WO2007035589A1 - Minimal growth medium for actinobacillus succinogenes - Google Patents

Abstract

A minimal defined medium for growth of A. succinogenes in an anaerobic environment is described. The medium expands the experimental opportunities for studying the metabolism of A. succinogenes, including 13C-labeling experiments . The medium comprises essential amino acids . Other ingredients are also essential such as glucose or another carbohydrate as the primary carbon source, minerals, and vitamins . The medium allows for 13C-labelling experiments that can reveal the metabolic pathways used by A. succinogenes and the extent to which they are used, including those pathways involved in succinate production

Description

MINIMAL GROWTH MEDIUM FOR ACTINOBACILLUS SUCCINOGENES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of Provisional Application No. 60/717,425, filed September 15, 2005, which is incorporated herein, by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was supported by National Science Foundation Grant No. BES-0224596. The U.S. Government has certain rights to this invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

[0003] In the present invention, a defined medium for growing wild-type Actinobacillus succinogenes is described. The reasons for glutamate auxotrophy are also examined, as they have important implications for the metabolic map of A. succinogenes. Metabolic rates were determined and compared at different NaHCO₃ concentrations. The minimal medium is most beneficial for metabolic and transcriptional studies of A. succinogenes. The minimal growth medium provides only essential amino acids and carbon dioxide sources for the production of succinic acid along with fermentable sugar, vitamins and minerals .

(2) Description of the Related Art

[0004] Bio-based chemical production is a growing multi- billion dollar industry converting renewable resources into valuable products (Wilke, D. 1999. Chemicals from biotechnology: molecular plant genetics will challenge the chemical and fermentation industry. Appl. Microbiol . Biotechnol. 52:135-145; Wilke, D. 1995. What should and what can biotechnology contribute to chemical bulk production? FEMS Microbiol. Rev. 16:89-100.). A US $15 billion market could be based on succinate for producing bulk chemicals such as 1,4~ butanediol (a precursor to ^wstronger-than-steel" plastics) , ethylene diamine disuccinate (a biodegradable chelator) , diethyl succinate (a green solvent for replacement of methylene chloride), and adipic acid (nylon precursor) (Zeikus, J. G-, M. K. Jain, and P. Elankovan. 1999. Biotechnology of succinic acid production and markets for derived industrial products. Appl. Environ. Microbiol. 51:545-552). However, the cost of bio- based succinate is not yet competitive with petrochemical-based alternatives such as maleic anhydride. The development of a cost-effective industrial succinate fermentation will rely on organisms able to produce high concentrations of succinate and at high rates .

[0005] Actinobacillus succinogenes {A. succinogenes) is a capnophilic, facultatively anaerobic, gram negative bacterium that naturally produces high concentrations of succinate as a fermentation endproduct in addition to formate, acetate, and ethanol (Guettler, M. V., D. Rumler, and M. K. Jain. 1999. Actinobacillus succinogenes sp. nov., a novel succinic-acid- producing strain from the bovine rumen. Int. J. Syst. Bacteriol. 49:207-216; Guettler, M. V., M. K. Jain, and B. K. Soni. 1996. Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms. U.S. Patent No. 5,504,004; Guettler, M. V., M. K. Jain, and D. Rumler. 1996. Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants. U.S. Patent No. 5,573,931; van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.). A. succinogenes converts glucose to phosphoenolpyruvate (PEP) , at which point metabolism splits into two branches: (i) the formate, acetate and ethanol-producing C3 pathway, and (ii) the succinate producing C4 pathway (Figure 1) . Metabolic engineering of A. succinogenes has begun, with the aim of achieving a homosuccinate fermentation. The most notable success has arisen from inactivation of pyruvate-formate lyase (PFL) by selecting mutants resistant to fluoroacetate (Guettler, M. V., M. K. Jain, and D. Rumler. 1996. Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants. U.S. Patent No. 5_/573,931; Park, D. H.₇ M. Laivenieks, M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1999. Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production. Appl . Environ. Microbiol. 65:2912-2917.). A. succinogenes PFL mutants have increased succinate yields, however significant amounts of pyruvate are also formed.

[0006] Modern, efficient metabolic engineering strategies rely on a thorough understanding of the metabolism under study, and of how metabolism responds to environmental and genetic perturbations (Fell, D. 1997. Understanding the control of metabolism. Portland Press, London and Miami; Stephanopoulos, G., A. A. Aristidou, and J. Nielsen. 1998. Metabolic Engineering: Principles and Methodologies. Academic Press, London) . This understanding can be obtained by using ¹³C- labeling experiments to measure intracellular metabolic fluxes. These experiments require a defined growth medium so that cell components (e.g., amino acids) are synthesized from labeled substrate (e.g., ¹³C-glucose) and not from complex media components such as yeast extract. Furthermore, an accurate metabolic map is essential for metabolic flux analyses. A defined medium, AM3, for growing wild-type A. succinogenes is described. A common experiment for succinate-producing capnophiles is conducted in AM3 with different NaHCO₃ concentrations that provides new insights into A. succinogenes metabolism. Finally, we improve the A. succinogenes metabolic map in the poorly characterized region of its TCA-cycle by using experiments made possible by one of A. succinogenes amino acid auxotrophies and by the advent of AM3.

SUMMARY OF THE INVENTION

[0007] The present invention provides a minimal defined growth medium composition for A. succinogenes comprising amino acids which consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof. In further embodiments, the amino acids consist essentially of cysteine, methionine, and glutamate. In still further embodiments, the minimal defined growth medium composition further comprises ammonium. In further embodiments, the glutamate supply is glutamine, α-ketoglutarate (αKG) and ammonium, αKG and aspartate, or mixtures thereof. In further embodiments, the minimal defined growth medium composition further comprises a sodium bicarbonate or carbon dioxide gas. In still further embodiments, the sodium bicarbonate is provided at a concentration of about 25 mM.

