes succinogenes (Actinobacillus succinogenes) use energy-conserving phosphoenolpyruvate carboxykinase for carboxylation during glucose fermentation to produce succinic acid as a major product (Kim et al, 2004; McKinlay et al, 2005; McKinlay and Vieille, 2008). The activity reported for this organism is 5 times that of KJ017 and half that obtained by KJ073 with a continuous growth-based selection (metabolic evolution). Thus, by using a combination of metabolic engineering (ldhA adhE ackA) and metabolic evolution (increased ATP production efficiency based on growth selection), the studies reported herein demonstrate the development of succinic acid-producing e.coli strains that resemble rumen organisms such as a. succinogenes, using only the natural repertoire of e.coli genes. Although it was previously reported that overexpression of E.coli phosphoenolpyruvate carboxylase (ppc) without mutations in phosphoenolpyruvate synthase did not help succinate production (Chao and Liao, 1993; Kim et al, 2004; Gokarn et al, 2000; Millard et al, 1996), KJ017 and derivatives were engineered to use phosphoenolpyruvate carboxykinase as the primary pathway for succinate and malate production.

Construction of KJ032 and KJ060

During growth with 10% (w/v) glucose, although the genes encoding the major lactate dehydrogenase (ldhA) and acetate kinase (ackA) activities were deleted, there were a large number of unwanted byproducts (acetic acid, formic acid, and lactic acid) in fermentations with KJ017(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT) (table 3). The production of lactate and acetate may also result in higher ATP yields, which are the basis for growth selection (fig. 1A).

The gene encoding pyruvate formate lyase (pflB) was deleted from KJ017 to eliminate the loss of reducing agent as formate and the excess of possible acetate source-acetyl-CoA. The upstream formate transporter (focA) in this operon was also deleted. As expected, the deleted strain (KJ032) did not grow without acetate, confirming that this is the major pathway for acetyl-CoA production in KJ017 (fig. 3C). It is well known that deletion of pflB under anaerobic conditions causes acetate auxotrophy (Sawers and Bock, 1988). Growth and succinate production of KJ032 were restored by addition of 20mM acetic acid (fig. 3C, fig. 4C and fig. 5). pflB (and focA) deletion resulted in a substantial reduction in formate and acetate production. Although this strain requires acetic acid for growth, additional acetic acid is also produced during fermentation. The same phenomenon was previously reported for pflB-deleted strains during the construction of pyruvate-producing E.coli K-12 biocatalysts (Causey et al, 2004). The lactic acid level in KJ032 was also reduced (Table 3; FIG. 4C). Subsequent passages were accompanied by improvements in growth and succinate production. During subsequent passages, the acetic acid addition was reduced, the inoculum size was reduced, and the glucose concentration was doubled (10% w/v) (FIG. 4D). After reducing the amount of acetic acid to 5mM, an unstable population was formed which consumed succinic acid to produce increased levels of malic acid. After further passages, acetic acid was omitted and a strain was developed which was no longer acetate auxotrophic, presumably due to increased expression of another gene. However, succinic acid yield decreased upon depletion of the added acetic acid, while malic and acetic acid levels increased. The origin of acetic acid and the basis for the increase in malic acid are unknown. Also, a small amount of pyruvic acid is produced. One clone isolated from the last passage was designated KJ060(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT Δ focA-pflB:: FRT). This strain produced 1 mole succinate per mole glucose metabolized in NBS mineral salts medium with 10% glucose.

Construction of KJ070 and KJ071 by deletion of methylglyoxal synthase (mgsA)

It was hypothesized that the small amounts of lactic acid present in the various strain broths were derived from the methylglyoxal synthase pathway (FIG. 6; Grabar et al, 2006). Although this represents a small loss in yield, lactic acid production through this pathway is indicative of the accumulation of the growth and glycolysis inhibitors methylglyoxal (Egyud and Szent-Gyorgyi, 1966; Grabar et al, 2006; Hopper and Cooper, 1971). The elimination of methylglyoxal and lactate production by deletion of the mgsA gene (methylglyoxal synthase) in KJ060 produced KJ070(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT Δ focA-pflB:: FRT Δ mgsA). Strain KJ070 was initially cultured on 5% (w/v) glucose (FIGS. 4E and 5). Deletion of mgsA is presumed to have an increased glycolytic flux as evidenced by accumulation of pyruvate in the culture medium (Table 3). This increase in glycolytic flux may also be responsible for a further decrease in the succinate/malate ratio due to increased production of oxaloacetate, an allosteric inhibitor of fumarate reductase (Iverson et al, 2002; Sanwal, 1970 c).

At passage 21, glucose was doubled to 10% (w/v) and passage continued. This higher glucose level and subsequent passage led to a further increase in malic acid production over succinic acid in subsequent passages (fig. 4E). The increased production of malic acid relative to succinic acid in 10% w/v glucose is also consistent with increased glycolytic flux and inhibition of fumarate reductase by oxaloacetate. At passage 50, 1.3 moles malic acid and 0.71 moles succinic acid were produced per mole of metabolized glucose (table 3). Significant amounts of acetic acid are also produced. A new strain was isolated from the final subculture and designated KJ071(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT Δ focA-pflB:: FRT Δ mgsA). The strain can be suitable for malic acid production.

Construction of KJ072 and KJ073 by deletion of poxB

Although the conversion of glucose to acetic acid is redox neutral, the assignment of carbon to acetic acid reduces the yield of succinic and malic acid. Pyruvate oxidase (poxB) represents acetic acid and CO during incubation under microaerophilic conditions₂Possible sources of (D) (Causey et al, 2004). Although it should not function to oxidize pyruvate under anaerobic conditions, poxB is the target of gene deletion (FIG. 6). As expected, deletion of poxB produced KJ072(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT Δ fo)cA-pflB: : FRT Δ mgsA Δ poxB) did not decrease acetate production indicating that an alternative pathway is involved in acetate production. However, elimination of poxB resulted in an unexpected change in the fermentation product, an increase in succinic acid and a decrease in malic acid (Table 3; FIG. 4F). This improved mechanism of succinate production is unknown, but may be associated with other activities of pyruvate oxidase such as 3-hydroxybutanone production, decarboxylation, and aldehyde ligation (carboligation) (Ajl and Werkman, 1948; Chang and Cronan, 2000).

Strain KJ072 was subjected to 40 more rounds of metabolic evolution in a lower salt medium, AM1 medium containing 10% (w/v) glucose (Table 3; FIG. 4F and FIG. 5). Improvements in growth, cell yield and succinate production were observed during these passages. Levels of malic acid, pyruvic acid and acetic acid were also increased. One clone was isolated from the final passage and was named KJ073(Δ ldhA:: FRT Δ adhE:: FRT Δ ackA:: FRT Δ pflB:: FRT Δ mgsA Δ poxB). This strain retains the phosphoenolpyruvate carboxykinase pathway for carboxylation (table 4). The in vitro activity of this strain was 45-fold higher than KJ012 and 10-fold higher than KJ017, providing further evidence of close coupling of energy conservation to succinic acid production and growth, and further establishing the basis for selection.

Fermentation of KJ060 and KJ073 in AM1 Medium containing 10% (w/v) glucose

FIG. 7 shows batch fermentation of KJ060 and KJ073, two of the best biocatalysts for succinic acid production. Although growth was completed within the first 48 hours of incubation, succinic acid production lasted 96 hours. One third of succinic acid production occurs when there is no cell growth. These strains produced 668-733mM succinate titres with molar yields of 1.2-1.6 based on metabolized glucose. The yield was generally higher when using AM1 medium than when using NBS mineral salts medium. Acetic, malic and pyruvic acids accumulated as undesirable by-products and were removed from the possible yields of succinic acid (table 3). From glucose and CO₂The maximum theoretical yield of succinic acid (in excess) is 1.71 moles per mole of glucose, based on the following equation:

7C₆H₁₂O₆+6CO₂→12C₄H₆O₄+6H₂O

however, the direct succinate pathway for this yield was not achieved in E.coli (FIG. 6).

