WO2011102305A2

WO2011102305A2 - A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE Enterobacteriaceae FAMILY HAVING A MUTANT ADENYLATE CYCLASE

Info

Publication number: WO2011102305A2
Application number: PCT/JP2011/052967
Authority: WO
Inventors: Natalia Sergeevne Eremina; Natalia Viktorovna Stoynova; Tatyana Abramovna Yampolskaya
Original assignee: Ajinomoto Co Inc
Current assignee: Ajinomoto Co Inc
Priority date: 2010-02-18
Filing date: 2011-02-04
Publication date: 2011-08-25
Anticipated expiration: 2012-08-18
Also published as: RU2471868C2; WO2011102305A3; RU2010105710A

Abstract

A method for producing an L-amino acid from ethanol or glycerol using a bacterium of the Enterobacteriaceae family, which contains mutant adenylate cyclase, is described.

Description

DESCRIPTION

A METHOD FOR PRODUCING AN L- AMINO ACID USING A BACTERIUM OF THE Enterobacteriaceae FAMILY HAVING A MUTANT ADENYLATE

CYCLASE

Technical field

The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the

Enterobacteriaceae family which has a mutant adenylate cyclase.

Background art

Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (U.S. Patent No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to feedback inhibition by the resulting L-amino acid (U.S. Patent Nos. 4,346,170, 5,661,012 and 6,040,160).

Mutants of E. coli K-12 deficient in adenylate cyclase (Cya) and catabolite activator protein (Crp) have been shown to grow more slowly than their parent strains in glucose-minimal medium. Their growth rate decreased markedly with increasing pH between 6 and 7.8. It was shown that this pH sensitivity is a direct consequence of the cya mutation, because a mutation to pH resistance also restored ability to ferment a variety of sugars. The proton motive force-dependent uptake of proline and glutamate was also reduced and sensitive to pH in the cya mutant. The membrane-bound ATPase activity was normal. The rate of oxygen uptake by cells, although reduced, was pH insensitive (Ahmad D, Newman EB. J Bacteriol.;170(8):3443-7 (1988)).

Adenylate cyclase deficient {cya) mutants of E. coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin. E. coli glpT mutants were resistant to fosfomycin and also to fosmidomycin. This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT. DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP (Sakamoto Y, et al., Biosci Biotechnol

Biochem.;67(9):2030-3(2003)).

But currently, there have been no reports of using bacterium having mutant adenylate cyclase for the purpose of producing L-amino acids.

SUMMARY OF THE INVENTION

Aspects of the present invention include enhancing the productivity of L-amino acid-producing strains and providing a method for producing non-aromatic or aromatic L-amino acids using these strains.

The above aspects were achieved by finding that mutation in adenylate cyclase can result in enhancing production of L-amino acids, such as L-threonine, L-lysine, L- cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L- serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L- proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

It is an aspect of the present invention to provide a bacterium of the

Enterobacteriaceae family having an increased ability to produce amino acids, such as L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L- histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L- glutamic acid, L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide mutant adenylate cyclase selected from the group consisting of:

(A) a protein in which L-lysine at a position corresponding to position 432 in SEQ ID NO: 2 is replaced with another L-amino acid residue; and

(B) a variant of protein (A), which has an activity of adenylate cyclase.

It is a further aspect of the present invention to provide adenylate cyclase as described above, wherein L-lysine at a position corresponding to position 432 in SEQ ID NO: 2 is replaced with L-glutamine.

It is a further aspect of the present invention to provide a DNA coding for mutant adenylate cyclase as described above.

It is a further aspect of the present invention to provide a bacterium of

Enterobacteriaceae family, which contains the DNA as described above.

It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has an ability to produce L-amino acids. It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium belongs to the genus Escherichia.

It is a further aspect of the present invention to provide a method for producing L-amino acid comprising cultivating the bacterium as described above in a culture medium containing glycerol or ethanol, and collecting the L-amino acid from the culture medium.

It is a further aspect of the present invention to provide the method as described above, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L- phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the method as described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

1. Mutant adenylate cyclase and DNA fragment encoding thereof of the present invention.

The cyaA gene (synonyms: ECK3800, b3806) encodes the Cya protein, adenylate cyclase (synonym B3806). The cyaA gene (nucleotides from 3,989,176 to 3,991,722; GenBank accession no. NC_000913.2; gi: 49175990) is located between the gene hemC, oriented in the opposite direction, and the gene cyaY, oriented in the opposite direction, on the chromosome of E. coli K-12. The nucleotide sequence of the cyaA gene and the amino acid sequence of Cya encoded by the cyaA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The phrase "adenylate cyclase activity" means the activity of catalyzing the synthesis of cyclic AMP (cAMP) by an intramolecular transfer of the adenylyl group of ATP to the 3 '-hydroxy group, releasing pyrophosphate. The adenylate cyclase activity assay can be carried out, for example, using the method described by Yang J.K. and Epstein W. (J Biol Chem.; 258(6):3750-8(1983)).

The adenylate cyclase in which L-lysine at a position corresponding to position 432 in SEQ ID NO: 2 is replaced with another L-amino acid residue, may be referred to as "the mutant adenylate cyclase", a DNA coding for the mutant adenylate cyclase may be referred to as "the mutant cyaAgene" or "mutant adenylate cyclase gene", and an adenylate cyclase without the substitution may be referred to as "a wild-type adenylate cyclase".

A position of an amino acid residue may change. For example, if an amino acid residue is inserted at an N-terminus portion, the amino acid residue inherently locates at the position 432 becomes position 433. In such a case, the amino acid residue corresponding to the original position 432 is designated as the amino acid residue at the position 432 in the present invention.

To determine the L-amino acid residue corresponding to the mutation 432 of adenylate cyclase, it is necessary to make alignment of the amino acid sequence of adenylate cyclase from E. coli and an amino acid sequence of adenylate cyclase from the bacterium of interest, and define the L-amino acid corresponding to number 432 in the adenylate cyclase from the bacterium of interest.

Another L-amino acid which can substitute for L-lysine at the position corresponding to position 432 is not particularly limited so long as the amino acid is other than L-lysine, and a bacterium containing the mutant adenylate cyclase having the mutation has a productivity of L-amino acid higher than that of a bacterium which does not contain the mutant adenylate cyclase. An example of the other amino acid is glutamine.

Since there may be some differences in DNA sequences between the genera, species or strains of the Enterobacteriaceae family, the cyaA gene is not limited to the gene shown in SEQ ID No: 1, but may include genes homologous to SEQ ID No: 1. Therefore, the protein variant encoded by the cyaA gene may have a homology of not less than 80%, or not less than 90%, or not less than 95 %, or not less than 98%, or even not less than 99% with respect to the entire amino acid sequence shown in SEQ ID NO. 2, as long as the activity of the Cya protein is maintained. The phrase "protein variant" as used in the present invention means proteins which have changes in the sequences, whether they are deletions, insertions, additions, or substitutions of amino acids. The number of changes in the variant proteins depends on the position in the three dimensional structure of the protein or the type of amino acid residues. It may be 1 to 30, 1 to 15, or even 1 to 5 in SEQ ID NO: 2. These changes in the variants can occur in regions of the protein which are not critical for the three dimensional structure of the protein. This is because some amino acids have high homology to one another so the three dimensional structure is not affected by such a change.

Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity, and similarity. The term "homology" can mean "identity". The substitution, deletion, insertion or addition of one or several amino acid residues can be conservative mutation(s) so that the activity is maintained. The representative conservative mutation is a conservative substitution. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution of Asn, Glu or Gin for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gin, substitution of Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for He, substitution of He, Met, Val or Phe for Leu, substitution of Asn, Glu, Gin, His or Arg for Lys, substitution of He, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, He or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, He or Leu for Val.

Therefore, the cyaA gene may be a variant which hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 1, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional Cya protein. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 80%, not less than 90%, not less than 95%), not less than 97%, not less than 98%, or even not less than 99% is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1 xSSC, 0.1% SDS, or 0.1 SSC, 0.1% SDS at 60°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times. The length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.

Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

2. Bacterium of the present invention

The bacterium of the present invention is an L-amino acid-producing bacterium of the Enter obacteriaceae family, wherein the bacterium has mutant adenylate cyclase. The bacterium according to the present invention can be obtained by introduction in the cell of the bacterium a DNA fragment encoding mutant adenylate cyclase according to the present invention.

"L-amino acid-producing bacterium" can mean a bacterium which has an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.

The term "L-amino acid-producing bacterium" as used herein can also mean a bacterium which is able to produce and cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of E. coli, such as E. coli K-12, and preferably means that the microorganism is able to cause accumulation in a medium of an amount, for example, not less than 0.5 g/L, or not less than 1.0 g/L, of the target L-amino acid. The term "L-amino acid" includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L- valine.

The term "aromatic L-amino acid" includes L-phenylalanine, L-tyrosine, and L-tryptophan. The term "non-aromatic L-amino acid" includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L- serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L- proline, and L-arginine. L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L- glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine are particularly examples.

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. Specifically, those classified into the Enterobacteriaceae according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database

(http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&lv l=3&keep^:=l&srchmode=l&unlock) can be used. A bacterium belonging to the genus Escherichia or Pantoea is an example.

The phrase "a bacterium belonging to the genus Escherichia!^'' means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. coli).

The bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited; however, for example, bacteria described by Neidhardt, F.C. et al. {Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) are encompassed by the present invention.

The phrase "a bacterium belonging to the genus Pantoea" means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)). A bacterium belonging to either Enterobacter or Pantoea can be used so long as it is classified as the family Enterobacteriaceae.

L-amino acid-producing bacteria

As a bacterium of the present invention which has mutation in the cyaA gene, bacteria which are able to produce either an aromatic or a non-aromatic L-amino acid may be used.

The bacterium of the present invention can be obtained by modifying a bacterium which inherently has the ability to produce L-amino acids so that the bacterium contains the mutant adenylate cyclase. Alternatively, the bacterium of present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium which already contains the mutant adenylate cyclase . The bacterium can contain a wild-type adenylate cyclase as long as the bacterium contains the mutant adenylate cyclase. However, it is preferable that the bacteirum does not contain the wild-type adenylate cyclase.

In order to modify a bacterium so that the bacterium contains the mutant adenylate cyclase, the following methods can be used. A gene encoding the mutant adenylate cyclase can be prepared by modifying a wild-type cyaA gene so that the L- amino acid residue corresponding to position 432 in SEQ ID NO: 2 is replaced by another amino acid residue, and then a bacterium of the Enterobacteriaceae family can be transformed with a DNA containing the mutant gene to cause recombination of a corresponding gene on the genome with the mutant gene to substitute the mutant gene for the objective gene on the genome. Examples of such gene substitution using homologous recombination include methods of using a linear DNA such as the method called Red-driven integration (Datsenko, K.A, and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97:6640-6645 (2000)), and the method utilizing the Red-driven integration in combination with an excisive system derived from λ phage (Cho, E.H., Gumport, R.I., Gardner, J.F., J. Bacteriol., 184:5200-5203 (2002)) (refer to WO2005/010175), a method of using a plasmid containing a temperature-sensitive replication origin (U.S. Patent No. 6,303,383, Japanese Patent Laid-open No. 05-007491), and so forth.

Furthermore, such site-specific mutagenesis based on gene substitution using homologous recombination can also be performed by using a plasmid which is not able to replicate in a host. In the bacterium of this invention, glycerol or ethanol

assimilability can be enhanced.

In the bacterium, in order to enhance glycerol assimilability, expression of the glpR gene (EP 1715056) may be attenuated, or expression of the glycerol metabolism genes (EP 1715055 A) such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, tpiA, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and talC genes may be enhanced. A gene which encodes a mutant glycerol kinase resistant to inhibition by fructose- 1 ,6-bisphosphate may be preferably enhanced (Pettigrew, D. W., Liu, W. Z., Holmes, C, Meadow, N. D., and Roseman, S., J. Bacteriol. 178, 10, 2846-52 (1996), Honisch, C. et.al., Genome Reseasch, 14: 2495-2502 (2004), WO2008/081959 and WO2008/107277). Furthermore, activities of glycerol dehydrogenase and

dihydroxyacetone kinase may be enhanced (WO2008/102861).

In the bacterium, in order to enhance etthanol assimilability, the gene encoding alcohol dehydrogenase (adhE) is modified to expressunder the control of a non-native promoter which functions under aerobic cultivation conditions. (WO2008/010565)

In the present invention, a bacterial strain used for producing an L-amino acid is modified so that expression of the adhE gene is controlled by a non-native promoter, i.e., a promoter that does not control the expression of the adhE gene in a wild-type strain. Such modification can be achieved by replacing the native promoter of the adhE gene on the choromosome with a non-native promoter which functions under an aerobic cultivation condition so that the adhE gene is operably linked with the non- native promoter. As a non-native promoter which functions under aerobic cultivation conditions, any promoter which can express the adhE gene above a certain level under aerobic cultivation conditions may be used. With reference to the level of the alcohol dehydrogenase (AdhE) protein in the present invention, the activity of alcohol dehydrogenase in the cell free extract measured according to the method by Clark and Cronan (J. Bacteriol. 141 177-183 (1980)) can be, for example, 1.5 units or more, 5 units or more, or 10 units or more, per mg of protein. Aerobic cultivation conditions can be those usually used for cultivation of bacteria in which oxygen is supplied by methods such as shaking, aeration and agitation. Specifically, any promoter which is known to express a gene under aerobic cultivation conditions can be used. For example, promoters of the genes involved in glycolysis, the pentose phosphate pathway, TCA cycle, amino acid biosynthetic pathways, etc. can be used. In addition, the P_tac promoter, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known to be strong promoters which function under aerobic cultivation conditions, and are preferably used.

