WO2008119009A2 - Materials and methods for efficient alanine production - Google Patents

Abstract

The subject application provides genetically engineered microorganisms that produce L-alanine as the primary fermentation product from sugars. Pentose sugars, such as xylose, and hexose sugars, such as glucose, can be effectively fermented to L-alanine. The strains described herein have the ability to metabolize all sugars that are constituents of lignocellulosic biomass and a variety of disaccharides, including lactose, maltose, sucrose and others.

Description

DESCRIPTION

MATERIALS AND METHODS FOR EFFICIENT ALANINE PRODUCTION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial No. 60/908,234, filed March 27, 2007, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

GOVERNMENT SUPPORT

This invention was made with government support under a grant awarded from the

Department of Energy under grant number USDOE-DE FG02-96ER20222 and Department of Energy in conjunction with the United States Department of Agriculture under grant number USDA & DOE Biomass RDI DE FG36-04GO14019. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Worldwide production of L-alanine has been estimated at 500 tons per year (Ikeda, 2003). L-alanine is produced commercially by the enzymatic decarboxylation of L-aspartic acid using immobilized cells or cell suspensions of Pseudomonas dacunhae as a biocatalyst with a yield >90% (Shibatani et ah, 1979). In pharmaceutical and veterinary applications, L- alanine is used with other L-amino acids as a pre- and post-operative nutrition therapy (Hols el ah, 1999). Alanine is also used as a food additive because of its sweet taste (Lee et ah, 2004).

Alanine is a central intermediate and an essential component of cellular proteins. Most microorganisms produce alanine for biosynthesis using a glutamate-pyruvate transaminase (Hashimoto and Katsumata, 1998). Some organisms such as Arthrobacter oxydans (Hashimoto and Katsumata, 1993; Hashimoto and Katsumata, 1998; Hashimoto and Katsumata, 1999), Bacillus sphaericus (Ohashima and Soda, 1979), and Clostridium sp. P2 (Orlygsson et ah, 1995) produce alanine from pyruvate and ammonia using an NADH-linked alanine dehydrogenase. However, fermentations are slow and yields from the best natural producers are typically 60% or less due to co-product formation (Hashimoto and Katsumata, 1998; Table 1).

Plasmid-borne genes encoding NADH-linked alanine dehydrogenase have been tested as an approach to develop improved biocatalysts with varying degrees of success (Table 1). Engineered strains of Zymomonas mobilis CP4 expressing the B. sphaericus cilaD gene produced low levels of racemic alanine during the anaerobic fermentation of 5% glucose (Uhlenbusch et ah, 1991). An WM-deleted strain of Lactococcus lactis containing a mutation in alanine racemase was engineered in a similar fashion and produced 12.6 g 1^-1 L-alanine from 1.8% glucose (Hols et ah, 1999). An E. coli aceF ldhA double mutant containing pTrc99A-<7/αZ⁾ plasmid produced 32 g 1^-1 racemic alanine in 27 h during a two-stage (aerobic and anaerobic) fermentation with a yield of 0.63 g alanine g^"1 glucose (Lee et ah, 2004). With further deletions and process optimization, the racemic alanine titer was increased to 88 g1^-1 in a more complex process with yields approaching the theoretical maximum (Smith et ah, 2006). However, this strain produced only racemic alanine, utilized multi-copy plasmids requiring antibiotic selection, and required complex media with a complex multi-stage fermentation process (Smith et ah, 2006; Table 1).

In this study, we developed novel biocatalysts that produce chirally pure L-alanine in simple batch fermentations without plasmids, antibiotics, or complex nutrients based on a derivative of E. coli W(strain SZl 94) that produces D-lactate (Zhou et ah, 2006a). The native chromosomal ldhA gene in SZ 194 was replaced with a single, chromosomally-integrated copy of the alanine dehydrogenase gene from the thermophile, Geobacillus stearothermophilus XL-65-6 (formerly Bacillus stearothermophilus; Lai and Ingram 1993). After additional deletions idadX and mgsA) and metabolic evolution, the resulting strain produced L-alanine at high titers and high yields in simple batch fermentations using mineral salts medium.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides novel microorganisms useful in the production of alanine. Additionally, the subject invention provides novel constructs for use in transforming any of numerous host organisms, for example, Escherichia coli, to express and/or suppress certain genes to produce alanine when the host organism is cultivated in a fermentable medium. Accordingly, the materials and methods of the subject invention can be used to enhance alanine production in host organisms thereby providing an increased supply of alanine for use in food and industrial applications.

In certain embodiments, derivatives of Escherichia coli (also referred to herein as E. coli) can be used for the construction of strains producing succinate, malate and alanine. In various embodiments, E. coli W (e.g., ATCC 27325) can be used. Additional advantages of this invention will become readily apparent from the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Integration vector used for chromosomal insertion of G. stear other mophilus alaD into the E. coli idhA. Sequence encoding the N-terminal and C- terminal regions are designated alaA ' and alaA ", respectively.

Figures 2 A and 2B. Plasmids used to delete mgsA. Plasmid pLOI4229 (left) was used to delete the mgsA gene and insert the cat-sacB cassette in the first recombination step.

Plasmid pLOI4230 (right) was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence. Sequence encoding the N-terminal and C-terminal regions are designated mgsA ' and mgsA ", respectively.

Figures 3 A and 3B. Plasmids used to delete dadX. Plasmid pLOI4218 (left) was used to delete the dadX gene and insert the cat-sacB cassette in the first recombination step.

Plasmid pLOI4220 (right) was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence. Sequence encoding the N-terminal and C-terminal regions are designated dadX ' and dadX", respectively.

Figures 4 A and 4B. Alignment of the nucleotide and translated amino acid sequences of alanine dehydrogenase genes from G stearotheromphillus XL-65-6 and B. sphaericus IFO3525. Figure 4A. Nucleotide sequence alignment (65% identity). Figure 4B. Alignment of translated amino acid sequences (73% identity).

Figures 5A and 5B. Alanine pathway in recombinant E. coli. Figure 5A. Native and recombinant fermentation pathways. The G. stearothermophilus alaD coding region and transcriptional terminator were integrated into the native IdhA gene under transcriptional control of the IdhA promoter. Solid stars represent gene deletions present in XZ 132. Note that the native biosynthetic route for alanine production is omitted for simplicity. Figure 5B. Coupling of ATP production and growth to NADH oxidation and L-alanine production.

Figures 6A and 6B. Metabolic evolution of XZl 11 to develop XZl 12. Strain XZl 12 was isolated after 10 serial transfers at 24 h intervals in NBS mineral salts medium containing 20 g1^-1 glucose and 1 niM betaine (inoculum of 0.03 CDW1^-1 ; pH controlled with 2N potassium hydroxide). Fermentation broths were sampled daily for 4 days. Figure 6 A. Cell mass (g1^-1 ). Figure 6B. Alanine production (4 days).

