RU2320719C2 - METHOD FOR PREPARING L-THREONINE USING MICROORGANISM BELONGING TO ESCHERICHIA GENUS WHEREIN ybiV GENE IS INACTIVATED - Google Patents

Abstract

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention represents a method for preparing L-threonine or L-arginine. Method involves culturing a modified microorganism belonging to Escherichia genus wherein ybiV gene is inactivated and isolation of L-threonine or L-arginine from cultural fluid. Invention provides preparing L-threonine or L-arginine with the high degree of effectiveness.

EFFECT: improved preparing method of amino acids.

3 cl, 3 tbl, 3 dwg, 13 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 ybiV gene of said bacterium is weakened.

Description of the Related Art

The ybiV gene of the bacterium Escherichia coli encodes a YbiV protein, a protein with a hypothetical function, the amino acid sequence of which is homologous to the proteins of the haloacidedehalogenase superfamily (HAD). Despite the fact that many members of this family have been identified, only some of them have known functions. Based on x-ray structural analysis, amino acid sequence analysis and biochemical analysis, YbiV protein was characterized as HAD phosphatase. Using X-ray structural analysis, it was shown that the YbiV protein consists of two domains: one domain with a secondary structure typical of HAD hydrolase, and another domain with an alpha / beta structure. To clarify the mechanism of action, we studied the complexes of YbiV protein with beryllium fluoride (BeF ₃ -) and aluminum trifluoride (AlF ₃ ). These complexes mimic, respectively, the phosphorylated intermediate and the transition compound during hydrolysis, by analogy with other HAD phosphatases. An analysis of these structures revealed a trench for binding the substrate, which has a hydrophilic nature. Based on the analysis of the structure of the YbiV protein and homology of the amino acid sequence, it was suggested that this protein may be sugar phosphatase. This assumption is also confirmed by biochemical analysis based on the measurement of free phosphate release using a number of sugar-like substrates (Roberts, A. et al. YbiV from Escherichia coli K12 is a HAD phosphatase. Proteins, 2005, 58 (4): 790- 801).

But there are currently no reports describing the use of ybiV 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 ybiV 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.

An object of the present invention is to provide an L-amino acid producing bacterium of the Enterobacteriaceae family modified in such a way that the expression of the ybiV gene of said bacterium is weakened.

It is also an object of the present invention to provide a bacterium as described above, wherein expression of said ybiV gene is attenuated by inactivation of said ybiV gene.

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

It is also an object of the present invention to provide a bacterium as 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 present the bacteria described above, wherein the 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 present the bacteria described above, wherein 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.

It is also an object of the present invention to provide a method for producing an L-amino acid, 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 present 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 present 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 a method as described above, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine , L-histidine, L-glycine, L-serine, L-alanine, L-asparagine, L-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

The bacterium of the present invention is an L-amino acid producing bacterium of the Enterobacteriaceae family, modified in such a way that the expression of the ybiV 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 L-amino acid and causes the accumulation of L-amino acid in 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 said microorganism is capable of accumulating in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l. The term “L-amino acid” includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine , L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

The term “aromatic L-amino acid” includes L-phenylalanine, L-tyrosine and L-tryptophan. The term “non-aromatic L-amino acid” includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, L-glycine, L-serine, L-alanine, L-asparagine, L-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) can be used.

(http://www.ncbi.nlm.nih.gov/htbinpost/Taxonomy/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, 1993, 43: 162-173).

The term “bacterium is modified in such a way that the expression of the ybiV gene is attenuated” means that said bacterium has been modified in such a way that, as a result of the modification, such a bacterium contains a reduced amount of YbiV protein compared to an unmodified bacterium or the specified bacterium is unable to synthesize YbiV protein.

The term "bacterium is modified in such a way that the expression of the ybiV gene is weakened" also means that the target gene is modified in such a way that it encodes a mutant protein (s) with reduced activity.

The term "inactivation of the ybiV 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 like promoter (s), enhancer (s), attenuator (s), ribosome binding site (s), etc.

