RU2366703C2 - METHOD FOR PREPARING L-THREONINE WITH USING Escherichia BACTERIUM WITH INACTIVATED tolC GENE - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns biotechnology and represents the method for preparing L-threonine with using Escherichia bacterium modified in such a way that tolC gene in specified bacterium is inactivated.

EFFECT: invention enables to prepare high-yield L-threonine.

3 cl, 2 dwg, 2 tbl, 12 ex

Description

Technical field

The present invention relates to the microbiological industry, in particular, to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, modified in such a way that the expression of the tolC gene in said bacterium is weakened.

Description of the Related Art

Traditionally, L-amino acids are produced on an industrial scale by fermentation using strains of microorganisms isolated from natural sources, or their mutants. Typically, microorganisms are modified to increase the production of L-amino acids.

Various techniques are used to increase L-amino acid production, including transformation of a microorganism of recombinant DNA (see, for example, US Pat. No. 4,278,765). Other methods for increasing product yield include increasing the activity of enzymes involved in the biosynthesis of amino acids, and / or eliminating the sensitivity of the target enzyme to inhibition by the amino acid produced by feedback (see, for example, PCT application WO 95/16042 or US patents 4346170; 5661012 and 6040160 )

Another way to increase the production of L-amino acids is to weaken the expression of one or more genes involved in the degradation of the target L-amino acid, genes that deviate the predecessors of the target L-amino acid from the biosynthesis of the L-amino acid, genes involved in the redistribution of carbon and nitrogen flows and phosphate, genes encoding toxins, cellular factors, etc.

TolC is the porin of the outer membrane involved in the flow of several hydrophobic and amphipathic molecules through the membrane. The TolC protein has a beta-folded structure and forms three cylinders, each of which consists of 18 membrane antiparallel beta strands. The TolC protein is a membrane component common to several excretion systems of various substances.

It was found that the tolC gene product is located on the outer membrane (Morona R., et al., J. Bacteriol. 153 (2): 693-699 (1983)). The TolC protein in the form of a trimmer was isolated from the preparation of the external membrane of the bacterium Escherichia coli and its quaternary structure was established using a two-dimensional projection with a resolution of 12 angstroms. It was found that the TolC protein is a protein of the outer membrane, each monomer of which is part of the membrane domain, presumably has a secondary structure of the beta barrel and the C-terminal periplasmic domain. The binding of the TolC protein to Sec-translocase for transfer through the inner membrane depends on the SecB protein (Baars L., et al., J. Biol. Chem., 281 (15): 10024-10034 (2006)).

Mutations in the tolC gene have been shown to slow down OmpF porin synthesis and accelerate OmpC porin synthesis. Reconstituational studies have confirmed that the TolC protein forms a channel for peptides in the outer membrane. It was found that the TolC protein is necessary for the functioning of the multicomponent ArcAB excretion system (Fralick J., J. Bacteriol, 178 (19): 5803-5805 (1996)) and its homologues AcrEF (Kobayashi K. et al. J. Bacteriol., 183 (8): 2646-2653 (2001)) and YhiUV (Nishino K., et al., J. Bacteriol., 184 (8): 2319-2323 (2002)), EmrAB excretion systems (Borges-Walmsley ML et al ., J. Biol. Chem., 278 (15): 12903-12912 (2003)) and its homologue EmrKY (Tanabe H., et al. J. Gen. Appt. Microbiol., 43: 257-263 (1997) ), MdtABC excretion systems (Nagakubo S., et al., J. Bacteriol., 184 (15): 4161-4167 (2002)) and MacAB macrolide extrusion systems (Kobayashi N., et al., J. Bacteriol., 183 (19): 5639-5644 (2001)).

Strains with mutations in the tolC gene become tolerant to colicin E1, their sensitivity to bacteriophages is impaired, the OmpF protein disappears, and hypersensitivity to detergents, paints, and certain antibiotics occurs.

However, there are currently no reports of inactivation of the tolC gene for the purpose of producing L-amino acids.

Description of the invention

The objectives of the present invention are to increase the productivity of strains producing L-amino acids and the provision of a method for producing L-amino acids using these strains.

These goals were achieved by detecting the fact that the weakening of the expression of the tolC gene can increase the 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.

The present invention provides a bacterium of the Enterobacteriaceae family that is capable of increased production of 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.

The aim of the present invention is the provision of bacteria producing L-amino acids of the Enterobacteriaceae family, wherein said bacterium has been modified so that the expression of the tolC gene in this bacterium is weakened.

It is also an object of the present invention to provide the bacteria described above, wherein expression of the tolC gene is attenuated by inactivation of the tolC gene.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide the bacteria described above, wherein said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide the bacteria 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 also an object of the present invention to provide the bacteria described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine and L-tryptophan.

It is also an object of the present invention to provide the bacteria 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.

Another objective of the present invention is the provision of a method for producing L-amino acids, which includes:

- growing the above bacteria in a nutrient medium for the purpose of production and isolation of L-amino acids in the culture fluid, and

- the allocation of the specified L-amino acids from the culture fluid.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is selected from the group consisting of aromatic L-amino acids and non-aromatic L-amino acids.

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

It is also an object of the present invention to provide the method 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.

In more detail, the present invention is described below.

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention

A bacterium according to the present invention is a bacterium belonging to the Enterobacteriaceae family with the ability to produce L-amino acids, while the bacterium is modified so that the expression of the E. coli gene in this bacterium is weakened.

According to the present invention, the term “bacterium with the ability to produce L-amino acids” means a bacterium having the ability to produce and release the L-amino acid in a nutrient medium when the bacterium of the present invention is grown in the specified nutrient medium.

The term “bacterium with the ability to produce L-amino acids” as used here also means a bacterium capable of producing and causing the accumulation of any L-amino acid in a nutrient medium in large quantities, compared with the wild type or parent strain of E. coli, such as a strain of E. coli K-12, and preferably means that the microorganism is capable of accumulating in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l. The term “L-amino acid” 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” 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, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline and L-arginine. Particularly preferred are L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L-proline and L-arginine.

The Enterobacteriaceae family includes bacteria belonging to the genus Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. More specifically, bacteria classified as belonging to the Enterobacteriaceae family according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = l & srchmode = l & unlock). A bacterium belonging to the genus Escherichia or Pantoea is preferred.

The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, but not limited to, the bacterium Escherichia coli (E. coli) may be mentioned.

The range of bacteria belonging to the genus Escherichia that can be used in the present invention is not limited in any way, however, for example, the bacteria described in Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1), can be included in the number of bacteria according to the present invention.

The term "bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea in accordance with the classification known to the specialist in the field of microbiology. Recently, several Enterobacter agglomerans species have been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like based on analysis of the 16S rRNA nucleotide sequence, etc. (Int. J. Syst. BacterioL, 43, 162-173 (1993)).

