RU2359029C2 - METHOD FOR OBTAINING L-THREONINE USING BACTERIUM RELATING TO Escherichia, IN WHICH rcsA GENE IS INACTIVATED - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention refers to biotechnology and represents the method for obtaining L-threonine using the bacterium relating to Escherichia, which is modified so that rcsA gene is inactivated in the above bacterium.

EFFECT: invention allows obtaining L-threonine with high efficiency degree.

3 cl, 2 dwg, 2 tbl, 11 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 so that the expression of the rcsA gene in said bacterium is weakened.

Description of the Related Art

Traditionally, L-amino acids on an industrial scale can be obtained by fermentation using strains of microorganisms obtained from natural sources, or their mutants, specially modified in order to increase the production of L-amino acids.

Many methods have been described to increase the production of L-amino acids, for example, by transforming a microorganism of recombinant DNA (see, for example, US patent 4,278,765). Other methods are based on increasing the activity of enzymes involved in the biosynthesis of amino acids, and / or reducing the sensitivity of the target enzyme to reverse inhibition produced by the L-amino acid (see, for example, US patents 4346170; 5661012 and 6040160).

Another method of increasing 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 whose expression leads to the distraction of the precursors of the target amino acid from the pathway of biosynthesis of L-amino acids; genes involved in the redistribution of carbon, nitrogen and phosphorus streams; genes encoding toxins, etc.

The rcsA gene encodes the RcsA protein, an unstable positive regulator required for the synthesis of colanic acid polysaccharide capsules in Escherichia coli. In vivo degradation of RcsA protein depends on the ATP-dependent Lon protease. The DNA and protein sequences are highly homologous to the rcsA gene and the protein Klebsiella pneumoniae and other organisms. The C-region of RcsA contains a probable DNA binding element in the helix-rotate-helix region, which resembles the sequence found in the C-terminus of RcsB, another positive capsule synthesis regulator, and several other transcriptional regulators, including members of the LuxR family . The in vivo stability of wild-type RcsA increases with the presence of multiple copies of RcsB, while RcsA degrades faster in the rcsB mutant host than in the wild-type host. These results suggest that RcsA and RcsB interact in vivo, and are consistent with genetic experiments that indicate an interaction between RcsA and RcsB (Stout, V. et.al., J Bacteriol. 1991 March; 173 (5): 1738- 1747)).

It was shown that RcsA functions as an activator of its own expression, since a 100-fold increase in the expression of rcsA :: lacZ transcriptional hybrid was revealed in strains with a high level of RcsA protein, either due to a mutation in Ion or due to overexpression of RcsA from a multicopy plasmid. Expression of the rcsA :: lacZ hybrid increases in the absence of RcsB. In addition, the effects of H-NS, histone-like protein, and RcsB on rcsA expression are independent of each other. The sequence conserved for the cps promoter of E. coli cps and the ams promoter of Erwinia amylovora and, as shown earlier, being the RcsA-RcsB binding site, has been identified in the rcsA promoter region and, as shown, is required for high level of rcsA expression. (Ebel, W. and Trempy, J.E., J Bacteriol., 181 (2): 577-84 (1999)).

The role of DnaJ, heat shock protein, in the degradation of RcsA was investigated. While the functional cycle of RcsA in the dnaJ mutant host is increased, the accumulating RcsA protein is largely aggregated. The simultaneous stabilization and aggregation of RcsA is consistent with a model in which DnaJ (and possibly also DnaK and GrpE) acts as an indirect stimulator of proteolysis, helping to maintain RcsA in soluble form. The effect of DnaJ on the solubility of RcsA is not limited to a protein overproduced by expression of a gene contained in the plasmid. It was found that the encoded protein gene on chromosome RcsA in lon cells ^- generally in the soluble form, but in cells lon ^- dnaJ ^{- -} in the insoluble; moreover, insolubility is associated with the inability of RcsA to function in dnaJ mutants (Jubete, Y., et.al., J. Biol. Chem., 271 (48), 30798-803 (1996)).