A wild-type alcohol dehydrogenase may be subject to metal catalyzed oxidation. Although such a wild-type alcohol dehydrogenase can be used, a mutant alcohol dehydrogenase which is resistant to aerobic inactivation is preferable in the present invention. The phrase "mutant alcohol dehydrogenase which is resistant to aerobic inactivation" means that the mutant alcohol dehydrogenase maintains its activity under aerobic conditions, or the activity is reduced by a negligible amount compared to the wild-type alcohol dehydrogenase.

For example, mutant alcohol dehydrogenase which is resistant to aerobic inactivation can be used in the following:

a) a protein in which Glutamic acid at a position corresponding to the position 568 in the E. coli AdhE is replaced with another amino acid residue, such as Lysine. (WO2008/010565)

b) a protein in which Glutamic acid at a position corresponding to the position 560 in the E. coli AdhE is replaced with another L-amino acid residue, such as Lysine. c) a protein in which phenylalanine at a position corresponding to the position 566 in the E. coli AdhE is replaced with another amino acid residue, such as Valine. d) a protein in which phenylalanine at a position corresponding to the position 566 in the E. coli AdhE is replaced with another amino acid residue, such as Valine. e) a protein in which the glutamic acid residue, the Methionine residue, the yyrosine residue, the isoleucine residue, and the alanine residues at positions 22, 236, 461, 554, and 786, respectively, in E. coli AdhE are replaced with another amino acid residue, e.g., a glycine residue, a valine residue, a cysteine residue, a serine residue, and a valine residue, respectively; and

f) combinations thereof.

L-threonine-producing bacteria

Examples of parent strains for deriving the L-threonine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5, 175, 107, U.S. Patent No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Patent No.5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP- 3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like. The strain TDH-6 is deficient in the thrC gene, as well as being sucrose- assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSF1010- derived vector. This mutant thrA gene encodes aspartokinase homoserine

dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on November 19, 1987 in the Ail-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on April 7, 1987 under the accession number B-3996.

E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain for deriving L-threonine-producing bacteria of the present invention. The strain B-5318 is prototrophic with regard to iso leucine, and a temperature-sensitive lambda-phage CI repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number of VKPM B-5318.

Preferably, the bacterium of the present invention is additionally modified to enhance expression of one or more of the following genes:

the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;

the thrB gene which codes for homoserine kinase;

the thrC gene which codes for threonine synthase;

the rhtA gene which codes for a putative transmembrane protein;

the asd gene which codes for aspartate-p-semialdehyde dehydrogenase; and

the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);

The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no.NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession no.NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession no.NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K-12. All three genes function as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808,

WO2003/097839).

A mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40 which is present in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is located between the pexB and ompX genes. The unit expressing a protein encoded by the ORF1 has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it was revealed that the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17^th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for

Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.

Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession no. NC_000913.1, gi: 16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.

L-lysine-producing bacteria

Examples of L-lysine-producing bacteria belonging to the genus Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine coexists in a medium. Examples of the L- lysine analogue include, but are not limited to, oxalysine, lysine hydroxamate, S-(2- aminoethyl)-L-cysteine (AEC), γ-methyllysine, a-chlorocaprolactam and so forth. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Patent No. 4,346,170) and Escherichia coli VL61 1. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.

The strain WC196 may be used as an L-lysine producing bacterium of

Escherichia coli. This bacterial strain was bred from the W3110 strain, which was derived from Escherichia coli K-12, by replacing the wild type lysC gene on the chromosome of the W31 10 strain with a mutant lysC gene encoding a mutant aspartokinase III desensitized to feedback inhibition by L-lysine in which threonine at position 352 had been replaced with isoleucine, and conferring AEC resistance to the resulting strain (U.S. patent No. 5,661 ,012).. The resulting strain was designated Escherichia coli AJ13069 strain and was deposited at the National Institute of

Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology,

International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December 6, 1994 and received an accession number of FERM P- 14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on September 29, 1995, and received an accession number of FERM BP-5252 (U.S. Patent No. 5,827,698).

Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Patent No.

6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde

dehydrogenase (asd), and aspartase (aspA) (EP 1253195 A). In addition, the parent strains may have increased expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof. Examples of parent strains for deriving L-lysine-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine. Examples of the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme

(WO2005/010175).

L-cysteine-producing bacteria

Examples of parent strains for deriving L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli JM15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Patent No. 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Patent No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity

(JP11155571 A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WOO 127307 A 1), and the like.

L-leucine-producing bacteria

Examples of parent strains for deriving L-leucine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Patent No. 6,124,121)) or leucine analogs including β-2- thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.

The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which are preferably represented by a mutant leuA gene coding for isopropylmalate synthase which is free from feedback inhibition by L- leucine (US Patent 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (EP 1239041 A2). L-histidine-producing bacteria

Examples of parent strains for deriving L-histidine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116 - B12121 (U.S. Patent No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U,S. Patent No. 6,258,554) and the like.

Examples of parent strains for deriving L-histidine-producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisl), phosphoribosyl-ATP pyrophosphohydrolase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation into ATP

phosphoribosyltransferase which imparts resistance to the feedback inhibition (Russian Patent Nos. 2003677 and 21 19536).

Specific examples of strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been introduced with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains introduced with rht, a gene for an amino acid-export (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL-l,2,4-triazole-3 -alanine, and streptomycin- resistance (VKPM B-7270, Russian Patent No. 21 19536), and so forth.

L-glutamic acid-producing bacteria

Examples of parent strains for deriving L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC⁺ (EP 1172433). E. coli VL334 (VKPM B- 1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in thrC and ilvA genes (U.S. Patent No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage PI which was grown on wild-type E. coli K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains which are deficient in a- ketoglutarate dehydrogenase activity, or strains in which one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of the genes involved in L-glutamic acid biosynthesis include genes encoding glutamate

dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, IpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (end), phosphoglyceromutase (pgmA, pgmT),

phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (g pA), triose phosphate isomerase (tpiA), fructose bisphosphate aldolase (fbp),

phosphofructokinase (pflcA, pflcB), and glucose phosphate isomerase (pgi).

Examples of strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced include those disclosed in EP1078989A, EP955368A, and EP952221A.

Examples of strains which have been modified so that expression of the citrate synthetase gene and/or the phosphoenolpyruvate carboxylase gene are reduced, and/or/are deficient in a-ketoglutarate dehydrogenase activity include those disclosed in EP1078989A, EP955368A, and EP952221A.

Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention also include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase (aceA), -ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvT), formate acetyltransferase (pfl), lactate dehydrogenase (Idh), and glutamate decarboxylase (gadAB). Bacteria belonging to the genus Escherichia deficient in the α-ketoglutarate dehydrogenase activity or having a reduced a- ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Patent Nos. 5,378,616 and 5,573,945. Specifically, these strains include the following:

E. coli W3110sucA::Km^R

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854) E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km^R is a strain obtained by disrupting the a-ketoglutarate dehydrogenase gene (hereinafter referred to as "sucA gene") of E. coli W3110. This strain is completely deficient in the a-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807) (U.S. Patent No. 5,908,768), FFRM P- 12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Patent No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Patent No. 6,1 10,714), and the like.