Conversion of other substrates to succinic acid

Although the study was primarily focused on the conversion of glucose to succinic acid. It is well known that E.coli has the natural ability to metabolize all the hexoses and pentoses that make up the plant cell wall (Asghari et al, 1996; Underwood et al, 2004). Some E.coli strains also metabolize sucrose (Moniruzzaman et al, 1997). The utilization of 2% hexose and pentose sugars by strain KJ073 was tested in serum tubes. In all cases, these sugars are converted mainly to succinic acid. Strain KJ073 also metabolises glycerol to succinic acid. During incubation with 2% glycerol, 143mM glycerol was metabolized yielding 127mM succinic acid in a molar yield of 0.89, which is 89% of the theoretical maximum.

Production of malic acid in NBS Medium containing 1mM betaine and 10% glucose

During growth-based selection, cultures were observed to differ in their production of malic acid, a potentially useful alternative product (table 3). Malic acid was the most abundant product when KJ071 used 10% glucose (Table 3; FIG. 4E), almost twice as much as succinic acid. This strain produced 1.44 molar yield of 516mM malic acid based on metabolized glucose (Table 3).

Conclusion

The fermentation metabolism of Escherichia coli shows very strong adaptability. Derivatives are engineered and developed to prevent substantial deletion of genes associated with the native fermentation pathway and to increase flux through the remaining enzymes to maintain redox balance, increase efficiency of ATP production and increase growth. Although more challenging, cells can make such adaptations on mineral salt media while balancing carbon allocation to provide all biosynthetic needs. After eliminating the basic pathways for NADH oxidation (lactate dehydrogenase, alcohol dehydrogenase) and acetate production (acetate kinase), growth and ATP production remain associated with NADH oxidation and malate or succinate production for redox balance (fig. 1B). Selection based on anaerobic growth ensures redox balance and selects for increased efficiency and increased ATP production rate, which is the basis for increased growth. This option for redox balance and ATP production cannot easily be distinguished between malic and succinic acid as end products, since both precursors act as electron acceptors. During these studies a strain was developed (KJ071) which produced more malic acid than succinic acid. The strain and other derivatives can be used for malic acid production. Other strains such as KJ073 and KJ060 produce succinic acid as a main product at a yield of 1.2 to 1.6 moles per mole of glucose.

Deletion of the major source of acetyl-CoA, pflB, during anaerobic growth leads to auxotrophs requiring acetate (Sawers and Bock, 1988). This need is eliminated by metabolic evolution, presumably due to the increased production of acetyl-CoA by other pathways such as pyruvate dehydrogenase (de Graef et al, 1999). The metabolic source of acetate or acetyl-CoA required to replace the auxotroph is unknown. Many permutations in metabolites are unexpected. The increase in malate during selection after deletion of mgsA was unexplained. Methylglyoxal is a metabolic inhibitor produced in response to a metabolic imbalance (Grabar et al, 2006). Elimination of methylglyoxal production may provide growth related advantages such as increased growth rate, shorter lag after inoculation, etc. The reduction of malic acid and shift to higher succinic acid production following poxB deletion was also surprising. Little change in acetate levels was observed, indicating that the enzyme is a secondary source of acetate, or its function was replaced by other pathways for acetate production. After deletion of poxB, succinic acid is again produced as the main dihydroxy acid. Malic acid and acetic acid were retained as abundant by-products when using the best strains KJ060 and KJ073 for succinic acid production (table 3; fig. 4D and 4F). The elimination of these represents yet another opportunity to improve yield.

All previously engineered E.coli developed for succinate production used complex media and plasmids with antibiotics for maintenance. Most of them only reach low succinate titers in single batch fermentations, requiring more complex processes to reach high titers (table 1). Various genetic pathways for increasing succinate production from glucose by recombinant E.coli in complex media have been reported. In our original constructs, growth and sugar metabolism were very poor in mineral salts medium, but very viable in complex medium (Luria medium). Complex media containing vitamins, amino acids and other macromolecular precursors may mask potential regulatory problems in metabolism and biosynthesis resulting from metabolic engineering.

Many other researchers also use heterologous genes and complex processes involving gases (CO)₂、H₂、O₂Or air) sparging and binary aerobic and anaerobic process steps. The complexity of the process and nutrients is expected to increase the cost of construction, materials, purification, and waste disposal. In contrast, strains KJ060 and KJ073 produced high titers of succinic acid (600- & gt, 700mM) in a single batch fermentation (10% sugar) using mineral salts medium without any complex nutrients or foreign genes.

Example 2

The microorganisms were deposited at the ARS culture Collection as follows:

culture of microorganisms	Strain name	Date of storage
			KJ073	B-50029	2007, 3, 15 days
KJ091	B-50110	2008, 20 days 2 month
			KJ098	B-50111	2008, 20 days 2 month
KJ104	B-50112	2008, 20 days 2 month
			KJ110	B-50113	2008, 20 days 2 month
KJ119	B-50114	2008, 20 days 2 month
			KJ122	B-50115	2008, 20 days 2 month
KJ134	B-50116	2008, 20 days 2 month

Materials and methods

Strains, media and growth conditions

Using unique combinations of gene deletions with growth-basedIn combination with the selection of (A), a novel derivative of E.coli C (ATCC 8739) was developed for succinic acid production. The strains, plasmids and primers used in this study are summarized in table 1. During strain construction, cultures were grown in modified Luria-Bertani (LB) liquid medium (10 g Difco tryptone, 5g Difco yeast extract, 5g sodium chloride per liter) (Miller, 1992) supplemented, as appropriate, with antibiotics (Jantama et al, 2008; Zhang et al, 2007) at 37 ℃. No genes, plasmids or foreign genes encoding antibiotic resistance were present in the final strains developed for succinic acid production. After construction the strains were cultured and maintained in AM1 medium (Martinez et al, 2007). With 100mM KHCO₃And glucose (as indicated) supplemented the medium. Betaine (1mM) was added when the initial glucose concentration was 5% (w/v) or higher.

Deletion of FRT marker in the adhE, ldhA and focA-pflB regions

Strategies for making continuous gene deletions and removing FRT markers from the adhE, ldhA and focA-pflB loci have been previously described (Datsenko and Wanner, 2000; Grabar et al, 2006; Jantama et al, 2008; Zhang et al, 2007). Plasmid pLOI4151 was used as the source of the cat-sacB cassette, and Red recombinase (pKD46) was used to facilitate double crossover (homologous recombination event). Chloramphenicol resistance was used to select for integration. The loss of sacB was selected using growth on sucrose. Successive deletions were constructed by this route, resulting in derivatives of KJ079 that eliminate all FRT sites. Primers and plasmids are listed in table 1.

To remove the FRT site in the Δ adhE region, for Δ adhE: : hybrid primers for FRT target regions (WMadhEA/C) were designed to contain a sequence similar to Δ adhE: : approximately 50bp homologous to the 5 'and 3' regions of the FRT site, and 20bp corresponding to the cat-sacB gene from pLOI 4151. These primers were used for PCR amplification of the cat-sacB cassette using pLOI4151 as template. The resulting PCR products were used to generate TG200 by double crossover (homologous recombination event) and selection for chloramphenicol resistance, replacing the FRT site in the Δ adhE region with the cat-sacB cassette.

The adhE gene and surrounding sequences were amplified from E.coli C using the up/downadhE primers. The PCR product containing ychE '-adhE-ychG' (3.44kb) was cloned into pCR2.1-TOPO to yield pLOI 4413. The products from the inside out were amplified using a second set of primers (IO-adhEup/down), and blunt-ended products were obtained using pLOI4413 as template and Pfu polymerase, in which the 2.6kb internal segment of the adhE sequence was deleted. The inside-out PCR product was subjected to kinase treatment and self-ligation to obtain pLOI 4419. By another double homologous recombination event and sucrose selection for sacB loss, the cat-sacB cassette in TG200 was replaced with the desired chromosomal sequence using PCR products amplified from pLOI4419 (up/downadhE primers). The resulting strain was designated TG201 (KJ 079 with FRT removed from. DELTA. adhE region)

Passage and deletion of adhE: : FRT sites in a similar manner, the FRT sites in the Δ ldhA and Δ (focA-pflB) regions were removed. The other primer sets (ldhAA/C and IO-ldhAUp/down) for removing FRT site in Δ ldhA are contained in Table 1 together with the corresponding plasmids (pLOI4430 and pLOI 4432). The strain TG202 was produced by replacing this region in TG201 with a PCR product from pLOI4151 (WMldhAA/C primer). The cat-sacB cassette in TG202 was replaced with PCR product from pLOI4432(ldhAA/C primer) and sucrose selection was performed for loss of sacB, yielding TG 203.

