# Patent

## Title
Method for producing an l-amino acid using a bacterium of the enterobacteriaceae family having a pathway of glycogen biosynthesis disrupted

## Abstract

There is provided a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to the genus Escherichia or Pantoea, having the glycogen biosynthesis pathway disrupted.

## Background
This application claims priority under 35 U.S.C. §119(a) to Russian patent application 2005101110, filed Jan. 19, 2005, and under 35 U.S.C. §119(e) to U.S. provisional patent application 60/703,426, filed Jul. 29, 2005, the entireties of both are hereby incorporated by reference. The Sequence Listing on Compact Disk filed herewith is also hereby incorporated by reference in its entirety (File Name: US-199 Seq List; File Size: 73 KB; Date Created: Jan. 17, 2006).


BACKGROUND OF THE INVENTION
1. Field of Invention
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, wherein the glycogen biosynthesis pathway has been disrupted.
2. Description of the Related Art
Glycogen represents the major form of stored carbon for Escherichia coli and many other prokaryotes, and provides a readily metabolized substrate for maintenance energy. Glycogen accumulation in Escherichia coli is inversely related to the growth rate, and occurs most actively when cells enter the stationary phase. The levels of the three biosynthetic enzymes undergo corresponding changes under these conditions, suggesting that genetic control of enzyme biosynthesis may account for at least part of the regulation (Preiss, J., Annu. Rev. Microbiol. 38, 419-458 (1984)). In Escherichia coli, the structural genes for glycogen biosynthesis are clustered on adjacent operons—glgBX and glgCAP. Interestingly, the glycogen biosynthetic (glgCA) and degradative (glgP) genes are localized together in a cluster, possibly to facilitate the regulation of these systems in vivo (Romeo, T., Gene. 70(2), 363-76 (1988)). The glgC gene is the structural gene for glucose-1-phosphate adenylyltransferase. Synonyms for glucose-1-phosphate adenylyltransferase are ADP-glucose synthase, ADP-glucose pyrophosphorylase, ADP: a-D-glucose-1-phosphate adenylyltransferase, GlgC protein.
Glucose-1-phosphate adenylyltransferase (EC 2.7.7.27) is an allosteric enzyme in the glycogen biosynthetic pathway of eubacteria. Among the enteric bacteria, glucose-1-phosphate adenylyltransferase is regulated by glycolytic intermediates with fructose 1,6-biphosphate as the activator and AMP, ADP, and Pi as inhibitors. The enzyme catalyzes the synthesis of ADP glucose and PPi from glucose 1-phosphate and ATP. This reaction is the first unique step in bacterial glycogen biosynthesis.
It is known that the carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action (Baker, C. S. et al, Mol. Microbiol., 44(6), 1599-610 (2002)).
The glgCAP operon is under the positive control of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). Both the cya gene encoding adenylate cyclase (EC 4.6.1.1) and the crp gene encoding CRP are required for optimal synthesis of glycogen (Fletterick, R. J. and Madsen, N. B., Annu. Rev. Biochem., 49, 31-61 (1980)). CRP binds to a site located upstream of the glgC gene. Glycogen synthesis in E. coli is also positively regulated by ppGpp, which stimulates the transcription of the glgCAP operon (Preiss. J., and Romeo, T., Prog. Nucleic Acid Res. Mol. Biol. 47, 299-329 (1994)).
By using a mini-Mu random chromosomal library and screening for glycogen overproduction, a novel gene (glgS) involved in glycogen synthesis was identified (Hengge-Aronis, R. and Fischer, D., Mol Microbiol. 6, 14, 1877-86 (1992)). It was also shown that the Escherichia coli protein GlgS is up-regulated in response to starvation stress and its overexpression was shown to stimulate glycogen synthesis (Kozlov, G. et al, BMC Biol., 2, 1, 10 (2004)).
The initial substrate for glycolysis biosynthesis is glucose-1-P, obtained from glucose-6-P, so said pathway competes with glycolysis for glucose-6-P. But currently, there have been no reports of inactivating the glgBX and/or glgCAP operons or inactivating the glgS gene for producing L-amino acids.
SUMMARY OF THE INVENTION
Objects of the present invention include enhancing the productivity of L-amino acid producing strains and providing a method for producing an L-amino acid using these strains.
The above objects were achieved by finding that inactivating the glgBX and/or glgCAP operons can enhance production of L-amino acids, such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine.
The present invention provides a bacterium of the Enterobacteriaceae family having an increased ability to produce amino acids, such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline, and L-arginine.
It is an object of the present invention to provide an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified so that the glycogen biosynthesis pathway is disrupted.
It is a further object of the present invention to provide the bacterium as described above, wherein the glycogen biosynthesis pathway is disrupted by attenuation of expression of the glgBX and/or glgCAP operons.
It is a further object of the present invention to provide the bacterium as described above, wherein the glycogen biosynthesis pathway is disrupted by inactivation of the glgBX and/or glgCAP operons.
It is a further object of the present invention to provide the bacterium as described above, wherein the inactivation of the glgBX and/or glgCAP operons is performed by deletion of a gene selected from a group consisting of glgB, glgX, glgC, glgA, glgP, and combinations thereof.
It is a further object of the present invention to provide the bacterium as described above, wherein the glycogen biosynthesis pathway is disrupted by attenuation of expression of the glgS gene.
It is a further object of the present invention to provide the bacterium as described above, wherein the glycogen biosynthesis pathway is disrupted by inactivation of the glgS gene.
It is a further object of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Escherichia. 
It is a further object of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Pantoea. 
It is a further object of the present invention to provide the bacterium 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 object of the present invention to provide the bacterium 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 object of the present invention to provide the bacterium 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, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
It is a further object of the present invention to provide a method for producing an L-amino acid comprising:

    
    
        cultivating the bacterium as described above in a medium to produce and excrete L-amino acid into the medium, and
        collecting said L-amino acid from the medium.
    
    


It is a further object 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 object 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 object 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, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the relative positions of primers glgCL and glgCR on plasmid pACYC184, which was used for amplification of the cat gene.
FIG. 2 shows the construction of the chromosomal DNA fragment containing the inactivated glgC gene.
FIG. 3 shows the construction of the chromosomal DNA fragment containing the inactivated glgBX and glgCAP operons.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is described in detail below.
1. Bacterium of the Present Invention
The bacterium of the present invention is an L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified so that the glycogen biosynthesis pathway is disrupted.
In the present invention, “L-amino acid-producing bacterium” means a bacterium which has an ability to produce and excrete L-amino acid into a medium, when the bacterium is cultured in the medium.
The term “L-amino acid-producing bacterium” as used herein also means 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 not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid. The term “L-amino acids” includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-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” comprises L-phenylalanine, L-tyrosine, and L-tryptophan. The term “non-aromatic L-amino acid” comprises L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-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 preferred.
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 in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/wgetorg?mode=Tree&id=1236&lvl=3& keep=1&srchmode=1&unlock) can be used. A bacterium belonging to the genus of Escherichia or Pantoea is preferred.
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 into 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 on the basis of nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)). Such strains are encompassed by “bacteria belonging to the genus Pantoea”. 
The phrase “bacterium has been modified so that the glycogen biosynthesis pathway is disrupted” means that the bacterium has been modified in such a way that the modified bacterium has a reduced ability to synthesize glycogen as compared with an unmodified bacterium or is unable to synthesize and accumulate glycogen. Disruption of glycogen biosynthetic pathway may be performed by attenuation of expression of genes encoding enzymes involved in glycogen biosynthesis, such as GlgB, GlgX, GlgC, GlgA, GlgP, and GlgS, and is preferably performed by inactivation of said genes.
The phrase “attenuation of expression of the glgBX and/or glgCAP operons” or “attenuation of expression of the glgS gene” means that the target operon or gene is modified in such a way that the modified operon or gene encodes a mutant protein(s) which has(have) a decreased activity. The phrase “inactivation of the glgBX and/or glgCAP operons” or “inactivation of the glgS gene” means that such modified operon or gene encodes a completely inactive protein(s). It is also possible that the modified DNA region is unable to naturally express the gene(s) due to the deletion of a part of the gene(s), the shifting of the reading frame of the gene(s), the introduction of missense/nonsense mutation(s), or the modification of an adjacent region of the gene(s), including sequences controlling gene expression, such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s), etc.
The level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis) and the like.
The glgC gene encodes glucose-1-phosphate adenylyltransferase (synonym—B3430). The glgC gene (gi: 16131304; nucleotides complementary to nucleotides 3566056 to 3567351 in the GenBank accession number NC—000913.2; SEQ ID NO: 1) is located between the glgA and glgX genes on the chromosome of E. coli K-12. The nucleotide sequence of the glgC gene and the amino acid sequence of the glucose-1-phosphate adenylyltransferase encoded by the glgC gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.
The glgA gene encodes a subunit of glycogen synthase (synonym—B3429). The glgA gene (nucleotide positions: 3,566,056 to 3,564,623; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glgP and glgC genes on the chromosome of E. coli K-12. The nucleotide sequence of the glgA gene and the amino acid sequence of the subunit of glycogen synthase encoded by the glgA gene are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.
The glgP gene encodes a subunit of glycogen phosphorylase/glycogen-maltotetraose phosphorylase (synonyms—B3428, GlgY). The glgP gene (nucleotide positions: 3,564,604 to 3,562,157; GenBank accession no. NC—000913.2; gi: 49175990) is located between the yzgL and glgA genes on the chromosome of E. coli K-12. The nucleotide sequence of the glgP gene and the amino acid sequence of the subunit of glycogen phosphorylase/glycogen-maltotetraose phosphorylase encoded by the glgP gene are shown in SEQ ID NO:13 and SEQ ID NO:14, respectively.