Figures 7 A and 7B. Effects of different bases for pH control on growth and alanine production of strain XZl 12 (NBS mineral salts medium containing 50 g 1^-1 glucose and 1 mM betaine; inoculum of 0.03 CDW 1^-1). Bases were automatically added to control pH. Figure 7A. Cell mass (g 1^-1). Figure 7B. Alanine production (4 days). Symbols: ■, 2N potassium hydroxide; A, 2N potassium hydroxide and IN ammonia bicarbonate; T, 5N ammonia hydroxide. Figures 8A and 8B. Metabolic evolution of XZl 12 to select XZl 13. Strain XZl 13 was isolated after 12 serial transfers at 24 h intervals in NBS mineral salts medium containing 50 g1^-1 glucose and 1 mM betaine (inoculum of 0.017 CDW 1^-1; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. Figure 8A. Cell mass (g1^-1 ). Figure 8B. Alanine production (3 days). Figures 9A and 9B. Metabolic evolution of XZl 13 to select XZl 15. Strain XZl 15 was isolated after 25 serial transfers at 24 h intervals in NBS mineral salts medium containing 80 g1^-1 glucose and 1 mM betaine (inoculum of 0.017 CDW 1^-1 ; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown. Figure 9 A. Cell mass (g 1^" ). Figure 9B. Alanine production (3 days).

Figures 1OA and 1OB. Metabolic evolution of XZ121 to select XZ123. Strain XZ123 was isolated after 40 serial transfers at 24 h intervals in NBS mineral salts medium containing 80 g 1^-1 glucose and 1 mM betaine (inoculum of 0.017 CDW1^-1 ; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown. Figure 1OA. Cell mass (g 1^-1 ). Figure 1OB. Alanine production (3 days).

Figures HA and HB. Effect of pH on cell growth and alanine production by strain XZl 23 (NBS mineral salts medium containing 80 g 1^-1 glucose and 1 mM betaine; inoculum of 0.017 CDW 1^-1). Broth pH was automatically controlled by the addition of 5N ammonium hydroxide. Figure HA. Cell Mass. Figure HB. Alanine production. Symbols: D, pH 6.5; Δ, pH 7.0; V, pH 7.5; and o pH 8.0.

Figures 12A and 12B. Effect of inoculum level on growth and alanine production by strain XZ 123 (NBS mineral salts medium containing 80 g1^-1 glucose and 1 mM betaine; pH controlled with 5 N ammonium hydroxide. Figure 12 A. Cell mass. Figure 12B. Alanine production. Symbols: D, 0.005 g CDW 1^-1; Δ, 0.008 g CDW 1-;¹ V, 0.017 g CDW 1;^-1 o, 0.035 g CDW r¹; O, 0.08 g CDW 1;^-1 X, 0.17 g CDW . 1^-1

Figures 13A and 13B. Metabolic evolution of XZ126 to develop XZ129. Strain XZl 29 was isolated after 30 serial transfers (1 :100 dilution) at 24 h intervals in NBS mineral salts medium containing 80 g 1^-1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown. Figure 13 A. Cell mass (g 1-)¹. Figure 13B. Alanine production (3 days). Figures 14A and 14B. Metabolic evolution of XZ129 to develop XZ130. Strain

XZl 30 was isolated after 7 serial transfers at a 1 :100 dilution and 7 transfers at 1:300 dilution. Cells were transferred at 24 h intervals in AMI mineral salts medium containing 80 g1^-1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. Figure 14A. Cell mass (g 1-)¹. Figure 14B. Alanine production (3 days).

Figures 15A and 15B. Metabolic evolution of XZ130 to develop XZ131. Strain XZl 31 was isolated after 16 serial transfers (1 :100 dilution) at 24 h intervals in AMI mineral salts medium containing 100 g 1^-1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown. Figure 15A. Cell mass (g 1^-1). Figure 15B. Alanine production (3 days).

Figures 16A and 16B. Metabolic evolution of XZ131 to develop XZ 132. Strain XZ 132 was isolated after 30 serial transfers (1 :100 dilution) at 24 h intervals in AMI mineral salts medium containing 12O g 1^-1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown. Figure 16A. Cell mass (g 1^-1). Figure 16B. Alanine production (3 days).

Figure 17. Diagram summarizing the construction of XZ132 for L-alanine production. Figure 18. Pathway for pyruvate production from L-alanine. L-alanine is first converted to D-alanine by an alanine racemase such as DADX (dadX) or ALR (air) and oxidized to pyruvate by a D-amino acid dehydrogenase such as DADA (dadA). FAD⁺ serves as an intermediate (FADH) and electron carrier. FADH is oxidized through the electron transport system (dotted arrow) to regenerate FAD⁺ with molecular oxygen serving as the ultimate electron acceptor.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the α/αD-forward primer; including the ribosomal binding region

(bold) and the amino terminus (underlined).

SEQ ID NO: 2 is the α/αD-reverse primer; downstream from the putative transcriptional terminator region.

SEQ ID NO: 3 is the idhA-forward primer. SEQ ID NO: 4 is the MhA -reverse primer.

SEQ ID NO: 5 is the ldhA-up (ydbH) primer. SEQ ID NO: 6 is the idhA-down (hslj) primer. SEQ ID NO: 7 is the JMcαtsαcBupNhel primer. SEQ ID NO: 8 is the JmcαtsacBdownNhel primer. SEQ ID NO: 9 is the cαt-up2 primer.

SEQ ID NO: 10 is the sαcB-down2 primer. SEQ ID NO: 11 is the mgsA-up primer. SEQ ID NO: 12 is the mgsA-down primer. SEQ ID NO: 13 is the mgsA-1 primer. SEQ ID NO: 14 is the mgsA-2 primer.

SEQ ID NO: 15 is the dαdX-up primer. SEQ ID NO: 16 is the dαdX-down primer. SEQ ID NO: 17 is the dαdX-4 primer. SEQ ID NO: 18 is the dαdX-5 primer.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides materials and methods wherein unique and advantageous combinations of gene mutations are used to direct carbon flow to a desired product, e.g., alanine. The techniques of the subject invention can be used to obtain products from native pathways as well as from recombinant pathways. Advantageously, the subject invention provides a versatile platform for the production of these products with only mineral salts and sugar as nutrients. The microorganism of the present invention can be obtained by modification of one or more target genes (for example, in a bacterium belonging to Escherichia), Thus, bacterium that can be used in the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter lumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium laclofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas βuorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, Xanthomonas citri and so forth.

In certain embodiments, the subject invention provides strains of E. coli suitable for the production of alanine. Unlike other microbial systems, the microorganisms of the subject invention can employ a single step process using sugars as substrates, high rates of production, high yields, simple nutrient requirements (e.g., mineral salts medium), and a robust metabolism permitting the bioconversion of hexoses, pentoses, and many dissacharides. Thus, microorganisms according to the instant disclosure can have one or more target genes inactivated by various methods known in the art. For example, target genes can be inactivated by the introduction of insertions, deletions, or random mutations 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 the target gene. Some aspects of the invention provide for the introduction 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 in order to introduce a frame shift mutation in a target gene. Other aspects of the invention provide for the insertion or of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or more bases in order to introduce a frame shift mutation in a target gene. Yet other embodiments of the subject application provide for the introduction of one or more point mutations (e.g., 1 to 30 or more) within a target gene. Other aspects of the invention provide for the total or complete deletion of a target gene from the microorganisms of the invention. In each of these aspects of the invention, metabolic pathways are inactivated and the enzymatic activity of the target gene is eliminated. Deletions provide maximum stability as there is no opportunity for a reverse mutation to restore function.