The term “bacterium modified in such a way that the tolC gene expression is weakened in the specified bacterium” means that the bacterium is modified in such a way that the modified bacterium contains less TolC protein than the unmodified bacterium, or that the modified bacterium becomes unable to synthesize the TolC protein.

The term "tolC gene inactivation" means that the modified gene encodes a fully non-functional protein. It is also possible that the modified portion of the DNA is unable to express the gene normally as a result of deletion of a portion of the gene, shift of the reading frame of the gene, introduction of the missense / nonsense mutation (s), or modification of the region adjacent to the gene, as a result of the inclusion of sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites, etc.

Gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using a number of known methods, including Northern blotting, quantitative RT-PCR, and the like. The amount of protein encoded by the gene can be measured by known methods, including SDS-PAGE followed by immunoblotting (Western blotting) and the like.

The tolC gene (synonyms - ESK3026, weeA, b3035, colE1-i, mtcB, mukA, refI, toc) encodes the TolC protein (synonyms: external membrane channel TolC, B3035, WeeA, Toc, RefI, MukA, MtcB). The tolC gene (nucleotides numbered 3,176,137 through 3,177,618 in sequence with accession number NC_000913.2; gi: 49175990; in the GenBank database) is located between the ycal and lpxK genes on the chromosome of E. coli K-12 strain. The nucleotide sequence of the tolC gene and the amino acid sequence of the TolC protein encoded by it, encoded by the tolC gene, are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Since there may be some differences in the DNA sequences of different genera or strains of the Enterobacteriaceae family, the nucleotide sequence of the tolC gene to be inactivated is not limited to the gene sequence shown in SEQ ID No: 1, but may also include gene sequences homologous to the sequence shown in SEQ ID No: 1. Therefore, a variant of the protein encoded by the tolC gene can be represented by a protein with a homology of at least 80%, more preferably at least 90%, and most preferably at least 95%, in terms of th to the entire amino acid sequence encoded by the gene and tolC shown in SEQ ID NO.2, as long as the TolC protein activity is maintained at the same level as before inactivation.

In addition, the tolC gene can be represented by a variant that hybridizes with the nucleotide sequence shown in the Sequence Listing under number 1 (SEQ ID NO: 1), or with a probe that can be synthesized based on the specified nucleotide sequence, under stringent conditions, under provided that the indicated variant encodes a functional TolC protein as it was before inactivation. "Stringent conditions" include those conditions under which specific hybrids are formed, for example hybrids having a homology of not less than 60%, more preferably not less than 70%, most preferably not less than 80%, even more preferably not less than 90%, and most preferably not less than 95%, and non-specific hybrids, for example, hybrids having less than the homology listed above, are not formed. For example, a demonstration of harsh conditions can be a single or multiple washing, preferably two or three washing, at a salt concentration of 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at a temperature of 60 ° C. Duration of washing depends on the type of membrane used for blotting, and, as a rule, is indicated by the manufacturers of sets in the instructions. For example, the recommended washing time for Hybond ™ N + nylon (Amersham) membranes under harsh conditions is 15 minutes. It is more preferable to repeat the washing 2-3 times. The length of the probe can be selected accordingly, depending on the conditions of hybridization, and usually varies in the range from 100 bp. up to 1 thousand p.p.

Homology between two amino acid sequences can be established using well-known methods, for example, the computer program BLAST 2.0.

The expression of the tolC gene can be attenuated by introducing a mutation into the gene on the chromosome so that the amount of the intracellular TolC protein encoded by this gene is reduced compared to the unmodified strain. Such a mutation can be achieved by introducing a drug resistance gene or by deleting a part of the gene or a full-sized gene (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon, DH et al J. Antimicrob Chemother. 46, 793-796 (2000)). The expression of the tolC gene can also be attenuated by modifying the expression of a regulatory sequence, such as a promoter, a Shine-Dalgarno (SD) sequence, etc. (WO 95/34672, Carrier, T.A. and Keasling, J.D., Biotechnol Prog 15, 58-64 (1999)).

For example, the following methods can be used to introduce mutations by gene recombination. A mutant gene is constructed and the bacterium that needs to be modified is transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome is replaced by a mutant gene using homologous recombination, and the resulting strain is selected by selection. Such gene substitution using homologous recombination can be carried out using linear DNA methods, such as, for example, the method known as “Red-dependent integration” or “Red-system integration” (Datsenko, KA, Wanner, BL, Proc. Natl. Acad. Sci. USA, 97, 12, 6640-6645 (2000)) and a method using a plasmid whose replication is temperature sensitive (US Pat. No. 6,303,383 or Japanese Application 05-007491 A). Further, the introduction of a site-specific mutation by replacing a gene using homologous recombination, as mentioned above, can also be carried out using a plasmid with reduced replication ability in the host cell.

Gene expression can also be attenuated by incorporating a transposon or IS factor into the coding region of the gene (US Pat. No. 5,175,107) or using conventional methods such as UV irradiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine ), site-directed mutagenesis, gene disruption by homologous recombination and / or due to a frame shift mutation (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83 and Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45), also called “Red-dependent Integration”.

The production of TolC protein in conjunction with MFP proteins (Membrane Fusion Proteins - Membrane Fusion Proteins) provides facilitated transport of substances through the outer membrane of Escherichia coli. Division strains in the tolC gene become hypersensitive to hydrophobic agents as a result of inactivation of the arcAB excretion system, which provides resistance to various drugs (Fralick J., J. Bacteriol., 178 (19): 5803-5805 (1996)). Therefore, a decrease or absence of activity of the TolC protein in the bacteria according to the present invention can be established by comparison with the parent unmodified strain of the bacterium.

The presence or absence of the tolC gene on the bacterial chromosome can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using a number of known methods, including Northern blotting, quantitative RT-PCR and the like. The amount of protein encoded by the gene can be measured by known methods, including SDS-PAGE followed by immunoblotting (Western blotting) and the like. For example, the recognition of the tolC gene product was performed by preparing autoradiographic images of gels after electrophoresis of labeled samples from a mini-cell strain of bacteria (Morona R., et al., J. Bacteriol., 153 (2): 693-699 (1983)).

Methods for preparing plasmid DNA, cutting and ligation of DNA, transformation, selection of oligonucleotides as primers and the like can be conventional methods well known to those skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, E.F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

Bacteria - L-amino acid producers

As a bacterium according to the present invention, which is modified in such a way that expression of the tolC gene is impaired, a bacterium capable of producing both aromatic and non-aromatic L-amino acids can be used.

The bacterium of the present invention can be obtained by inactivating the tolC gene in a bacterium already possessing the ability to produce L-amino acids. Alternatively, the bacterium of the present invention can be obtained by imparting a bacterium in which the tolC gene is already inactivated to produce L-amino acids.