[0008] The present invention provides a minimal defined growth medium composition for A. succinogenes in an anaerobic environment which comprises in admixture: an inorganic phosphate and mineral based medium; an antibiotic which maintains A. succinogenes in preference to other microorganisms; a carbohydrate assimilated by A. succinogenes; vitamins; and amino acids which consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof. In further embodiments, the composition further comprises a sodium bicarbonate or carbon dioxide gas . In further embodiments, the sodium bicarbonate is provided at a concentration of about 25 irtM. In still further embodiments, the A. succinogenes strain is deposited as ATCC 55618 or a mutant thereof. In further still embodiments, the vitamins comprise biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, cyanocobalamin, p- aminobenzoic acid and thioctic acid. In still further embodiments, the antibiotic is kanamycin. In still further embodiments, the anaerobic environment is provided by nitrogen gas. In further embodiments, the anaerobic environment is provided by carbon dioxide. In some embodiments, the medium is incorporated into a semi-solid gel. In further embodiments, the medium further comprises MgCU3 as a source of carbon dioxide gas. In further embodiments, the carbohydrate is glucose.

[0009] The present invention relates to an anaerobic environment in a closed container and to a minimal defined growth medium composition for A. succinogenes_r which comprises in admixture: (a) a sterilized inorganic phosphate and mineral based medium; (b) optionally a filter sterilized, sodium bicarbonate or carbon dioxide gas; (c) a filter sterilized antibiotic which maintains A. succinogenes in preference to other microorganisms; (d) a filter sterilized carbohydrate assimilated by A. succinogenes; (e) vitamins; and (f) filter sterilized amino acids consisting essentially of glutamate or glutamine, optionally glutamine and glutamate, cysteine and methionine. Preferably, the invention relates to a composition wherein the A. succinogen.es strain is deposited as ATCC 55618 or a mutant thereof. The minerals are as described by Lovely (Lovely, D. 2000, posting date. Dissimilatory Fe (III) -and Mn ( IV) -Reducing Prokaryotes . Springer-Verlag http://141.150.157.117:8080/prokPϋB/index.htm) and contained per liter: 1.5 g nitrilotriacetic acid, 3 g MgSO₄*H₂O, 0.1 g FeSO₄*7H₂O, 0.1 g CaCl₂*2H₂O, 0.1 g CoCl₂*6H₂0, 13 mg ZnCl₂, 10 rag CuSO₄*5H₂O, 10 mg AlK(SO₄) ₂*12H₂O, 10 mg H₃BO₃, 25 mg Na₂MoO₄, 25 mg NiCl₂*6H₂O, 25 mg Na₂WO₄*2H₂O, and 10 mg NaSeO₃. It is to be understood that the minerals can be in the form of other salts than those listed above so as to provide equivalent amounts of the minerals. Preferably, the vitamins are biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, cyanocobalamin, p-aminobenzoic acid and thioctic acid. Preferably the antibiotic is kanamycin. The anaerobic environment can be provided by nitrogen gas in the container. Also preferably the anaerobic environment is provided by carbon dioxide in the container. The medium can also be incorporated into a semi-solid gel. Preferably, the carbonate further comprises MgCO₃. Also preferably the anaerobic environment is provided by the carbon dioxide gas . Preferably the carbohydrate is glucose .

[0010] The present invention provides a method of producing succinate comprising: providing a minimal defined growth medium composition for A. succinogenes comprising amino acids that consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof; providing A. succinogenes; and culturing A. succinogenes in the minimal defined growth medium composition to produce the succinate. In further embodiments of the method, the A. succinogenes strain is deposited as ATCC 55618 or a mutant thereof. [0011] Further, the present invention relates to an improved method for use in determination of pathways involved in or affecting succinate production. The present invention provides a method for use in determination of a pathway to succinate production, the improvement which comprises using an anaerobic environment in a closed container and a minimal defined medium composition for A. succinogenes which comprises in admixture amino acids consisting essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, one or more glutamate precursors, and mixtures thereof. In further embodiments, A. succinogenes strains are selected for increased use of pathways leading to succinate production and for decreased use of pathways leading to other fermentation products. In further embodiments, the carbohydrate is labeled with ³C to determine active metabolic pathways in A. succinogenes. In further embodiments, the defined medium is used to determine expression of A. succinogenes genes. In still further embodiments, the genes are modified to select for enzymes in a pathway to thereby overproduce succinate. In still further embodiments, sodium bicarbonate is used as a source of carbon dioxide. [0012] Preferably the medium is used for determination of pathways leading to or affecting succinate production and then genetically engineer the organism based on what we learn about the roles of each pathway in succinate production. This comprises using an anaerobic environment in a closed container and a minimal defined medium composition for A. succinogenes that comprises in admixture: (a) sterilized inorganic phosphate and mineral based medium; (b) optionally a filter sterilized sodium bicarbonate or carbon dioxide gas; (c) a filter sterilized antibiotic which maintains A. succinogenes in preference to other microorganisms; (d) a filter sterilized carbohydrate assimilated by the A. succinogenes; (e) vitamins; and filter sterilized amino acids consisting essentially of glutamate or glutamine, optionally glutamine and glutamate, cysteine and methionine. Preferably A. succinogenes strains are selected for increased use of pathways leading to succinate production and for decreased use of pathways leading to other fermentation products. Most preferably the carbohydrate is labeled with ¹³C to determine active metabolic pathways used by A. succinogenes and the extent to which the pathways are used (i.e., the metabolic fluxes) during different growth conditions .

[0013] Further, the present invention relates to a defined medium which is used to determine expression of A. succinogenes genes. Preferably the expressed genes are modified to select for enzymes in the C4 pathway to thereby overproduce succinate. Determining gene expression is done through RT-PCR and microarrays of gene segments. Gene expression can be affected by any number of environmental factors including known and unknown compounds in the medium. Thus, a defined medium is preferred for gene expression studies because the user knows all of the components in the medium, making it possible to examine whether any of the components affect gene expression. £0014] Sodium bicarbonate is used as a source of carbon dioxide. Sodium bicarbonate can be labeled with ¹³C, which provides information on the pathways being used and the extent to which they are used for succinate production.

BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 is a simplified metabolic map of A. succinogenes central metabolism. Thin black arrows are glucose uptake, pentose phosphate pathway (PPP), and Embden-Meyerhoff- Parnas pathway (EMP) reactions. Grey arrows are C3 pathway reactions . Thick black arrows are C4 pathway reactions . Dashed arrows are TCA-associated reactions that have not been tested. 1. hexokinase or PEP: glucose phosphotransferase; 2. PPP; 3. EMP; 4. pyruvate kinase and PEP: glucose phosphotransferase; 5. pyruvate-formate lyase; 6. acetaldehyde dehydrogenase and alcohol dehydrogenase; 7. phosphotransacetylase and acetate kinase; 8. PEP carboxykinase; 9. malate dehydrogenase, fumarase, and fumarate reductase; 10 succinyl-CoA synthetase, cxKG dehydrogenase, and αKG synthase; 11. isocitrate dehydrogenase and aconitase; 12. citrate lyase and citrate synthase. Metabolites: GIc, glucose; G6P, glucose-6-phosphate; Pyr, pyruvate; For, formate; AcCoA, acetyl-CoA; EtOH, ethanol; Ace, acetate; OAA, oxaloacetate; Sue, succinate; Cit, citrate. [0016] Figure 2 is a schematic drawing showing possible enzyme activities leading to glutamate synthesis. 1, aconitase; 2, isocitrate dehydrogenase; 3, glutamate dehydrogenase; 4, succinyl-CoA synthetase; 5, αKG dehydrogenase; 6, aspartate: glutamate transaminase; 7, glutamate synthase; 8, glutamine synthetase. Metabolites: Cit, citrate;- let, isocitrate; Suc> succinate; S-CoA, succinyl-CoA; Oaa, oxaloacetate.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0017] All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control.

[0018] A defined medium, AM3, for growing wild-type A. succinogenes is described. A common experiment for succinate- producing capnophiles is conducted in AM3 with different NaHCO₃ concentrations that provides new insights into A. succinogenes metabolism. Finally, we improve the A. succinogenes metabolic map in the poorly characterized region of its TCA-cycle by using experiments made possible by one of A. succinogenes amino acid auxotrophies and by the advent of AM3. Chemically defined media allow for a variety of metabolic studies that are not possible in undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate- producing A. succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH₄Cl as the main nitrogen source, and glutamate, cysteine and methionine as required amino acids . A. succinogenes growth trends and endproduct distributions in AM3 and rich medium fermentations were compared. The effect of NaHCO₃ concentration in AM3 on endproduct distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at 25 mM NaHCO₃ than at any other NaHCO₃ concentration, likely because both energy-producing metabolic branches (i.e., the succinate-prόducing branch and the formate, acetate, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or αKG. The inability to synthesize αKG from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes. [0019] The term "auxotrophic" as used herein refers to the requirement of an organism such as A. succinogenes for one or more compounds that are necessary for growth and metabolism. The minimal medium contains several compounds that are required for growth and metabolism in Actinobacillus succinogenes. For example, A. succinogenes was found herein to be auxotrophic for cysteine, methionine and glutamate.

[0020] The term "capnophilic" as used herein refers to an organism that grows best when carbon dioxide (CO₂) concentrations exceed those in normal air (0.033% by volume). In the present invention, the CO2 is in a non-oxidizing atmosphere.

[0021] The term "C4 pathway chemical" as used herein refers to any chemical of the C4 pathway. The term refers to C4 pathway chemicals such as succinate, and also other commercially important chemicals that are intermediates of, or can be derived from_., the C4 pathway. C4 pathway chemicals include, but are not limited to, malate, fumarate, succinate, and 5-aminolevulinate.

[0022] As used herein, the term "glutamate supply" refer to any compound or mixture of compounds that can be used by A. succinogenes as a source of glutamate. Examples of compounds that can be a glutamate supply include glutamate, one or more glutamate precursors, and mixtures thereof.

[0023] As used herein, the term "glutamate precursors" refer to any compound or set of compounds capable of being converted by A. succinogenes into glutamate. Examples of glutamate precursors include glutamine, αKG with ammonium (NH4⁺) , and αKG with aspartate, as set forth in Table 4, or mixtures thereof. [0024] Chemicals, bacteria, and culture conditions: All chemicals were purchased from Sigma-Aldrich (St. Louis,, MO) unless otherwise stated. Escherichia coli K12 (ATCC 10798) and A. succixiogenes type strain 130Z (ATCC 55618) were obtained from the American Type Culture Collection. All liquid cultures were incubated at 37 "C and shaken at 250 rpm. Cultures were inoculated with cell suspensions that were harvested in late log phase, washed twice in sterile saline, and resuspended in an appropriate volume of sterile saline to give a starting OD₆6o of 0.1 after inoculation.

[0025] Identification of a defined growth medium, AM3: The defined medium was based on the phosphate buffer of the rich medium, Medium A, commonly used to grow A. succinogenes (Park, D. H., M. Laivenieks, M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1999. Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production. Appl. Environ. Microbiol. 65:2912-2917; Park, D. H., and J. G. Zeikus. 1999. Utilization of electrically reduced neutral red by Actinobacillus succinogenes : physiological function of neutral red in membrane-driven fumarate reduction and energy conservation. J. Bacterid. 181:2403-2410; van der Werf, M. J.., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.), and it contained per liter: 15.5 g K₂HPO₄, 8.5 g Na₂HPO₄*H₂O, 1 g NaCl, 2 g NH₄Cl, and 10 ml mineral mix. This basal solution was aliquoted into 28-ml anaerobic test tubes. The tubes were then sealed with rubber bungs and aluminum crimps, repeatedly flushed and evacuated with N₂. After autoclaving, each tube received filter- sterilized vitamin mix, kanamycin, amino acids, glucose, and NaHCθ3 to final respective concentrations of 2 ml/L, 10 μg/ml, 0.08 %, 50 mM, and 30 mM. Concentrations of basal solution and supplement stocks were adjusted to give total culture volumes of 10 ml. Soluble NaHCO₃ was used instead of insoluble MgCO₃ (Guettler, M. V., M. K. Jain, and B. K. Soni. 1996. Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms. U.S. Patent No. 5,504,004; Guettler, M. V., M. K. Jain, and D. Rumler. 1996. Method for making succinic acid, bacterial variants for use in the process, and methods for obtaining variants. U.S. Patent No. 5,573,931.; van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus . 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.) to facilitate growth measurements by optical density. One M NaHCO3 stock solutions under 100 % CO₂ atmosphere were prepared as described (Widdel, F., and F. Bak. 1992. Gram-negative mesophilic sulfate-reducing bacteria., p. 3358-3378. In A. Balows, H. G. Truper, M. Dworkin, W. Harder, and K. H. Schleifer (ed. ) , The Prokaryotes, 2nd ed, vol. 4. Springer-Verlag, New York) . An equal volume of sterile 100% CO₂ was used to replace any volume of NaHCO₃ taken from the stock solutions. The final medium pH was 6.9 - 7.1, depending on the amount of NaHCO₃ added. The mineral mix was based on Lovley (Lovely, D/ 2000, posting date. Dissimilatory Fe(III)- and Mn (IV) -Reducing Prokaryotes. Springer-Verlag (Online) http ://141.150.157.117 :8080/prokPUB/index.htm. (Online)) and contained per liter: 1.5 g nitrilotriacetic acid, 3 g MgSO₄*7H₂O, 0.5 g MnSO₄*H₂O, 0.1 g FeSO₄*7H₂O, 0.1 g CaCl₂*2H₂O, 0.1 g CoCl₂*6H₂0, 13 mg ZnCl₂, 10 mg CuSO₄*5H₂O, 10 mg AlK (SO₄) ₂*12H₂O, 10 mg H₃BO₃, 25 mg Na₂MoO₄, 25 mg NiCl₂*6H₂O, 25 mg Na₂WO₄*2H₂θ, and 10 mg NaSeO₃. The vitamin mix was based on Wolin et al. (Wolin, E., M. Wolin, and R. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886.) and contained per liter: 10 mg biotin, 10 mg folic acid, 50 mg pyridoxine HCl, 25 mg thiamine HCl, 25 mg riboflavin, 25 mg nicotinic acid, 25 mg pantothenic acid, 0.5 mg cyanocobalamin, 25 mg P-aminobenzoic acid, and 25 mg thioctic acid.