"Target gene(s)" as used herein, refer(s) to one or more of the fumarate reductase subunit genes (e.g., the frdA, frdB, frdC, frdD, various combinations of the subunit genes or all of the subunit genes (e.g., frdABCD)), the alcohol dehydrogenase gene (e.g., adhE), the pyruvate formatelyase gene (e.g., pflB), the acetate kinase gene (e.g., ackA); the lactate dehydrogenase gene (e.g., idhA), the methylglyoxal synthase gene (e.g., mgsA); and/or the alanine racemase gene (e.g., dadX). The selection process for strains with improved growth and alanine production is referred to as "metabolic evolution" (examples of which are provided within the disclosed examples). A "heterologous alanine dehydrogenase gene" is to be understood to be a gene obtained from any microorganism other than E. coli. For example, the alanine dehydrogenase gene from Geobacillus stearothermophilus or any other microorganism (e.g., thermophilic microorganisms) can be used. Non-limiting embodiments of the invention include: 1. A genetically modified microorganism that comprises the inactivation or deletion of a lactate dehydrogenase gene and the integration of an alanine dehydrogenase gene and one or more of the following genetic modifications: a) the optional inactivation or deletion of a pyruvate formatelyase gene; b) the optional inactivation or deletion of an alcohol dehydrogenase gene; c) the optional inactivation or deletion of an acetate kinase gene; d) the optional inactivation of one or more fumarate reductase subunit genes by insertion or deletion; e) the optional inactivation or deletion of an alanine racemase gene; and/or f) the optional inactivation or deletion of a methylglyoxal synthase gene.

2. A genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxyloca casAB gene or other genes encoding cellobiose utilizing enzyme II and phospho-β-glucosidase behind the stop codon of lacY; b) integration of an Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene into one or more fumarate reductase subunit genes or inactivation of one or more fumarate reductase subunit genes by insertion or deletion; c) inactivation or deletion of an alcohol dehydrogenase gene; d) inactivation or deletion of a pyruvate formatelyase gene; e) inactivation or deletion of an acetate kinase gene; f) inactivation or deletion of a lactate dehydrogenase gene; g) integration of an alanine dehydrogenase gene; h) inactivation or deletion of a methylglyoxal synthase gene; and i) inactivation or deletion of an alanine racemase gene.

3. The genetically modified microorganism according embodiment 1 or 2, wherein said genetically modified microorganism further comprises inactivated or deleted antibiotic resistance genes.

4. The genetically modified microorganism according to embodiment 1, 2 or 3, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and Erwinia chrysanthemi eel Y gene or any other endoglucanase or cellulase encoding gene are inactivated or deleted in said genetically modified microorganism after insertion. 5. The genetically modified microorganism according to embodiment 1 or 2 or 3, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and/or Erwinia chrysanthemi celY gene have not been inserted into said microorganism and one or more of the fumarate reductase subunit genes is inactivated by insertion or deletion.

6. The genetically modified microorganism according to embodiment 1, 2, 3, 4, or 5 wherein the lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene.

7. The genetically modified organism according to embodiment 1, 2, 3, 4, 5 or 6, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

8. The genetically modified organism according to embodiment 1, 2, 3, 4, 5 or 6 wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

9. The genetically modified microorganism according to embodiment 1, 2, 3, 4, 5, 6, 7 or 8, wherein said genetically modified microorganism is metabolically evolved.

10. The genetically modified microorganism according to embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein antibiotic genes are deleted with FLP recombinase.

11. The genetically modified organism according to embodiment 1, 3, 4, 5, 6, 7, 8, 9 or 10, wherein said microorganisms comprises the following additional genetic modifications set forth in embodiment Ia though If: Ia only; Ib only; Ic only; Id only; Ie only; Ia and Ib only; Ia and Ic only; Ia and Id only; Ia and Ie only; Ia and If only; Ib and Ic only; Ib and Id only; Ib and Ie only; Ib and If only; Ic and Id only; Ic and Ie only; Ic and If only; Id and Ie only; Id and If only; Ie and If only; Ia, Ib, Ic, Id, and Ie only; Ia, Ib, Ic, Id and If only; Ia, Ib, Ic, Ie and If only; Ia, Ib, Id, Ie, If and only; Ia, Ic, Id, Ie and If only; Ib, Ic, Id, Ie and If only; Ia, Ib, Ic and Id only; Ia, Ib, Ic and Ie only; Ia, Ib, Ic and If only; Ia, Ib, Id and Ie only; Ia, Ib, Id and If only; Ia, Ib, Ie and If only; Ia, Ic, Id and Ie only; Ia, Ic, Id and I f only; Ia, Ic, Ie and If only; Ia, Id, Ie and If only; Ib, Ic, Id and Ie only; Ib, Ic, Id and If only; Ib, Ic, Ie and If only; Ib, Id, Ie and If only; Ic, Id, Ie and If only; Ia, Ib and Ic only; Ia, Ib and Id only; Ia, Ib and Ie only; Ia, Ib and If only; Ia, Ic and Id only; Ia, Ic and Ie only; Ia, Ic and If only; Ia, Id and Ie only; Ia, Id and If only; Ia, Ie and If only; Ib, Ic and Id only; Ib, Ic and Ie only; Ib, Ic and If only; Ib, Id and Ie only; Ib, Id and If only; Ib, Ie and If only; Ic, Id and Ie only; Ic, Id and If only; Ic, I e and If only; Id, Ie and If only; or Ia, Ib, Ic, Id, Ie and If.

12. A method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and culturing or growing said genetically modified microorganism.

13. A method of producing L-alanine comprising culturing one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 under conditions that allow for the production of alanine.

14. The method according to any one of embodiments 12 or 13, wherein said genetically modified microorganism is cultured in a mineral salts medium.

15. The method according to embodiment 14, wherein the mineral salts medium comprises between 2% and 20% (w/v) of a sugar.

16. The method according to embodiment 15, 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.

17. The method according to embodiment 15 or 16, wherein the sugar is glucose or sucrose or a combination of glucose, sucrose, pentose and xylose.

18. The method according to any one of embodiment 11, 12, 13, 14 or 15, wherein chirally pure alanine is produced. 19. The method according to embodiment 12, 13, 14, 15, 16, 17 or 18, wherein the yield of alanine is at least 90%.

20. The method according to embodiment 19, 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%.

21. The method according to embodiments 13, 14, 15, 16, 17, 18, 19 or 20, wherein there is no detectable contamination of one stereoisomeric form of alanine with the other stereoisomeric form or the chiral purity of L-alanine is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

22. A composition comprising one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and medium.

23. The genetically modified microorganism of any one of embodiments 1-11, the methods of any one of embodiments 12-21 or the composition of embodiment 22, wherein said genetically modified microorganism is a strain of E. coli.

24. The method according to embodiments 13, 14 ,15, 16, 17, 18, 19, 20 or 21, wherein there is no detectable contamination of one stereoisomeric form of alanine with the other stereoisomeric form (e.g., the chiral purity of the specified stereoisomer (L-alanine) is at least, greater than (or greater than or equal to) 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%).

Other aspects of the invention provide the following additional non-limiting embodiments.

1. A genetically modified microorganism that comprises the inactivation or deletion of a lactate dehydrogenase gene and the integration of an alanine dehydrogenase gene and the following genetic modifications: a) the inactivation or deletion of a pyruvate formatelyase gene; b) the inactivation or deletion of an alcohol dehydrogenase gene; c) the inactivation or deletion of an acetate kinase gene; d) the inactivation of one or more fumarate reductase subunit genes by insertion or deletion; e) the inactivation or deletion of an alanine racemase gene; and f) the inactivation or deletion of a methylglyoxal synthase gene.

2. The genetically modified microorganism according to embodiment 1, wherein the integrated alanine dehydrogenase gene is a heterologous alanine dehydrogenase gene.