L-threonine producing bacterium

Examples of the parent strain for the production of the L-threonine-producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain TDH-6 / pVIC40 (VKPM B-3996) (US Pat. Nos. 5,175,107 and 5705371), E. coli strain 472T23 / pYN7 (ATCC 98081) (US patent 5631157), E. coli strain NRRL-21593 (US patent 5939307), E. coli strain FERM BP-3756 (US patent 5474918), strains E. coli FERM BP-3519 and FERM BP-3520 (US patent 5376538), E. coli strain MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), E. coli strains VL643 and VL2055 (European patent application EP 1149911 A) and the like.

Strain TDH-6 is deficient in the thrC gene, is able to assimilate sucrose, and contains the ilvA gene with a leaky mutation. The specified strain contains a mutation in the rhtA gene, which causes resistance to high concentrations of threonine and homoserine. Strain B-3996 contains the plasmid pVIC40, which was obtained by introducing into the vector derived from the RSF1010 vector the thrA * BC operon including the thrA mutant gene encoding aspartokinase-homoserine dehydrogenase I, which has a significantly reduced feedback sensitivity to threonine inhibition. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 117105 Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. The strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF , 117545 Moscow, 1st Road passage, 1) with inventory number B-3996.

E. coli VKPM B-5318 (European patent EP 0593792 B) can also be used as the parent strain for producing the L-threonine producing bacterium according to the present invention. Strain B-5318 is prototrophic for isoleucine and contains the plasmid pVIC40, in which the regulatory region of the threonine operon is replaced by a temperature-sensitive C1 phage λ repressor and PR promoter. The strain VKPM B-5318 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) on May 3, 1990 with accession number VKPM B-5318.

Preferably, the bacterium of the present invention is further modified so as to have increased expression of one or more of the following genes:

- mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I, resistant to threonine inhibition by feedback type;

- thrB gene encoding homoserine kinase;

- thrC gene encoding threonine synthase;

- rhtA gene, presumably encoding a transmembrane protein;

the asd gene encoding aspartate β-semialdehyde dehydrogenase, and

- aspC gene encoding aspartate aminotransferase (aspartate transaminase).

The nucleotide sequence of the thrA gene encoding aspartokinase-homoserine dehydrogenase I from Escherichia coli is known (nucleotide numbers 337 to 2799 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrA gene is located on the chromosome of E. coli K-12 strain between the thrL and thrB genes. The nucleotide sequence of the thrB gene encoding homoserine kinase from Escherichia coli is known (nucleotide numbers 2801 to 3733 in sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrB gene is located on the chromosome of E. coli K-12 strain between the thrA and thrC genes. The nucleotide sequence of the thrC gene encoding a threonine synthase from Escherichia coli is known (nucleotide numbers 3734 to 5020 in the sequence with accession number NC_000913.2 in the GenBank database, gi: 49175990). The thrC gene is located on the chromosome of E. coli K-12 strain between the thrB gene and the open yaaX reading frame. All three of these genes function as one threonine operon. To enhance the expression of the threonine operon, it is desirable to remove the region of the attenuator from the operon that affects transcription (PCT application WO 2005/049808, PCT application WO 2003/097839).

The mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I, resistant to threonine inhibition by feedback type, as well as the thrB and thrC genes can be obtained as a single operon from the well-known plasmid pVIC40, which is represented in the E. coli producer strain VKPM B-3996. Plasmid pVIC40 is described in detail in US Pat. No. 5,705,371.

The rhtA gene is located at the 18th minute of the E. coli chromosome near the glnHPQ operon, which encodes the components of the glutamine transport system, the rhtA gene is identical to ORF1 (ybiF gene, nucleotide numbers 764 to 1651 in sequence with accession number AAA218541 in the GenBank database, gi: 440181) , is located between the genes PEX and OMPX. A region of DNA expressed to form the protein encoded by the ORF1 reading frame was named the rhtA gene (rht: resistance to homoserine and threonine). The rhtA23 mutation has also been shown to represent an A-to-G substitution at position -1 with respect to the start of the ATG codon (Abstracts of the 17 ^th International Congress of Biochemistry and Molecular Biology, Abstracts of the 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology , San Francisco, California August 24-29, 1997, abstract No.457, European application EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in the sequence with accession number NC_000913.1 in the GenBank database, gi: 161313 07) and can be obtained using PCR (polymerase chain reaction; link to White, TJ et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on the nucleotide sequence of the gene. Asd genes from other microorganisms can be obtained in a similar way.

Also, the nucleotide sequence of the aspC gene from E. coli is known (nucleotide numbers 983742 to 984932 in the sequence with accession number NC_000913.1 in the GenBank database, gi: 16128895) and can be obtained by PCR. AspC genes from other microorganisms can be obtained in a similar way.

L-lysine producing bacterium

Examples of bacteria producing L-lysine belonging to the genus Escherichia include mutants that are resistant to the L-lysine analogue. The L-lysine analogue inhibits the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially removed when L-lysine is also present in the medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine, lysine hydroxyamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysis, α-chlorocaprolactam, and so on. Mutants that are resistant to these lysine analogues can be obtained by treating bacteria belonging to the genus Escherichia with traditional mutagens. Specific examples of bacterial strains used to produce L-lysine include Escherichia coli strain AJ 11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli strain VL611. In these microorganisms, aspartokinase is resistant to feedback inhibition by L-lysine.

Strain WC 196 can be used as a bacterium producer of L-lysine Escherichia coli. This bacterial strain was obtained by selection of the phenotype of resistance to AEC in strain W3110, derived from the strain Escherichia coli K-12. The resulting strain was named Escherichia coli AJ 13069 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry), currently called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan (National I nstitute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), December 6, 1994 and received accession number FERM P -14690. Then, the strain was internationally deposited in accordance with the terms of the Budapest Treaty on September 29, 1995, and the strain received accession number FERM BP-5252 (US patent 5827698).

Examples of parental strains for producing L-lysine producing bacteria of the present invention also include strains in which the expression of one or more genes encoding L-lysine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-lysine include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate carboxylase (lysA), diaminopromenodel 60 ppc), aspartate semialdehyde dehydrogenase (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1 253 195 A). In addition, parental strains may have an increased level of expression of the gene involved in the respiration process (suo) (European application EP 1170376 A), the gene encoding nicotinamide nucleotranshydrogenase (pntAB) (US patent 5830716), the ybjE gene (PCT application WO 2005/073390) , or combinations of these genes.

Examples of parent strains for producing bacteria producing L-lysine according to the present invention also include strains in which enzyme activity is reduced or absent which catalyze the formation of compounds other than L-lysine branching from the main pathway of L-lysine biosynthesis. Examples of enzymes that catalyze the formation of non-L-lysine compounds branching off from the main L-lysine biosynthesis pathway include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698) and malate dehydrogenase (PCT application WO 2005/010175).