Primer sets (upfocA/MidpfA and IO-ycaOup/IO-midpFlaDOwn) for removing FRT sites in Δ (focA-pflB) and corresponding plasmids (pLOI4415 and pLOI4421) are included in Table 1. The strain TG204 was produced by replacing this region in TG203 with a PCR product from pLOI4151(WMpflBA/C primer). The cat-sacB cassette in TG204 was replaced with PCR product from pLOI4421(upfocA/MidpflA primer) and sucrose selection was performed for loss of sacB, yielding KJ 091. KJ091 is a derivative of KJ073 in which all FRT sites have been deleted from the chromosomal Δ adhE, Δ ldhA and Δ focA-pflB regions.

Construction of pLOI4162 containing cat-sacB cassette for marker-free Gene deletion

To facilitate the continuous deletion of chromosomal DNA, plasmid pLOI4162 (FIG. 1) with a removable cat-sacB cassette, optionally containing an 18-bp synthetic DNA segment with a stop codon in all reading frames, was constructed. This plasmid consists of synthetic sequences and portions of plasmids pLOI2228(Martinez-Morales et al 1999), pLOI2511(Underwood et al 2002) and pEL04(Lee et al 2001; Thomason et al 2005). PCR was performed from the inside out using pEL04 as a template, with JMpEL04F1/R1 primers, to eliminate the unwanted SmaI and BamHI sites between the cat and sacB genes. The amplified product was digested with BglII (within two primers) and self-ligated, yielding pLOI 4152. Plasmid pLOI4131 was constructed by ligating the FRT-cat-FRT fragment from pLOI2228 (BanI, ClaI treated with Klenow) into the compatible site of pLOI2511 (NheI, ClaI treated with Klenow). Plasmid pLOI4131 was subsequently digested with EcoRI and self-ligated to remove the FRT-cat-FRT fragment, resulting in pLOI4145 which retains a single KasI and XmaI site. A polylinker segment (SfPBXPS) was prepared by annealing complementary oligonucleotides (SfPBXPSsense and SfPBXPScomp). After digestion with KasI and XmaI, the segment was ligated into the corresponding site of pLOI4145, resulting in pLOI 4153. The modified cat-sacB cassette of pLOI4152 was amplified by PCR using the JMcAtsacBup3/down3 primer set. After digestion with BamHI and XhoI, the cassette was ligated into the corresponding site of pLOI4153, yielding pLOI 4146. To create an 18-bp region (5 'GCCTAATTAATTAATCCC 3') (SEQ ID NO: 1) containing a stop codon in all six reading frames, pLOI4146 was digested with PacI and self-ligated to generate pLOI4154 (not shown), removing the cat-sacB cassette. Two additional bases (T and A) were inserted between the SfoI and PacI sites of pLOI4154 using mutagenic primers (JM4161sense/comp) and linear plasmid amplification, resulting in pLOI 4161. Finally, the PacI-digested fragment from pLOI4146 containing the cat-sacB cassette was ligated into the PacI-digested site of pLOI4161, yielding pLOI4162(GenBank accession No. EU 531506).

Construction of tdcDE and aspC Gene deletions

The tdcDE gene and adjacent 1000bp region (tdcG '-tdcFED-tdcC', 5325bp) were amplified using tdcDEup/down primers and cloned into pCR2.1-TOPO vector, yielding plasmid pLOI 4515. This 1000-fold diluted preparation of plasmid DNA acts as a template in the inside-out amplification using tdcDEF7/R7 primer (both primers are within the tdcDE gene and facing outward). The resulting replicon-containing 6861bp fragment was ligated with the SmaI/SfoI-digested cat-sacB cassette amplified from pLOI4162 (JMcatsacBup3/down3 primer), yielding pLOI 4516. This 6861bp fragment was also used to construct a second plasmid, pLOI4517 (kinase treated, self-ligated) containing tcdD and tdcE deletions. The tdcDE region in KJ091 was replaced by a PCR fragment amplified (tdcDEup/down primer) from pLOI4516 and pLOI 4517. The resulting clones were tested for loss of ampicillin and chloramphenicol resistance and were designated KJ 098.

The aspC gene was deleted from KJ104 in a similar manner to that used for deletion of the tdcDE gene. The other primer sets used to construct the aspC deletion (aspup/down and aspC1/2) are included in table 1 along with the corresponding plasmids (pLOI4280, pLOI4281 and pLOI 4282). The resulting strain was named KJ 110. Neither KJ098 nor KJ110 contained any intervening sequences in the respective deleted regions (tdcDE and aspC).

Removal of FRT site in ackA region and construction of deletions of the citF, sfcA and pta-ackA genes

To eliminate the FRT site in the ackA region of KJ073, a plasmid containing the desired mutated sequence was constructed as follows. Coli C genomic DNA was used as a template for PCR amplification of ackA with JMackAF1/R1 primers, which bind approximately 200bp upstream and downstream of the ackA gene. The linear product was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, Calif.) yielding pLOI 4158. Then, using plasmid pLOI4158 as a template, inside-out PCR using JMackAup1/down1 primer and Pfu polymerase was performed to obtain blunt-ended products of the 808-bp internal segment lacking ackA. The cat-sacB cassette flanked by PacI (SmaI/SfoI fragment from pLOI 4162) was then ligated into the blunt-ended PCR product, resulting in pLOI 4159. Plasmid pLOI4159 served as a template for PCR amplification (JMackAF1/R1 primer). This PCR product was used in place of the FRT site in the ackA region of KJ073 by double crossover homologous recombination and selection for chloramphenicol resistance. The resulting clone was designated KJ 076.

Plasmid pLOI4159 was also digested with PacI to remove the cat-sacB cassette and self-ligated to generate pLOI4160 retaining the 18-bp translational termination sequence. Plasmid pLOI4160 served as a template for PCR (JMackAF1/R1 primer). This amplified fragment was used to replace the cat-sacB cassette in KJ076 by double crossover homologous recombination and selection for loss of sacB. After removal of pKD46 by growth at elevated temperature, the resulting strain was named KJ 079. In this strain, the deleted region was replaced by an 18-bp translational termination sequence.

The strategy used above to remove the FRT site from the ackA region was used to make a continuous deletion of citF, sfcA or pta-ackA and to replace the deleted region with an 18-bp translational termination sequence. The other primer sets used to construct the citF deletion (citFup/down and citF2/3) are included in Table 1 along with the corresponding plasmids (pLOI4629, pLOI4630 and pLOI 4631). The resulting strain was named KJ 104.

The sfcA gene was deleted from the strains KJ104 and KJ110 to give strains designated KJ119 and KJ122, respectively. The other primer sets used to construct the sfcA deletion (sfcAup/down and sfcA1/2) are included in Table 1 along with the corresponding plasmids (pLOI4283, pLOI4284 and pLOI 4285).

The ackA-pta operon (including synthetic translational termination sequences) was deleted from KJ122, yielding strain KJ 134. The other primer sets used to construct this deletion (ackAup/ptabrown and ackA2/pta2) are included in Table 1 along with the corresponding plasmids (pLOI4710, pLOI4711, and pLOI 4712). Strain KJ134 does not contain any FRT sites or foreign genes.

Fermentation of

In a medium containing 10% (w/v) glucose (555mM), 100mM KHCO₃The inoculum cultures and fermentations were incubated at 37 ℃ (100rpm) in AM1 mineral salt medium (Martinez et al, 2007) with 1mM betaine HCl. Addition of 3M K₂CO₃And 6N KOH and provides CO₂. Differences in the alkali composition (1: 1, 4: 1, 6: 1 mixtures) had little effect on the fermentation. The fermentation was carried out in a small fermentor with a working volume of 350 ml. Unless otherwise stated, at 0.01(3.3mg CDW l)^-1) Initial OD of₅₅₀And (5) inoculating and fermenting. Except for 16 meters serving as air vents and sampling portsThe fermenter was sealed except for the measuring needle. Anaerobic life is rapidly achieved during growth. The added bicarbonate exerts an effect to ensure atmospheric CO₂The function of (1).