3. The genetically modified microorganism according to embodiment 1 or 2, wherein the genetically modified microorganism is Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum,

Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetogluiamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Escherichia coli, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca,

Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolylicus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella scholtmuller,ov Xanthomonas citri.

4. The genetically modified microorganism according to embodiment 3, wherein said genetically modified microorganism is Escherichia coli.

5. The genetically modified microorganism according embodiment 1, 2, 3 or 4, wherein said genetically modified microorganism further comprises inactivated or deleted antibiotic resistance genes.

6. The genetically modified microorganism according to embodiment 1, 2, 3, 4 or 5, wherein the lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene.

7. The genetically modified organism according to embodiment 1, 2, 3, 4, 5 or 6, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

8. The genetically modified organism according to embodiment 1, 2, 3, 4, 5 or 6, wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

9. The genetically modified microorganism according to embodiment 1, 2, 3, 4, 5, 6, 7 or 8, wherein said genetically modified microorganism is metabolically evolved.

10. The genetically modified microorganism according to embodiment 1, 2, 3, 4, 5, 6, 7, 8 or 9, wherein antibiotic genes are deleted with FLP recombinase.

11. A genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxytoca casAB gene or other genes encoding cellobiose utilizing enzyme II and phospho-β-glucosidase behind the stop codon of lacY; b) integration of an Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene into one or more fumarate reductase subunit genes or inactivation of one or more fumarate reductase subunit genes by insertion or deletion; c) inactivation or deletion of an alcohol dehydrogenase gene; d) inactivation or deletion of a pyruvate formatelyase gene; e) inactivation or deletion of an acetate kinase gene; f) inactivation or deletion of a lactate dehydrogenase gene; g) integration of an alanine dehydrogenase gene; h) inactivation or deletion of a methylglyoxal synthase gene; and i) inactivation or deletion of an alanine racemase gene.

12. The genetically modified microorganism according to embodiment 11, wherein the integrated alanine dehydrogenase gene is a heterologous alanine dehydrogenase gene.

13. The genetically modified microorganism according to embodiment 11 or 12, wherein the genetically modified microorganism is Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter parqffϊneus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Escherichia coli, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streplomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibiolicus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmuller, or Xanlhomonas citri.

14. The genetically modified microorganism according to embodiment 13, wherein said genetically modified microorganism is Escherichia coli.

15. The genetically modified microorganism according to embodiment 11, 12, 13 or 14, wherein said genetically modified microorganism further comprises inactivated or deleted antibiotic resistance genes.

16. The genetically modified microorganism according to embodiment 11, 12, 13,

14 and 15, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene are inactivated or deleted in said genetically modified microorganism after insertion.

17. The genetically modified microorganism according to embodiment 11, 12, 13,

14, 15 or 16, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and/or Erwinia chrysanthemi celY gene have not been inserted into said microorganism and one or more of the fumarate reductase subunit genes is inactivated by insertion or deletion.

18. The genetically modified microorganism according to embodiment 11, 12, 13,

14, 15, 16 or 17, wherein the lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene. 19. The genetically modified organism according to embodiment 11, 12, 13, 14, 15, 16, 17 or 18, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

20. The genetically modified organism according to embodiment 11, 12, 13, 14, 15, 16, 17 or 18, wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

21. The genetically modified microorganism according to embodiment 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, wherein said genetically modified microorganism is metabolically evolved.

22. The genetically modified microorganism according to any one of embodiments 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, wherein antibiotic genes are deleted with FLP recombinase.

23. A method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21 or 22 and culturing or growing said genetically modified microorganism.

24. A method of producing L-alanine comprising culturing one or more genetically modified microorganism according to any one of embodiments 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 under conditions that allow for the production of alanine.

25. The method according to any one of embodiments 23 or 24, wherein said genetically modified microorganism is cultured in a mineral salts medium. 26. The method according to embodiment 25, wherein the mineral salts medium comprises between 2% and 20% (w/v) of a sugar.

27. The method according to embodiment 26, 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%, 1 1.5%, 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.

28. The method according to embodiment 26 or 27, wherein the sugar is glucose or sucrose or a combination of glucose, sucrose, pentose and xylose.

29. The method according to any one of embodiments 23, 24, 25, 26, 27 or 28, wherein chirally pure alanine is produced.

30. The method according to embodiment 23, 24, 25, 26, 27, 28 or 29, wherein the yield of alanine is at least 90%.

31. The method according to embodiment 30, 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%.

32. The method according to embodiments 23, 24, 25, 26, 27, 28, 29, 30 or 31, wherein there is no detectable contamination of one stereoisomeric form of alanine with the other stereoisomeric form or the chiral purity of L-alanine is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

33. A composition comprising one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 and medium.

Thus, the subject invention provides microorganisms that have been engineered to enhance cell growth and alanine production in various media. The following microorganisms were deposited with the Agricultural Research Service Culture Collection, 1815 N. University Street, Peoria, Illinois, 61604 U.S.A. These cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

The accession numbers and deposit dates are as follows;

Following are examples that illustrate procedures 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 noted. EXAMPLE 1 Materials and Methods

Strains, plasmids, media and growth conditions

The strains and plasmids used in this study are listed in Table 2. Strain SZ 194 was previously engineered from a derivative of E. coli W (ATCC 27325) and served as a starting point for constructions (Zhou et ah, 2006b). Geobacillus stearothermophilus XL-65-6

(formerly Bacillus stearothermophilus; Lai and Ingram, 1993) was used for cloning alanine dehydrogenase gene. The primers used in this study are listed in Table 3.

During strain construction, cultures were grown aerobically at 3O⁰C, 37°C, or 39⁰C in Luria broth (1O g 1- D¹ ifco tryptone, 5 g D1i-f¹co yeast extract and 5 g NaCl1)^-1 containing 2% (w/v) glucose or 5% (w/v) arabinose. Ampicillin (50 mg l^"1), tetracycline (12.5 mg 1^-1), kanamycin (50 mg 1-)¹, or chloramphenicol (40 mg ) w1^-1ere added as needed. For initial tests of fermentative alanine production, strains were grown without antibiotics at 37°C in NBS mineral salts medium (Causey et ah, 2004) supplemented with 100 mM ammonia sulfate, 1 mM betaine and 2% (w/v) glucose. Fermentation experiments (2%-12% sugar) were carried out in NBS medium and AMI medium (Martinez et ah, 2007; Table 4). Broth was maintained at pH 7 by the automatic addition of 5 M NH₄OH.

Genetic methods Standard methods were used for genomic DNA extraction (Qiagen), PCR amplification (Stratagene and Invitrogen), transformation, plasmid extraction (Qiagen), and restriction endonuclease digestion (New England Biolabs). Methods for foreign gene (alaD) integration and for chromosomal gene (mgsA and dadX) deletion are described below. DNA sequencing was provided by the University of Florida Interdisciplinary Center for Biotechnology Research. The Biocyc and Metacyc databases (Karp et ah, 2005) were instrumental in the design and completion of these studies.

Cloning the alanine dehydrogenase gene alaD from G. stearothermophilus XL-65-6, and detection of the enzyme activity The primers for amplifying alaD from G. stearothermophilus XL-65-6 were designed based on the alaD sequence of G. stearothermophilus strain 10. The forward primer (5 '-3' GGAAAAAGGAGGAAAAAGTGATGAAGATCGGCATT) (SEQ ID NO: 1) included the ribosomal binding region (bold) and the amino terminus (underlined). The reverse primer (5'- 3' GAAGGAGTTGATCATTGTTTAACGAGAGAGG) (SEQ ID NO: 2) was downstream from the putative transcriptional terminator region.