Examples of L-glutamic acid-producing bacteria include mutant strains belonging to the genus Pantoea which are deficient in the a-ketoglutarate

dehydrogenase activity or have a decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains include Pantoea ananatis AJ13356. (U.S. Patent No. 6,331,419). Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on February 19, 1998 under an accession number of FERM P-16645. It was then converted to an international deposit under the provisions of Budapest Treaty on January 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in the α-ketoglutarate dehydrogenase activity as a result of disruption of the ocKGDH-El subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter

agglomerans, for the purposes of this specification, they are described as Pantoea ananatis.

L-phenylalanine-producing bacteria

Examples of parent strains for deriving L-phenylalanine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::Tnl0, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Patent No. 5,354,672); E. coli MWEClOl-b (KR8903681); E. coli NRRL B-12141, NRRL B- 12145, NRRL B-12146 and NRRL B-12147 (U.S. Patent No. 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12

[W3110 (tyrA)/pPHAD] (FERM BP- 12659), E. coli K-12 [W31 10 (tyrA)/pPHATerm] (FERM BP- 12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488424 Bl). Furthermore, L- phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 Al and 2003/0157667 Al).

L-tryptophan-producing bacteria

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) which is deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Patent No. 5,756,345); E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Patent No. 6,180,373); E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Patent No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a

phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Patent No. 6,319,696), and the like may be used. L-tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene may also be used (U.S. patent applications

2003/0148473 Al and 2003/0157667 Al).

Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains in which one or more activities of the enzymes selected from anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include a E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase. Examples of parent strains for deriving the L-tryptophan-producing bacteria of the present invention also include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Patent No. 4,371,614). Moreover, L-tryptophan- producing ability may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of a and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon

(WO2005/103275).

L-proline-producing bacteria

Examples of parent strains for deriving L-proline-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline (EP 1172433). The bacterium of the present invention may be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes for L-proline producing bacteria which are preferred include the proB gene coding for glutamate kinase of which feedback inhibition by L-proline is desensitized (DE Patent 3127361). In addition, the bacterium of the present invention may be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from bacterial cell. Such genes are exemplified by b2682 and b2683 genes (ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B- 12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F.R. et al (The 15^th Miami winter symposium, 1983, p. 34), and the like.

L-arginine-producing bacteria

Examples of parent strains for deriving L-arginine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus

Escherichia, such as E. coli strain 237 (VKPM B-7925) (U.S. Patent Application 2002/058315 Al) and its derivative strains harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B- 7926) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EP1 170361A1), and the like.

Examples of parent strains for deriving L-arginine producing bacteria of the present invention also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase (carAB).

L-valine-producing bacteria

Examples of parent strains for deriving L-valine-producing bacteria of the present invention include bacteria belonging to the genus Escherichia such as H-81 (VKPM B- 8066), NRRL B-12287 and NRRL B-12288 (US patent No. 4,391 ,907), VKPM B-441 1 (US patent No. 5,658,766), VKPM B-7707 (European patent application EP1016710A2), or the like.

Example of parent strains for deriving L-valine-producing bacteria of the present invention include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon (U.S. Patent No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.

Examples of parent strains for deriving L-valine-producing bacteria of the present invention also include mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Patent No. 5,658,766). For example, E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL1970 has been deposited in the Russian National Collection of Industrial

Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on June 24, 1988 under accession number VKPM B-441 1.

Furthermore, mutants requiring lipoic acid for growth and/or lacking H⁺- ATPase can also be used as parent strains ( WO96/06926).

L-isoleucine-producing bacteria

Examples of parent strains for deriving L-isoleucine producing bacteria of the present invention include, but are not limited to, mutants having resistance to 6- dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L- isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, can also be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Patent No.

5,998,178).

L-Methionine-producing bacteria

Examples of L-methionine-producing bacteria and parent strains for deriving L-methionine producing bacteria include, but are not limited to, L-threonine- auxotrophic mutant strain and norleucine-resistant mutant strain (JP 2000-139471 A). Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine transsuccinylase and cystathionine γ-synthase (JP 2000-139471 A) can also be used as parent strains.

2. Method of the present invention

The method of the present invention is a method for producing an L-amino acid comprising cultivating the bacterium of the present invention in a culture medium to produce and excrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.

In the method of the present invention, Glycerol and/or Ethanol can be used as a carbon source in the fermentation medium.

"Glycerol" refers to a substance of the nomenclatural name of propane- 1,2,3- triol. Crude glycerol refers to industrially produced glycerol which also contains impurities. Crude glycerol is industrially produced by hydrolyzing fats and oils with water at a high temperature and under high pressure, or by the esterification reaction for biodiesel fuel production. Biodiesel fuel includes aliphatic acid methyl esters produced from fats and oils and methanol produced by a transesterification. Crude glycerol is produced as a by-product of this reaction (refer to Fukuda, H., Kondo, A., and Noda, H., 2001 , J. Biosci. Bioeng., 92, 405-416). In the biodiesel fuel production process, the alkaline catalyst method is used for the transesterification in many cases, and acids are added for neutralization. Therefore, crude glycerol of a purity of about 70 to 95% by weight, and also containing water and impurities, is produced. Crude glycerol produced in the biodiesel fuel production process contains, in addition to water, residual methanol, alkali salts such as NaOH which act as a catalyst, and impurities including an acid such as K₂S0₄ which is used for neutralizing the alkali. Although it depends on manufacturers and production methods, the amount of these salts and methanol can reach several percent. The crude glycerol preferably contains ions from the alkali and the neutralization acid, such as sodium ions, potassium ions, chloride ions, and sulfate ions in an amount of, for example, 2 to 7%, 3 to 6%, or 4 to 5.8%, based on the weight of the crude glycerol. Although methanol may not be present, it can be present in an amount of 0.01% or less.

The crude glycerol may further contain trace amounts of metals, organic acids, phosphorus, aliphatic acids, and so forth. Examples of the organic acids include formic acid, acetic acid, and so forth, and although they may not be present, they are preferably present in an amount of 0.01% or less. Trace metals required for growth of the microorganism are preferred, and examples include, for example, magnesium, iron, calcium, manganese, copper, zinc, and so forth. Magnesium, iron, and calcium are preferably present in an amount of for example, 0.00001 to 0.1%, 0.0005 to 0.1%, 0.004 to 0.05%, or 0.007 to 0.01%, in terms of the total amount based on the weight of the crude glycerol. Manganese, copper, and zinc are preferably present in an amount of, for example, 0.000005 to 0.01%, 0.000007 to 0.005%, or 0.00001 to 0.001%, in terms of the total amount.

The purity of the crude glycerol may be 10% or higher, 50% or higher, 70% or higher, or 80% or higher. As long as the impurities are within the aforementioned range, the purity of the glycerol may be 90% or higher.