But there are currently no reports describing the use of rcsA gene inactivation to produce 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 provide a method for producing L-amino acids using these strains.

The above objectives were achieved by establishing the fact that the weakening of the expression of the rcsA gene can lead to increased production of L-amino acids, such as 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, 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, L- 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, modified so that the expression of the rcsA gene in the specified bacteria is weakened.

It is also an object of the present invention to provide a bacterium as described above, wherein expression of said rcsA gene is attenuated by inactivation of said rcsA 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 the aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine and L-tryptophan.

Another objective of the present invention is the provision of the bacteria described above, while the 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-aspartate, 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 accumulation of L-amino acids in a nutrient medium, 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 an aromatic L-amino acid and a non-aromatic L-amino acid.

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, L-glycine, L-serine, L-alanine, L-asparagine, L-aspartate, L-glutamine, L-glutamic acid, L-proline and L-arginine.

In more detail, the present invention is described below.

Detailed Description of the Best Mode for Carrying Out the Invention

1. The bacterium according to the present invention

A bacterium according to the present invention is an L-amino acid producing bacterium of the Enterobacteriaceae family, modified so that the expression of the rcsA gene in said bacterium is weakened.

According to the present invention, “L-amino acid producing bacterium” means a bacterium capable of producing and secreting an L-amino acid into a culture medium when the bacterium of the present invention is grown in said culture medium.

As used herein, the term “L-amino acid producing bacterium” also means a bacterium that is capable of producing the L-amino acid and causes the accumulation of the L-amino acid in the fermentation medium in large quantities compared to the natural or parent E. coli strain, such as strain E. coli K-12, and preferably means that the microorganism is able to accumulate 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-aspartate, L-glutamine, L-glutamic acid, L-proline and L-arginine. Most 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 genera 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) (https://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = 1 & srchmode = 1 & unlock). A bacterium belonging to the genera 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, 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 classified 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 is modified in such a way that the expression of the rcsA gene is attenuated” means that the bacterium has been modified in such a way that, as a result of the modification, the bacterium contains a reduced amount of RcsA protein compared to an unmodified bacterium, or the modified bacterium is not able to synthesize RcsA protein.

The term "inactivation of the rcsA gene" means that the specified gene is modified in such a way that such a modified gene encodes a completely inactive protein. It is also possible that natural expression of the modified DNA region is not possible due to deletion of the target gene or part of it, shift of the reading frame of this gene, introduction of missense / nonsense mutations (mutations), or modification of regions adjacent to the gene that include sequences that control gene expression, such as promoter (s), enhancer (s), attenuator (s), ribosome binding site (s), etc.

Gene expression can be estimated by measuring the amount of mRNA transcribed from the target gene using various known techniques, including Northern blotting, quantitative RT-PCR and the like. The amount of protein encoded by this gene can be measured using known methods, including the SDS-PAGE method followed by Western blotting and the like.

The rcsA gene (synonyms - ESK1949, cpsR, b1951) encodes the RcsA protein (synonyms - CpsR, B 1951). The rcsA gene (nucleotide numbers from 2,021,992 to 2,022,615 in the nucleotide sequence with inventory number NC_000913.2 in the GenBank database; gi: 49175990; SEQ ID NO: 1) is located on the chromosome of strain E. coli K-12 between the fliR gene and the dsrB gene. The nucleotide sequence of the rcsA gene and its corresponding amino acid sequence of the RcsA protein encoded by the rcsA gene are shown in the Sequence Listing Numbers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2), respectively.

Since representatives of various genera and strains of the Enterobacteriaceae family may experience some variations in the nucleotide sequences, the concept of the inactivated rcsA gene is not limited to the gene shown in the Sequence Listing No. 1 (SEQ ID No: 1), but may also include genes homologous to it, coding variants of the RcsA protein. The term "protein variant", as used in the present invention, means a protein with changes in the sequence, be it deletion, insertion, addition or replacement of amino acids, while maintaining the activity of the RcsA protein. The number of changes in a protein variant depends on the position or type of amino acid residue in the tertiary structure of the protein. Perhaps from 1 to 30, preferably from 1 to 15, more preferably from 1 to 5, changes in the sequence shown in the List of sequences under number 2 (SEQ ID No: 2). These changes in protein variants can occur in areas of the protein that are not critical to the functioning of the protein. Such changes are allowed due to the fact that some amino acids have a high homology to each other, therefore, such changes do not affect the tertiary structure or activity. Thus, a variant of the protein encoded by the rcsA gene can be represented by a protein with a homology of at least 80%, preferably at least 90% and most preferably at least 95%, with respect to the complete amino acid sequence shown in Sequence Listing No. 2 (SEQ ID NO.2).