[0026] The inoculum was A. succinogenes 130Z grown from glycerol frozen stocks in 10 ml of BBL trypticase soy broth

(TSB; Becton Dickinson, Sparks, MD) containing 50 mM glucose, 30 mM NaHCθ₃, and 10 μg/ml kanamycin in 15-ml screw cap glass tubes with air as headspace. The defined medium was inoculated with 0.5 ml of washed cell suspension. The original defined medium supporting growth contained 12 amino acids (i.e., glutamate, aspartate, cysteine, tyrosine, phenylalanine, serine, alanine_/ isoleucine, valine/ argininβ_/ leucine_/ and methionine) that were chosen based on literature for Haemophilus influenzae defined media (Herroitt, R. M., E. M. Meyer, and M. Vogt. 1970. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101:517-524; Herroitt, R. M., E. Y. Meyer, M. Vogt, and M. Modan. 1970. Defined medium for growth of Haemophilus influenzae. J. Bacteriol. 101:513-516.). Cells grown in this medium were washed and used to inoculate various defined media containing eleven (11) amino acids, each medium missing one of the initial twelve (12) amino acids. This procedure was repeated with fewer and fewer amino acids until the amino acids required for growth were identified. The defined medium with the fewest amino acids still supporting growth was called AM3. [0027] Growth of A. succinogenes on AM3 solid agar medium: AM3 agar was prepared as for the liquid medium, with the addition of 1.5% Bacto agar (Becton Dickinson) and with or without 10 g/L MgCθ₃ prior to autoclaving. NaHCU₃ was added to some preparations after autoclaving to a final concentration of 30 mM. A. succinogenes was grown in liquid AM3 and washed as described. After aerobic inoculation, plates were incubated at 37° C in an anaerobic jar with a CO₂ headspace . [0028] Determination of growth trends and fermentation balances in AM3 and Medium A: Anoxic Media A and AM3 (11 ml final volume in 28-ml test tubes) were inoculated with 0.25 ml of washed cells grown in identical media. Medium A differs from AM3 by having 5 g/L yeast extract in place of the vitamins, minerals, amino acids, NaCl, and NH4CI in AM3. Both media contained 150 mM NaHCO₃. Growth was monitored throughout log phase by measuring OD660 with a Spectronic 20 (Bausch and Lomb, Rochester, NY) , which does not require culture sampling. Growth rates were determined from 4-6 measurements. Samples (< 1 ml) were collected at the beginning of incubation, once during log phase (0.6-1.0 OOββo) _r ^ⁿd at the end of log phase. The optical densities of these samples were determined using a Dϋ 650 spectrophotometer (Beckman, Fullerton, CA) . These OD₆₆o values (more precise than those obtained with the Spectronic 20) were used to calculate final OD, and in carbon and electron balances . Glucose and metabolic endproducts in the sample supernatants were separated by HPLC (Waters, MiIford, MA) on a 300 x 7.8 mm Aminex HPX-87H column (Bio-Rad, Hercules, CA) at 23° C with 4 mM H₂SO₄ as the eluent, at a flow rate of 0.6 ml/min. Glucose and ethanol were quantified using a Waters 410 differential refractometer, and organic acids were quantified using a Waters 2487 UV detector at 210 nm.

[0029] Determination of fermentation balances, growth rates, and product formation rates in AM3 with different NaHCθ₃ concentrations: Anoxic AM3 was prepared as described but with NaHCθ₃ concentrations ranging from 5 to 150 mM. The inoculum was 0.25 ml of washed culture grown in AM3 of identical NaHCθ₃ concentration. Sample collection and determination of cell densities, growth rates, and endproduct concentrations were performed as described above. CO₂ was detected by transferring 1 ml of culture headspace and 0.3 ml of liquid cultures to separate bung-sealed 13-ml serum vials. The liquid sample was acidified with 50 μl of 3.2 N H₂SO₄. Vial headspaces were sampled using a pressure syringe and injected into a Series 750 gas chromatograph (GOW-MAC, Bethlehem, PA) equipped with a Carbosphere column, methanizer, and flame ionization detector. Specific rates were calculated as described for batch cultures (Sauer, U., D. R. Lasko, J. Fiaux, M. Hochuli, R. Glaser, T. Szyperski, K. Wurthrich, and J. E. Bailey. 1999. Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J. Bacter±ol. 181:6679-6688; Stephanopoulos, G., A. A. Aristidou, and J. Nielsen. 1998. Metabolic Engineering: Principles and Methodologies. Academic Press, London.). For example, to calculate a specific product formation rate the equation £_P = Y_XP P_/ was used, where r_p is the specific product formation rate, Y_XP is the amount of product produced per gram of biomass, and μ is the growth rate.