Alanine dehydrogenase was verified in clones using an activity stain (Sakamoto et ah, 1990). E. coli TOPlOF' harboring plasmids containing alaD was grown on LB plates at

37°C, then transferred to a Whatman 7.0 cm filter paper. The filter was immersed in 1OmM potassium phosphate buffer (pH 7.2), and incubated for 20 min at 8O⁰C for lysis of the cells and denaturation of the E. coli proteins. The dried filter paper was assayed in a reaction mixture containing 50 mM L-alanine, 50 mM Tris-HCl buffer (pH 9.0), 0.625 mM NAD⁺, 0.064 mM phenazine methosulfate, and 0.24 mM nitro blue tetrazolium. The cells with alanine dehydrogenase appeared as blue spots on the filter.

Integration of alaD into E. coli SZl 94

The alaD gene was integrated into the chromosomal ldhA gene of SZl 94. The fragment (Smal-Kpnl, 1.7 kb) containing a tet gene flanked by two FRT sites was isolated from pLOI2065 and cloned into pLOI4211 between a unique BarήΑl site (Klenow-treated) and Kpnl site to produce plasmid pLOI4213 (6.0 kb). In this plasmid, transcription of alaD and tet are oriented in the same direction.

The Apal (treated with T4 DNA polymerase to produce a blunt end)-ΛjrøI fragment (2.2 kb) containing alaD and tet was isolated from pLOI4213 and cloned into pLOI2395 {Hindi to Kpnl sites) to produce pLOI4214 (6.5 kb). In this plasmid, idhA, alaD and let genes are transcribed in the same direction. The Ascl fragment (4.3 kb) containing these three genes was isolated from pLOI4214 and cloned into the R6K integration vector pLOI2224 to produce pLOI4215 (6.2 kb). Plasmid pLOI4215 contains resistance genes for both tetracycline and kanamycin (Figure 1).

The Ascl fragment (4.3 kb) containing IdhA, alaD and tet genes was isolated from pLOI4215, further cut by Xmnl to eliminate any remaining uncut plasmid DNA, and electroporated into SZ 194 containing the Red recombinase plasmid pKD46 (Datsenko and Wanner, 2000). Integrants were selected for tetracycline resistance, confirmed by sensitivity to kanamycin and ampicillin and by PCR analysis using the primers of IdhA and its neighboring genes ydbH and hslJ (Table 3). Deletion ofmgsA and dadX genes

A modified method for deleting E. coli chromosomal genes was developed using two steps of homologous recombination (Thomason et ah, 2005). With this method, no antibiotic genes or scar sequences remain on the chromosome after gene deletion. In the first recombination, part of the target gene was replaced by a DNA cassette containing a chloramphenicol resistance gene {cat) and levansucrase gene (sacB). In the second recombination, the cat-sacB cassette was removed by selection for resistance to sucrose. Cells containing the sacB gene accumulate levan during incubation with sucrose and are killed. Surviving recombinants are highly enriched for loss of the cat-sacB cassette. A new cassette was constructed as a template to facilitate gene deletions. The cat- sacB region was amplified from pEL04 (Lee et ah, 2001; Thomason el ah, 2005) by PCR using the JMcatsacBwpNhel and JMcatsacBdov/nNhel primers (Table 3), digested with Nhe\, and ligated into the corresponding site in pLOI3421 to produced pLOI4151. The cat-sacB cassette was amplified by PCR using pLOI4151 as a template with the cat-up2 and sacB- down2 primers (EcoRV site included in each primer), digested with EcoRY, and used in subsequent ligations.

The mgsA gene and neighboring 500 bp regions (yccT'-mgsA-helD' , 1435 bp) were amplified using the mgsA-xφ and mgsA-down primers and cloned into the pCR 2.1-TOPO vector (Invitrogen) to produce plasmid pLOI4228. A 1000-fold diluted plasmid preparation of this plasmid served as a template for inside-out amplification using the mgsA-X and mgsA- 2 primers (both within the mgsA gene and facing outward). The resulting 4958 bp fragment containing the replicon was ligated to the Zscoi?V-digested cat-sacB cassette from pLOI4151 to produce pLOI4229. This 4958 bp fragment was also used to construct a second plasmid, pLOI 4230, by phosphorylation and self-ligation. In pLOI4230, the central region of mgsA is deleted (yccT'-mgsA '-mgsA "- helD ').

After digestion of pLOI4229 and pLOI4230 with Xmnl (within the vector), each served as a template for amplification using the mgsA-up and mgsA-down primers to produce linear DNA for integration step 1 (yccT'-mgsA '- cat-sacB- mgsA "- helD') and step II iyccT'- mgsA '-mgsA "- helD'), respectively. After electroporation of the step 1 fragment into XZl 15 containing pKD46 (Red recombinase) and 2 h of incubation at 3O⁰C to allow expression and segregation, recombinants were selected for chloramphenicol (40 mg ) a1-n¹d ampicillin (20 mg I^" ) resistance in Luria broth at 3O⁰C (18 h). Three clones were selected, grown in Luria broth containing Ampicillin and arabinose (5% w/v), and prepared for electroporation. After electroporation with the step 2 fragment, cells were incubated at 37⁰C for 4 h and then transferred into a 250-ml flask containing 100 ml of modified LB (100 mM MOPS buffer added and NaCl omitted) containing 10% sucrose. After overnight incubation (37⁰C), clones were selected on modified LB plates (no sodium chloride; 100 mM MOPS added) containing 6% sucrose (39⁰C, 16 h). Resulting clones were tested for loss of ampicillin and chloramphenicol resistance. Construction was confirmed by PCR using the mgsA up/down primer set. A clone with a deletion in the central region of mgsA was selected and designated XZ121.

The dadX gene was deleted in a manner analogous to that used to delete the mgsA gene. Primers for dadX deletion are shown in Table 3, and the corresponding plasmids shown in Table 2 and Figure 3.

Fermentation

NBS mineral salts medium (Causey et al., 2004) with 1 mM betaine (Zhou et al., 2006a) was used in the initial fermentation (pH 7.0). Pre-inoculum was grown by inoculating three colonies into a 250 ml flask (100 ml NBS medium, 2% glucose, and 100 mM ammonium sulfate). After 16h (37⁰C, 120 rpm), this pre-inoculum was diluted into 500 ml fermentation fieakers containing 300 ml NBS medium (2-8% glucose, 100 mM ammonium sulfate and 1 mM betaine) with 33 mg cell dry wt (CDW) 1-.¹ In early experiments, pH was maintained at 7.0 by automatically adding 2 M potassium hydroxide. In later experiments 5 M ammonium hydroxide was used to maintain pH and a low salt medium, AMI (Martinez et al., 2007; Table 4), was used to replace the NBS medium for fermentation (8-12% glucose). AMI medium contains much less salt and has been optimized for E. coli B.