The glgX gene encodes glycogen phosphorylase-limit dextrin α-1,6-glucohydrolase (synonyms—B3431, GlyX). The glgX gene (nucleotide positions: 3,569,342 to 3,567,369; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glgC and glgB genes on the chromosome of E. coli K-12. The nucleotide sequence of the glgX gene and the amino acid sequence of the glycogen phosphorylase-limit dextrin α-1,6-glucohydrolase encoded by the glgX gene are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively.
The glgB gene encodes a glycogen-branching enzyme (synonym—B3432). The glgB gene (nucleotide positions: 3,571,525 to 3,569,339; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glgX and asd genes on the chromosome of E. coli K-12. The nucleotide sequence of the glgB gene and the amino acid sequence of the glycogen-branching enzyme encoded by the glgB gene are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively.
The glgS gene encodes a rpoS-dependent protein of glycogen biosynthesis (synonym—B3049). The glgS gene (nucleotide positions: 3,189,961 to 3,189,761; GenBank accession no. NC—000913.2; gi: 49175990) is located between the yqiI and yqiJ ORFs on the chromosome of E. coli K-12. The nucleotide sequence of the glgS gene and the amino acid sequence of the rpoS-dependent protein of glycogen biosynthesis encoded by the glgS gene are shown in SEQ ID NO:19 and SEQ ID NO:20, respectively.
Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the glgC gene to be deleted on the chromosome is not limited to the gene shown in SEQ ID No:1, but may include a homologous gene of SEQ ID No:1. Therefore, the protein variant encoded by the glgC gene may have a homology of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, with respect to the entire amino acid sequence encoded shown in SEQ ID NO. 2, as long as the activity of glucose-1-phosphate adenylyltransferase prior to inactivation is maintained.
Moreover, the glgC 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 1-phosphate adenylyltransferase prior to inactivation. “Stringent conditions” include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, and still more preferably not less than 90%, and most preferably not less than 95% 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, preferably two or three times at a salt concentration corresponding to 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS at 60° C. The length of the probe may be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
The similar expression as described above for the glgC gene can be applied for the variants of other genes of the glgBX and glgCAP operons.
Inactivation of the gene can be performed by conventional methods, such as mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) treatment, site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83) and (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45) also called “Red-driven integration”.
Activity of glucose-1-phosphate adenylyltransferase encoded by the glgC gene can be measured by the method described by, for example, Haugen, T. H. et al (J. Biol. Chem. 251 (24), 7880-5 (1976)). So, the decreasing or absent activity of glucose-1-phosphate adenylyltransferase in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
Activity of glycogen synthase encoded by the glgA gene can be measured by the method described by, for example, Fox, J. et al (Methods Enzymol., 28, 539-544 (1972)). So, the decreasing or absent activity of glycogen synthase in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
Activity of glycogen phosphorylase/glycogen-maltotetraose phosphorylase encoded by the glgP gene can be measured by the method described by, for example, Graves, D. J. and Wang, J. H. (The Enzymes, 7 (3rd ed.), 435-482 (1972)). So, the decreasing or absent activity of glycogen phosphorylase/glycogen-maltotetraose phosphorylase in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
Activity of glycogen phosphorylase-limit dextrin α-1,6-glucohydrolase encoded by the glgX gene can be measured by the method described by, for example, Jeanningros, R. et al (Biochim Biophys Acta, 438(1), 186-199 (1976)). So, the decreasing or absent activity of glycogen phosphorylase-limit dextrin α-1,6-glucohydrolase in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
Activity of glycogen-branching enzyme encoded by the glgB gene can be measured by the method described by, for example, Illingworth, B. B. and Brown, D. H. (Methods Enzymol., 8, 395-403 (1966)). So, the decreasing or absent activity of glycogen-branching enzyme in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
Activity of rpoS-dependent protein of glycogen biosynthesis encoded by the glgS gene can be detected by complementation of glgS-null mutation which inhibits glycogen synthesis as described by, for example, Hengge-Aronis, R, and Fischer, D. (Mol. Microbiol., 6, 14, 1877-1886 (1992)). So, the decreasing or absent activity of rpoS dependent protein of glycogen biosynthesis in the bacterium according the present invention can be determined when compared to the parent unmodified bacterium.
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).
L-Amino Acid-Producing Bacteria