Crude glycerol produced in the production of biodiesel fuel is preferred.

Furthermore, crude glycerol which enables production of more L-amino acid is preferred, compared with that when using an equal weight of reagent glycerol. To produce more L-amino acid as compared with reagent glycerol means to increase the amino acid production amount by, for example, 5% or more, 10% or more, or 20% or more, as compared with when reagent glycerol is used as the carbon source. "Reagent glycerol" means glycerol marketed as regent grade or having a purity equivalent to the purity of glycerol marketed as regent grade. Preferably, the glycerol has a purity of 99% by weight or higher, and pure glycerol is particularly preferred. The expression "reagent glycerol in the same amount as crude glycerol" means that the reagent glycerol is the same weight as crude glycerol except for water, when the crude glycerol contains water.

Crude glycerol may be diluted with a solvent such as water. In such a case, the aforementioned descriptions concerning the amount of glycerol and impurities can be applied to crude glycerol before dilution. That is, when a solution of crude glycerol in a solvent is used and the solvent is reduced so that it is 30% by weight or less, 20% by weight or less, or 10% by weight or less, if the content of glycerol and any impurities are within the aforementioned ranges, this crude glycerol corresponds to the "crude glycerol" used in the present invention.

Ethanol is a monohydric alcohol represented by the molecular formula C₂H₅OH, and may be used alone, or may be present as a mixture in the medium, such as the ethanol which is produced in ethanol fermentation in the medium etc..

Ethanol may be present in the medium at any concentration so long as the chosen bacterium can assimilate it as the carbon source. When it is used as the sole carbon source in the medium, it is present in an amount of 20% w/v or less, 10% w/v or less, or 2%> w/v or less. Furthermore, ethanol may be present in the medium at any concentration so long as it can be assimilated as the carbon source by the chosen bacterium. When it is used as the sole carbon source in the medium, it is desirably present in the medium in an amount of 0.001% w/v or more, 0.05% w/v or more, or 0.1%) w/v or more.

As for the feed medium, when ethanol is used as the sole carbon source, it can be present in the medium in an amount of 10% w/v or less, 5% w/v or less, or 1% w/v or less.

Although the concentration of ethanol can be measured by various methods, the enzymatic method is convenient and common (Swift R., 2003, Addiction, 98:73-80). The concentration of aliphatic acid can be measured by known methods such as gas chromatography and HPLC (TrAC Trends Anal. Chem., 2002, 21 :686-697; Lin J.T., Snyder L.R., and McKeon, T.A., 1998, J. Chromatogr. A., 808:43-49).

Furthermore, the medium may contain a mixture of ethanol. The

concentrations of ethanol which are added may be any concentration so long as the chosen bacterium can assimilate them as the carbon source. However, when a mixture of ethanol is used as the sole carbon source in the medium, it can be present in an amount of 20% w/v or less, 10% w/v or less, or 2% w/v or less, in terms of the total concentration. Furthermore, a mixture of ethanol may be present in the medium at any concentration so long as it can be assimilated as the carbon source by the bacterium. However, when a mixture of ethanol is used as the sole carbon source in the medium, it can be contained in the medium in an amount of 0.001 ) w/v or more, 0.05% w/v or more, or 0.1% w/v or more, in terms of the total concentration of ethanol .

In addition to ethanol and/or glycerol, other carbon sources may also be added to the medium, for example, such as saccharides such as glucose, fructose, sucrose, lactose, galactose, blackstrap molasses, and starch hydrolysate, polyhydric alcohols, and organic acids such as fumaric acid, citric acid, and succinic acid. Glucose, sucrose, and fructose are especially preferred.. The carbon source may be one kind of substance or a mixture of two or more kinds of substances. When other carbon sources are used, the ratio of ethanol, aliphatic acid, or a mixture of ethanol and glycerol in the carbon source can be 10% by weight or more, 30% by weight or more, or 50%» by weight or more.

In the present invention, the cultivation, collection, and purification of an L- amino acid from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.

A medium used for culture may be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. Ethanol or glycerol is used as the carbon source. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium

monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.

The cultivation is preferably performed under aerobic conditions, such as by shaking and/or stirring with aeration, at a temperature of 20 to 40 °C, preferably 30 to 38 °C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to

accumulation of the target L-amino acid in the liquid medium.

After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.

The collected L-amino acid may contain bacterial cells, medium components, moisture, and by-product metabolites of the bacterium in addition to the L-amino acid. Purity of the collected L-amino acid can be 50% or higher, 85% or higher, or even 95% or higher (Japanese Patent No. 1214636, U.S. Patent Nos. 5,431,933, 4,956,471, 4,777,051, 4,946,654, 5,840,358, 6,238,714, U.S. Patent Published Application No. 2005/0025878).

Examples

The present invention will be more concretely explained below with reference to the following non-limiting examples. Example 1. Preparation of the E. coli strain MG1655 cyaA^K432Q Pi,_tar,-adhE^*.

To obtain an E. coli strain, which can utilize ethanol more effective, the breading according to adaptive evolution method described by Fong S. et al.

(http://www.genome.Org/cgi/doi/10.l 101/gr.3832305) has been carried out. The culture of strain MG1655 P_L-tacadhE* (WO2008010565) was maintained in prolonged exponential growth by daily passage into fresh minimal medium (M9) supplemented with 2% (v/v) ethanol. Each 100 hours of experiment, probes of culture were taken and growth rate on the same medium was measured. The strain MG1655 PL-tacadhE* was derived from the strain MG1655. In the strain MG1655 PL-tacadhE*, the wild-type adhE gene is replaced with a mutant adhE gene which encodes a mutant alcohol dehydrogenase having Glu568Lys (E568K) mutation. The mutant adhE gene is expressed by P_L-tac promoter. The strain MG1655 (ATCC47076, ATCC700926) can be available from the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). At the 700 hours of experiment no further improvement of growth rate was observed. The cell suspension of this "fitness" culture was spread on the LB plates to form the individual colonies. The growth rate of seven independent clones was measured. It was shown that all these clones and "fitness" culture has the same growth rate on ethanol. One from these clones, Fit73, was chosen for further investigation.

Comparative Genome Sequencing analysis (NimbleGen) of the above selected clone Fit73 of "fitness" strain have reveled 16 mutations: 3 synonymous and 13 non- synonymous. One of non-synonymous substitution was founded in gene cyaA: A→G in 1294 position. As the result, lysine residue at the 432 position of adenylate cyclase was replaced by glutamine.

Then this strain was marked with chloramphenicol resistance marker (Cm^R) downstream cyaA^K432Q gene as follows. The DNA fragment carrying chloramphenicol resistance marker (Cm^R) encoded by the cat gene was integrated into the chromosome of strain Fit73 downstream cyaA^K432Q gene by the method described by Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration".