Analysis of

By using Bausch&Lomb Spectronic 70 Spectrophotometer, the amount of cells was evaluated from the optical density at 550nm (OD 1.0: 333mg dry cell weight l)^-1). Organic acids and sugars were determined by using high performance liquid chromatography (Grabar et al, 2006).

Results and discussion

Construction of marker-free strains for succinic acid production

The central anaerobic fermentation gene in E.coli C wild-type was deleted consecutively with PCR products and removable antibiotic markers (by using FRT recognition sites and FLP recombinase) by the strategy of Datsenko & Wanner (2000). These constructs were used in combination with metabolic evolution (increased ATP production efficiency based on growth selection) to select mutant strains that restored phosphoenolpyruvate carboxykinase (pck) with conserved energy to increase growth and succinate production (fig. 9). The resulting strain KJ073 metabolizes to produce 1.2 moles succinate per mole glucose (Jantama et al, 2008) and now uses a succinate pathway very similar to the rumen bacteria Actinomyces succinogenes (van der Werf et al, 1997) and Mannheimia succiniciproducens (Song et al, 2007). However, the method used to construct these gene deletions left a single 82 to 85-nt genetic scar or FRT site in each region of the deleted gene (ackA, ldhA, adhE, ackA, focA-pflB). These FRT sites function as recognition sites for FLP recombinase during the removal of the antibiotic gene (Storici et al, 1999). All of these foreign sequences were removed sequentially from KJ073 using the previously described method and replaced with native DNA containing only the desired gene deletion (Grabar et al, 2006; Zhang et al, 2007; Jantama et al, 2008). The resulting strain KJ091 contains specific deletions in ackA, ldhA, adhE, focA-pflB, ackA, mgsA and poxB, and lacks all FRT sites. This strain is devoid of all foreign and synthetic DNA, except for the 18-bp translational termination sequence in ackA. Succinate production by strain KJ091 was equal to KJ073 (table 7). This strain was used as a parent for further improvement of succinic acid production.

Reduction of acetic acid during succinic acid production by deletion of tdcD and tdcE

During the anaerobic fermentation of glucose by E.coli, pyruvate formate lyase (pflB) acts as the major source of acetyl-CoA (acetyl-P precursor) and acetate kinase (ackA) acts as the major pathway for the production of acetate from acetyl-P (Karp et al, 2007; Kessler & Knappe, 1996). The acetic acid abundance as fermentation product in strains KJ073 and KJ091 was surprising, since these strains contained deletions in both ackA and pflB (fig. 9). This residual acetic acid at the end of the fermentation represents an opportunity to further alter metabolism for improved succinic acid production.

An enzyme associated with acetate kinase (and propionate kinase) activity is encoded by tdcD and is generally only produced for degradation of threonine (Hesslinger et al, 1998; Reed et al, 2003.) mutations that occur during possible selection increase the expression of tdcD as shown in FIG. 10. during anaerobic growth using 10% (w/v) glucose, the expression of tdcD can functionally replace ackA, increasing acetyl-P to acetate production. adjacent tdcE genes in the same operon are similar to pflB and encode pyruvate (and α -ketobutyrate) formate lyase activity that is co-expressed during threonine degradation (Hesslinger et al, 1998). during anaerobic growth, possibly using 10% (w/v) glucose, the increased expression of the genes can increase acetyl-CoA (direct precursor of acetyl-P) and waste products such as formate (FIG. 10) during anaerobic growth, the simultaneous deletion from tdKJ td and cE (adjacent) leads to an increase in pyruvate production, the two alternative pyruvate production pathways-the metabolic pathways of which are also predicted to decrease, the two alternative pyruvate production pathways for the deletion of pyruvate kinase (KcJ 098) and elimination of pyruvate kinase, the increased pyruvate production of the two alternative pyruvate production pathways-098. the increased pyruvate production of direct precursor and elimination of pyruvate-098-the two alternative pathways are also predicted to be eliminated during simultaneous deletion of the metabolic pathways-the two alternative pyruvate production of pyruvate-pyruvate production pathway.

Effect of citrate lyase (citDEF) deletion on acetic acid yield during succinate production

Under anaerobic conditions oxaloacetate is partitioned into reduced product (malate) and oxidized intermediate (citrate) (fig. 9) citrate can be converted back into oxaloacetate and acetate by citrate lyase (citDEF), thereby recycling the intracellular OAA pool for another metabolic function (Nilekani et al, 1983) the bulk expression of citrate lyase is associated with growth on citrate (lutgenes and Gottschalk, 1980; Kulla and Gottschalk, 1977.) the citrate lyase is a multienzyme complex composed of three different polypeptide chains α subunit or macrosubunit is a citrate-ACP transferase subunit β subunit catalyzing the first step, β subunit is a citrate-ACP transferase subunit catalyzing the second step, γ subunit of the citrate-ACP transferase subunit or small subunit, and the subunit is a citrate-coa transferase subunit catalyzing the second step, which is a promoter of the first step, which is a promoter of the production of succinate, which is responsible for the loss of acetate or succinate produced by the acetate lyase, which is responsible for the loss of acetate production of acetate, and the loss of succinate production of acetate, which results in a loss of the acetate production of the acetate by the acetate lyase (kotj 098) and the pyruvate lyase, which results in the loss of the production of acetate by the enzyme, whereas the deletion of the acetate synthase promoter, which is not due to the loss of acetate production of acetate, the acetate production of the loss of the acetate, the acetate production of the acetate, which is not the acetate production of the acetate, the acetate production of acetate, which is not the acetate production of the acetate, which is not the acetate, which is due to the release of the acetate, the release of the acetate, the release of the acetate, which was not the release of the acetate, which was not observed gene, which was.

Effect of aspC and sfcA deletions on succinic acid yield

Aspartate aminotransferase (aspC) is a multifunctional enzyme that catalyzes the synthesis of aspartate, phenylalanine, and other compounds by an transamination reaction. L-aspartic acid is synthesized from oxaloacetate, an intermediate product of PEP carboxylation, by transamination reaction with L-glutamic acid in this reaction. Aspartic acid is a component of proteins and is involved in several other biosynthetic pathways. It is estimated that about 27% of the cell nitrogen flows through aspartate (Reitzer, 2004). Aspartate biosynthesis and succinate production share a common intracellular pool of oxaloacetate. Deletion of aspC results in increased succinate production, but can also create an auxotrophic need to prevent anaerobic growth on minimal salt media such as AM 1.

The aspartate aminotransferase gene (aspC) was deleted from KJ104, yielding KJ 110. Unexpectedly, the deletion of aspC in KJ110 had no effect on succinate yield or cell yield compared to KJ104 (table 7). Thus, in our strains, aspartase does not appear to shift significant levels of oxaloacetate conversion away from succinate production. It appears that alternative enzymes are available which replace the biosynthetic requirements previously catalyzed by aspartate aminotransferases.

At the end of the fermentation using KJ104 and other E.coli strains engineered for succinate production, a large amount of pyruvate was present (Table 7). This pyruvate represents an unwanted product and a further opportunity to increase the yield of succinic acid. This high level of pyruvate in the fermentation broth may result from the decarboxylation of malate to pyruvate by malic enzyme (sfcA), as illustrated in fig. 10. The enzyme is thought to act primarily during gluconeogenesis rather than during anaerobic metabolism of glucose (Unden and Kleefeld, 2004; Stols and Donnelly, 1997; Oh et al, 2002). Although reductive carboxylation of pyruvate to form malate is thermodynamically favored, the kinetic parameters of the enzyme favor dehydrogenation and decarboxylation under physiological conditions (Stols and Donnelly, 1997). Overexpression of this enzyme to carboxylate pyruvate has previously been used as a basis for the construction of E.coli strains for succinate production (Stols and Donnelly 1997).