As a bacterium of the present invention, which is modified to inactivate the glgBX and/or glgCAP operons or the glgS gene, bacteria which are able to produce either aromatic or non-aromatic L-amino acids may be used.
The bacterium of the present invention can be obtained by inactivating the glgBX and/or glgCAP operons or the glgS gene in a bacterium which inherently has the ability to produce L-amino acids. Alternatively, the bacterium of the present invention can be obtained by imparting the ability to produce L-amino acids to a bacterium already having the glgBX and/or glgCAP operons or the glgS gene inactivated.
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. Pat. No. 5,175,107, U.S. Pat. No. 5,705,371), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Pat. 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 Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russian Federation) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd., 1) on Apr. 7, 1987 under the accession number B-3996.
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-β-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 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 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 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 functions as a single threonine operon.
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 presented in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Pat. 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 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 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 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 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 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, α-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. Pat. No. 4,346,170) and Escherichia coli VL611. 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 by conferring AEC resistance to the strain W3110, which was derived from Escherichia coli K-12. 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 Dec. 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 Sep. 29, 1995, and received an accession number of FERM BP-5252 (U.S. Pat. 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 the enzymes involved in L-lysine biosynthesis include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (U.S. Pat. No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB), and aspartase (aspA) (EP 1253195 A).
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 and lysine decarboxylase (U.S. Pat. No. 5,827,698).
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. Pat. 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. Pat. No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JP11155571A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.
L-Leucine-Producing Bacteria
Examples of parent strains 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. Pat. No. 6,124,121)) or leucine analogs including as 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 freed from feedback inhibition by L-leucine (U.S. Pat. No. 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. Pat. No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347); E. coli H-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U.S. Pat. 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 the L-histidine-biosynthetic enzymes include ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), 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 genes encoding the L-histidine biosynthetic enzyme (hisG, hisBHAFI) are inhibited by L-histidine, and therefore an L-histidine-producing ability can also be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase (hisG) (Russian Patent Nos. 2003677 and 2119536).
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-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), 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. Pat. No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage P1 grown on the wild-type E. coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC+ (VKPM B-8961) was obtained. This strain is able to produce L-glutamic acid.
Examples of parent strains for deriving the L-glutamic acid-producing bacteria of the present invention include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of the enzymes involved in L-glutamic acid biosynthesis include glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, isocitrate dehydrogenase, aconitate hydratase, citrate synthase, phosphoenolpyruvate carboxylase, pyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phophate dehydrogenase, triose phosphate isomerase, fructose bisphosphate aldolase, phosphofructokinase, and glucose phosphate isomerase.
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 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, and branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes include isocitrate lyase, α-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, formate acetyltransferase, lactate dehydrogenase, and glutamate decarboxylase. Bacteria belonging to the genus Escherichia deficient in the α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Pat. Nos. 5,378,616 and 5,573,945. Specifically, these strains include the following:

    
    
        E. coli W3110sucA::Kmr
        E. coli AJ12624 (FERM BP-3853)
        E. coli AJ12628 (FERM BP-3854)
        E. coli AJ12949 (FERM BP-4881)
    
    


E. coli W3110sucA::Kmr is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucA gene”) of E. coli W3110. This strain is completely deficient in the α-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. Pat. No. 5,908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Pat. No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), and the like.
Examples of L-glutamic acid-producing bacteria, include mutant strains belonging to the genus Pantoea which are deficient in the α-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. Pat. 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 Feb. 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 Jan. 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 αKGDH-E1 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::Tn10, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the pheA34 gene (U.S. Pat. No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat. 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 [W3110 (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 B1). 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 A1 and 2003/0157667 A1).
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) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No. 5,756,345); E. coli SV164 (pGH5) having serA allele encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine and the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan (U.S. Pat. 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. Pat. No. 4,371,614); E. coli AGX17/pGX50, pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Pat. No. 6,319,696), and the like may be used.
Previously, it was identified that the yddG gene encoding a membrane protein, which is not involved in biosynthetic pathway of any L-amino acid, and imparts to a microorganism resistance to L-phenylalanine and several amino acid analogues when the wild-type allele of the gene was amplified on a multi-copy vector in the microorganism. Besides, the yddG gene can enhance production of L-phenylalanine or L-tryptophan when additional copies are introduced into the cells of the respective producing strain (WO03044192). So it is desirable that the L-tryptophan-producing bacterium be further modified so that expression of the yddG open reading frame is enhanced.
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. Pat. 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 α and β subunits which are encoded by trpA and trpB, respectively.
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 15th 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 A1) 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 (JP 57-5693 A), 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 the L-arginine biosynthetic enzymes include 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.
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 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. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the used microorganism, alcohol, including ethanol and glycerol, may be used. 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 a shaking culture, and a stirring culture 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.
EXAMPLES
The present invention will be more concretely explained below with reference to the following non-limiting Examples.
Example 1
Construction of a Strain with an Inactivated glgC Gene
1. Deletion of the glgC Gene
A strain having deletion of the glgC gene can be constructed by the method initially developed by Datsenko, K. A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called “Red-driven integration”. According to this procedure, the PCR primers glgCL (SEQ ID NO: 3) and glgCR (SEQ ID NO: 4) complementary to both the region adjacent to the glgC gene and the gene conferring antibiotic resistance in the template plasmid can be constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession number X06403) can be used as a template in PCR reaction. Conditions for PCR can be as follows: denaturation step at 95° C. for 3 min; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1093-bp PCR product (FIG. 1) can be obtained and purified in agarose gel and can be used for electroporation of E. coli MG1655 (ATCC 700926), which contains the plasmid pKD46 having temperature-sensitive replication. The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide (31088-33241) DNA fragment of phage λ (GenBank accession No. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.