Metabolic evolution

Cells from pH-controlled (5 M ammonium hydroxide) fermentations were serially transferred at 24 h intervals to facilitate metabolic evolution through competitive, growth- based selection. At the beginning, sequentially transferred cultures were inoculated with an initial density of 33 mg CDW 1-.¹ As growth increased, the inoculum was changed to a 1 :100 dilution and subsequently to a 1:300 dilution. Periodically, clones were isolated from these experiments, assigned new strain designations, and frozen for storage. Analysis

Cell mass was estimated by measuring the optical density at 550 nm (OD550). Organic acids and glucose concentrations were measured by HPLC (Underwood et al, 2002). Fermentation products were determined by using mass spectroscopy and an amino acid analyser at the University of Florida Interdisciplinary Center for Biotechnology Research. Alanine was found to be the predominant product. The alanine concentration and isomeric purity were further measured by HPLC using the Chiralpak MA(+) chiral column (Chiral Technologies Inc).

Result

Cloning of the alanine dehydrogenase gene

Alanine dehydrogenase (ALD) is found in Bacillus (and Geobacillus) species where it plays a pivotal role in energy generation during sporulation (Ohashima and Soda, 1979;

Sakamoto et al., 1990). ALD from B. sphaericus IFO3525 has been widely used with varying degrees of success to engineer alanine production in heterologous bacteria (Uhlenbusch et al.,

1991; Hols et al, 1999; Lee et al, 2004; Smith et al, 2006). Selection of the B. sphaericus

IFO3525 is presumed to be due in part to the high specific activity (Ohashima and Soda,

1979). In contrast, we have selected a thermostable ALD from the thermophile, G. stearothermophilus XL-65-6, based on our prior experience in expressing genes from this organism in recombinant E. coli (Burchhardt and Ingram, 1992; Lai and Ingram, 1993; Lai and Ingram, 1995).

The ribosomal-binding region, coding region and transcriptional terminator of alaD were amplified from G. stearothermophilus XL-65-6 and sequenced. The deduced amino acid sequence was identical to that reported for Geobacillus kaustophilus HTA426 and very similar to G. stearothermophilus strain 10 (99% identity), and G. stearothermophilus strain

IFO 12550 (94% identity). The nucleotide sequence (65% identity) and the deduced ALD amino acid sequence (74% identity) were quite different from the B. sphaericus IFO3525 gene (Figure 4A and 4B), the gene previously used for alanine production in heterologous bacteria.

Modification of E. coli W strain for homoalanine production

E. coli W strain SZ 194 (pflB frdBC adhE ackA) was previously constructed to produce only D-lactic acid. AU major fermentation pathways except lactate have been blocked in this strain by gene deletions. In order to convert this strain to the production of alanine, part of the native ldhA coding region was replaced by a DNA fragment containing the ribosomal-binding region, coding region and transcriptional terminator of alaD from G. stear other mophϊlus XL-65-6 (Figure 5A). The promoterless alaD was oriented in the same direction as ldhA to allow expression from the native ldhA promoter (Figure 1).

After electroporation, approximately 500 colonies were recovered with tetracycline- resistance and sensitivity to kanamycin, consistent with a double crossover event. These colonies were further examined by PCR using ldhA forward and reverse primer set (Table 3). Only 8 colonies were correct based on an analysis of PCR fragments. These 8 colonies were further verified using primer sets for alaD, ldhA forward and alaD reverse, alaD forward and ldhA reverse, ldhA outside primers (Table 3) and designated XZ103, XZ104, XZ105, XZ106, XZ107, XZ108, XZ109, and XZI lO, respectively. These 8 strains were initially tested in 15 ml screw-cap tubes containing NBS medium with 2% glucose and 100 mM ammonium sulfate, which were filled to the brim. Strain XZl 05 grew faster than the other strains (37⁰C for 48 h) and was selected for further development.

XZ 105 was transformed with pFT-A, which contains an inducible FLP recombinase (Martinez-Morales el al., 1999; Posfai et ah, 1997). The chromosomal FRT-flanked tet gene in XZ 105 was removed by inducing the FLP recombinase. After growing in 39°C to eliminate the temperature sensitive plasmid pFT-A, resulting strain was designated XZl I l. Expression of G. stearolhermophilus alaD in XZl I l is transcriptionally regulated by the ldhA promoter, the same promoter that regulates the production of lactate dehydrogenase (dominant fermentation pathway) in native E. coli.

pH-contr oiled batch fermentation for alanine production Alanine production by strain XZl I l was tested in 500 ml fermentation vessels containing 300 ml NBS medium, 20 g 1^-1 glucose, 100 mM ammonium sulfate and 1 mM betaine. Broth pH was automatically controlled by adding 2 N potassium hydroxide. After 96 h, 181 mM alanine was produced. The alanine yield from the glucose (total) was 81% (g/g), and the yield was 84% based on consumed glucose. The chiral purity of L-alanine was 96.1% (Table 5). Very low levels of other products (lactate, succinate, acetate, ethanol) were present, typically below 1 mM. This result demonstrated that the integrated G. stearothermophihis alaD gene as a single chromosomal copy under the control of the native ldhA promoter can provide sufficient levels of ALD to support E. coli growth from the production of alanine as the sole fermentation product.

Metabolic evolution of strain XZlIl Although XZl 11 could accumulate alanine as the primary product, incubation times were long and volumetric productivity was limited. When using a high glucose concentration (80 g 1^-1), growth and alanine productivity were further reduced (Table 5). In this strain, ATP production and growth are tightly coupled to NADH oxidation and alanine production by ALD (Figure 5B). This coupling provided a basis for strain improvement by selecting for increased growth during serial cultivation, ie. metabolic evolution. Cells with increased growth due to spontaneous mutations will successively displace their parents while co- selecting for increased alanine productivity.

Serial transfers of XZl I l were carried out at 24 h intervals in NBS mineral salts medium with 1 mM betaine. Cultures were first transferred in the medium containing 20 g 1^"' glucose, and the pH was controlled by automatically adding 2N potassium hydroxide. However, after 10 transfers to strain XZl 12, little improvement was observed (Figure 6). Since ammonia is essential for alanine production, it was thought that ammonia may be limiting for fermentation. 2 N potassium hydroxide containing 1 N ammonia carbonate and 5 N ammonia hydroxide alone were each tested for pH control during fermentations with XZl 12 in NBS medium with 50 g 1^" glucose. 5 N ammonia hydroxide was superior for cell growth and alanine production (Figure 7). The maximum cell mass was 0.41 g whe1n^-1 using 2 N potassium hydroxide and 0.397 g 1^-1 for 2 N potassium hydroxide with 1 N ammonia carbonate. The alanine concentration was 174 mM and 170 mM respectively after 4 days. However, when using 5 N ammonia hydroxide, the maximum cell mass was 0.767 g an1d^-1 the alanine concentration after 4 days was 387 mM (Figure 7). Thereafter, 5 N ammonia hydroxide was used in all the fermentations.

Strain XZl 12 was then transferred in NBS medium with 50 g 1^-1 glucose. The cell yield and alanine production increased continuously with the transfers (Figure 8). After transferring for 12 times, one single colony was isolated and designated XZl 13. Strain XZl 13 could complete the fermentation of 50 g 1^-1 glucose in 48 h, producing 483 mM alanine. The alanine yield from glucose was 86% (g/g), and the L-alanine purity was 97.0%.

Strain XZl 13 was then transferred in NBS medium with 80 g 1- g¹ lucose. After 25 transfers, XZl 15 was obtained (Figure 9). This strain completed the fermentation of 80 g 1 glucose in 60 h, producing 836 niM alanine. The alanine yield from glucose was 93% (g/g), and the L-alanine purity was 97.5%. Although succinate and ethanol were below 1 inM, low levels of lactate (5 mM), acetate (7 niM) were now present in the broth (Table 5).

