RU2337956C2 - METHOD OF L-THREONINE RECEIVING WITH USAGE OF BACTERIA BELONGING TO GENUS Escherichia, IN WHICH INACTIVATED GENE lrhA - Google Patents

Abstract

FIELD: chemistry, biotechnology.

SUBSTANCE: invention concerns biotechnology and corresponds method of L-threonine receiving with usage bacteria belonging to genus Escherichia, which is modified such as gene lrhA in mentioned bacteria is inactivated.

EFFECT: receiving of L-threonine with high level of efficiency.

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 in such a way that the expression of the lrhA gene in said bacterium is weakened.

Description of the Related Art

The lrhA gene in Escherichia coli encodes the lrhA protein, a transcriptional regulator known to repress flagellar protein genes, genes whose products provide movement and chemotaxis. The function of E. coli LrhA, a LysR type regulator, was determined by comparison using the DNA microarray method of mRNA profiles of the wild-type E. coli complete genome and the isogenic mutant lrhA. In the lrhA mutant, a large number (48) of genes whose products are involved in flagellation, movement, and chemotaxis showed a relative excess of mRNA, an increase for different genes ranged from 3 to 80 times (Lehnen, D. et al., Mol. Microbiol., 45 , 2, 521-532 (2002)). The LrhA transcription regulator regulates biofilm formation and expression of type 1 fibrils by binding to the promoter regions of two fim recombinases (FimB and FimE), which catalyze the inversion of the fimA promoter (Blumer, C. et al., Microbiol., 151, 10, 3287-3298 ( 2005)). Enhanced expression of type 1 fibrils as a result of lrhA destruction was confirmed by mannose sensitive agglutination of yeast cells. Biofilm formation was stimulated by inactivation of lrhA and completely suppressed with increased production of lrhA. The influence of LrhA on the formation of biofilms was manifested through altered levels of surface molecules, most likely both flagella and type 1 fibrils. These data show the role of LrhA as a repressor of expression of type 1 fibrils and, thus, a regulator of the initial stages of biofilm formation and, presumably, also adhesion bacteria to host epithelial cells (Blumer, C. et al., Microbiol., 151, 10, 3287-3298 (2005)).

There are currently no reports describing the use of lrhA 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 goals were achieved by establishing the fact that inactivation of the lrhA gene can lead to increased production of L-amino acids such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L -phenylalanine, L-tryptophan, L-proline and L-arginine.

The present invention provides a bacterium of the Enterobacteriaceae family that is capable of increased production of amino acids such as L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamic acid, L-phenylalanine, L-tryptophan, L Proline and L-Arginine.

The 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 lrhA 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 lrhA gene is attenuated by inactivation of said lrhA 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 aromatic L-amino acids and non-aromatic L-amino acids.

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 bacteria described above 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 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, 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.

BEST MODE FOR CARRYING OUT THE INVENTION

1. The bacterium according to the present invention

The bacterium of the present invention is an L-amino acid producing bacterium of the Enterobacteriaceae family, modified in such a way that lrhA gene expression 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.

The term “L-amino acid producing bacterium” as used herein also means a bacterium that is capable of producing an L-amino acid and causes the accumulation of an L-amino acid in a fermentation medium in large quantities compared to a natural or parent E. coli strain such as 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) (http://www.ncbi.nlm.nih.gov/htbinpos/Taxonomy/ can be used. wgetorg? mode = Tree & id = 1236 & lvl = 3 & keep = 1 & 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, 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 lrhA gene is weakened” means that the bacterium has been modified so that, as a result of the modification, the bacterium contains a reduced amount of LrhA protein compared to an unmodified bacterium, or the specified bacterium is unable to synthesize the LrhA protein.

The term "inactivation of the lrhA 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 a modified DNA region is not possible due to deletion of a given gene or part of it, shift of the reading frame of a given gene, introduction of missense / nonsense mutations (mutations) or modification of regions adjacent to a gene that include sequences that control gene expression, such like promoters, enhancers, attenuators, ribosome binding sites, etc.

The lrhA gene encodes the LrhA protein, a transcriptional regulator (synonyms - b2289, genR). The lrhA gene (nucleotides complementary to nucleotides numbered 2403725 to 2404663 in sequence with accession 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 nuoA and yfbQ. The nucleotide sequence of the lrhA gene and the amino acid sequence of the LrhA protein encoded by the lrhA 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 lrhA gene is not limited to the gene shown in the Sequence Listing Number 1 (SEQ ID No: 1), but may also include genes homologous to it. Thus, a variant of the protein encoded by the lrhA 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), provided that the ability of the LrhA protein to repress transcription prior to its inactivation is maintained.