Electrocompetent cells can be prepared as follows: a night culture of E. coli MG1655 can be grown at 30° C. in LB medium supplemented with ampicillin (100 mg/l) and then diluted 100-fold with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)) containing ampicillin and L-arabinose (1 mM). The obtained culture can be grown with aeration at 30° C. to an OD600 of ≈0.6 and then can be made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation can be performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation can be incubated with 1 ml of SOC medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)) at 37° C. for 2.5 hours and after that can be plated onto L-agar and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm at 42° C. can be performed and the obtained colonies can be tested for sensitivity to ampicillin.
2. Verification of the glgC Gene Deletion by PCR
The mutants, containing the deletion of the glgC gene, marked with Cm resistance gene, can be verified by PCR. Locus-specific primers glgCl (SEQ ID NO: 5) and glgC2 (SEQ ID NO: 6) can be used in PCR for verification. Conditions for PCR verification can be as follows: denaturation step at 94° C. for 3 min; profile for the 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product, which can be obtained in the reaction with the parental strain MG1655 glgC+ as a template, should be 1471 bp in length. The PCR product, which can be obtained in the reaction with the mutant MG1655 ΔglgC::cat strain as a template, should be 1197 bp in length (FIG. 2).
Example 2
Production of L-Glutamate By E. coli VL334thrC+-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-glutamate producing strain VL334thrC+ (EP 1172433) can be performed by an ordinary well-known method, for example, by P1 transduction from MG1655 ΔglgC::cat strain, to obtain the strain VL334thrC+-ΔglgC. The strain VL334thrC+ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Dec. 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on Dec. 8, 2004.
Both strains, VL334thrC+ and VL334thrC+-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2 ml of a fermentation medium. The fermentation medium (pH 7.2) should contain glucose (60 g/l), ammonium sulfate (25 g/l), KH2PO4 (2 g/l), MgSO4 (1 g/l), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO3 (25 g/l). Glucose and CaCO3 should be sterilized separately. Cultivation can be carried out at 30° C. for 3 days with shaking. After the cultivation, the amount of L-glutamic acid produced can be determined by paper chromatography (liquid phase composition: butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl2.
Example 3
Production of L-Proline by E. coli 702ilvA-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-proline-producing strain 702ilvA (VKPM B-8012, Russian patent application 2000124295, EP1172433) can be performed by an ordinary well-known method as described above to obtain the strain 702ilvA-ΔglgC. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Jul. 18, 2000 under the accession number VKPM B-8012.
Both E. coli strains, 702ilvA and 702ilvA-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above.
Example 4
Production of L-Arginine By E. coli 237-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-arginine-producing strain 237 (VKPM B-7925) can be performed by an ordinary well-known method as described above to obtain the strain 237-ΔglgC. The strain 237 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1 Dorozhny proezd, 1) on Apr. 10, 2000 under accession number VKPM B-7925 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
Both E. coli strains, 237 and 237-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above.
Example 5
Production of L-Leucine By E. coli 57-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-leucine-producing strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121) can be performed by an ordinary well-known method as described above to obtain the strain 57-pMW-ΔglgC. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on May 19, 1997 under the accession number VKPM B-7386.
Both E. coli strains, 57 and 57-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above without addition of isoleucine into the medium.
Example 6
Production of L-Cysteine By E. coli JM15(ydeD)-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-cysteine-producing strain JM15(ydeD) can be performed by an ordinary well-known method as described above to obtain the strain JM15(ydeD)-ΔglgC.