Homology between two amino acid sequences can be determined using known methods, for example, the BLAST 2.0 computer program, which counts three parameters: amino acid number, identity, and similarity.

In addition, the rcsA gene can be represented by a variant that hybridizes under stringent conditions 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 indicated nucleotide sequence, provided that this option encodes a functional protein RcsA. "Stringent conditions" include those conditions under which specific hybrids, for example hybrids with a degree of homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% are formed, and non-specific hybrids, for example hybrids with a degree of homology lower than the above, are not formed. A practical example of harsh conditions is a one-time washing, preferably two or three times, at a salt concentration corresponding to standard washing conditions for Southern hybridization, for example, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C . The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended wash time for Hybond ™ N + nylon membrane (Amersham) under stringent conditions is 15 minutes. Two to three times washing is preferred. The length of the probe can be selected depending on the hybridization conditions, usually it is from 100 bp up to 1 kb

Expression of the rcsA gene can be attenuated by the introduction of such a mutation into the gene on the chromosome that the activity of the protein encoded by the gene is reduced compared to the unmodified strain. Such a mutation may be a rearrangement of one or more bases in order to obtain an amino acid substitution in a protein encoded by the gene (“missense” mutation), insertion of a stop codon (“nonsense” mutation), deletion of one or more bases for reading frame shift, gene insertion antibiotic resistance or deletion of a part of a gene or deletion of a gene completely (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 rcsA gene can also be attenuated by modifying the expression control sequence, such as a promoter, a Shine-Dalgarno (SD) sequence, etc. (PCT Application 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 that encodes a mutant protein with reduced activity, the bacterium is transformed for modification by a DNA fragment containing the mutant gene. Then the native gene on the chromosome is replaced using homologous recombination with the mutant gene, and the resulting strain is selected. Such gene substitution using homologous recombination can be carried out using a linear DNA method known as “Red-dependent integration” (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 97, 12. p 6640- 6645 (2000), PCT application WO 2005/010175), or methods using a plasmid whose replication is temperature sensitive (Proc. Natl. Acad. Sci., USA, 97, 12, p. 6640-6645 (2000), patents U.S. 6303383 and 5616480). Further, the introduction of a site-specific mutation by gene replacement using homologous recombination, such as described above, can also be carried out using a plasmid that is unable to replicate in the host cell.

Gene expression can also be attenuated by inserting a transposon or IS factor gene into the coding region (US Pat. No. 5,175,107) or by conventional methods such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) .

Gene inactivation can also be carried out by traditional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination and / or mutagenesis deletion insert (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 presence of the RcsA protein can be determined using polyclonal antibodies by the method described in (Ebel, W. and Trempy, J.E., J Bacteriol., 181 (2): 577-84 (1999)).

The presence or absence of the rcsA gene on the bacterial chromosome can be determined by known methods, including PCR, Southern blotting, and the like. In addition, the level of gene expression can be estimated by measuring the amount of RNA transcribed from the gene using known methods, including Northern blotting, quantitative RT-PCR, etc. The amount or molecular weight of the protein encoded by the gene can be measured by known methods, including SDS-PAGE electrophoresis followed by Western blotting analysis, etc.

Methods for obtaining plasmid DNA, cutting and donating 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).

L-amino acid producing bacterium

As a bacterium according to the present invention, modified so that the expression of the rcsA gene is impaired, a bacterium capable of producing an aromatic or non-aromatic L-amino acid can be used.