A DNA fragment containing a Cm^R marker encoded by the cat gene was obtained by PCR using the pMWl 18-attL-Cm-attR plasmid (WO 05/010175) as a template, and primers PI (SEQ ID NO: 3) and P2 (SEQ ID NO: 4). PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems). The reaction mixture (total volume - 50 μΐ) consisted of 5 μΐ of lOx PCR-buffer with 25 mM MgCl₂ ("Fermentas", Lithuania), 200 μΜ each of dNTP, 25 pmol each of the exploited primers and 1 U of Taq-polymerase ("Fermentas", Lithuania).

Approximately 5 ng of the plasmid DNA was added in the reaction mixture as a template DNA for the PCR amplification. The temperature profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at +72 °C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.

The obtained DNA fragment was used for electroporation and Red-mediated integration into the bacterial chromosome of the selected E. coli "fitness" strain Fit73.

Cells of strain Fit73 were grown overnight at 30 °C in liquid LB-medium containing ampicillin (100 μg/ml), then diluted 1 : 100 by SOB-medium (Yeast extract, 5 g/1; NaCl, 0.5 g/1; Tryptone, 20 g/1; KC1, 2.5 mM; MgC12, 10 mM) containing ampicillin (100 μg/ml) and L-arabinose (10 mM) (arabinose is used for inducing the plasmid encoding genes of Red system) and grown at 30 °C to reach the optical density of the bacterial culture OD600=0.4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times by the ice-cold de-ionized water, followed by suspension in 100 μΐ of the water. 10 μΐ of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No. 165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells were added to 1-ml of SOC medium (Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)), incubated for 2 hours at 37 °C, and then were spread onto L-agar containing 25 μg/ml of chloramphenicol. Colonies grown for 24 hours were tested for the presence of Cm^R marker downstream cyaA^K432Q gene by PCR using primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6). For this purpose, a freshly isolated colony was suspended in 20μ1 water and then Ιμΐ of obtained suspension was used for PCR. The temperature profile follows: initial DNA denaturation for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec and elongation at 72 °C for 30 sec; the final elongation for 5 min at 72 °C. A few Cm^R colonies tested contained the desired 2076 bp DNA fragment, confirming the presence of Cm^R marker DNA downstream cyaA^K432Q gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46 by culturing at 37 °C.

Using PI transduction cyaA *-cat fragment from the resulting strain was introduced into the strain MG1655 P_L-tacadhE* (WO2008010565). The growth of the obtained strain on the M9 medium supplemented with 2% of ethanol was tested. It was shown, that cyaA^K432Q mutation has a great influence on the growth: mutant cyaA * gene improved the growth of MG 1655 P_L-_tac dhE* strain exhibiting approximately 50% of growth phenotype of Fit73 strain.

Thus, a mutant strain containing mutant cyaA^K432Q gene was obtained. The strain was named MG1655 cyaA^K432Q P_Ltac-adhE*.

Example 2. The effect of mutation in the cyaA gene on L-threonine production. To evaluate the effect of K432Q mutation in the cyaA gene on threonine production, the E. coli strain MG1655Atdh, rhtA*, cyaA^K432Q P_L-_tac dhE* was obtained by transferring the DNA fragment from the chromosome of the E. coli strain MG1655 cyaA^K432Q?u∞-adhE* to the E. coli strain MG1655 Atdh rhtA* P_Ltac-adhE*

(WO2008010565) by PI transduction. The strain MG1655 Atdh rhtA* P_Ltac-adhE* was obtained from the MG1655Atdh rhtA* strain. The MG1655Atdh rhtA* strain corresponds to the MG1655 strain, but the tdh gene encoding threonine dehydrogenase is disrupted by the method of Datsenko and Wanner and a rhtA23 mutation is introduced therein, which imparts resistance to high concentrations of threonine in a minimal medium to the rhtA gene (Livshits, V.A., Zakataeva, N.P., Aleshin, V.V., Vitushkina, M.V., 2003, Res. Microbiol., 154:123-135). Then E. coli strains

MG1655Atdh, rhtA*, P_L-ta_CadhE* and MG1655Atdh, rhtA*, c^^^{i e}P_L-tacadhE* were transformed with the plasmid pVIC40 (the plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371). pVIC40 contains a thrA*BC operon including a mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized feedback inhibition by threonine. pVIC40 can be obtained from the strain E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Patent No. 5,175,107), The strain E. coli TDH-6/pVIC40 was deposited at the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1 , Moscow 1 17545, Russian Federation) on April 7, 1987 under the accession number VKPM B- 3996.

Thus threonine-producing strains MG1655Atdh, rhtA*, P_L-ta_CadhE*/ pVIC40 and MG1655Atdh, rhtA*, cy^^^2eP_L-tacadhE*/ pVIC40 were constructed.

The E. coli strains MG1655Atdh, rhtA*, P_L-ta_CadhE*/ pVIC40 and MG1655Atdh, rhtA*,

pVIC40 were cultivated at 37 °C for 18 hours in a nutrient broth and 0.1 ml of each of the obtained cultures was inoculated into 2 ml of fermentation medium in a 20x200 mm test tube and cultivated at 32 °C for 48 hours with a rotary shaker. After cultivation the amount of L-threonine, which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution 2% of ninhydrin in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted in 0.5 % water solution of CdCl₂, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of 8 independent test tube fermentations for each medium are shown in Table 1.

Table 1

As follows from Table 1, MG1655Atdh, rhtA*, cyaA^K4S2Q PL-_tacadhE*/ pVIC40 caused accumulation of a higher amount of L-threonine, as compared with

MG1655Atdh, rhtA*, PL-_tacadhE*/ pVIC40 both in the medium containing ethanol and in the medium containing glycerol.

Fermentation medium composition (g/1):

Ethanol /Glycerol 20/40

(NH₄)₂S0₄ 22

K₂HP0₄ 2

MgS0₄-7H₂0 0.8

MnS0₄-5H₂0 0.02

FeS0₄-7H₂0 0.02

Thiamine hydrochloride 0.002

Yeast extract 1.0

CaC0₃ 30

K₂HP0₄ and CaC0₃ were sterilized separately. The pH was adjusted to 7.0.

Example 3. The effect of mutation in the cyaA gene on on L-lysine production.

To evaluate the effect of K432Q mutation in the cyaA gene on L-lysine production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-ta_CadhE* (US2009203090 (Al) can be transferred to the lysine-producing E. coli strain WC196 (pCABD2) by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain WC196 PL-_ta_c dhE* (pCABD2). pCABD2 is a plasmid including the dapA gene coding for a dihydrodipicolinate synthase having a mutation which desensitizes feedback inhibition by L-lysine, the lysC gene coding for aspartokinase III having a mutation which desensitizes feedback inhibition by L-lysine, the dapB gene coding for a dihydrodipicolinate reductase, and the ddh gene coding for diaminopimelate dehydrogenase (U.S. Patent No. 6,040, 160).

Then the E. coli strain WC196 P_L-ta_CadhE* cat- cyaA^K432Q (pCABD2) can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 cyaA^K432Q P_Ltac-adhE* into the E. coli strain WC196 P_L-tacadhE* (pCABD2) by PI transduction.