[0030] Preparation of crude cell extracts and enzyme assays: A. succinogenes 130Z was grown in 450 ml Medium A containing 33 mM glucose and 15 mM NaHCθ₃ in 1 L spherical flasks with an N₂ headspace. Cultures were harvested in log phase by centrifugation, washed once with 200 ml 0.1 M Tris-HCl (pH 7.7), and resuspended in 20 ml 0.1 M Tris-HCl (pH 7.7). Cells were lysed by two passages through a French press at 1,200 - 1,400 Ib under an N₂ headspace. Cell extracts were stored at - 2O⁰C before assays. The cell extract protein concentration (i.e., 1.8 mg protein /ml) was quantified by the bicinchoninic acid assay (Pierce, Rockford, IL) with bovine serum albumin as the standard (McKinlay, J. B., and J. G. Zeikus. 2004. Extracellular iron reduction is mediated in part by neutral red and hydrogenase in Escherichia coli. Αppl. Environ. Microbiol. 70:3467-3474.) .

[0031] Enzyme activities were assayed by measuring the oxidation or reduction of NADP(H) using a Cary 300 spectrophotometer (Varian, Palo Alto, CAJ . An extinction coefficient of 6.23 cm^"1 mM^"1 at 340 nm was used for NADPH (van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342). Reagents were dissolved in 0.1 M Tris-HCl (pH 8.0), and reactions were carried out in triplicate in 1 ml volumes at 37° C. The reaction mixture for the glutamate dehydrogenase assay contained 40 mM NH₄CI, 5 mM α-ketoglutarate (αKG) , 0.3 inM NADPH, 1 mM CaCl₂, and 25 μl cell extract. The reaction was started by the addition of αKG. Glutamate synthase activity was tested in the presence of 5 mM glutamine, 5 mM αKG, 0.3 mM NADPH, 1 mM CaCl₂, and 25 μl cell extract. The reaction was started by the addition of glutamine. Isocitrate dehydrogenase was assayed using anoxic reagents in rubber-stoppered cuvettes that were evacuated and flushed with N₂ as described (Zeikus, J. G.,^' G. Fuchs, W. Kenealy, and R. K. Thauer. 1977. Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum. J. Bacterial . 132:604- 613.) . The reaction mixture contained 0.1 M NaCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.3 mM NADP⁺, 50 μl cell extract, and 5 mM isocitrate (Thorsness, P. E., and D. E. Koshland, Jr. 1987. Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem 262:10422-10425.). Cell extracts (15.2 mg protein/ml) from E. coli K12 aerobically grown in LB with 25 mM glucose were used as a positive control. The reaction was started with the addition of isocitrate. No enzyme activity was detected in any assay when NAD(H) was used in place of NADP(H) .

[0032] Test of glutamate precursors to support growth: Anoxic defined medium was prepared as described for AM3, but glutamate and/or NH₄CI were omitted where appropriate. Filter- sterilized stock solutions of potential glutamate precursors

(i.e., αKG, glutamine, aspartate, isocitrate, and citrate) were added to the autoclaved medium to 15 mM final concentration. The inoculum was 0.25 ml of A. succinogenes grown in AM3 and washed as described. The culture volume was 12 ml. The turbidity was monitored until stationary phase was reached or for five days using a Spectronic 20. If growth occurred, cells were washed as before and used to inoculate identical medium to ensure that growth was not due to nutrient carry over.

[0033] Results and Discussion.

[0034] Creation of the defined growth medium, AM3: A. succinogenes grew slowly (0.06 hr^"1) when first transferred from TSB to defined medium containing the initial 12 amino acids, with final ODββo values ranging from 0.7 to 1.1. After several transfers in defined medium, growth rates increased to 0.14 hr^'1. This improvement could be due to a slow response in gene regulation to suit the new growth conditions or to genetic drift. After removing amino acids from the defined medium one at a time, the amino acid requirements of A. succinogenes were determined to be cysteine, glutamate, and methionine. To improve the A. succinogenes growth rate and final OD in defined medium, concentrations of amino acids, NH₄Cl, vitamin mix, and mineral mix were varied, and their effects on growth rate and final OD were determined. Mineral mix, vitamin mix, and amino acids were required for anaerobic growth on glucose. Increasing the vitamin concentration from 2 ml/L to 10 ml/L doubled the growth rate and tripled the final OD. A. succinogenes grew without NH₄Cl when glutamate, cysteine, and methionine were present but the growth rate (0.03 ± 0.00 hr ^~τ) and final OU660 (0.44 ± 0.07) were poor. The improved medium, called AM3, contained per liter: 15.5 g K₂HPO₄, 8.5 g Na₂HPO₄*H₂O, 1 g NaCl, 2 g NH₄Cl, 0.15 g L-glutamate, 0.08 g L- cysteine-HCl, 0.08 g L-methionine, 10 ml mineral mix, 10 ml vitamin mix, 30 mmol NaHCO₃, and 50 mmol glucose. [0035] A. succinogenes also grew on solid AM3 agar. One mm sized colonies developed after 2-4 days of incubation under CO₂ gas phase at 37 °C. Colonies developed with and without MgCθ₃ or NaHCO₃-

[0036] Growth trends and fermentation balances in AM3 and Medium A: In a defined medium, bacteria are forced to synthesize a number of cellular building blocks that would otherwise be available from rich medium components . For this reason, growth rates were lower in AM3 (0.24 ± 0.01 hr^"1) than in Medium A (0.43 ± 0.01 hr^'1) . The final OD₆₆O in AM3 (2.82 ± 0.05) was slightly lower than in Medium A (3.03 ± 0.14). Since most of the succinate is produced during log phase, fermentation balances were based on log phase samples . While carbon and electron recoveries for cultures grown in AM3 were near 100%, recoveries for cultures grown in Medium A exceeded 100% (Table 1) likely because carbon and electron recoveries only take into account the glucose consumed. The yeast extract carbon in Medium A is ~50% that of the supplied glucose, according to the BD Diagnostic Systems website (http: //www.bd. com/diagnostics/microservices/regulatory) and Doyle et al. (Doyle, A., M. N. Weintraub, and J. P. Schimel. 2004. Persulfate digestion and simultaneous colorimetric analysis of carbon and nitrogen in soil extracts. Soil Sci. Soc. Am. J. 68:669-676.). Thus, there is ample carbon in yeast extract to explain a 117% carbon recovery in Medium A. Yeast extract may also have contributed to the higher formate and. acetate yields and to the lower succinate product ratio in Medium A compared to AM3. With no undefined carbon sources to track in AM3, the comparison of fermentation balances in AM3 and Medium A illustrates how a chemically defined medium facilitates metabolic studies.