Deletion of the methylglyoxal synthase gene (mgsA) to eliminate lactate and improve cell growth

The methylglyoxal bypass is an alternative pathway that produces lactate in E. coli

(Totemeyer et al, 1998; Weber et al, 2005; Grabar et al, 2006). Deletion of the mgsA gene encoding the first committed step, methylglyoxal synthase, has been shown to improve product yields and productivity and to increase the cell yield (Grabar et al., 2006). This pathway represented the most likely source of lactate in the fermentation broth of XZl 15.

Strain XZ121 was constructed by deletion of the mgsA gene. After 40 rounds of metabolic evolution to co-select additional mutations that increase growth and product formation

(Figure 10), a further improved strain was isolated and designated XZ123. XZ 123 completed the fermentation of 80 g 1^"' glucose in 48 h (832 mM alanine), 12 h faster than the parent strain XZl 15. The lactate concentration was below 1 mM although the acetate and succinate levels were slightly higher than in XZl 15 (Table 5). The alanine yield from glucose was 93%, and the L-alanine purity was 97.7%.

The effect of pH was tested with XZ 123 fermentations. Cell growth and alanine production were best at pH 7 (Figure 11). Cell growth was slower at pH 6.5 and pH 7.5 although fermentations were complete for all within 60 h. XZ123 did not grow at pH 8.0 in this medium with ammonium hydroxide for pH control.

Different inoculation levels were also tested. Cell growth and alanine production were similar when inocula were varied between 0.006 and 0.03 g (1F^-1igure 12). However, when the initial inoculation increased to 0.083 and 0.17 g , f1-e¹rmentations were completed within

36 h (Figure 12).

Deletion of the major alanine racemase gene (dadX) to increase L-alanine purity

E. coli has two distinct alanine racemase genes, which are responsible for the conversion between L-alanine and D-alanine (Wild et al., 1985). One racemase encoded by the air gene is constitutive (Wild et ah, 1985) and less abundant than the racemase encoded by dadX (Neidhardt et al., 1996). DADX is a catabolic enzyme that is induced by D- or L- alanine in the absence of glucose. To increase the chiral purity of L-alanine, dadX was deleted in XZ 123 to make strain XZ 126. After 30 transfers to allow metabolic evolution, strain XZ129 was isolated (Figure 13). Strain XZ129 completed the fermentation of 80 g 1^-1 glucose within 42 h, and accumulated 833 mM alanine with a yield of 93%. The chiral purity of L-alanine increased to 99.4% (Table 5). Cell yield and volumetric productivity of alanine were 6-fold and 11 -fold greater, respectively, than with the parent strain XZl 11 (Table 6).

Fermentation on AMI medium

A low salts mineral medium, AMI, was recently developed in our laboratory that is optimized for anaerobic fermentations of E. coli (Martinez et ah, 2007). This medium was tested for alanine production after serially transferring strain XZ 129 for 14 times (Figure 14). The resulting strain, designated XZl 30, completed the fermentation of 80 g gluc1-o¹se within 42 h, and accumulated 847 mM alanine with the yield of 94%. The cell yield and volumetric productivity was 33% and 3% greater, respectively, than XZ 129 in NBS medium (Table 6).

Strain XZl 30 was serially transferred in AMI medium with 100 g 1^-1 glucose for 16 times and the resulting strain designated XZl 31 (Figure 15). This strain was serially transferred 30 times in 120 g 1^-1 glucose for 30 times to produce XZ132 (Figure 16). Strain XZ132 completed the fermentation of 120 g 1^" glucose within 48 h, and accumulated 1279 mM alanine (114 g 1^-1) with a yield of 95% (Table 3). Cell yield and alanine volumetric productivity were 10 and 30 times greater, respectively than the parent strain XZl I l (Table 6).

Fermentation of the pentose sugar, xylose (50 g )1 w^-1as also tested in AMI medium. With xylose, strain XZl 32 produced 483 mmol of L-alanine (99.5% chiral purity) in 72 h with a yield of 86% by weight. Further improvement for xylose fermentation to L-alanine would be expected based on metabolic evolution although this was not investigated. Thus strain XZ 132 could be used as a biocatalyst for the conversion of biomass-derived hexose and pentose sugars to L-alanine.

Discussion

Previous microorganisms engineered for L-alanine production have many disadvantages. In early strains (Table 1), fermentation times were quite long with low yields of L-alanine and high levels of co-products. Recently, E. coli ALS929(pTrc99A-α/αD) was constructed which lacked pyruvate formatelyase, phosphoenolpyruvate synthase, pyruvate oxidase, lactate dehydrogenase, components of the pyruvate dehydrogenase complex and expressed B. sphaericus IFO3525 alciD (Smith et ah, 2006). This strain produced high titers and yields of alanine but required complex processes (aerobic growth phase and anaerobic production phase) and produced only racemic alanine. In this strain, multiple copies of the plasmid-borne B. sphaericus alaD gene are expressed from a /αc/-regulated artificial promoter (Vtrc). All prior biocatalysts that produced high titers and yields of alanine used multi-copy plasmids for the high level expression of recombinant alanine dehydrogenase genes from mesophilic bacteria, antibiotics for plasmid maintenance, and required complex medium supplements such as casamino acids and yeast extract. Strains that produced chirally pure L-alanine typically required supplementation with D-alanine. These media requirements increase the cost of materials, purification, chiral separations, and waste disposal. Multi-step aerobic and anaerobic (fed batch) processes may also increase capital costs.

We have investigated an alternative approach for the production of chirally pure L- alanine using a thermophilic ALD {alaD) from G. stearothermophilus and a highly engineered E. coli biocatalyst designed for lactate production as the host for this heterologous gene. The deduced amino acid and nucleotide sequences of the B. sphaericus and G. stearothermophilus alaD genes are quite different. For the conversion of pyruvate to alanine, the kinetic properties of the G. stearothermophilus ALD would appear less favorable than those of the B. sphaericus ALD. The G. stearothermophilus enzyme is only half as active with an 8-fold higher Km for NADH (0.08 mM) and 2-fold higher Km for pyruvate (5.0 mM) in comparison to the B. sphaericus enzyme (Ohashima and Soda, 1979; Sakamoto et ah, 1990). G. stearothermophilus XL-65-6 was selected as a source of alaD based on our previous successes in stably expressing genes from this organism in E. coli with minimal effects on growth (Lai and Ingram, 1993, Lai and Ingram, 1995). Unlike all prior biocatalysts for alanine, the alaD gene was integrated under the control of the chromosomal ldhA promoter, the promoter which controls expression of the dominant fermentative enzyme in native E. coli. In this way, alaD expression would be expected to mimic native regulation of fermentation processes. In this construct, ATP production for growth was coupled to NADH oxidation using alanine dehydrogenase, providing a growth-based selection for strain improvements by metabolic evolution (Figure 5B). Additional mutations were added to improve growth (mgsA deletion) and chiral purity (dadX deletion) that have not previously been employed in the development of biocatalysts for L-alanine production. Figure 17 provides a summary of the steps involved in strain construction. The combination of different construction strategies, deliberate mutations, use of the thermostable alaD gene, absence of plasmids, and the extensive use of metabolic evolution and growth-based selection have resulted in a uniquely productive biocatalyst, strain XZl 32. During simple batch fermentation in mineral salts medium (~4 g 1 s^-1alts) , this strain can produce over 1200 mmol of L-alanine from 12% glucose in 48 h with a maximum volumetric productivity of over 4 g alanine T¹Ii^"1 (Table 6). After cell removal, dried broth from this fermentation would be approximately 95% (w/w) L-alanine without further purification.