The E. coli strains WC 196 P_L-tacadhE* cat- cyaA^K432Q (pC ABD2) and WC 196 PL-_tacadhE* (pCABD2) can be separately cultured in L-medium containing 20 mg/1 of streptomycin at 37 °C, and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500 ml-flask. The cultivation can be carried out at 37° C for 16 hours by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual ethanol in the medium can be measured by a known method (Biotech-analyzer AS210, manufactured by Sakura Seiki Co.). Then, the yield of L-lysine relative to consumed ethanol can be calculated for each of the strains.

The composition of the fermentation medium (g/1):

Ethanol 20.0

(NH₄)₂S0₄ 24.0

K₂HP0₄ 1.0

MgS0₄ 7H₂0 1.0

FeS0₄ 7H₂0 0.01

MnS0₄ 5H₂0 0.01

Yeast extract 2.0

pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115°C for 10 min. Ethanol and MgS0₄ 7H₂0 are sterilized separately. CaC0₃ is dry-heat sterilized at 180°C for 2 hours and added to the medium at a final concentration of 30 g/1.

Example 4. The effect of mutation in the cyaA gene on L-histidine production.

To evaluate the effect of K432Q mutation in the cyaA gene on L-histidine production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-tacadhE* (US2009203090 (Al) can be transferred to the histidine- producing E. coli strain 80 by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 80 PL-_tacadhE*. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(GNII genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on October 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.

Then the E. coli strain 80 P_L-tacadhE* cat- cyaA^K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 cyaA^K4S2Q P ac-adhE* into the E. coli strain 80 P_L-_tacadhE* by PI transduction.

The E. coli strains 80 P_L-tacadhE* cat- cyaA^K432Q and 80 P_L-_taCadhE* can be separately cultivated in L broth for 6 hours at 29 °C. Then, 0.1 ml of obtained culture can each be inoculated into 2 ml of fermentation medium in a 20x200mm test tube and cultivated for 65 hours at 29 °C with a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase: n-butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.

The composition of the fermentation medium (pH 6.0) (g/1):

Ethanol 20.0

Mameno (soybean hydrolyzate) 0.2 as total nitrogen

L-proline 1.0

(NH₄)₂S0₄ 25.0

KH₂P0₄ 2.0

MgSO₄ 7H₂0 1.0

FeS0₄ 7H₂0 0.01

MnS0₄ 0.01

Thiamine 0.001

Betaine 2.0

CaC0₃ 60.0

Ethanol, proline, betaine and CaC0₃ are sterilized separately. pH is adjusted to 6.0 before sterilization.

Example 5. The effect of mutation in the cyaA gene on on L-leucine production.

To evaluate the effect of K432Q mutation in the cyaA gene on L-leucine production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-taeadhE* (US2009203090 (Al) can be transferred to the L-leucine - producing E. coli strain NS1391 by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain NS 1391 P_L-ta_CadhE* . Then the E. coli strain NS 1391 P_L-tacadhE* cyaA^K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli MG1655 Atdh rhtA* cy A^K432QP_Uac- adhE* to the E. coli strain NS1391 P_L-ta_CadhE* (WO2008010565) by PI transduction.

The E. coli strains NS1391 P_L-ta_CadhE* cyaA^K432Q and NS1391 P_L-ta_CadhE*, can be separately cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed in 2 ml of a minimal fermentation medium in 20x200-mm test tubes. Cells can be grown for 48-72 hours at 32°C with shaking at 250 rpm. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol - acetic acid - water = 4:1 :1).

The composition of the fermentation medium (g/1) (pH 7.2):

Ethanol 0/10.0/20.0/30.0

(NH₄)₂S0₄ 25.0

K₂HP0₄ 2.0

MgS0₄ 7H₂0 1.0

Thiamine 0.01

CaC0₃ 25.0

Glucose, ethanol and CaC0₃ are sterilized separately.

Example 6. The effect of mutation in the cvaA gene on L-phenylalanine production.

To evaluate the effect of K432Q mutation in the cyaA gene on L- phenylalanine production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-tacadhE* (US2009203090 (Al) can be transferred to the L- phenylalanine -producing E. coli strain AJ12739 (tyrA::Tnl0, tyrR) (WO2003/044191) by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain AJ12739 PL-tacadhE*.

The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1 , Moscow 117545, Russian Federation) on November 6, 2001 under accession number VKPM B- 8197 and then converted to a deposit under the Budapest Treaty on August 23, 2002.

Then the E. coli strain AJ 12739 P_L-_ta_CadhE* cyaA^K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA^K432Q Puac-adhE* to the strain E. co//.AJ12739 P_L-ta_CadhE* by PI transduction. The E. coli strains AJ12739 P_L-tacadhE* cat- cyaA^K432Q and AJ12739 P_L- _tacadhE* can be separately cultivated at 37°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in a 20 x 200 mm test tube and cultivated at 37 °C for 48 hours with a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. 10 x 15 cm TLC plates coated with 0.1 l.mra layers of Sorbfil silica gel without fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase: propan- 2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40 : 7 : 16 (v/v). A solution (2%) of ninhydrin in acetone can be used as a visualizing reagent.

The composition of the fermentation medium (g/1)

Ethanol 20.0

(NH^SC 16.0

K₂HP0₄ 0.1

MgS0₄ 7H₂0 1.0

FeS0₄ 7H₂0 0.01

MnS0₄ 5H₂0 0.01

Thiamine HC1 0.0002

Yeast extract 2.0

Tyrosine 0.125

CaC0₃ 20.0

Ethanol and magnesium sulfate are sterilized separately. CaC0₃ dry-heat sterilized at 180°C for 2 hours. pH is adjusted to 7.0.

Example 7. The effect of mutation in the cyaA gene on L-arginine production.

To evaluate the effect of K432Q mutation in the cyaA gene on L-arginine production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-tacadhE* (US2009203090 (Al) can be transferred to the L- arginine - producing E. coli strain 382 (EP1170358A1) by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 382 PL-_tacadhE*. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNII genetika, 1

Dorozhny proezd, 1, Moscow 117545, Russian Federation) on April 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001. Then the E. coli strain 382 P_L-_tac dhE* cyaA^K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA^K432Qcat PL_tac- adhE* into the strain E. coli 382 P_L-_ta_CadhE* by PI transduction.

The E. coli strains 382 P_L-tacadhE* cat- cyaA^K432Q and 382 P_L-_tacadhE* can be separately cultivated with shaking at 37 °C for 18 hours in 3 ml of nutrient broth, and 0.3 ml of the obtained cultures can be inoculated into 2 ml of a fermentation medium in 20 x 200-mm test tubes and cultivated at 32 °C for 48 hours on a rotary shaker.

After the cultivation, the amount of L-arginine which accumulates in the medium can be determined by paper chromatography using the following mobile phase:

butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent. A spot containing L-arginine can be cut out, L- arginine can be eluted with 0.5% water solution of CdCl₂, and the amount of L- arginine can be estimated spectrophotometrically at 540 nm.