[0037] Effect of AM3 NaHCOe concentration on fermentation balances, growth rates, and metabolic rates: Succinate production by A. succinogenes requires CO₂, presumably as a substrate for PEP carboxykinase (Kim, P., M. Laivenieks, C. Vieille, and J. G. Zeikus. 2004. Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Appl. Environ. Microbiol. 70:1238-1241; van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.) (Figure 1). We previously showed that A. succinogenes produces more succinate and less alternative endproducts when the MgCO₃ concentration was increased in Medium A (van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.). To confirm that this trend holds in AM3, we compared endproduct distributions in AM3 with different NaHCO₃ concentrations . Indeed, increasing the NaHCO₃ concentration in AM3 increased the succinate product ratio (Table 2) . In addition to confirming this trend, we also wanted to determine an optimal NaHCO₃ concentration for succinate production in AM3. In our previous study, the maximum MgCO₃ concentration tested was equimolar to that of the supplied glucose (i.e.₇ 55 inM)^' (van der Werf, M. J., M. V. Guettler, M. K. Jain, and J. G. Zeikus. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 167:332-342.). Here we tested NaHCO₃ concentrations up to 3 times the molar concentration of supplied glucose. As seen in Table 2, the succinate production ratio plateaued at NaHCO₃ concentrations above 75 inM. Although not quantitatively precise, CO₂ was detected in all cultures at the time of log phase sampling (data not shown) . Therefore, the reported succinate yields are not due to complete CO₂ consumption. While CO₂ was not completely consumed, the endproduct distributions suggest that NaHCO₃, at 5 and 25 inM, may be limiting succinate production.

[0038] As shown in Table 2, the average growth rate was statistically higher at 25 inM NaHCO₃ than at any other NaHCO₃ concentration (t-test, 2-tail, equal variance, p = 0.001). This high growth rate can be explained by the metabolic rates shown in Table 3. At 5 mM NaHCO₃, C3 flux (estimated from specific acetate and ethanol formation rates) is high, while C4 flux (i.e., the specific succinate formation rate) is low. At 25 mM NaHCO₃, C3 flux remains high, with a maximum acetate formation rate, and C4 flux reaches a maximum. At 75 mM NaHCO₃ and above, C4 flux remains high, while C3 flux decreases by about half. ATP can be derived from PEP conversion to succinate via the C4 pathway (1.67 mol ATP), and from ethanol (1 mol ATP) or acetate (2 mol ATP) productions via the C3 pathway. With high C3 and C4 fluxes at 25 mM NaHCO₃, more ATP is generated for biosynthesis, explaining the high growth rate in these conditions . The maximum succinate formation rate at 25 mM NaHCO₃ also indicates that NaHCO₃ is not limiting succinate production, unlike what Table 2 endproduct distributions suggest. MSU 4.1-758 9/14/2006

Table 1. Log phase fermentation balances of A. succinogenes in AM3 and Medium A mmol product / 100 mmol glucose consumed

Electron Succinate

Carbon

Medium Recovery⁰ product ^"Recovery¹³ (%) ratio^d

Succinate Formate Acetate Ethanol Biomass^a

Defined

70 ± 1 61 ± 3 64 ± 2 9 + 2 166 ± 5 97 + 2 106 ± 2 0.97 + 0.02

(AM3)

Rich

70 ± 1 99 ± 6 80 ± 3 16 + 4 199 ± 5 117 + 1 117 + 0 0.73 ± 0.02

(Medium A)

Data are means + standard deviation from triplicate cultures . ^a mmol Biomass was determined using assumed values of 567 mg dry cell weight / ml per OD₆₆₀ and a cell composition of CH₂O₀₁₅N_0-2 (24.967 g/mol) (van der Werf et al. Arch. Microbiol. 167:332-342.). ^b Carbon in product / carbon in glucose consumed. An assumption was made that one mole of CO₂ was fixed per mole succinate produced (van der Werf et al. Arch. Microbiol. 167:332-342.). Therefore, C₃H₆O₂WaS used as the chemical composition of succinate derived from glucose consumed.

⁰ Electron recoveries are based on available hydrogen (Gottschalk, G. 1986. Bacterial Metabolism, 2nd ed.

Springer-Verlag, New York) . ^d Succinate / (acetate + ethanol) . It was assumed that all formate is formed from PFL and is accounted for in the sum of acetate and ethanol. Production of less formate than the sum of acetate and ethanol may be due to pyruvate dehydrogenase activity, however this has not yet been proven for A. succinogenes.

Table 2. Effect NaHCO₃ concentration on endproduct distribution and growth rate in AM3

mmol product / 100 mmol glucose consumed

NaHCO₃ concn• ' Carbon Electron Succinate (πiM) .Recovery Recovery product Growth rate

Succinat Chr^"1)

Formate Acetate Ethanol Biomass (%) (%} ratio

5 33 ± 1 117 ± 1 72 ± 1 51 ± 7 146 + 6 101 ± 1 IQQ ± 2 0.27 + 0.02 0.25 ± 0.01

25 52 ± 2 91 ± 1 68 + 1 28 ± 3 169 ± 4 101 + 2 110 + 2 0.54 ± 0.03 0.32 ± 0.01

75 67 ± 1 58±5 61 + 3 14 + 1 _lg9 + ₁₀ 102 ± 4 113 + 4 0.90 + 0.03 0.25 ± 0.00

125 71± 1 60 + 2 64 ± 1 10 + 3 176 + 1 100 ± 2 110 + 2 0.96 ± 0-05 0.23 + 0.01

150 70 + 0 61 + 3 64 + 2 9 + 2 166 + 5 97 + 2 106 + 2 0.97 ± 0.02 0.24 + 0.01

Data are means + standard deviation from triplicate cultures. Biomass, carbon recovery, electron recovery, and succinate product ratio were calculated as described in Table 1.