If malic enzyme (sfcA) carboxylates in KJ104 (and related strains) and contributes to succinate production, deletion of this gene is expected to reduce succinate yield and increase the levels of other products such as pyruvate. Alternatively, if malic enzyme (scfA) decarboxylates in KJ104 and turns malate to pyruvate, deletion of the gene encoding the enzyme would be expected to increase succinate yield and decrease pyruvate levels. Unexpectedly, deletion of sfcA gene from KJ104 to produce KJ119 had no measurable effect on succinic acid production, growth, pyruvate levels, etc., compared to KJ104 (table 7). These results clearly demonstrate that malic enzyme (sfcA) is not important for succinic acid production in KJ104 and related strains. This result is in direct contrast to the succinic acid-producing strains developed by Stols et al, (1997) in which increased malic enzyme production was used as the primary pathway for succinic acid production.

Although no significant benefit was observed from either sfcA deletion or aspC deletion in KJ104, studies were conducted to test the effect of deleting both genes in combination. This was achieved by deleting the sfcA gene in KJ110 to produce KJ122, and no benefit was expected. However, the combined deletion of both sfcA and aspC (strain KJ122) resulted in an unexpected increase in succinic acid yield and titer, and a small decrease in acetic acid, compared to the parent strain KJ110 and related strains (KJ104 and KJ119) (table 7). The combined deletions (aspC and sfcA) in KJ122 resulted in significant increases in succinic acid yield, succinic acid titer and average productivity of 18%, 24% and 24%, respectively, compared to KJ 104. Although the mechanism is unknown, it is possible that single mutations are ineffective because they are partially compensated by increased flux through retention activity, malic enzyme or aspartate aminotransferase (fig. 10), buffering any possible benefit. It is speculated that the increase in succinate yield and titer is caused by an increase in oxaloacetate availability, allowing a larger fraction to proceed to succinate. The malic acid level also remained very low.

Strain KJ122 (table 7) produced 1.5 moles of succinic acid per mole of glucose, which is 88% of the maximum theoretical yield (1.71 moles per mole of glucose). To produce this high level of succinic acid and to completely reduce malic acid, additional reducing agents are required. Although the source of this additional reducing agent is unknown, these results are consistent with an increase in pyruvate flow through pyruvate dehydrogenase. The enzyme is thought to act primarily during aerobic metabolism (Guest et al, 1989), but has also been reported to act at low levels during fermentation (de Graef et al, 1999).

Reduction of pyruvate and acetate by deletion of pta

KJ122 gave excellent succinic acid yields (1.5 mol)^-1Glucose) plus smaller amounts of acetic acid and pyruvic acid. The maximum theoretical yield of succinic acid is 1.71mol^-1Glucose, and these 3-carbon intermediates represent an opportunity to further improve yields. Pyruvate is presumed to accumulate from glycolysis as a metabolic overflow and may be associated with acetate accumulation. acetyl-CoA is an isomeric regulator of many enzymes. The source of acetate and acetate kinase activity was unknown because the genes encoding the two major activities of acetate kinase (tdcD and ackA) had been deleted (fig. 9 and 10). Assuming that acetate is produced from acetyl-P, the product of phosphotransacetylase, a further deletion was constructed in KJ122 to inactivate the pta gene. The resulting strain, KJ134, produced succinic acid at levels close to theoretical levels (table 7). In this strain, pyruvate and acetate levels were significantly reduced. Volumetric productivity was also reduced by 17%. The succinic acid yield of strain KJ134 was equal to or better than all other strains, regardless of the complexity of the fermentation process, the medium or the growth conditions.

^aAbbreviations: CSL, corn steep liquor; YE, yeast extract; NR, not reported.

^bThe average volumetric productivity is shown in parentheses at the succinic acid titer [ g l^-1h^-1]。

^cMolar yields were calculated based on succinic acid produced from metabolized sugars under aerobic and anaerobic conditions. Biomass is produced primarily during aerobic growth. Succinic acid mainly uses CO₂、H₂Or a mixture of both during anaerobic incubation.

^aData from van der Werf et al, 1997

^bIt cannot be measured in wild type E.coli C due to the presence of lactate dehydrogenase.

^aNBS +1mM betaine: NBS medium modified with betaine (1 mM).

^bThe KOH included for neutralization of the betaine-HCl stock solution was calculated.

^cTrace metal stock solutions (1000X) were prepared in 120mM HCl.

Reference to the literature

U.S.Patent No.5,723,322

U.S.Patent No.5,869,301

U.S.Patent No.5,143,834,Glassner et al.,1992

U.S.Patent No.5,723,322,Guettler et al.,1998

U.S.Patent No.5,573,931,Guettler et al.,1996a

U.S.Patent No.5,505,004,Guettler et al.,1996b

Ajl,S.J.,Werkman,C.H.(1948)“Enzymatic fixation of carbon dioxide in-ketoglutaric acid”Proc.Natl.Acad.Sci.USA 34:491-498.

Andersson,C.,Hodge,D.,Berglund,K.A.,Rova,U.(2007)“Effect of differentcarbon sources on the production of succinic acid using metabolicallyengineered Escherichia coli.”Biotechnol Prog 23(2):381-388.

Asghari,A.,Bothast,R.J.,Doran,J.B.,Ingram,L.O.(1996)“Ethanolproduction from hemicellulose hydrolysates of agricultural residues usinggenetically engineered Escherichia coli strain KO11”J.IndustrialMicrobiol.16:42-47.

Causey,T.B.,Shanmugam,K.T.,Yomano,L.P,Ingram,L.O.(2004)“EngineeringEscherichia coli for efficient conversion of glucose to pyruvate”Proc.Natl.Acad.Sci.USA 101:2235-2240.

Chao,Y and Liao,J.C.(1993)“Alteration of growth yield byoverexpression of phosphoenol pyruvate carboxylase and phosphoenolpyruvatecarboxykinase in Escherichia coli”Appl Environ Microbiol.59:4261-4265.

Chang,Y.Y.,Cronan,J.E.,Jr.(2000)“Conversion of Escherichia colipyruvate decarboxylase to an‘alpha-ketobutyrate oxidase’”Biochem.J.352:717-724.

Chatterjee,R.,Cynthia,S.M.,Kathleen,C.,David,P.C.,Donnelly,M.I.(2001)“Mutation of the ptsG gene results in increased production of succinate infermentation of glucose by Escherichia coli.”Appl.Environ.Microbiol.67:148-154.

Cox,S.J.,Levanon,S.S.,Sanchez,A.M.,Lin,H.,Peercy,B.,Bennett,G.N.,San,K.Y.(2006)“Development of a metabolic network design and optimizationframework incorporating implement constraints:A succinate production casestudy”Metab.Engin.8:46-57.

Datsenko,K.A.,Wanner,B.L.(2000)“One-step inactivation of chromosomalgenes in Escherichia coli K-12using PCR products”Proc.Natl.Acad.Sci.USA 97:6640-6645.

de Graef,M.R.,Alexeeva,S.,Snoep,J.L.,de Mattos,M.J.T.(1999)“Thesteady-state internal redox state(NADH/NAD)reflects the external redox stateand is correlated wit catabolic adaptation in Escherichia coli.”J.Bacteriol.181:2351-235.

Delbaere,L.T.J.,Sudom,A.M.,Prasad,L.,Leduc,Y.,Goldie,H.(2004)“Structure/function studies of phosphoryl transfer by phosphoenolpyruvatecarboxykinase”Biochimica et Biophysica Acta.1679:271-278.

Du,C.,Lin,S.K.,Koutinas,A.,Wang,R.,Webb,C.(2007)“Succinic acidproduction from wheat using a biorefining strategy”Appl MicrobiolBiotechnol.76(6):1263-1270.

Egyud,L.G.,Szent-Gyorgyi,A.(1966)“On the regulation of cell division”Proc.Natl.Acad.Sci.USA 56:203-207.

Farmer,W.and Liao,J.C.(1997)“Reduction of aerobic acetate productionby Escherichia coli”Appl.Environ.Microbiol.63:3205-3210.

Fraenkel,D.G.(1996)Chapter 14,Glycolysis.In A.R.Curtiss III,J.B.Kaper,F.C.Neidhardt,T.K.E.Rudd,and C.L.Squires(ed.),EcoSal—Escherichia coli and Salmonella:cellular and molecular biology.[Online.]http://www.ecosal.org.ASM Press,Washington,D.C.