The composition of the fermentation medium (g/l):

Ethanol 20

Glucose 0 / 5

(NH₄)₂S0₄ 25

K₂HP0₄ 2

MgS0₄-7H₂0 1.0

Thiamine hydrochloride 0.002

Yeast extract 5.0

CaC0₃ 33

MgS0₄-7H₂0, ethanol and CaC0₃ are each sterilized separately.

Example 8. The effect of mutation in the cyaA gene on L-tryptophan production.

To evaluate the effect of K432Q mutation in the cyaA gene on L- tryptophan production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-tacadhE* (US2009203090 (Al) can be transferred to the L- tryptophan - producing E. coli strain SV164 (pGH5) by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain SV164 P_L-t_acadhE*(pGH5) . The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SV164 (pGH5) is described in detail in U.S. Patent No. 6,180,373. Then the E. coli strain SV164 P_L-tacadhE* cyaA^K432Q (pGH5) can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA^K432Q P_Ltac-adhE* into the E. coli strain SV164 P_L-tacadhE*(pGH5) by PI transduction.

The E. coli strains SV164 P_L-ta_CadhE* cat- cyaA^K432Q (pGH5) and SV164 P_L- t_acadhE* (pGH5) can be separately cultivated with shaking at 37 °C for 18 hours in a 3 ml of nutrient broth supplemented with 20 mg/1 of tetracycline (marker of pGH5 plasmid). 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium containing tetracycline (20 mg/1) in 20 x 200 mm test tubes, and cultivated at 37 °C for 48 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 6. The fermentation medium components are set forth in Table 2, but should be sterilized in separate groups A, B, C, D, E, F, and H, as shown, to avoid adverse interactions during sterilization.

Table 2

Solution A had a pH of 7.1, adjusted by NH₄OH. Example 9. The effect of mutation in the cyaA gene on L- glutamic acid production.

To evaluate the effect of K432Q mutation in the cyaA gene on L- glutamic acid production, at first the DNA fragments from the chromosome of the E. coli strain MG1655::P_L-tacadhE* (US2009203090 (Al) can be transferred to the glutamic acid- producing E. coli strain VL334thrC⁺ (EP1172433) by PI transduction (Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain VL334thrC⁺ P_L-tacadhE* . The strain VL334thrC⁺ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 1 17545, Russian Federation) on December 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on December 8, 2004.

Then the E. coli strain VL334thrC⁺ PL-_tacadhE* cyaA^K432Q can be obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA^K432Q Puac-adhE* to the E. coli strain VL334thrC⁺ P_L-ta_CadhE* by PI transduction.

The E. coli strains VL334thrC⁺ P_L-tacadhE* cyaA^K432Q and VL334thrC⁺ PL- _ta_cadhE* can be separately cultivated with shaking at 37 °C for 18 hours in a 3 ml of nutrient broth. 0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation medium in 20 x 200 mm test tubes, and cultivated at 37 °C for 48 hours with a rotary shaker at 250 rpm.

The composition of the fermentation medium (pH 7.2) (g/1):

Ethanol 20.0

Ammonium sulfate 25.0

KH₂P0₄ 2.0

MgS0₄ 7H₂0 1.0

Thiamine 0.0001

L-isoleucine 0.05

CaC0₃ 25.0

Ethanol and CaC0₃ are sterilized separately.

Example 10. The effect of mutation in the cyaA gene on L-valine production To evaluate the effect of K432Q mutation in the cyaA gene on L-valine production, at first the L-valine producing strain E. coli H-81 PL-t_acadhE* was obtained by transferring DNA fragments from the chromosome of the E. coli strain

MG1655::P_L-_tacadhE* (US2009203090 (Al) to the valine-producing E. coli strain H-81 by PI transduction (Miller, J.H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, NY). The H-81 strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM) (GNU genetika, 1 Dorozhny proezd, 1, Moscow 117545, Russian Federation) on January 30, 2001 under the accession number VKPM B-8066. Then the strain E. coli H-81 P_L-_tac dhE* cyaA^K432Q was obtained by transferring DNA fragments from the chromosome of the E. coli strain MG1655 cyaA^K432Qcat P_Ltac- adhE* into the strain E. coli H-81 P_L-taCadhE* by PI transduction.

The strains H-81 P_L-tacadhE* cyaA^K432Q and H-81 P_L-tacadhE* were cultivated at 37 °C for 18 hours in a nutrient broth and 0.1 ml of each of the obtained cultures was inoculated into 2 ml of fermentation medium in a 20x200 mm test tube and cultivated at 32 °C for 72 hours with a rotary shaker. After cultivation for 24 hours in

fermentation medium containing ethanol and for 18 hours in fermentation medium containing glycerol, accumulated amounts of L-valine were measured by TLC as described in Example 6. The results of 8 independent test tube fermentations for each medium are shown in Table 3.

Table 3

As follows from Table 3, H-81 P_L-t_acadhE* cyaA^{K43 Q} caused accumulation of a higher amount of L-valine, as compared with H-81 P_L-_tacadhE*, both in the medium containing ethanol and in the medium containing glycerol.

Fermentation medium composition (g/1):

Ethanol/Glycerol 20/80

(NH₄)₂S0₄ 15

KH₂P0₄ 1.5

MgS0₄-7H₂0 1

Mameno (TN) 0.4

CaCOs 25

CaC0₃ was dry-heat sterilized at 180 °C for 2 hours. The pH was adjusted to 7.0.

While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents is incorporated by reference herein in its entirety.

Industrial Applicability

According to the present invention, in a method for producing an L-amino acid from by fermentation using a microorganism belonging to the family

Enter obacteriaceae, productivity of L-amino acid can be improved.

Claims

1. A mutant adenylate cyclase selected from the group consisting of:

(A) a protein in which L-lysine at a position corresponding to the position 432 in SEQ ID NO: 2 is replaced with another L-amino acid residue; and

(B) a variant of protein (A), which has an activity of adenylate cyclase.

2. The mutant adenylate cyclase according to claim 1, wherein L-lysine at a position corresponding to the position 432 in SEQ ID NO: 2 is replaced with L-glutamine.

3. A DNA coding for mutant adenylate cyclase according to claim 1 or 2.

4. A bacterium of Enterobacteriaceae family, which contains the DNA

according to claim 3.

5. The bacterium according to claim 4, wherein said bacterium has an ability to produce L-amino acids.

6. The bacterium according to claim 4 or 5, wherein said bacterium belongs to the genus Escherichia.

7. The bacterium according to claim 6, wherein said bacterium is Escherichia coli.

8. A method for producing L-amino acid comprising cultivating the bacterium according to any one of claims 5 to 8 in a culture medium containing ethanol or grycerol as a carbon source, and collecting the L-amino acid from the culture medium.

9. The method according to claim 8, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

10. The method according to claim9, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L- tryptophan.

11. The method according to claim 9, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L- methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L- proline, and L-arginine.