E. coli JM15(ydeD), a derivative of E. coli JM15 (U.S. Pat. No. 6,218,168), can be transformed with DNA having the ydeD gene, which encodes a membrane protein and is not involved in a biosynthetic pathway of any L-amino acid (U.S. Pat. No. 5,972,663).
Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of U.S. Pat. No. 6,218,168.
Example 7
Production of L-Threonine by E. coli B-3996-ΔglgC
The deletion of the glgC gene in the chromosome of E. coli L-threonine-producing strain B-3996 (VKPM B-3996) can be performed by an ordinary well-known method as described above to obtain strain B-3996-ΔglgC.
Both E. coli strains, B-3996 and B-3996-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above.
Example 8
Production of L-Lysine By E. coli AJ11442-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-lysine-producing strain AJ11442 (FERM BP-1543, NRRL B-12185) can be performed by an ordinary well-known method as described above to obtain strain AJ11442-ΔglgC. The strain AJ11442 was deposited at the Fermentation Research Institute, 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 May 1, 1981 and received an accession number of FERM P-5084. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on Oct. 29, 1987, and received an accession number of FERM BP-1543.
Both E. coli strains, AJ11442 and AJ11442-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above.
Example 9
Production of L-Phenylalanine By E. coli AJ12739-ΔglgC
The deletion of the glgC gene in the chromosome of the E. coli L-phenylalanine-producing strain AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197) can be performed by an ordinary well-known method as described above to obtain the strain AJ12739-ΔglgC. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Nov. 6, 2001 under accession no. VKPM B-8197.
Both E. coli strains, AJ12739 and AJ12739-ΔglgC, can be grown for 18-24 hours at 37° C. on L-agar plates. Then these strains can be cultivated under the same conditions as described above.
Example 10
Construction of a Strain with the glgBX and glgCAP Operons Inactivated
1. Deletion of the glgBX and glgCAP Operons
A strain having deletion of the glgBX and glgCAP operons was constructed by the method initially developed by Datsenko, K. A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called “Red-driven integration”. According to this procedure, the PCR primers glgBXCAPL (SEQ ID NO: 7) and glgBXCAPR (SEQ ID NO: 8) complementary to both the region adjacent to the glgBX and glgCAP operons and the gene conferring antibiotic resistance in the template plasmid were constructed. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (GenBank/EMBL accession number X06403) was used as a template in PCR. Conditions for PCR were described in detail in Example 1.
A 1152-bp PCR product purified in agarose gel was used for electroporation of E. coli MG1655 (ATCC 700926) containing the plasmid pKD46 having temperature -sensitive replication, as described in Example 1.
2. Verification of the Deletion of the glgBX and glgCAP Operons by PCR
The mutants containing the deletion of the glgBX and glgCAP operons and marked with Cm resistance gene were verified by PCR. Locus-specific primers glgBXCAP1 (SEQ ID NO: 9) and glgBXCAP2 (SEQ ID NO: 10) were used in PCR for the verification. Conditions for PCR verification were described in Example 1. The PCR product obtained in the reaction with the parental strain MG1655 glgBXCAP+ as a template was 9425 bp in length. The PCR product obtained in the reaction with the mutant strain MG1655 ΔglgBXCAP::cat as a template was 1208 bp in length (FIG. 3).
Example 11
Production of L-Threonine By E. coli Strain B-3996-ΔglgBXCAP
To test the effect of inactivation of the glgBX and glgCAP operons on threonine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔglgBXCAP::cat were transferred to the threonine-producing E. coli strain B-3996 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.).
Both E. coli strains, B-3996 and B-3996-ΔglgBXCAP, were grown for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells were grown for 65 hours at 32° C. with shaking at 250 rpm.
After cultivation, the amount of L-threonine 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 of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut off, L-threonine was eluted with 0.5% water solution of CdCl2, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of ten independent test-tube fermentations are shown in Table 1.
The composition of the fermentation medium (g/l) was as follows:
