In addition, the lrhA gene can be represented by a variant that hybridizes under stringent conditions with the nucleotide sequence shown in Sequence Listing number 1 (SEQ ID NO: 1), or with a probe that can be synthesized based on the specified nucleotide sequence, provided that this option encodes a functional LrhA protein prior to inactivation. "Stringent conditions" include those conditions under which specific hybrids are formed and non-specific hybrids 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 length of the probe can be selected depending on the hybridization conditions, usually it is from 100 bp up to 1 kb

Examples of methods for attenuating lrhA gene expression include those such as mutations or deletions of the lrhA gene, in which the intracellular activity of the protein encoded by the lrhA gene is reduced or absent compared to a non-mutated or naturally occurring strain. For example, this can be achieved using recombination in order to inactivate the lrhA gene on the chromosome or to modify the expression of regulatory sequences such as a promoter or Shine-Dalgarno (SD) sequence (PCT application WO95 / 34672; Carrier, TA and Keasling, JD, Biotechnol Prog 15, 58-64 (1999)). This can also be achieved by introducing an amino acid substitution ("missense" mutation) in the region of the chromosome encoding the enzyme; the introduction of a stop codon ("nonsense" mutation); the introduction or deletion of one or two bases to create a frameshift mutation; deletion of a region of a gene or the entire gene (Qiu, Z. and Goodman, MF, J. Biol. Chem., 272, 8611-8617 (1997); Kwon. DH et al, J. Antimicrob. Chemother., 46, 793-796 (2000)).

Enzyme activity can also be reduced or removed by constructing a gene encoding a mutant enzyme with an incomplete coding region, replacing a normal gene on the chromosome with this gene using homologous recombination, introducing a transposon or IS factor into the gene.

For example, the following methods can be used to introduce mutations by gene recombination that cause a decrease or removal of the activity of the above enzyme. Part of the sequence of the target gene is modified, a mutant gene is constructed from which a normally functioning enzyme is not synthesized; this DNA gene is used to transform Enterobacteriaceae bacteria; a mutant gene is being constructed for recombination with a chromosome gene, and the target chromosome gene is replaced with a mutant gene.

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 “Integration via the Red system” (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 05-007491A). 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.

Inactivation of the indicated gene can be carried out by traditional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine).

Since the LrhA protein affects the formation of flagella, the presence or absence of LrhA protein activity can be determined, for example, by electron microscopy, as described by Blumer, C. et al. (Microbiol., 151, 10, 3287-3298 (2005)). Reduced activity or lack of activity of the protein according to the present invention can be determined by comparison with cells of the parent unmodified strain. In addition, the level of gene expression can be estimated by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting, quantitative RT-PCR, etc. The amount of protein encoded by this gene can be measured by well known methods, including SDS-PAGE electrophoresis followed by Western blot analysis, etc.

Further, confirmation of a decrease in the activity of LrhA protein can be obtained using the method of immunofluorescence antibodies, increasing the level of coagulation in the presence of D-mannose, or the degree of ability to coagulate (Pallesen, L. et al, Microbiology, 141, 11, 2839-2848 (1995)) .

Methods for obtaining 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).

L-amino acid producing bacterium

As a bacterium according to the present invention, modified so that the expression of the lrhA 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 inactivating the lrhA gene in a bacterium already capable of producing L-amino acids. On the other hand, a bacterium according to the present invention can be obtained by giving a bacterium in which the lrhA gene is already inactivated 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)), 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.

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

a mutant thrA gene encoding aspartokinase-homoserine dehydrogenase I,

feedback-resistant threonine inhibition;

- 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, 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 gInHPQ 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 pexB and ompX genes. The DNA region 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 ATG start codon (abstract 17 ^th International Congress of Biochemistry and Molecular Biology, abstract 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 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-methyllysine, 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 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, Ministry of International Trade and Industry), currently called the National Institute of Advanced Industrial Science and Technology, International Depository of Organizations for Patenting Purposes, Central 6.1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan ( National ins titute 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), diaminopromedolate ), aspartate semialdehyde dehydrogenase (asd), nicotinamide adenine dinucleotide transhydrogenase (pntAB) and aspartase (aspA) (European application EP 1253195 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 5,830,716), 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 parental strains used to produce L-cysteine-producing bacteria according to 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 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 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 that are 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 (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 strain 24 (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 the production of 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 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 is introduced, for the export of amino acids (European application EP1016710A), 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 K 12 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 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 expression of the citrate synthetase gene, phosphoenolpyruvate carboxylase gene and / or glutamate dehydrogenase gene is enhanced include those described in European applications EP 1078989A, EP 955368A and EP 952221A.