L-cysteine producing bacterium

Examples of parent strains used to produce L-cysteine-producing bacteria of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain JM15 transformed with various cysE alleles encoding inhibition resistant cysE feedback type of serine acetyltransferase (US patent 6218168; RF patent application 2003121601); E. coli strain W3110 containing overexpressed genes encoding a protein capable of secretion of compounds toxic to the cell (US patent 5972663); E. coli strains containing cysteine desulfohydrase with reduced activity (Japanese patent JP 11155571 A2); E. coli strain W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (international PCT application WO 0127307 A1) and the like.

L-Leucine Producer Bacteria

Examples of parental strains used to produce the L-leucine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (e.g., E. coli strain 57 ( VKPM B-7386, US Pat. No. 6,124,121)) or to leucine analogs including, for example, β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine (Japanese Patent Laid-open No. 62-34397 and 8 -70879), E. coli strains obtained using the genetic engineering methods described in PCT application 96/06926; E. coli strain H-9068 (JP8-70879 A2) and the like.

The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-leucine. Examples of such genes include the leuABCD operon genes, and are preferably represented by the mutant leuA gene encoding feedback feedback depleted L-leucine isopropyl malate synthase (US Pat. No. 6,333,342). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that export the L-amino acid from a bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes) (European application EP 1239041 A2).

L-histidine producing bacterium

Examples of parent strains used to produce L-histidine producing bacteria of the present invention include, but are not limited to, L-histidine producing bacteria belonging to the genus Escherichia, such as E. coli 24 strain (VKPM B-5945, patent RF 2003677); E. coli strain 80 (VKPM B-7270, RF patent 2119536); E. coli NRRL B-12116-B12121 strains (US Pat. No. 4,388,405); E. coli strains H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (US patent 6344347); E. coli strain H-9341 (FERM BP-6674) (European Patent 1085087); E. coli strain AI80 / pFM201 (US Pat. No. 6,258,554) and the like.

Examples of parent strains for producing bacteria producing L-histidine according to the present invention also include strains in which the expression of one or more genes encoding L-histidine biosynthesis enzymes is enhanced. Examples of enzymes involved in the biosynthesis of L-histidine include ATP-phosphoribosyltransferase (hisG), phosphoribosyl-AMP-cyclohydrolase (hisI), phosphoribosyl-ATP-phosphohydrolase (hisIE), phosphoribosylformimino-5-aminamide amide amide amide amide azide (his) histidinolphosphate aminotransferase (hisC), histidinolphosphatase (hisB), histidinol dehydrogenase (hisD), etc.

It is known that genes encoding L-histidine biosynthesis enzymes (hisG, hisBHAFI) are inhibited by L-histidine, therefore, the ability to produce L-histidine can also be significantly enhanced by introducing a feedback inhibition mutation into the ATP-gene phosphoriboside transferase (hisG) (RF patents. 2003677 and 2119536).

Specific examples of strains having the ability to produce L-histidine include E. coli FERM-P 5038 and 5048, into which a vector containing DNA encoding the L-histidine biosynthesis enzyme (Japanese application 56-005099 A), strains E. coli into which the rht gene has been introduced for export of the amino acid (European application EP 1016710 A), strain E. coli 80, which is given resistance to sulfaguanidine, DL-1,2,4-triazole-3-alanine and streptomycin (VKPM B- 7270, RF patent 2119536), etc.

L-glutamic acid producing bacterium

Examples of parental strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain VL334 thrC ⁺ European patent EP 1172433). Strain E. coli VL334 (VKPM B-1641) is an auxotroph of L-isoleucine and L-threonine with mutations in the thrC and ilvA genes (US patent 4278765). The natural allele of the thrC gene was transferred to this strain by the general transduction method using bacteriophage P1 grown on cells of the natural E. coli K12 strain (VKPM B-7). The result was a strain, auxotroph for L-isoleucine, VL334thrC ⁺ (BKPM B-8961). This strain has the ability to produce L-glutamic acid.

Examples of parent strains used to produce the L-glutamic acid producing bacterium of the present invention include, but are not limited to, strains in which the expression of one or more genes encoding L-glutamic acid biosynthesis enzymes is enhanced. Examples of the enzymes involved in the biosynthesis of L-glutamic acid 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-phosphate dehydrogenase, triose phosphate isomerase, fructose, phosphofructoninase and glucose phosphatisomerase.

Examples of strains modified in such a way that the expression of the citrate synthetase gene, phosphoenolpyruvate carboxylase gene and / or glutamate dehydrogenase gene is enhanced include EP 1078989 A, EP 955368 A and EP 952221 A described in European applications.

Examples of parental strains for producing L-glutamic acid producing bacteria according to the present study also include strains in which enzyme activity is reduced or absent that catalyzes the synthesis of compounds other than L-glutamic acid branching from the main pathway of L-glutamic acid biosynthesis. Examples of such enzymes include isocitrate lyase, α-ketoglutarate dehydrogenase, phosphotransacetylase, acetate kinase, acetohydroxy acid synthase, acetolactate synthase, format acetyl transferase, lactate dehydrogenase and glutamate decarboxylase. Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having a decreased activity of α-ketoglutarate dehydrogenase and methods for their preparation are described in US patents 5378616 and 5573945. Specifically, examples of such strains include the following strains:

E.coli W311sucA :: Km ^R

E.coli AJ 12624 (FERM BP-3853)

E.coli AJ 12628 (PERM BP-3854)

E.coli AJ 12949 (FERM BP-4881)

E.coli W3110sucA :: Km ^R is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as the “sucA gene”) in the E. coli W3110 strain. In this strain, the activity of α-ketoglutarate dehydrogenase is completely absent.

Other examples of bacteria producing L-glutamic acid include bacteria belonging to the genus Escherichia and resistant to aspartic acid antimetabolites and deficient in the activity of α-ketoglutarate dehydrogenase, for example, strain AJ 13199 (FERM BP-5807), US patent 5908768), or strain FERM P-12379, additionally having low activity for the cleavage of L-glutamic acid (US patent 5393671); E. coli strain ALZ 138 (FERM BP-5565) (US patent 6110714) and the like.

Examples of bacteria producing L-glutamic acid include mutant strains belonging to the genus Pantoea that are devoid of α-ketoglutarate dehydrogenase activity or have reduced α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Examples of such strains are Pantoea ananatis AJ 13356 strain (US Pat. No. 6,331,419), Pantoea ananatis AJ 13356 strain deposited at the National Institute of Biological Sciences and Human Technologies, Agency for Industrial Science and Technology, Department of International Trade and Industry (National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry) (currently called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting, Central 6, 1-1, Higash 1-Ch Ome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan - 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) February 19, 1998 and received FERM P-16645. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. The Pantoea ananatis strain AJ13356 does not have α-KGDH activity as a result of the destruction of the αKGDH-E1 gene subunit (sucA). The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13356 strain. However, it was later classified as Pantoea ananatis based on the 16S rRNA nucleotide sequence and other evidence. Although strain AJ 13356 has been deposited as Enterobacter agglomerans at the above depository, for the purposes of this description it will be referred to as Pantoea ananatis.