These results demonstrate that a chromosomally integrated copy of the alaD gene can provide sufficient levels of expression to support high levels of glycolytic flux (>4 g glucose T¹Ii^"1) without the need for multi-copy plasmids and selection agents for plasmid retention. Such approaches could prove generally useful for the development of new biocatalysts for many other products. This strain like other derivatives of E. coli can effectively ferment both hexose and pentose sugars.

With minor modifications, the L-alanine producing strains in this study could be used as a highly efficient route to produce pyruvate using enzymatic hydrolysis. In E. coli, L- alanine is catabolized to pyruvate by a two step process. The DADX racemase interconverts the L and D forms, followed by conversion to pyruvate plus ammonia by a membrane-bound D-amino acid dehydrogenase (Denu and Fitzpatrick, 1992; Lobocka et ah, 1994). This reaction could be accelerated and driven to completion by stripping with air and/or reduced pressure to facilitate removal of ammonia (Figure 18). In this way, sugars would be converted to pyruvate with higher efficiencies (using alanine as an intermediate) than previously achieved during direct fermentation of sugars to pyruvate (Causey et ah, 2004).

All patents, patent applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

^a NBS + 1 mM betaine: NBS media amended with betaine (1 mM). ^b Calculation includes KOH used to neutralize betaine-HCl stock. ^c Trace metal stock (1000X) was prepared in 120 mM HCl. OO

1 mM betaine was added to all media.

Yields are based on grams of alanine produced from total glucose; the maximum theoretical yield for alanine is 0.989 gram per gram of glucose (2 mol of alanine per mol of glucose).

Yields in parenthesis are based on grams of alanine produced per gram of glucose consumed.

^a 1 raM betaine was added to all media. b Maximum volumetric productivity and maximum specific productivity were calculated based on the most productive 12 h or 24 h period.

Average volumetric productivity was calculated based on total alanine produced per total fermentation time. d The maximum volumetric productivity and specific productivity of XZl 11 in NBS medium was based on 0-24 h.

The maximum volumetric productivity and specific productivity of XZ129 and XZ132 was based on 24-36 h.

The maximum volumetric productivity of XZl 30 was based on 12-24 h. g The maximum specific productivity of XZl 30 was based on 0-12 h.

The maximum volumetric productivity and specific productivity of XZl 11 in AMI medium was based on 72-96 h.

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Claims

CLAIMSWe claim:

1. A genetically modified microorganism that comprises the inactivation or deletion of a lactate dehydrogenase gene and the integration of an alanine dehydrogenase gene and the following genetic modifications: a) the inactivation or deletion of a pyruvate formatelyase gene; b) the inactivation or deletion of an alcohol dehydrogenase gene; c) the inactivation or deletion of an acetate kinase gene; d) the inactivation of one or more fumarate reductase subunit genes by insertion or deletion; e) the inactivation or deletion of an alanine racemase gene; and f) the inactivation or deletion of a methylglyoxal synthase gene.

2. The genetically modified microorganism according to claim 1, wherein the integrated alanine dehydrogenase gene is a heterologous alanine dehydrogenase gene.

3. The genetically modified microorganism according to claim 1 or 2, wherein the genetic ally modified microorganism is Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibaclerium ammoniagenes, divaricalum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophihim, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Escherichia coli, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus retlgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformcms, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kilasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmuller ; or Xanthomonas citri.

4. The genetically modified microorganism according to claim 3, wherein said genetically modified microorganism is Escherichia coli.

5. The genetically modified microorganism according to claim 3, wherein said genetically modified microorganism further comprises inactivated or deleted antibiotic resistance genes.

6. The genetically modified microorganism according to claim 3, wherein the lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene.

7. The genetically modified organism according to claim 3, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

8. The genetically modified organism according to claim 3, wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

9. The genetically modified microorganism according to any preceding claim, wherein said genetically modified microorganism is metabolically evolved.

10. The genetically modified microorganism according to any preceding claim, wherein any antibiotic resistance genes are deleted with FLP recombinase.

11. A genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxytoca casAB gene or other genes encoding cellobiose utilizing enzyme II and phospho-β-glucosidase behind the stop codon of lacY; b) integration of an Erwinia chrysanlhemi celY gene or any other endoglucanase or cellulase encoding gene into one or more fumarate reductase subunit genes or inactivation of one or more fumarate reductase subunit genes by insertion or deletion; c) inactivation or deletion of an alcohol dehydrogenase gene; d) inactivation or deletion of a pyruvate formatelyase gene; e) inactivation or deletion of an acetate kinase gene; f) inactivation or deletion of a lactate dehydrogenase gene; g) integration of an alanine dehydrogenase gene; h) inactivation or deletion of a methylglyoxal synthase gene; and i) inactivation or deletion of an alanine racemase gene.

12. The genetically modified microorganism according to claim 11, wherein the integrated alanine dehydrogenase gene is a heterologous alanine dehydrogenase gene.

13. The genetically modified microorganism according to claim 11 or 12, wherein the genetic ally modified microorganism is Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacler lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, A∑otobacler indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glulamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Escherichia coli, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmuller,or Xanthomonas citri.

14. The genetically modified microorganism according to claim 13, wherein said genetically modified microorganism is Escherichia coli.

15. The genetically modified microorganism according claim 13, wherein said genetically modified microorganism further comprises inactivated or deleted antibiotic resistance genes.

16. The genetically modified microorganism according to claim 11, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene are inactivated or deleted in said genetically modified microorganism after insertion.

17. The genetically modified microorganism according to claim 13, wherein the cellobiose utilizing enzyme II, phospho-β-glucosidase and/or Erwinia chrysanthemi celY gene have not been inserted into said microorganism and one or more of the fumarate reductase subunit genes is inactivated by insertion or deletion.

18. The genetically modified microorganism according to claim 13, wherein the lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene.

19. The genetically modified organism according to claim 13, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

20. The genetically modified organism according to claim 13, wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.

21. The genetically modified microorganism according to any one of claims 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein said genetically modified microorganism is metabolically evolved.

22. The genetically modified microorganism according to any one of claims 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21, wherein antibiotic genes are deleted with FLP recombinase.

23. A method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to any one of the preceding claims and culturing or growing said genetically modified microorganism.

24. A method of producing L-alanine comprising culturing one or more genetically modified microorganism according to any one of the preceding claims under conditions that allow for the production of alanine.

25. The method according to any one of claims 23 or 24, wherein said genetically modified microorganism is cultured in a mineral salts medium.

26. The method according to claim 25, wherein the mineral salts medium comprises between 2% and 20% (w/v) of a sugar.

27. The method according to claim 26, 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.

28. The method according to claim 26 or 27, wherein the sugar is glucose or sucrose or a combination of glucose, sucrose, pentose and xylose.

29. The method according to any one of claims 23, 24, 25, 26, 27 or 28, wherein chirally pure alanine is produced.

30. The method according to claim 23, 24, 25, 26, 27, 28 or 29, wherein the yield of alanine is at least 90%.

31. The method according to claim 30, 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%.

32. The method according to claims 23, 24, 25, 26, 27, 28, 29, 30 or 31, wherein there is no detectable contamination of one stereoisomeric form of alanine with the other stereoisomeric form or the chiral purity of L-alanine is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

33. A composition comprising one or more genetically modified microorganism according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 and medium.