_ 03 —

[0039] It is also worth noting that the high specific product formation rates at 25 mM NaHCU₃ are allowed by a high glucose consumption rate (Table 3) . This high glucose uptake rate (and expected corresponding high glycolytic flux) should be able to support a succinate formation rate higher than the observed 4 mmol x g biomass^"1 x hr^"1. Not enough is known at this stage to identify the regulatory mechanism or mechanisms behind this observation. Among the possible explanations are that (i) high NaHCθ3 concentrations inhibit the C3 pathway and that the C4 pathway cannot process substrate faster than 4 mmol x g biomass^"1 x hr^"1. This bottleneck would in turn inhibit glucose uptake and glycolysis, (ii) Another possibility is that NaHCO₃ (or CO₂) inhibits glucose uptake and/or glycolysis. Any of these mechanisms would have important implications for the metabolic engineering of A. succinogenes succinate production. Future metabolic flux analyses, in which genetic or environmental perturbations individually affect individual pathway branches, will focus on identifying what limits the succinate production rate in A. succinogenes.

[0040] A. succinogenes is missing at least two TCA cycle- associated enzyme activities: A. succinogenes was found to be auxotrophic for cysteine, methionine and glutamate. Glutamate auxotrophy was initially surprising since A. succinogenes cell extracts have aspartate : glutamate transaminase activity (van der Werf et al. Arch. Microbiol. 167:332-342.). Figure 2 shows possible enzyme activities leading to glutamate, not all of which are known to be present in A. succinogenes. Several possible glutamate precursors (i.e., αKG, isocitrate, citrate, and succinate) are TCA cycle intermediates. It is still unclear whether A. succinogenes has a complete TCA cycle (Fig. 1) . Because a complete TCA cycle would mean at least two pathways for succinate production and/or consumption, we used A. succinogenes 's glutamate auxotrophy to our advantage to study a poorly characterized region of the A. succinogenes central metabolic map. Table 4 shows that αKG can replace glutamate in the growth medium when NH4CI is present, indicating in vivo glutamate dehydrogenase activity. Aspartate plus αKG also supported growth, while aspartate alone did not . These results suggest that aspartate: glutamate transaminase is functional in vivo. Alternatively, aspartase activity could convert aspartate to fumarate and NH₄ ⁺, then NH₄ ⁺ could be used with αKG by glutamate dehydrogenase to produce glutamate. Growth on glutamine indicates the presence of a glutamine deaminating activity (e.g., glutamine synthetase or carbamoyl phosphate synthetase) . In vitro enzyme activity assays suggested that glutamate dehydrogenase (1,100 ± 180 nmol x min^{" 1} x mg protein^"1) and glutamate synthase (30 ± 10 nmol x min^"1 x mg protein^"1) are also functional in A. succinogenes. Taken together, these results suggest that all the enzyme activities (i.e., 3, 6, 7, and 8) below αKG in Figure 2 are present in A. succinogenes .

MSU 4. 1-758 9/14/2006

TABLE 3 . Effect of NaHCO₃ concentration on specific metabolic rates and estimated fluxes

Specific rates (mmol x g biomass ^-1 x hr^'1)

NaHCO₃ Glucose Succinate Formate Acetate Ethanol Estimated Estimated Estimated concn. consumption formation or formation formation formation C3 flux* net ATP glycolytic (niM) C4 flux formation¹³ flux^c

5 6.6 ± 0.0 2.2 ± 0.1 7.8 ± 0.1 4.8 ± 0.0 3.4 ± 0.4 8.2 ± 0.5 16.6 ± 0.4 10.4 ± 0.4

25 7.7 ± 0.0 4.0 ± 0.2 7.0 ± 0.1 5.3 ± 0.1 2.2 ± 0.2 7.4 ± 0.2 19.4 ± 0.5 11.4 ± 0.3

75 5.0 ± 0.2 3.4 ± 0.1 2.9 ± 0.1 3.1 ± 0.0 0.7 ± 0.1 3.8 ± 0.1 12.5 ± 0.2 7.2 ± 0.1

125 5.3 ± 0.2 3.7± 0.1 3.2 ± 0.1 3.4 ± 0.1 0.5 ± 0.1 3.9 ± 0.2 13.5 ± 0.3 7.6 ± 0.2

150 5.7 ± 0.3 4,0 ± 0.1 3.5 ±0.1 3.6 ±0.1 0.5 ± 0.1 4.2 ± 0.2 14.5 ±0.4 8.2 ± 0.4

^a Estimated C3 flux = specific acetate formation rate + specific ethanol formation rate. ^b Estimated net ATP formation: Estimated ATP formation - estimated ATP consumption flux in central metabolism. The assumptions of ATP consumption and formation by central metabolic pathways are as follows: (i) glucose uptake consumes 1 ATP either by ATP-utilizing hexokinase or by PEP:glucose phosphotransferase system preventing ATP production by pyruvate kinase, (ϋ) 1 ATP is consumed by phosphofructokinase, (iii) pyruvate kinase, acetate kinase, and PEP carboxykinase each produce 1 ATP, and (iv) fumarate reductase produces 0.67 ATP per reaction (Kroger, A., S. Biel, J. Simon, R. Gross, G.

Unden, and C. R. D. Lancaster.2002. Fumarate respiration of Wolinella succinogenes: enzymology, energetics and coupling mechanism. Biochim. Biophys.

Acta. 1553:23-28.).

° Estimated glycolytic flux = specific succinate production rate + estimated C3 flux.

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TABLE 4. Ability of glutamate precursors to support growth of A. succinogenes in AM3.

Supplement

NH₄ ⁺ NH₄ ⁺ Asp NH₄ ⁺ + + GIn Asp + GIu αKG αKG

Growth^* + + + - +

^a Growth (+) is defined as > 1.5 ODsβo within 24 hours. No growth (-) is defined as no OD₆₆O increase within 5 days after inoculation. Tests were performed at least in duplicate.