L-phenylalanine producing bacterium

Examples of parental strains used to produce the L-phenylalanine-producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as strain AJ12739 (tyrA :: Tn10, tyrR) (VKMP B-8197); strain HW1089 (ATCC-55371) containing the pheA34 gene (US patent 5354672); mutant strain MWEC101-b (KR8903681); strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patent 4407952) and the like. Also, bacteria belonging to the genus Escherichia, producers of L-phenylalanine, such as E. coli K-12 strain [W3110 (tyrA) / pPHAB] (FERM BP-3566), E. coli K strain, can be used as parent strains. -12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 strain [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 strain [W3110 (tyrA ) / pBR-aroG4, pACMAB], named as AJ12604 (FERM BP-3579) (European patent EP 488424 B1). In addition, L-phenylalanine producing bacteria belonging to the genus Escherichia with increased activity of proteins encoded by the yedA gene or yddG gene can also be used (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

L-tryptophan producing bacterium

Examples of parental strains used to produce L-tryptophan-producing bacteria of the present invention include, but are not limited to, L-tryptophan-producing bacteria belonging to the genus Escherichia, such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) lacking the activity of tryptophanyl tRNA synthetase encoded by the mutant trpS gene (US patent 5756345); E. coli strain SV164 (pGH5) containing an allele of the serA gene encoding the phosphoglycerate dehydrogenase enzyme not feedback inhibitable by serine and a trpE allele encoding the anthranilate synthase enzyme not feedback inhibitory tryptophan (US Pat. No. 6,18037); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase activity (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, which enhances the ability to synthesize phosphoenolpyruvate (PCT application WO9708333, US patent 6319696), and the like. L-tryptophan-producing bacteria belonging to the genus Escherichia and having increased activity of the identified protein encoded by the yedA gene or yddG gene can also be used as parent strains (US patent applications 2003/0148473 A1 and 2003/0157667 A1).

Examples of parent strains used to produce the L-tryptophan producing bacterium of the present invention also include strains in which the activity of one or more enzymes selected from the group consisting of anthranilate synthase, phosphoglycerate dehydrogenase and tryptophan synthase is increased. Both anthranilate synthase and phosphoglycerate dehydrogenase are feedback inhibited by L-tryptophan and L-serine, so mutations can be introduced into these enzymes that reduce the sensitivity to feedback inhibition. Specific examples of strains with such a mutation include E. coli SV164, whose anthranilate synthase is not susceptible to feedback inhibition, and a transform strain obtained by introducing pGH5 plasmid in E. coli SV164 (PCT application WO 94/08031), which contains the mutant gene serA encoding phosphoglycerate dehydrogenase, which is not sensitive to feedback inhibition.

Examples of parental strains used to produce the L-tryptophan producing bacterium of the present invention also include strains that have introduced a tryptophan operon containing a gene encoding anthranilate synthase that is not sensitive to feedback inhibition (Japanese application 57-71397 A, Japanese Patent Application 62-244382 A, U.S. Patent 4,371,614). In addition, the ability to produce L-tryptophan can be imparted by enhancing the expression of a gene (from the tryptophan operon) encoding tryptophan synthase (trpBA). Tryptophan synthase consists of two subunits α and β, which are encoded by trpA and trpB, respectively. In addition, the ability to produce L-tryptophan can be increased by enhancing the expression of the isocytratliase-malatesynthase operon (PCT application WO 2005/103275).

L-proline producing bacterium

Examples of L-proline producing bacteria used as the parent strain of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 702ilvA (VKPM B-8012), deficient in the ilvA gene and capable of producing L-proline (European patent EP 1172433). The bacterium of the present invention can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-proline. Preferably, examples of such genes for bacterium-producing L-proline include the proB gene encoding glutamate kinase with desensitized feedback regulation of L-proline (German patent 3127361). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that secrete the L-amino acid from a bacterial cell. Examples of such genes are the b2682 and b2683 genes (ygaZH genes) (European Patent Application EP 1239041 A2).

Examples of bacteria belonging to the genus Escherichia and having the ability to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (UK patent GB 2075056), VKPM B-8012 (RF patent application 2000124295), plasmid mutants described in German patent DE 3127361, plasmid mutants described in Bloom FR et al. (The 15 ^th Miami winter symposium, 1983, p. 34), and the like.

L-arginine producing bacterium

Examples of parent strains used to produce the L-arginine producing bacterium of the present invention include, but are not limited to, strains belonging to the genus Escherichia, such as strain E. coli 237 (VKPM B-7925) (US Patent Application 2002 / 058315 A1) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP 1170358A1), arginine producing strain into which the argA gene encoding the gene encoding N-acetylglutamate synthetase (European Patent Application EP 1170361 A1), and like them.

Examples of parent strains used to produce the L-arginine producing bacterium of the present invention also include strains in which the expression of one or more genes encoding L-arginine biosynthesis enzymes is enhanced. Examples of such genes include genes encoding N-acetylglutamylphosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetyl glutamate kinase (argB), acetylornithine transaminase (argD), ornithinecarbamoyl transferase (argF), arginic acid synthase carbamoylphosphate synthetase (carAB).

L-valine producing bacterium

Examples of parental strains used to produce the L-valine producing bacterium of the present invention include, but are not limited to, strains modified to overexpress the ilvGMEDA operon (US Pat. No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon that is necessary to attenuate expression so that the expression of the operon is not attenuated by the resulting L-valine. Furthermore, it is desirable to destroy the ilvA gene in the operon in order to reduce the activity of threonine deaminase.

Examples of parental strains used to produce the L-valine producing bacterium of the present invention also include mutant strains having a mutation of the aminoacyl tRNA synthetase (US Pat. No. 5,658,766). For example, E. coli strain VL1970, which has a mutation in the ileS gene encoding isoleucine-tRNA synthetase, can be used. The strain E.coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1st Road passage) on June 24, 1988 with accession number VKPM B-4411.

Further, mutant strains that require lipoic acid and / or with an insufficient amount of H ⁺ -ATPase (PCT application WO 96/06926) can also be used as parent strains.

L-isoleucine producing bacterium

Examples of parent strains used to produce the L-isoleucine producing bacterium of the present invention include, but are not limited to, mutant strains resistant to 6-dimethylaminopurine (Japanese application 5-304969A), mutant strains resistant to isoleucine analogue, such as thiaisoleucine and isoleucine hydroxamate, and mutant strains additionally resistant to DL-ethionine and / or arginine hydroxamate (Japanese application 5-130882A). In addition, recombinant strains transformed with genes encoding proteins involved in the biosynthesis of L-isoleucine, such as threonine deaminase and acetohydroxate synthase (Japanese application 2-458A, French patent 0356739 and US patent 5998178) can also be used as parent strains.