The bacterium of the present invention can be obtained by attenuating the expression of the rcsA 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 expression of the rcsA gene is already impaired to the ability to produce L-amino acids.

L-threonine producing bacterium

Examples of the parent strain for producing 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 5,705,737) , E. coli strain NRRL-21593 (US patent 5939307), E. coli strain FERM BP-3756 (US patent 5474918), E. coli strains FERM BP-3519 and FERM BP-3520 (US patent 5376538), strain E. coli MG442 (Gusyatiner et al., Genetics, 14, 947-956 (1978)), strains of E. coli VL643 and VL2055 (European patent application EP 1149911 A) and the like.

The TDH-6 strain is defective in the thrC gene, is capable of assimilating 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.

The E. coli strain VKPM B-5318 (European application 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 relative to isoleucine, the regulatory region of the threonine operon on plasmid pVIC40 is replaced by a temperature-sensitive C1 phage λ repressor and PR promoter. VKPM strain V-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 uaaX reading frame. All three of these genes function as one threonine operon. To enhance the expression of the threonine operon, the region of the attenuator that affects transcription is removed from the operon (PCT application WO 2005/049808, WO 2003/097839).

The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I, which is resistant to threonine inhibition by feedback, 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) , located between the genes pXB 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 17 ^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No.457, EP 1013765 A).

The nucleotide sequence of the asd gene from E. coli is known (nucleotide numbers 3572511 to 3571408 in sequence with accession number NC_000913.1 in the GenBank database, gi: 16131307) 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 hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllyzine, 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 AJ11442 (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 WC196 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 AJ13069 and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, currently called the National Institute of Progressive Industrial Science and Technology, International Organizational Depository for Patenting, Central 6, 1-1, Higashi 1-Chome, 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, Ibara ki-ken, 305-8566, Japan), December 6, 1994 and received 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 (see US Pat. No. 5,827,698).

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), asparticemialdehyde dehydrogenase (asd, nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1253195 A). In addition, parent strains may have an increased level of expression of the gene involved in respiration i (suo) (European application EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US patent 5830716), the ybjE gene (PCT application WO 2005/073390) or a combination 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 the L-cysteine producing bacterium 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 serine acetyltransferase (US patent 6218168, patent application of the Russian Federation 2003121601); E. coli strain W3110 containing genes with increased expression 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 application PCT WO 0127307 A1) and the like.

L-Leucine Producer Bacteria

Examples of parent 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 analogs, including, for example, β-2 -thienylalanine, 3-hydroxyleucine, 4-azaleucine and 5,5,5-trifluoroleucine (Japanese Patent Laid-open 62-34397 and 8-70879), E. coli strains obtained using the genetic engineering methods described in PCT 96 / 06926; E. coli strain H-9068 (JP 08-70879A), 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 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 strains NRRL B-12116-B12121 (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 such genes include genes encoding ATP-phosphoribosyltransferase (hisG), phosphoribosyl-AMP-cyclohydrolase (hisI), phosphoribosyl-ATP-phosphohydrolase (hisIE), phosphoribosylformimino-hisimonosulfonamide (isomerizide aminoside) isomerase 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 with 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 the export of amino acids (European application EP 1016710A), 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 VL334thrC ⁺ (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 ⁺ (VKPM 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 such enzymes include glutamate dehydrogenase (gdh), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydrate, acnA, acnu zarzudenosefruzidazufarzudenosephase (glutamine), phosphate synthase pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglycerometase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phosphate dehydrogen phosphate dehydrogenase (gapA) isofluosophosphate pfkB) and glucose phosphatisomerase (pgi).

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 those described in European applications EP 1078989 A, EP 955368 A and EP 952221 A.

Examples of strains modified in such a way that the expression of the citrate synthetase gene and / or phosphoenolpyruvate carboxylase gene is reduced and / or lacking in activity of α-cotoglutarate dehydrogenase include those described in European applications EP 1078989 A, EP 955368 A and EP 952221 A.