Glucose
80.0



(NH4)2SO4
22.0



NaCl
0.8



KH2PO4
2.0



MgSO4•7H2O
0.8



FeSO4•7H2O
0.02



MnSO4•5H2O
0.02



Thiamine HCl
0.0002



Yeast extract
1.0



CaCO3
30.0










Glucose and magnesium sulfate were sterilized separately. CaCO3 was sterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to 7.0. The antibiotic was added to the medium after sterilization.
As follows from Table 1, B-3996-ΔglgBXCAP caused accumulation of a higher amount of L-threonine, as compared with B-3996.
Example 12
Production of L-Lysine By E. coli WC196-ΔglgBXCAP
To test the effect of inactivation of the glgBX and glgCAP operons on L-lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔglgBXCAP::cat were transferred to the L-lysine producing E. coli strain WC196 (FERM BP-5252) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.).
To obtain a seed culture, both E. coli strains, WC196 and WC196-ΔglgBXCAP, were grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of medium diluted two times compare to the fermentation medium described below. Then the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells were grown for 24 hours at 32° C. with shaking at 250 rpm.
After cultivation, the amount of L-lysine 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 of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-lysine was cut off, L-lysine was eluted with 0.5% water solution of CdCl2, and the amount of L-lysine was estimated spectrophotometrically at 540 nm. The results of ten independent test-tube fermentations are shown in Table 2.
The composition of the fermentation medium (g/l) was as follows:
















Glucose
40.0



(NH4)2SO4
24.0



KH2PO4
1.0



MgSO4•7H2O
1.0



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Yeast extract
2.0



CaCO3
30.0










Glucose, potassium phosphate and magnesium sulfate were sterilized separately. CaCO3 was sterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to 7.0.
As follows from Table 2, WC196-ΔglgBXCAP caused accumulation of a higher amount of L-lysine, as compared with WC196.
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. All the cited references herein are incorporated as a part of this application by reference.











TABLE 1









Amount of



Strain
OD540
L-threonine, g/l









B-3996
23.5 ± 0.3
27.5 ± 0.4



B-3996-ΔglgBXCAP
22.1 ± 0.2
28.2 ± 1.2





















TABLE 2









Amount of



Strain
OD540
L-lysine, g/l









WC196
26.2 ± 0.5
2.1 ± 0.1



WC196-ΔglgBXCAP
26.9 ± 0.3
2.4 ± 0.1










INDUSTRIAL APPLICABILITY
According to the present invention, production of an aromatic L-amino acid or a non-aromatic L-amino acid of a bacterium of the Enterobacteriaceae family can be enhanced.

## Claims

1. An Escherichia coli bacterium which is able to produce L-threonine or L-lysine and which has been modified so that the glycogen biosynthetic pathway is disrupted by attenuation of expression of the glgBX and glgCAP operons.

2. The bacterium according to claim 1, wherein said glycogen biosynthetic pathway is disrupted by inactivation of the glgBX and/or glgCAP operons.

3. The bacterium according to claim 2, wherein the inactivation of the glgBX and/or glgCAP operons comprises deletion of a gene selected from the group consisting of glgB, glgX glgC, glgA, glgP, and combinations thereof.

4. The bacterium according to claim 1, wherein said glycogen biosynthetic pathway is disrupted by attenuation of expression of the glgS gene.

5. The bacterium according to claim 1, wherein said glycogen biosynthetic pathway is disrupted by inactivation of the glgS gene.

6. A method for producing L-amino acid selected from the group consisting of L-threonine. L-lysine, and combinations thereof, comprising:
cultivating the bacterium according to claim 1 in a medium to produce and excrete said L-amino acid into the medium, and
collecting said L-amino acid from the medium.

7. A method for producing an L-amino acid selected from the group consisting of L-threonine, L-lysine, and combinations thereof, comprising:
cultivating the bacterium according to claim 2 in a medium to produce and excrete said L-amino acid into the medium, and
collecting said L-amino acid from the medium.

8. A method for producing an L-amino acid selected from the group consisting of L-threonine, L-lysine, and combinations thereof, comprising:
cultivating the bacterium according to claim 3 in a medium to produce and excrete said L-amino acid into the medium, and
collecting said L-amino acid from the medium.

9. A method for producing an L-amino acid selected from the group consisting of L-threonine, L-lysine, and combinations thereof, comprising:
cultivating the bacterium according to claim 4 in a medium to produce and excrete said L-amino acid into the medium, and
collecting said L-amino acid from said medium.

10. A method for producing an L-amino acid selected from the group consisting of L-threonine, L-lysine, and combinations thereof, comprising:
cultivating the bacterium according to claim 5 in a medium to produce and excrete said L-amino acid into the medium, and
collecting said L-amino acid from the medium.