Gokarn,R.R.,Eiteman,M.A.,Altman,E.(2000)“Metabolic analysis ofEscherichia coli in the presence and absence of carboxylating enzymesphosphoenolpyruvate carboxylase and pyruvate carboxylase”Appl.Environ.Microbiol.66:1844-1850.

Goldie,A.H.and Sanwal,B.D.(1980a)“Genetic and physiologicalcharacterization of Escherichia coli mutants deficient in phosphoenolpyruvatecarboxykinase activity”J Bacteriol.141(3):1115-1121.

Goldie,A.H.and Sanwal,B.D.(1980b)“Allosteric control by calcium andmechanism of desensitization of phosphoenolpyruvate carboxykinase ofEscherichia coli.”J Biol Chem.255(4):1399-1405.

Gottschalk,G.(1985)Bacterial metabolism.2nd ed.Springer-Verlag,NewYork.

Grabar,T.B.,Zhou,S.,Shanmugam,K.T.,Yomano,L.P.,Ingram,L.O.(2006)“Methylglyoxal bypass identified as source of chiral contamination in L(+)andD(-)lactate fermentations by recombinant Escherichia coli.”Biotechnol.Lett.28:1527-1535.

Guest,J.R.,Angier,S.J.,Russell(1989)“Structure,expression,and proteinengineering in the pyruvate dehydrogenase complex of Escherichia coli.”Ann.N.Y.Acad.Sci.573:76-99.

Hesslinger,C.,Fairhurst,S.A.,Sawers,G.(1998)“Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway inEscherichia coli that degrades L threonine to propionate”Mol Microbiol 27(2):477-492.

Hopper,D.J.,Cooper,R.A.(1971)“The regulation of Escherichia colimethylglyoxal synthase:a new control site in glycolysis”FEBS Lett 13:213-216.

Iverson,T.M.,Luna-Chavez,C.,Croal,L.R.,Cecchini,G.,Rees,D.C.(2002)“Crystallographic studies of the Escherichia coli quinol-fumarate reductasewith inhibitors bound to the quinol-binding site.2002”J.Biol.Chem.277:16124-16130.

Jantama,K.,Haupt,M.J.,Svoronos,S.A.,Zhang,X.,Moore,J.C.,Shanmugam,K.T.,Ingram,L.O.(2008)“Combining metabolic engineering and metabolicevolution to develop nonrecombinant strains of E.coli C that producesuccinate and malate”Biotech.Bioeng.99(5):1140-1153.

Jarboe,L.R.,Hyduke,D.R.,Tran,L.M.,Chou,K.J.,Liao,J.C.(2008)“Determination of the Escherichia coli S-nitrosoglutathione response networkusing integrated biochemical and systems analysis”J Biol Chem.283:5148-5157.

Kao,K.C.,Tran,L.M.,Liao,J.C.(2005)“A global regulatory role ofgluconeogenic genes in Escherichia coli revealed by transcriptome networkanalysis”J Biol Chem 280:36079-36087.

Karp,P.D.,Keseler,I.M.,Shearer,A.,Latendresse,M.,Krummenacker,M.,Paley,S.M.,Paulsen,I.T.,Collado-Vides,J.,Gamma-Castro,S.,Peralta-Gil,M.,Santos-Zavaleta,A.,Penaloza-Spinola,M.,Bonavides-Martinez,C.,Ingraham,J.(2007)“Multidimensional annotation of Escherichia coli K-12genome”Nucl AcidsRes.35(22):7577-7590.

Keseler,I.M.,Collado-Vides,J.,Gamma-Castro,S.,Ingraham,J.,Paley,S.,Paulsen,I.T.,Peralta-Gil,M.,Karp,P.D.(2005)“Ecocyc:A comprehensive databaseresource for Escherichia coli”Nucl Acids Res 33:D334-D337.

Kessler,D.,and Knappe,J.(1996)Anaerobic dissimilation of pyruvate.InNeidhardt FC,Curtiss III R,Ingraham JL,Lin ECC,Low KB,Magasanik B,ReznikoffWS,Riley M,Schaechter M,Umbarger HE,editors.Escherichia coli and Salmonella:cellular and molecular biology.ASM Press,Washington,D.C.p.199-205.

Kim,P.,Laivebieks,M.,Vieille,C.,Zeikus,J.G.(2004)“Effect ofoverexpression of Actinobacillus succinogenes phosphoenolpyruvatecarboxykinase on succinate production in Escherichia coli”Appl EnvironMicrobiol.70(2):1238-41.

Krebs,A.,Bridger,W.A.(1980)“The kinetic properties ofphosphoenolpyruvate carboxykinase of Escherichia coli”Can J Biochem 58(4):309-318.

Kulla,H.,and Gottschalk,G.(1977)“Energy-dependent inactivation ofcitrate lyase in Enterobacter aerogenes”J.Bacteriol.132(3):764-770.

Laivenieks,M.,Vieille,C.,Zeikus,J.G.(1997)“Cloning,sequencing,andoverexpression of the Anaerobiospirillum succiniciproducensphosphoenolpyruvate carboxykinase(pckA)gene”Appl Environ Microbiol 63(6):2273-2280.

Lee,E-C.,Yu,K.,Martinez de Velasco,J.,Tessarollo,L.,Swing,D.A.,Court,D.L.,Jenkins,N.A.,and Copeland,N.G.(2001)“A highly efficient Escherichiacoli-based chromosome engineering system adapted for recombinogenic targetingand subcloning of BAC DNA”Genomics 73:56-65.

Lee,S.Y.,Hong,S.H.,Lee,S.H.,Park,S.J.(2004)“Fermentative productionof chemicals that can be used for polymer synthesis”Macromol.Biosci.4:157-164.

Lee,S.J,Lee,D.Y.,Kim,T.Y.,Kim,B.H.,Lee,J.,Lee,S.Y.(2005)“Metabolicengineering of Escherichia coli for enhanced production of succinic acid,based on genome comparison and in silico gene knockout simulation”ApplEnviron Microbiol 71:7880-7887.

Lee,S.J.,Song,H.,Lee,S.Y.(2006)“Genome-based metabolic engineering ofMannheimia succiniciproducens for succic acid production”Appl EnvironMicrobiol 72(3):1939 1948.

Lin,H.,Bennett,G.N.,San,K.Y.(2005a)“Chemostat culturecharacterization of Escherichia coli mutant strains metabolically engineeredfor aerobic succinate production:A study of the modified metabolic networkbased on metabolite profile,enzyme activity,and gene expression profile”Metab.Engin.7:337-352.

Lin,H.,Bennett,G.N.,San,K.Y.(2005b)“Metabolic engineering of aerobicsuccinate production systems in Escherichia coli to improve processproductivity and achieve the maximum theoretical succinate yield”Metab.Engin.7:116-127.

Lin,H.,Bennett,G.N.,San,K.Y.(2005c)“Effect of carbon sourcesdiffering in oxidation state and transport route on succinate production inmetabolically engineered Escherichia coli”J.Ind.Microbiol.Biotechnol.32:87-93.

Lin,H.,Bennett,G.N.,San,K.Y.(2005d)“Fed-batch culture of ametabolically engineered Escherichia coli strain designed for high-levelsuccinate production and yield under aerobic conditions”Biotechnol Bioeng 90:775-779.

Lutgens,M.,and Gottschalk,G.(1980)“Why a co-substrate is required forthe anaerobic growth of Escherichia coli on citrate”J.Gen Microbiol.119:63-70.

Martinez,A.,Grabar,T.B.,Shanmugam,K.T.,Yomano,L.P.,York,S.W.,Ingram,L.O.(2007)“Low salt medium for lactate and ethanol production by recombinantEscherichia coli”Biotechnol.Lett.29:397-404.

Martinez-Morales,F.,Borges,A.C.,Martinez,A.,Shanmugam,K.T.,Ingram,L.O.(1999)“Chromosomal integration of heterologous DNA in Escherichia coliwith precise removal of markers and replicons used during construction”JBacteriol.181:7143-7148.