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 reduced activity of α-ketoglutarate dehydrogenase and methods for their preparation are described in US patents 5,378,616 and 5,573,945. Specifically, examples of such strains include the following strains:

E.coli W3110sucA :: Kmr

E. coli AJ 12624 (FERM BP-3853)

E. coli AJ 12628 (FERM BP-3854)

E. coli AJ 12949 (FERM BP-4881)

E. coli W3110sucA :: Kmr 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 L-glutamic acid producing bacteria include bacteria belonging to the genus Escherichia and resistant to aspartic acid antimetabolites, and α-ketoglutarate dehydrogenase deficient in activity, for example, strain AJ 13199 (FERM BP-5807), (US patent 5,908,768), or strain FERM P-12379, additionally having low activity for the cleavage of L-glutamic acid (US patent 5,393,671); E. coli strain AJ13138 (FERM BP-5565) (US patent 6,110,714) and the like.

Examples of the L-glutamic acid producing bacterium 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 patent 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) (now called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting Purposes, 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, Ibaraki-ken, 305-8566, Japan) February 19, 1998 and received accession number FERM P-16645. Then, this strain was internationally deposited in accordance with the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. The strain Pantoea ananatis AJ 13356 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 AJ 12604 (FERM BP-3579) (European patent EP488424 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 parent 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 the rena serA allele encoding an enzyme not inhibited by serine by feedback (US Pat. No. 6,180,373); 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.

It was previously shown that the natural allele of the yddG gene, which encodes a membrane protein that is not involved in the biosynthesis of any of the L-amino acids, amplified on a multi-copy vector in a microorganism, gives this microorganism resistance to L-phenylalanine and several analogues of this amino acid. In addition, the introduction of additional copies of the yddG gene into the cells of bacteria producing L-phenylalanine or L-tryptophan can positively affect the production of the corresponding amino acids (PCT international application WO03044192). Thus, it is desirable that the bacterium producing L-tryptophan be further modified so that expression of the open reading frame yddG is enhanced in this bacterium.

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 (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 WO2005 / 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 EP1239041A2).

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 E. coli strain 237 (VKPM B-7925) and mutant derivatives thereof N-acetylglutamate synthase (RF patent application 2001112869), an arginine producing strain into which the argA gene encoding N-acetylglutamate synthetase (Japanese Patent Application Laid-Open No. 57-5693) has been introduced and the like.

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 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.

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 5998178). 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 culture medium in order to produce and accumulate the L-amino acid in the culture medium and isolate 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 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 the primers lrhAL and lrhAR on the plasmid pACYC184 used to amplify the cat gene.

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

1. Deletion of the lrhA gene

The strain containing the deletion of the lrhA gene was constructed using the method 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, lrhAL PCR primers (SEQ ID NO: 3) and lrhAR (SEQ ID NO: 4) were homologous to the regions adjacent to the lrhA gene and the antibiotic resistance reporting gene on the plasmid used as template 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 (Figure 1), purified on an agarose gel, can be used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, K.A. 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 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 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 lrhA gene by PCR.

Mutants with the deleted lrhA gene containing the Cm resistance gene were verified by PCR. Locus-specific primers lrhAl (SEQ ID NO: 5) and lrhA2 (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 lrhA ⁺ MG1655 as a matrix of cells is 1615 bp The length of the PCR product obtained by the reaction using the mutant strain MG1655 ΔlrhA :: cat as a matrix of cells is 1580 bp (Figure 2). Since these PCR reaction products were practically indistinguishable using agarose gel electrophoresis, the cat fragment containing the EcoR1 restriction site was additionally identified by the restriction enzyme EcoR1, resulting in the formation of two DNA fragments (469 bp and 1120 bp long). m.).

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

To assess the effect of inactivation of the lrhA gene on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔlrhA :: cat described above were transferred to the E. coli B-3996 L-threonine producing 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-ΔlrhA.

Both strains of E. coli B-3996 and B-3996-ΔlrhA 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 10 independent in vitro fermentations are shown in Table 1.

Used the following composition of the fermentation medium, g / l:

Glucose 80.0 (NH ₄ ) ₂ SO ₄ 22.0 NaCl 0.8 KN 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.