[0041] These results also point to a single reason for A. succinogenes's glutamate auxotrophy: A. sυccinogenes cannot synthesize αKG from glucose. This inability means that enzymes are absent or inactive in two pathways: (i) between succinate and αKG in the reverse TCA cycle (especially since A. succinogenes produces ample succinate) , and (ii) in the TCA cycle from acetyl-CoA and oxaloacetate to citrate to αKG (Fig. 1) . This conclusion is supported in part by the fact that no in vitro isocitrate dehydrogenase activity could be detected in anaerobically and aerobically grown A. succinogenes cell extracts, while it was detected in E. coli cell extracts as a positive control (70 ± 10 nmol NADP(H) min^"1 mg protein^"1). Growth experiments on citrate or isocitrate were not informative (A. succinogenes did not grow when citrate or isocitrate were supplied with NH₄Cl or asparate, data not shown) for at least two reasons: (i) it is not known whether citrate and isocitrate are taken up by A. succinogenes cells; and (ii) citrate prevented A. succinogenes growth at concentrations above 3 mM in the presence of glutamine or glutamate (data not shown) . This inhibition was countered by adding extra minerals (data not shown) , suggesting that citrate binds essential minerals (e.g., iron) and prevents mineral acquisition.

[0042] A. succinogenes is a promising catalyst for bio-based production of succinate and potentially other chemicals (e.g., malate, fumarate, 5-aminolevulinate, αKG, and glutamate) . We have described a chemically defined medium for growing A. succinogenes and studying its metabolism. NaHCO₃ concentration had pronounced effects on fermentation end product distributions between 5 and 75 mM but not at higher concentrations. A. succinogenes had an optimal growth rate at 25 mM NaHCC>₃, where both energy producing pathways displayed their highest fluxes . αKG could be used in place of glutamate to support growth, indicating that at least two TCA cycle- associated enzyme activities are absent. The defined medium made testing growth on glutamate precursors possible. The discovery that A. succinogenes lacks a full TCA cycle is a key information for the construction of an accurate A. succinogenes metabolic map that will be essential in future metabolic flux analyses and practical metabolic engineering designs for A. succinogenes-based chemical production.

[0043] We have described a chemically defined medium for growing and studying A. succinogenes 130Z. A. succinogenes auxotrophy for αKG indicates that at least two enzyme activities are absent, resulting in an incomplete TCA cycle. The absence of these activities is important for designing an accurate metabolic map for A. succinogenes, which in turn will guide metabolic engineering of the organism. However, in terms of actually producing succinate, having a medium without unknown compounds should be beneficial for succinate purification because it is devoid of unknown compounds that can interfere with the purification process.

[0044] This application is related to U.S. Application Serial No. 10/991,961 filed August 5, 2004, which describes Actinobacillus succinogenes useful in the production of succinic acid. This application is also related to PCT Application No. PCT/US06/30425 to Zeikus et al., filed August 04, 2006, which teaches the use of genes from Actinobacillus succinogenes 130Z (ATCC 55618) for production of chemicals from the A. succinogenes C4-pathway. Both of these applications are incorporated herein by reference in their entirety. [0045] While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the Claims attached herein.

Claims

WE CLAIM :

1. A minimal defined growth medium composition for Actinobacillus succinogenes comprising amino acids, which consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof.

2. The minimal defined growth medium composition of Claim 1 wherein the amino acids consist essentially of cysteine, methionine, and glutamate.

3. The minimal defined growth medium composition of Claim 2, further comprising ammonium.

4. The minimal defined growth medium composition of Claim 1 wherein the glutamate supply is glutamine, αKG and ammonium, ocKG and aspartate, or mixtures thereof.

5. The minimal defined growth medium composition of Claim 4 further comprising sodium bicarbonate or carbon dioxide gas .

6. The minimal defined growth medium composition of Claim 5 wherein the sodium bicarbonate is provided at a concentration of about 25 mM.

7. A minimal defined growth medium composition for A. succinogenes in an anaerobic environment, which comprises in admixture :

(a) an inorganic phosphate and mineral based medium;

(b) an antibiotic that maintains A. succinogenes in preference to other microorganisms;

(c) a carbohydrate assimilated by A. succinogenes;

(d) vitamins; and

(e) amino acids that consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof.

8. • The composition of Claim 7 further comprising sodium bicarbonate or carbon dioxide gas .

9. The composition of Claim 8 wherein the sodium bicarbonate is provided at a concentration of about 25 mM.

10. The composition of Claim 7 wherein the vitamins comprise biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid/ cyanocobalamin, p- aminobenzoic acid, and thioctic acid.

11. The composition of Claim 7 wherein the antibiotic is kanamycin.

12. The composition of Claim 7 wherein the anaerobic environment is provided by nitrogen gas .

13. The composition of Claim 7 wherein the anaerobic environment is provided by carbon dioxide.

14. The composition of Claim 7 wherein the medium is incorporated into a semi-solid gel.

15. The composition of Claim 8 wherein the medium further comprises MgCO₃ as a source of carbon dioxide gas.

16. The composition of any one of Claims 7 wherein the carbohydrate is glucose.

17. A method of producing a C4 pathway chemical comprising:

(a) providing a minimal defined growth medium composition for A. succinogenes comprising amino acids, which consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, a glutamate precursor, and mixtures thereof;

(b) providing A. succinogenes; and

(c) culturing A. succinogenes in the minimal defined growth medium composition to produce the C4 pathway chemical.

18. The method of Claim 17, wherein the C4 pathway chemical is succinate.

19. The method of Claim 17, wherein the A. succinogenes is deposited as strain ATCC 55618 or a mutant thereof.

20. In a method for use in determination of a pathway to succinate production, the improvement which comprises using an anaerobic environment in a closed container and a minimal defined medium composition for A. succinogenes that comprises in admixture amino acids which consist essentially of cysteine, methionine, and a glutamate supply selected from the group consisting of glutamate, one or more glutamate precursors, and mixtures thereof .

21. The method of Claim 20 wherein A. succinogenes strains are selected for increased use of pathways leading to succinate production and for decreased use of pathways leading to other fermentation products .

22. The method of Claim 20 wherein the carbohydrate is labeled with C to determine active metabolic pathways in A. succinogenes .

23. The method of Claim 20 wherein the defined medium is used to determine expression of A. succinogenes genes.

24. The method of Claim 20 wherein the genes are modified to select for enzymes in a pathway to thereby overproduce succinate.

25. The method of Claim 20 wherein the sodium bicarbonate is used as a source of carbon dioxide.