2. The method according to the present invention.

The method according to the present invention is a method for producing an L-amino acid, comprising the steps of growing a bacterium according to the present invention in a nutrient medium for the purpose of producing and accumulating an L-amino acid in a nutrient medium and isolating the L-amino acid from the culture fluid.

According to the present invention, the cultivation, isolation and purification of an L-amino acid from a culture or similar liquid may be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a bacterium.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation of the microorganism used, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron 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 carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 40 ° C, preferably in the range from 30 to 38 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffers. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the L-amino acid can be isolated and purified by ion exchange chromatography, concentration and / or crystallization.

A brief description of the figures.

Figure 1 shows the relative positions of primers P1 and P2 on the plasmid pMW118-attL-Cm-attR used to amplify the cat gene.

Figure 2 shows the construction of a chromosomal DNA fragment containing an inactivated tolC gene.

Examples

The present invention will be described in more detail below with reference to the following non-limiting Examples.

Example 1. Construction of a strain with inactivated tolC gene

1. Division of the tolC gene

The strain containing the tolC gene deletion was constructed using a technique developed by Datsenko, K.A. and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-Dependent Integration." The DNA fragment containing the drug resistance marker Cm ^R encoded by the cat gene was obtained by PCR using primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) and plasmid pMW118-attL-Cm-attR (WO 05 / 010175) as a matrix. Primer P1 contains both a region complementary to the region located at the 5'end of the tolC gene and a region complementary to the attR region. Primer P2 contains both a region complementary to the region located at the 3'end of the tolC gene and a region complementary to the attL region. The following temperature profile for PCR was used: denaturation at 95 ° C for 3 min; the first two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; and the following 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C.

The resulting PCR product with a length of 1699 bp (Figure 1), purified on an agarose gel, was used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) contains a 2154 nucleotide phage λ fragment (positions 31088 through 33241 of the nucleotide sequence with accession number J02459 in the GenBank database), and also contains the λ genes of the Red-homologous recombination system (γ, β, exo genes) under the control of the P _araB promoter; induced by arabinose. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain MG1655.

Electrocompetent cells were obtained as follows: an overnight culture of E. coli strain MG1655 / pKD46 was grown at 30 ° C in LB medium supplemented with ampicillin (100 mg / L), diluted 100 times by adding 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 resulting culture was grown with stirring at 30 ° С until OD ₆₀₀ ≈0.6 was reached, after which the cells were made electrocompetent by 100-fold concentration and three times washing with ice-cold deionized H ₂ O. Electroporation was performed using 70 μl of cells and ≈100 ng of PCR product. After electroporation, cells were incubated in 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, after which they were plated on L plates agar containing chloramphenicol (30 μg / ml) and grown at 37 ° C to select Cm ^R recombinants. Then, to remove plasmid pKD46, 2 passages were performed on L-agar with Cm at 42 ° C, and the obtained colonies were tested for sensitivity to ampicillin.

2. Confirmation of deletion of the tolC gene by PCR.

Mutants with a tolC delegated gene containing the Cm resistance gene were verified by PCR. Locus-specific primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6) were used to verify deletion by PCR. The following temperature profile was used for PCR testing: denaturation at 94 ° C for 3 min; profile for 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 length of the PCR product obtained by the reaction using the parental strain MG1655tolC ⁺ as a matrix of cells is ~ 2.0 kb. The length of the PCR product obtained as a result of the reaction using a mutant strain as a matrix of cells is ~ 1.7 kb. (Figure 2). The mutant strain was named MG1655 ΔtolC :: cat.

Example 2. Production of L-threonine by E. coli strain B-3996-ΔtolC

To assess the effect of tolC gene inactivation on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔtolC :: cat described above were transferred to the E. coli B-3996 L-threonine producer strain (VKPM B-3996) using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), resulting in strain B-3996-Δ tolC. Strain B-3996 was deposited on November 19, 1987 at the All-Union Scientific Center for Antibiotics (RF, 117105 Moscow, Nagatinskaya St., 3-A) with inventory number RIA 1867. The strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) ( Russian Federation, 117545 Moscow, 1st Road passage, 1) with inventory number B-3996.

Both strains of E. coli B-3996 and B-3996-ΔtolC were grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, these strains were grown at 32 ° C for 18 hours on a rotary shaker (250 rpm) in 20 × 200 mm test tubes containing 2 ml of L-broth with 4% glucose. Then, 0.21 ml (10%) of the inoculum was added to the fermentation medium. Fermentation was carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells were grown for 65 hours at 32 ° C with stirring (250 rpm).

After growing, the amount of L-threonine accumulated in the medium was determined by paper chromatography using the mobile phase of the following composition: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution (2%) of ninhydrin in acetone was used for visualization. The spot containing L-threonine was excised, L-threonine was eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-threonine was estimated spectrophotometrically at a wavelength of 540 nm. The results of five independent in vitro fermentations are shown in Table 1. As can be seen from Table 1, strain B-3996-ΔtolC accumulated a greater amount of L-threonine compared to strain B-3996. Used the following composition of the fermentation medium (g / l):

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 0.8 FeSO ₄ · 7H ₂ O 0.02 MnSO ₄ · 5H ₂ O 0.02 Thiamine hydrochloride 0.0002 Yeast extract 1.0 CaCO ₃ 30.0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0. The antibiotic was added to the medium after sterilization.

Example 3. Production of L-lysine by E. coli strain AJ11442-ΔtolC

To assess the effect of tolC gene inactivation on lysine production, DNA fragments of the chromosome of the E. coli MG1655 ΔtolC :: cat strain described above can be transferred to E. coli AJ11442 L-lysine producer strain using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby AJ11442-ΔtolC strain can be obtained.

Both strains of E. coli, the parent AJ11442 and the resulting AJ11442-ΔtolC, can be grown in L-medium at 37 ° C, and 0.3 ml of the obtained cultures can be introduced into 20 ml of fermentation medium containing the necessary antibiotics in 500 ml flasks. Cultivation can be carried out at 37 ° C for 16 hours using a reciprocating rocking chair with a stirring speed of 115 rpm. After growing, the amounts of L-lysine and residual glucose in the medium can be measured in a known manner (Biotech-analyzer AS210, manufacturer - Sakura Seiki Co.). Then, for each of the strains, the yield of L-lysine can be calculated in terms of glucose consumed. The composition of the fermentation medium (g / l):

Glucose 40 (NH ₄ ) ₂ SO ₄ 24 K ₂ NRA ₄ 1.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Yeast extract 2.0

The pH was adjusted to 7.0 with KOH and the medium was autoclaved at 115 ° C for 10 minutes. Glucose and MgSO ₄ · 7H ₂ O are sterilized separately. Also add 30 g / l CaCO ₃ pre-sterilized by dry heat at 180 ° C for 2 hours.