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 (ACEA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate transfluenzoase (ILV), ) Bacteria belonging to the genus Escherichia, lacking the activity of α-ketoglutarate dehydrogenase or having reduced 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 W3110 sucA :: Km ^R

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

The E. coli strain W3110 sucA :: Km ^R was obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as the “sucA gene") in E. coli strain W3110. 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 AJ13199 (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 AJ13138 (FERM BP-5565) (US Pat. No. 6,110,714) 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 AJ13356 strain (US patent 6,331,419), Pantoea ananatis AJ13356 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 Purposes, Central 6, 1-1, Higashi 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 accession number 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 AJ13356 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 parent 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 E. coli strain AJ12739 (tyrA :: Tn10, tyrR) (VKMP B- 8197); E. coli strain HW1089 (ATCC-55371) containing the pheA34 gene (US patent 5354672); mutant E. coli strain MWEC101-b (KR8903681); E. coli strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US 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), strain E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and strain E. coli K-12 [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 the 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 a serA allele encoding a phosphoglycerate dehydrogenase non-feedback inhibitable serine and a trpE allele encoding anthranilate synthase non feedback inhibitory tryptophan (US Pat. No. 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) in which tryptophanase activity is absent (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, which enhances the ability to synthesize phosphoenolpyruvate (PCT application WO 9708333, US patent 6319696), and the like. Can also be used bacteria-producers of L-tryptophan belonging to the genus Escherichia, in which the activity of the protein encoded by the yedA gene or yddG gene is increased (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 sensitive to feedback inhibition, and a transformant strain obtained by introducing the plasmid pGH5 into 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. 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 L-proline producing bacteria 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 possessing 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 parental 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 E. coli strain 237 (VKPM B-7925) and mutant derivatives thereof N-acetylglutamate synthase (RF patent application 2001112869), E. coli strain 382 (VKPM B-7926) (European patent application EP 1170358 A1), arginine producing strain into which the argA gene encoding N-acetylglutamate synthetase (European patent application E) was introduced 1170361 A1), and the like.

Examples of parental 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 L-arginine biosynthesis enzymes include N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetyl glutamate kinase (argB), acetylornithine transaminase (argD), ornithinecarbamoyltransurase arginic acid, argN, argin, argin, argin, argin, 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 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. Further, 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, a strain of E. coli 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 5-304969 A), 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 5-130882 A). 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 2-458 A, 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. Thiamine and yeast extract can be used as vitamins.

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 buffer solutions. 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.

Brief Description of the Drawings

Figure 1 shows the relative positions of primers P1 and P2 on plasmid pACYC184 used to amplify the cat gene.

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

1. Division of the rcsA gene

The strain containing the deletion of the rcsA gene was constructed using the technique developed by Datsenko, K.A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-dependent Integration". In accordance with this technique, PCR primers P1 (SEQ ID NO: 3) and P2 (SEQ ID NO: 4) homologous to the region adjacent to the rcsA gene and to the gene reporting antibiotic resistance on the plasmid used as matrices for PCR. The plasmid pACYC184 (NBL Gene Sciences Ltd., UK) (accession number X06403 in the GenBank / EMBL database) was used as a template for PCR. 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 1152 bp (Fig. 1), purified on an agarose gel, can be used for electroporation in E. coli 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 _{araB inducer} P 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: E. coli MG1655 night culture 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 Н 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 plates with L-agar containing chloramphenicol (30 μg / ml) and grown at 37 ° C for selection of 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 the division of the rcsA gene using PCR.

Mutants with a delegated rcsA 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 parent strain rcsA ⁺ MG1655 as a matrix of cells is 722 bp The length of the PCR product obtained by the reaction using the mutant strain MG1655 ΔrcsA :: cat as a matrix of cells is 1214 bp (figure 2). The mutant strain was named MG1655 ΔrcsA :: cat.

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

To assess the effect of inactivation of the rcsA gene on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: 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) to obtain strain B-3996-ΔrcsA. 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 accession number RIA 1867. The strain was also deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (RF , 117545 Moscow, 1st Dorozhniy proezd, 1) April 7, 1987 with inventory number B-3996.