McKinlay,J.B.,Zeikus,J.G.,Vieille,C.(2005)“Insights intoActinobacillus succinogenes fermentative metabolism in a chemically definedgrowth medium”Appl Environ Microbiol.71(11):6651-6656.

McKinlay,J.B.,Vieille,C.(2008)“¹³C-metabolic flux analysis ofActinobacillus succinogenes fermentative metabolism at different NaHCO₃andH₂concentrations”Metab.Eng.10:55-68.

McKinlay,J.B.,Vieille,C.,Zeikus,J.G.(2007)“Prospects for a bio-basedsuccinate industry”Appl Microbiol Biotechnol.76(4):727-740.

Meynial-Salles,I.,Dorotyn,S.,Soucaille,P.(2007)“A new process for thecontinuous production of succinic acid from glucose at high yield,titer andproductivity”Biotechnol Bioeng.99(1):129-135.

Miller,J.H.(1992)A short course in bacterial genetics:A laboratorymanual and handbook for Escherichia coli and related bacteria,Cold SpringHarbor Press.

Millard,C.S.,Chao,Y.P.,Liao,J.C.,Donnelly,M.I.(1996)“Enhancedproduction of succinic acid by overexpression of phosphoenolpyruvatecarboxylase in Escherichia coli”Appl.Environ.Microbiol.62:1808-1810.

Morikawa,M.,K.Izui,et al.(1980)“Regulation of Escherichia coliphosphoenolpyruvate carboxylase by multiple effectors in vivo.Estimation ofthe activities in the cells grown on various compounds”J Biochem (Tokyo)87(2):441-449.

Moniruzzaman,M.et al.(1997)“Isolation and molecular characterizationof high-performance cellobiose-fermenting spontaneous mutants ofethanologenic Escherichia coli K011containing the Klebsiella oxytoca casABoperon”Appl.Environ.Microbiol.63:4633-4637.

Nilekani,S.,Sivaraman,C.(1983)“Purification and properties of citratelyase from Escherichia coli”Biochemistry 22(20)；4657-63.

Oh,M.K.,Rohlin,L.,Kao,K.C.,Liao,J.C.(2002)“Global expressionprofiling of acetate-grown Escherichia coli”J Biol Chem 277:13175-13183.

Okino,S.,Inui,M.,Yukawa,H.(2005)“Production of organic acids byCorynebacterium glutanicum under oxygen deprivation”Appl Microbiol Biotechnol68:475-480.

Posfai,G.,Koob,M.D.,Kirkpatrick,H.A.,Blattner,F.C.(1997)“Versatileinsertion plasmids for targeted genome manipulations in bacteria:Isolation,deletion,and rescue of the pathogenicity island LEE of the Escherichia coliO157:H7genome”J.Bacteriol.179:4426-4428.

Quentmeier,A.,Holzenburg,A.,Mayer,F.,Antranikian,G.(1987)“Reevaluation of citrate lyase from Escherichia coli”Biochim Biophys Acta 913(1):60-5.

Reed,J.L.,Vo,T.D.,Schilling,C.H.,Palsson,B.O.(2003)“An expandedgenome-scale model of Escherichia coli K-12(iJR904GSM/GPR)”Genome Biol.4(9):R54.

Reitzer,L.2004.6July 2004,posting date.Chapter 3.6.1.3,Biosynthesisof Glutamate,Aspartate,Asparagine,L-Alanine,and D-Alanine.In A.R.Curtiss III,J.B.Kaper,F.C.Neidhardt,T.K.E.Rudd,and C.L.Squires(ed.),EcoSal—Escherichia coli and Salmonella:cellular and molecular biology.[Online.]http://www.ecosal.org.ASM Press,Washington,D.C.

Samuelov,N.S.,Lamed,R.,Lowe,S.,Zeikus,J.G.(1991)“Influence of CO₂-HCO₃levels and pH on growth,succinate production,and enzyme-activities ofAnaerobiospirillum succiniproducens”Appl.Environ.Microbiol.57:3013-3019.

Sanchez,A.M.,Bennett,G.N.,San,K.Y.(2005a)“Novel pathway engineeringdesign of the anaerobic central metabolic pathway in Escherichia coli toincrease succinate yield and productivity”Metabolic Engineering 7:229-239.

Sanchez,A.M.,Bennett,G.N.,San,K.Y.(2005b)“Efficient succinic acidproduction from glucose through overexpression of pyruvate carboxylase in anEscherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant”Biotechnol.Prog.21:358-365.

Sanchez,A.M.,Bennett,G.N.,San,K.Y.(2006)“Batch culturecharacterization and metabolic flux analysis of succinate-producingEscherichia coli strains”Metabolic Engineering 8:209-226.

Sanwal,B.D.and Smando,R.(1969a)“Malic enzyme of Escherichia coli:Possible mechanism for allosteric effects”J Biol Chem.244(7):1824-1830.

Sanwal,B.D.and Smando,R.(1969b)“Malic enzyme of Escherichia coli:Diversity of the effectors controlling enzyme activity”J Biol Chem.244(7):1817-1823.

Sanwal,B.D.(1970a)“Allosteric controls of amphibolic pathways inbacteria”Bacteriol.Rev.34:20-39.

Sanwal,B.D.(1970b)“Regulatory characteristics of thediphosphopyridine nucleotide-specific malic enzyme of Escherichia coli”J BiolChem.245(5):1212-1216.

Sanwal,B.D.(1970c)“Regulatory mechanisms involving nicotinamideadenine nucleotide as allosteric effectors:A control of glucose 6-phosphatedehydrogenase”J Biol Chem 245(7):1625-1631.

Sawers,G.,Bock,A.(1988)“Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12”J.Bacteriology.170:5330-5336.

Song,H.,Lee,J.W.,Choi,S.,You,J.K.,Hong,W.H.,Lee,S.Y.(2007)“Effects ofdissolved CO2levels on the growth of Mannheimia succiniciproducens andsuccinic acid production”Biotechnol Bioeng.98(6):1296-1304.

Storici,F.,Coglievina,M.,Bruschi,C.V.(1999)“A 2-μm DNA-based makerrecycling system for multiple gene disruption in the yeast Saccharomycescerevisiae”Yeast 15:271-283.

Stols,L.,Donnelly,M.I.(1997)“Production of succinic acid throughoverexpression of NAD dependent malic enzyme in an Escherichia coli mutant”Appl.Environ.Microbiol.63:2695-2701.

Thomason,L.,Court,D.L.,Bubunenko,M.,Constantino,N.,Wilson,H.,Datta,S.,Oppenheim,A.(2005)“Recombineering:Genetic Engineering in Bacteria UsingHomologous Recombination”,sections 1.16.1-1.16.21.In F.M.Ausubel,R.Brent,R.E.Klingston,D.D.Moore,J.G.Deidman,J.A.Smith,and K.Struhl(Eds.),CurrentProtocols in Molecular Biology.John Wiley&Sons Inc.,New York.

Underwood,S.A.,Buszko,M.L.,Shanmugam,K.T.,Ingram,L.O.(2004)“Lack ofprotective osmolytes limits final cell density and volumetric productivity ofethanologenic Escherichia coli KO11during xylose fermentation”Appl.Environ.Microbiol.70(5):2734-40.

Underwood,S.A.,Buszko,M.L.,Shanmugam,K.T.,Ingram,L.O.(2002)“Geneticchanges to optimize carbon partitioning between ethanol and biosynthesis inethanologenic Escherichia coli”Appl Environ Microbiol.68(12):6263-6272.

Unden,G.and Kleefeld,A.30July 2004,posting date.Chapter 3.4.5,C4-Dicarboxylate Degradation in Aerobic and Anaerobic Growth.In A.R.Curtiss III,J.B.Kaper,F.C.Neidhardt,T.K.E.Rudd,and C.L.Squires(ed.),EcoSal—Escherichia coli and Salmonella:cellular and molecular biology.[Online.]http://www.ecosal.org.ASM Press,Washington,D.C.

van der Werf,M.J.,M.V.Guettler,M.K.Jain,and J.G.Zeikus(1997)“Environmental and physiological factors affecting the succinate productratio during carbohydrate fermentation by Actinobacillus sp.130Z”Arch.Microbiol.167:332-342.