Example 4. Production of L-cysteine by E. coli strain JM15 (ydeD) -ΔtolC

To assess the effect of tolC gene inactivation on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔtolC :: cat described above can be transferred to the E. coli JM15 L-cysteine producer strain (ydeD) using P1 transduction (Miller , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby the JM15 (ydeD) -ΔtolC strain can be obtained.

The strain E. coli JM15 (ydeD) is a derivative of the strain E. coli JM15 (US patent 6218168), which can be transformed with DNA containing the ydeD gene encoding a membrane protein not involved in the biosynthesis of any of the L-amino acids (US patent 5972663 ) Strain JM15 (CGSC # 5042) can be obtained from the genetic collection of the University of New Haven, USA (The E. coli Genetic Resource Center, Yale University, New Haven, USA (http://cgsc.biology.yale.edu/)) .

Fermentation conditions for evaluating L-cysteine production are described in detail in Example 6 of US Pat. No. 6,218,168.

Example 5. The production of L-leucine strain E. coli 57-ΔtolC

To assess the effect of tolC gene inactivation on the production of L-leucine, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔtolC :: cat can be transferred to the E. coli 57 L-leucine producer strain (VKPM B-7386, US patent 6124121) by Pl transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby 57-pMW-ΔtolC strain can be obtained. The strain E.coli 57 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1st Dorozhniy proezd, 1) on May 19, 1997 with accession number VKPM B-7386.

Both strains of E. coli, 57 and 57-ΔtolC, can be grown for 18-24 hours at 37 ° C on plates with L-agar. To obtain a seed culture, these strains can be 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 with 4% sucrose. Then, 0.21 ml (10%) of the seed culture can be added to the fermentation medium. Fermentation can be carried out in 2 ml of minimal fermentation medium in test tubes measuring 20 × 200 mm. Cells can be grown for 48-72 hours at 32 ° C with stirring (250 rpm).

The amount of L-leucine can be measured using paper chromatography (composition of the mobile phase: butanol - acetic acid - water = 4: 1: 1)

The following composition of the fermentation medium (g / l) (pH 7.2) can be used:

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 25.0 K ₂ HPO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine 0.01 CaCO ₃ 25.0

Glucose and chalk should be sterilized separately.

Example 6. Production of L-histidine by E. coli strain 80-ΔtolC.

To assess the effect of tolC gene inactivation on L-histidine production, DNA fragments of the chromosome of the E. coli MG1655 ΔtolC :: cat strain described above can be transferred to the E. coli 80 L-histidine producing strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby an 80-ΔtolC strain can be obtained. Strain 80 is described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) October 15, 1999 with accession number VKPM B-7270, and then deposited in July 12, 2004 in accordance with the Budapest Treaty.

Both strains of E. coli, parent 80 and obtained 80-ΔtolC, can be grown in L-broth at 29 ° C for 6 hours. Then 0.1 ml of the obtained cultures can be introduced into 2 ml of fermentation medium in 20 × 200 mm test tubes and the cultures can be grown at 29 ° C for 65 hours on a rotary shaker (350 rpm). After growing, the amount of histidine accumulated in the medium can be determined by paper chromatography. The mobile phase of the following composition can be used: n-butanol - acetic acid - water = 4: 1: 1 (v / v). A solution of ninhydrin (0.5%) in acetone can be used for visualization.

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

Glucose 100.0 Mameno (soybean hydrolyzate) 0.2 total nitrogen L-proline 1.0 (NH ₄ ) ₂ SO ₄ 25.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ 0.01 Thiamine 0.001 Betaine 2.0 CaCO ₃ 60.0

Glucose, proline, betaine and CaCO _{3 are} sterilized separately. The pH was adjusted to 6.0 before sterilization.

Example 7. The production of L-glutamic acid strain E. coli VL334thrC ⁺ -ΔtolC

To assess the effect of inactivation of the tolC gene on the production of L-glutamic acid, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔtolC :: cat can be transferred to the producer strain of L-glutamic acid E. VL334thrC ⁺ (EP 1172433) using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain VL334thrC ⁺ -ΔtolC can be obtained. Strain VL334thrC ^{+ was} deposited on December 6, 2004 at the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) with accession number B-8961, and then deposited on December 8, 2004 in accordance with Budapest The contract.

Both strains, the parent VL334thrC ⁺ and the resulting VL334thrC ⁺ -ΔtolC, can be grown on L-agar plates at 37 ° C for 18-24 hours. Further, one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium should contain glucose - 60 g / l, ammonium sulfate - 25 g / l, KH ₂ PO ₄ - 2 g / l, MgSO ₄ - 1 g / l, thiamine - 0.1 mg / ml, L-isoleucine - 70 μg / ml and chalk - 25 g / l (pH 7.2). Glucose and chalk should be sterilized separately. Cultivation can be carried out at 30 ° C for 3 days with stirring. After growing, the amount of L-glutamic acid obtained can be determined using paper chromatography (composition of the mobile phase: butanol - acetic acid - water = 4: 1: 1), followed by staining with ninhydrin (1% solution in acetone) and further eluting the obtained compounds in 50% ethanol with 0.5% CdCl ₂ .

Example 8. The production of L-phenylalanine strain E. coli AJ12739-ΔtolC

To assess the effect of tolC gene inactivation on the production of L-phenylalanine, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔtolC :: cat can be transferred to the L-phenylalanine producing strain E.coli AJ12739 using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby AJ12739-ΔtolC strain can be obtained. Strain AJ12739 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Road passage, 1) on November 6, 2001 with accession number VKPM B-8197, and then on August 23, 2002 was deposited in accordance with the Budapest Treaty.

Both strains, the parent AJ12739 and the obtained AJ12739-ΔtolC, can be grown at 37 ° C for 18 hours in nutrient broth, 0.3 ml of the obtained cultures can be introduced into 3 ml of fermentation medium in 20 × 200 mm tubes, and the cultures can be grown at 37 ° C for 48 hours on a rotary shaker. At the end of the fermentation, the amount of phenylalanine accumulated in the medium can be determined by thin layer chromatography (TLC). For this purpose, 10 × 15 cm TLC plates coated with a 0.11 mm layer of Sorbfil silica gel without a fluorescent indicator can be used (Sorbpolymer Joint-Stock Company, Krasnodar, Russia). Sorbfil plates can be exhibited in the mobile phase of the following composition: propan-2-ol: ethyl acetate: 25% aqueous ammonia: water = 40: 40: 7: 16 (v / v). A solution (2%) of ninhydrin in acetone can be used for visualization.