Both strains of E. coli, B-3996 and B-3996-ΔrcsA, 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 seed culture was introduced into 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 72 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). For visualization, a solution (2%) of ninhydrin in acetone was used. A spot containing L-threonine was excised; L-threonine was suirable 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 eight independent in vitro fermentations are shown in table. 1. As can be seen from the table. 1, strain B-3996-ΔrcsA accumulated a greater amount of L-threonine compared to strain B-3996. Used fermentation medium of the following composition (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 WC196-ΔrcsA.

To assess the effect of rscA gene inactivation on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrscA :: cat described above can be transferred to the E. coli L-lysine producer strain AJ11442) using P1 transduction (Miller, JH (1972 ) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain AJ11442-ΔrscA. The plasmid pCABD2 contains the dapA gene encoding a dihydrodipicolinate synthase with a mutation that induces resistance to inhibition by feedback type L-lysine, the lysC gene encoding aspartokinase III with a mutation that causes resistance to inhibition by feedback type L-lysine, the dapB gene encoding dihydrodicol and the ddh gene encoding diaminopimelate dehydrogenase (US patent 6040160).

Both strains of E. coli, AJ11442 and AJH442-ΔrscA, can be grown in L-medium containing 20 mg / l streptomycin at 37 ° C; and 0.3 ml of the obtained cultures can be introduced into 20 ml of a fermentation medium containing the necessary antibiotics into 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 amount 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 in terms of glucose consumed can be calculated. A fermentation medium of the following composition (g / l) can be used:

Glucose 40 (NH ₄ ) ₂ SO ₄ 24 K ₂ HPO 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. CaCO _{3 is} also added to a concentration of 30 g / l, previously sterilized by dry heat at 180 ° C for 2 hours.

Example 4. Production of L-cysteine by E. coli strain JM15 (vdeD) -ΔrcsA.

To assess the effect of rcsA gene inactivation on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: 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) -ΔrcsA 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 Coli Genetic Collection at the E. coli Genetic Resource Center, MCD Biology Department, Yale University (https://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. Production of L-leucine by E. coli strain 57-ΔrcsA.

To assess the effect of rcsA gene inactivation on L-leucine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the E. coli 57 L-leucine producer strain (VKPM B-7386, US patent 6124121) by P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby 57-pMW-ΔrcsA strain can be obtained. Strain 57 was deposited in the All-Russian collection of industrial microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on May 19, 1997 with accession number VKPM B-7386.

Both strains of E. coli, 57 and 57-ΔrcsA, 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% glucose. 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 (mobile phase composition: butanol - acetic acid - water = 4: 1: 1).

Can be used in a fermentation medium of the following composition (g / l) (pH 7.2):

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 CaCO ₃ should be sterilized separately.

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

To assess the effect of rcsA gene inactivation on L-histidine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the E. coli 80 L-histidine producer strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain the strain 80-ΔrcsA. 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, then July 12, 2004 the international deposit of this strain in accordance with the terms of the Budapest Treaty. Both strains of E. coli, 80 and 80-ΔrcsA, can be grown in L-broth at 29 ° C for 6 hours. Then 0.1 ml of each 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 (soy 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 is adjusted to 6.0 before sterilization.

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

To assess the effect of inactivation of the rcsA gene on the production of L-glutamic acid, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the L-glutamic acid producing strain E. VL334thrC ⁺ (EP 1172433) using P1- transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY), whereby strain VL334thrC ⁺ -ΔrcsA can be obtained. The strain VL334thrC ^{+ was} deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on December 6, 2004 with accession number B-8961, then on December 8, 2004 this strain was internationally deposited in accordance with the conditions of the Budapest Of the contract.

Both strains, VL334thrC ⁺ and VL334thrC ⁺ -ΔrcsA, 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 CaCO ₃ 25 g / l (pH 7.2). Glucose and CaCO ₃ 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. Production of L-phenylalanine by E. coli strain AJ12739-ΔrcsA.