Vemuri,G.N.,Eiteman,M.A.,Altman,E.(2002a)“Effects of growth mode andpyruvate carboxylase on succinic acid production by metabolically engineeredstrains of Escherichia coli.”Appl.Environ.Microbiol.68:1715-1727.

Vemuri,G.N.,Eiteman,M.A.,Altman,E.(2002b)“Succinate production indual-phase Escherichia coli fermentations depends on the time of transitionfrom aerobic to anaerobic conditions”J.Ind.Microbiol.Biotechnol.28,325-332.

Wang,Q.,Chen,X.,Yang,Y.,Zhao,X.(2006)“Genome-scale in silico aidedmetabolic analysis and flux comparisons of Escherichia coli to improvesuccinate production”Appl Microbiol Biotechnol 73:887-894.

Wendisch,V.F.,Bott,M.,Eikmanns,B.J.(2006)“Metabolic engineering ofEscherichia coli and Corynebacterium glutamicum for biotechnologicalproduction of organic acids and amino acids”Curr Opin Microbiol 9:1-7.

Wood,B.E.,Yomano,L.P.,York,S.W.,Ingram,L.O.(2005)“Development ofindustrial medium required elimination of the 2,3-butanediol fermentationpathway to maintain ethanol yield in an ethanologenic strain of Klebsiellaoxytoca”Biotechnol.Prog.21:1366-1372.

Wright,J.A.and Sanwal,B.D.(1969)“Regulatory mechanisms involvingnicotinamide adenine nucleotide as allosteric effectors:Control ofphosphoenolpyruvate carboxykinase”J Biol Chem.244(7):1838-1845.

Yun,N.R.,San,K.Y.,Bennett,G.N.(2005)“Enhancement of lactate andsuccinate formation in adhE or pta-ackA mutants of NADH dehydrogenase-deficient Escherichia coli”J.Appl.Microbiol.99:1404-1412.

Zhang,X.,Jantama,K.,Moore,J.C.,Shanmugam,K.T.,Ingram,L.O.(2007)“Production of L-alanine by metabolically engineered Escherichia coli”Appl.Microbiol.Biotechnol.77:355-366.

Zhou,S.,Shanmugam,K.T.,Ingram,L.O.(2003)“Functional replacement ofthe Escherichia coli D-(-)-lactate dehydrogenase gene(ldhA)with the L-(+)-lactate dehydrogenase gene(ldhL)from Pediococcus acidilactici”Appl.Environ.Microbiol.69:2237-2244.

Zhou,S.,Grabar,T.B.,Shanmugam,K.T.,Ingram,L.O.(2006)“Betaine tripledthe volumetric productivity of D-(-)-lactate by Escherichia coli strainSZ132in mineral salts medium”Biotechnol.Lett.28:671-676.

Claims

1. A genetically modified escherichia coli strain, wherein said strain comprises genetic modifications to a target gene encoding: a) acetate kinase ackA, b) ldhA of lactate dehydrogenase, c) alcohol dehydrogenase adhE, d) pyruvate-formate lyase pflB, e) formate transporter focA, f) methylglyoxal synthase mgsA, said genetic modification inactivating the enzymatic activity of the polypeptide encoded by said target gene, and said escherichia coli strain overexpressing phosphoenolpyruvate carboxykinase pck, wherein said escherichia coli strain produces at least 200mM succinate.

2. The genetically modified escherichia coli strain of claim 1, wherein the escherichia coli strain further comprises a genetic modification to a target gene encoding pyruvate oxidase and the genetic modification inactivates the enzymatic activity of a polypeptide encoded by the gene.

3. The genetically modified escherichia coli strain of claim 1, wherein the escherichia coli strain further comprises a genetic modification to a target gene encoding a) pyruvate oxidase, b) propionate kinase, and c) α -ketobutyrate formate lyase, and the genetic modification inactivates an enzymatic activity of a polypeptide of the target gene.

4. The genetically modified Escherichia coli strain of claim 1, wherein said Escherichia coli strain further comprises a genetic modification to a target gene encoding a) pyruvate oxidase, b) propionate kinase, c) α -ketobutyrate formate lyase, and d) citrate lyase, and said genetic modification inactivates an enzymatic activity of a polypeptide of said target gene.

5. The genetically modified Escherichia coli strain of claim 1, wherein said Escherichia coli strain further comprises a genetic modification to a target gene encoding a) pyruvate oxidase, b) propionate kinase, c) α -ketobutyrate formate lyase, d) citrate lyase, e) aspartate aminotransferase, and f) malic enzyme, and said genetic modification inactivates the enzymatic activity of a polypeptide of said target gene.

6. The genetically modified Escherichia coli strain of claim 1, wherein said Escherichia coli strain further comprises a genetic modification to a target gene encoding a) pyruvate oxidase, b) aspartate transaminase, c) malic enzyme, d) propionate kinase, e) α -ketobutyrate formate lyase, f) citrate lyase, and g) phosphate acetyltransferase, and said genetic modification inactivates the enzymatic activity of a polypeptide of said target gene.

7. The genetically modified Escherichia coli strain of any one of claims 1 to 6, wherein the gene or portion thereof, or the target gene or portion thereof, is inactivated by deletion, frameshift mutation, point mutation, insertion of a stop codon, or a combination thereof.

8. The genetically modified Escherichia coli strain of any one of claims 1-6, wherein the genetically modified Escherichia coli strain has no foreign gene or fragment thereof or only native gene.

9. The genetically modified Escherichia coli strain of any one of claims 1 to 6, with the proviso that: 1) the genetically modified E.coli strain has not been inactivated by one or more of the following enzymes: a) fumarate reductase; b) an ATP synthase; c) 2-ketoglutarate dehydrogenase; d) a succinate dehydrogenase; e) a repressor of isocitrate lyase; and/or 2) the genetically modified strain does not contain plasmids encoding and/or overexpressing malate dehydrogenase, phosphoenolpyruvate carboxylase, pyruvate carboxylase and/or citrate synthase.

10. The genetically modified escherichia coli strain of claim 9, wherein the plasmid is a multicopy plasmid.

11. The genetically modified Escherichia coli strain of any one of claims 1-6, wherein said genetically modified Escherichia coli strain is metabolically evolved.

12. A method for producing succinic acid, the method comprising culturing one or more genetically modified Escherichia coli strain of any one of claims 1-6 in a culture medium comprising 2% to 20% w/v of a carbohydrate that is glucose, fructose, xylose, arabinose, galactose, mannose, rhamnose, sucrose, cellobiose, hemicellulose or a combination thereof.

13. The process of claim 12, wherein the yield of succinic acid is greater than or equal to 90%.

14. The method of claim 12 or 13, wherein the culture medium comprises glycerol as a substrate for the production of succinic acid.

15. A genetically modified Escherichia coli strain according to claim 2, further comprising a genetic modification to genes encoding a propionate kinase and α -ketobutyrate formate lyase, said genetic modification inactivating the enzymatic activity of a polypeptide produced by said genes, wherein said strain is characterized by Δ mgsA, Δ poxB, Δ ackA:, translation termination sequence, Δ adhE, Δ ldhA, Δ (focA-pflB), Δ tdcDE.

16. The genetically modified escherichia coli strain of claim 15, wherein said strain further comprises a genetic modification to a gene encoding citrate lyase that inactivates the enzymatic activity of a polypeptide encoded by said gene.

17. The genetically modified escherichia coli strain of claim 16, wherein the strain further comprises a genetic modification of a gene encoding malic enzyme that inactivates the enzymatic activity of a polypeptide produced by the gene.

18. The genetically modified escherichia coli strain of claim 16, wherein said strain further comprises a genetic modification of a gene encoding aspartate aminotransferase, said genetic modification inactivating the enzymatic activity of a polypeptide produced by said gene.

19. The genetically modified escherichia coli strain of claim 18, wherein said strain further comprises a genetic modification of a gene encoding malic enzyme that inactivates the enzymatic activity of a polypeptide produced by said gene.

20. The genetically modified escherichia coli strain of claim 19, wherein said strain further comprises a genetic modification of a gene encoding a phosphate acetyltransferase, said genetic modification inactivating the enzymatic activity of a polypeptide produced by said gene.