As can be seen from Table 1, strain B-3996-ΔlrhA accumulated a larger amount of L-threonine compared to strain B-3996.

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

To assess the effect of inactivation of the lrhA gene on lysine production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔlrhA :: cat can be transferred to E. coli WC196 L-lysine producer strain (pCABD2) using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). pCABD2 is a plasmid containing the dapA gene encoding a mutant dihydropicolinate synthase that is resistant to feedback inhibition by L-lysine, the lysC gene encoding mutant aspartokinase III, resistant to feedback by L-lysine inhibition, the dapB gene encoding digiducidase ddh gene encoding diaminopimelate dehydrogenase (US patent 6040160).

Both strains of E. coli, the parent WC 196 (pCABD2) and the obtained WC196 (pCABD2) ΔlrhA :: cat, can be grown in L medium containing 20 mg / L streptomycin at 37 ° C, and 0.3 ml of the resulting cultures can be added to 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 in terms of glucose consumed can be calculated.

The composition of the fermentation medium, g / l:

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. Also add 30 g / l CaCO ₃ , previously sterilized by dry heat at 180 ° C for 2 hours.

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

To assess the effect of inactivation of the lrhA gene on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔlrhA :: 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 strain JM15 (ydeD) -ΔlrhA can be obtained. The E. coli strain JM15 (ydeD) is a derivative of the E. coli strain 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 )

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-ΔlrhA

To assess the effect of inactivation of the lrhA gene on L-leucine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔlrhA :: 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-ΔlrhA strain can be obtained.

Both strains of E. coli, 57 and 57-ΔlrhA, 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 (mobile phase composition: 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-ΔlrhA.

To assess the effect of inactivation of the lrhA gene on L-histidine production, DNA fragments of the chromosome of the E. coli MG1655 ΔlrhA :: 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). Strain 80 is described in RF patent 2119536 and deposited in the All-Russian collection of industrial microorganisms (Russia, 117545 Moscow, 1st Dorozhniy passage, 1) with accession number VKPM V-7270.

Both strains of E. coli, parent 80 and obtained 80-ΔlrhA, 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 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 is adjusted to 6.0 before sterilization.

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

To assess the effect of inactivation of the lrhA gene on the production of L-glutamic acid, DNA fragments of the chromosome of the above E. coli strain MG1655 ΔlrhA :: 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 ⁺ -ΔlrhA can be obtained.

Both strains, the parent VL334thrC ⁺ and the resulting VL334thrC ⁺ -ΔlrhA, 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 (mobile phase composition: 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-ΔlrhA

To assess the effect of inactivation of the lrhA gene on L-phenylalanine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔlrhA :: 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). 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.

Both strains, the parent AJ12739 and the obtained AJ12739-ΔlrhA, 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 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) -ΔlrhA

To assess the effect of lrhA gene inactivation on L-tryptophan production, DNA fragments of the chromosome of the E. coli strain MG1655 described above ΔlrhA :: cat can be transferred to the E. coli L-tryptophan producing strain SV164 (pGH5) using P1 transduction (Miller , JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY). 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) -ΔlrhA 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) (20 mg / ml). 0.3 ml of the obtained cultures can be introduced into 3 ml of fermentation medium containing tetracycline (20 mg / ml) in 20 × 200 mm tubes and 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 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-ΔlrhA

To assess the effect of inactivation of the lrhA gene on L-proline production, DNA fragments from the chromosome of the E. coli MG1655 ΔlrhA :: cat strain 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). Strain 702ilvA was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) with accession number VKPM V-8012.

Both strains of E. coli 702ilvA and 702ilvA-ΔlrhA 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-ΔlrhA

To assess the effect of inactivation of the lrhA gene on L-arginine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔlrhA :: cat 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). 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 V-7926.

Both strains, obtained 382-ΔlrhA and 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, using the following composition of the mobile phase: butanol: acetic acid: water = 4: 1: 1 (v / v). A solution of ninhydrin (2%) in acetone was 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 KN 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 for 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 21.4 ± 0.2 24.2 ± 0.5 B-3996-ΔlrhA 25.2 ± 1.2 25.0 ± 0.9

table 2 Solutions Component Final concentration, g / l BUT KN 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 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.

Claims (3)

1. A bacterium belonging to the genus Escherichia is a producer of L-threonine, modified in such a way that the lrhA gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said lrhA gene is inactivated due to deletion of the lrhA gene in the bacterial chromosome. 3. A method of producing L-threonine, comprising growing a bacterium 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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