To assess the effect of inactivation of the rcsA gene on L-phenylalanine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the E. coli L-phenylalanine producing strain AJ12739 using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain AJ12739-ΔrcsA. Strain AJ12739 was deposited in the All-Russian collection of industrial microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) November 6, 2001 with accession number VKPM B-8197, then on August 23, 2002, this strain was internationally deposited according to terms of the Budapest Treaty.

Both strains, AJ12739-ΔrcsA and AJ12739, can be grown at 37 ° C for 18 hours in nutrient broth; 0.3 ml of each of the obtained cultures can be introduced into 3 ml of fermentation medium in 20 × 200 mm tubes, and 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 exposed 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 is} sterilized by dry heat at 180 ° C for 2 hours. The pH was adjusted to 7.0.

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

To assess the effect of rcsA gene inactivation on L-tryptophan production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the E. coli SV164 L-tryptophan producer strain (pGH5) using P1 transduction (Miller) , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain SV164 (pGH5) -ΔrcsA. Strain SV164 contains the trpE allele encoding anthranilate synthase, which is not susceptible to tryptophan inhibition by feedback. Plasmid pGH5 contains the mutant gene serA, encoding phosphoglycerate dehydrogenase, which is not susceptible to serine inhibition by feedback. Strain SV164 (pGH5) is described in detail in US Pat. No. 6,180,373 and in European Patent No. 0,662,143.

Both strains, SV164 (pGH5) -ΔrcsA4 and SV164 (pGH5), can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth with tetracycline (plasmid marker pGH5, 20 mg / ml). 0.3 ml of the obtained cultures can be introduced into 3 ml of a fermentation medium containing tetracycline (20 mg / ml) in 20 × 200 mm test tubes; and cultures can be grown at 37 ° C. for 48 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 presented in table. 1, but the groups of components A, B, C, D, E, F and H should be sterilized separately, as shown in table. 1, to avoid unwanted interactions during sterilization.

Example 10. Production of L-Proline by E. coli strain 702ilvA-ΔrcsA.

To assess the effect of inactivation of the rcsA gene on L-proline production, DNA fragments from the chromosome of the E. coli strain MG1655 ΔrcsA :: cat described above can be transferred to the E. coli 702ilvA L-proline producer strain using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 702ilvA-ΔrcsA. Strain 702ilvA was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) July 18, 2000 with accession number VKPM B-8012, then on May 18, 2001 this strain was internationally deposited according to the terms of the Budapest Treaty.

Both strains of E. Coli, 702ilvA and 702ilvA-ΔrcsA, 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-ΔrcsA.

To assess the effect of rcsA gene inactivation on L-arginine production, DNA fragments of the chromosome of the E. coli MG1655 ΔrcsA :: cat strain described above can be transferred to the E. coli 382 L-arginine producing strain using P1 transduction (Miller, JH ( 1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) to obtain strain 382-ΔrcsA. Strain 382 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhny proezd, 1) on April 10, 2000 with accession number VKPM B-7926, then on May 18, 2001, this strain was internationally deposited according to terms of the Budapest Treaty. Both strains, 382-ΔrcsA and 382, can be grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth; 0.3 ml of the obtained cultures can be introduced into 3 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 stain 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 was adjusted to 7.0.

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 L-threonine, g / l B-3996 29.4 ± 0.7 25.9 ± 0.7 B-3996-ΔrcsA 29.8 ± 0.5 27.7 ± 0.8

table 2 Solutions Component Final concentration, g / l BUT KN ₂ PO ₄ 1.5 NaCl 0.5 (NN ₄ ) ₂ SO ₄ 1.5 L-methionine 0.05 L-phenylalanine 0.1 L-tyrosine 0.1 Mameno (total N) 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 IN ₃ 0.0025 CoCl 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 HCl 0.005 G CaCO ₃ 30.0 H Pyridoxine 0.03

The pH of solution A was adjusted to 7.1 with NH ₄ OH. Each solution was sterilized separately, mixed and then the solutions were mixed.

Claims (3)

1. A bacterium belonging to the genus Escherichia is a producer of L-threonine, modified in such a way that the rcsA gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said rcsA gene is inactivated due to deletion of the rcsA 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 the isolation of L-threonine from the culture fluid.

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