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

Glucose 40.0 (NH ₄ ) ₂ SO ₄ 16.0 K ₂ HPO ₄ 0.1 MgSO ₄ · 7H ₂ O 1.0 FeSO ₄ · 7H ₂ O 0.01 MnSO ₄ · 5H ₂ O 0.01 Thiamine HCl 0.0002 Yeast extract 2.0 Tyrosine 0.125 CaCO ₃ 20.0

Glucose and magnesium sulfate are sterilized separately. CaCO ₃ sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Example 9. Production of L-tryptophan by E. coli strain SV164 (pGH5) -ΔtolC

To assess the effect of tolC gene inactivation on L-tryptophan production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔtolC :: cat described above can be transferred to the E. coli SV164 L-tryptophan producing strain (pGH5) using P1 transduction (Miller) , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain SV164 (pGH5) -ΔtolC can be obtained. Strain SV164 has an allele of the trpE gene encoding the anthranilate synthase enzyme, which is not inhibited by tryptophan in a feedback manner. Plasmid pGH5 contains a mutant gene, sera, which encodes a phosphoglycerate dehydrogenase that is not inhibited by serine by feedback. Strain SV164 (pGH5) is described in detail in US Pat. No. 6,180,373 or European Patent No. 0,662,143.

Both strains obtained SV164 (pGH5) -ΔtolC and parent SV164 (pGH5) can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth with tetracycline (plasmid marker pGH5) (10 mg / ml). 0.3 ml of the obtained cultures can be introduced into 3 ml of fermentation medium containing tetracycline (10 mg / ml) in 20 × 200 mm test tubes and can be grown at 32 ° C for 72 hours on a rotary shaker at 250 rpm. After growing, the amount of tryptophan accumulated in the medium can be determined using TLC, as described in Example 8. The components of the fermentation medium are shown in Table 2, but the groups of components A, B, C, D, E, F and H should be sterilized separately, as shown in the Table to avoid unwanted interactions during sterilization.

Example 10. The production of L-proline strain E. coli 702ilvA-ΔtolC

To assess the effect of tolC gene inactivation on L-proline production, DNA fragments from the chromosome of the E. coli MG1655 ΔtolC :: cat strain described above can be transferred to the E. coli 702ilvA L-proline producer strain by Pl transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain 702ilvA-ΔtolC can be obtained. Strain 702ilvA was deposited on July 18, 2000 at the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, ^1st Dorozhniy proezd, 1) with accession number VKPM B-8012, and then on May 18, 2001 was deposited in accordance with the Budapest Treaty .

Both strains of E. coli 702ilvA and 702ilvA-Δ tolC can be grown for 18-24 hours at 37 ° C on plates with L-agar. Then fermentation using these strains can be carried out under the same conditions as described in Example 7.

Example 11. The production of L-arginine strain E. coli 382-ΔtolC

To assess the effect of tolC gene inactivation on L-arginine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔtolC :: cat described above can be transferred to the E. coli 382 L-arginine producing strain using Pl transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain 382-ΔtolC can be obtained. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on April 10, 2000 with accession number VKPM B-7926, and then on May 18, 2001 was deposited in accordance with the Budapest Treaty .

Both strains obtained by 382-ΔtolC and the parent 382 can be grown at 32 ° C for 18 hours in 2 ml of LB broth, 0.3 ml of the resulting cultures can be introduced into 2 ml of fermentation medium in 20 × 200 mm tubes, and cultures can be grown at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium can be determined using paper chromatography, and the following composition of the mobile phase can be used: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone can be used for visualization. A spot containing L-arginine can be excised, L-arginine can be eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine can be determined spectrophotometrically at a wavelength of 540 nm.

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

Glucose 48.0 (NH ₄ ) ₂ SO ₄ 35.0 KH ₂ PO ₄ 2.0 MgSO ₄ · 7H ₂ O 1.0 Thiamine HCl 0.0002 Yeast extract 1.0 L-isoleucine 0.1 CaCO ₃ 5.0

Glucose and magnesium sulfate are sterilized separately. CaCO _{3 is} sterilized by dry heat at 180 ° C for 2 hours. The pH is adjusted to 7.0.

Example 12. Removing the gene for resistance to chloramphenicol Cm (cat gene) from the chromosome of E. coli strains producing L-amino acids.

The Cm resistance gene (cat gene) can be removed from the chromosome of strains producing L-amino acids by using the int-xis system. To this end, the L-amino acid producing strain to which DNA fragments from the above MG1655 ΔtolC :: cat strain were transferred by P1 transduction can be transformed with the plasmid pMWts-Int / Xis. Selection of transformed clones can be performed using LB medium containing ampicillin (100 μg / ml). Cells can be incubated at 30 ° C overnight. Transformed clones can be freed from the cat gene by growing individual colonies at 37 ° C (at this temperature, the CIts repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removal of the cat gene from the chromosome of strains can be confirmed by PCR. Locus-specific primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6) can be used for this confirmation by PCR. PCR conditions may be the same as described above. The PCR product obtained in the case of using cells with the deleted cat gene as a matrix should be much shorter (~ 0.3-0.5 kbp in length) than when using a strain with an undeleted cat gene as a matrix of cells.

Although the invention has been described in detail with reference to the best mode of carrying out the invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention. Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1 Strain OD ₅₄₀ The amount of L-threonine, g / l B-3996 25.3 ± 0.8 26.2 ± 0.2 B-3996-ΔtolC 19.6 ± 1.2 27.2 ± 0.6

Table 2 Groups Component Final concentration, g / l BUT KH ₂ PO ₄ 1.5 NaCl 0.5 (NH ₄ ) ₂ SO ₄ 1.5 L-methionine 0.05 L-phenylalanine 0.1 L-tyrosine 0.1 Mameno (total nitrogen) 0.07 AT Glucose 40.0 MgSO ₄ 7H ₂ O 0.3 FROM CaCl ₂ 0.011 D FeSO ₄ · 7H ₂ O 0.075 Sodium citrate 1.0 E Na ₂ MoO ₄ · 2H ₂ O 0.00015 H ₃ BO ₃ 0.0025 CoC ₂ · 6H ₂ O 0.00007 CuSO ₄ · 5H ₂ O 0.00025 MnCl ₂ · 4H ₂ O 0.0016 ZnSO ₄ · 7H ₂ O 0.0003 F Thiamine HC1 0.005 G CaCO ₃ 30.0 N Pyridoxine 0.03

Group A has a pH of 7.1, which is adjusted with ammonia. Each group is sterilized separately, cooled and then mixed with each other.

Claims (3)

1. A bacterium belonging to the genus Escherichia is a producer of L-threonine, modified in such a way that the tolC gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said tolC gene is inactivated due to deletion of the tolC gene in the bacterial chromosome. 3. A method of obtaining L-threonine, including:
growing bacteria according to any one of claims 1 and 2 in a nutrient medium, causing production and accumulation of L-threonine in the culture fluid, and
isolation of L-threonine from the culture fluid.

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