The ybiV gene encodes a protein YbiV, HAD-phosphatase (synonym - b0822). The ybiV gene (nucleotide numbers 859251 through 858436 in the nucleotide sequence with accession number NC_000913.2 in the GenBank database; gi: 49175990; SEQ ID NO: 1) is located on the chromosome of E. coli strain K-12 between the ybiU and ybiW genes. The nucleotide sequence of the ybiV gene and the corresponding amino acid sequence of the YbiV protein encoded by the ybiV 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 inactivable ybiV gene is not limited to the gene shown in the Sequence Listing Number 1 (SEQ ID No: 1), but may also include genes that are homologous to the sequences shown in the List of sequences under the number 1 (SEQ ID No: 1). Thus, a variant of the protein encoded by the ybiV 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 the Sequence List under number 2 (SEQ ID NO. 2), provided that prior to inactivation, the ability of the YbiV protein to regulate the transcription of a number of genes is preserved.

In addition, the ybiV 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 the indicated variant encodes the functional protein YbiV before 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

Inactivation of the indicated gene can be performed by conventional methods, such as mutagenesis using UV radiation or treatment with nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, inactivation of the gene by homologous recombination, and / or insertion-deletion mutagenesis (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 YbiV protein activity can be determined by complementing the ybiV mutation, for example, using the method described by Roberts, A. et al. YbiV from Escherichia coli K12 is a HAD phosphatase. Proteins, 2005, 58 (4): 790-801. Thus, a decrease or lack of activity of the YbiV protein in the bacteria according to the present invention can be determined by comparing the specified bacteria with the parent unmodified bacterium. In addition, the level of 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.

Methods for producing plasmid DNA, cutting and DNA leader, 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 ybiV gene is weakened, 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 ybiV 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 the bacterium in which the ybiV 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 / p VIC40 (VKPM B-3996) (US Pat. Nos. 5,175,107 and 5,705,5371 ), 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.

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.

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_00913.2 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.

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 producer strain E. coli 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. 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 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 by 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 patent 4346170) 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 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), 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 (see US Pat. No. 5,827,698).

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 11155571A2); 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 08-70879 A), 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) (RF patent application 2001117632).

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.

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 parental strains used to produce the L-glutamic acid producing bacterium of the present invention include mutant strains lacking α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity. 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 W3110sucA :: Kmr

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

The E. coli strain W3110sucA :: Kmr 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 Pantoea that are devoid of α-ketoglutarate dehydrogenase activity or have decreased α-ketoglutarate dehydrogenase activity and can be obtained as described above. Examples of such strains are Pantoea ananatis AJ13356 strain (US Pat. No. 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), now called the National Institute of Advanced Industrial Science and Technology, International Organizational Depository for Patenting Purposes, Central 6, 1-1, Higashi 1-Cho me, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6.1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), February 19, 1998 and received FERM P-16645. Then, the international deposit of this strain was made according to the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. In the Pantoea ananatis strain AJ13356, α-KGDH activity is absent due to the destruction of the αKGDH-E1 subunit gene (sucA). The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13355 strain. However, it was later classified as Pantoea ananatis based on the nucleotide sequence of 16S rRNA and other evidence (see the Examples section). Although both strains — AJ13355 and the strain AJ13356 derived from it — were deposited as Enterobacter agglomerans in the above depository, for the purposes of this description they 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). E. coli strain K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli strain 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 an allele of the serA gene encoding an enzyme not inhibited by serine in a feedback manner (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 (international application 9708333, 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 WO 03044192). 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.

L-proline producing bacterium

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

Examples of bacteria belonging to the genus Escherichia and 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 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) (US Patent Application US 2002058315) ) and its derivatives containing mutant N-acetylglutamate synthase (RF patent application 2001112869), E. coli 382 strain (VKPM B-7926) (European patent application EP 1170358), arginine producing strain into which the argA gene encoding N- acetylglutamate synthetase (Japanese Patent Laid-Open 57 -5693 A), and the like.

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 the L-amino acid from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using bacteria.

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 construction of the plasmid pMW118-attL-Cm-attR used as a template for PCR.

Figure 2 shows the relative positions of the primers P17 and P18 on the plasmid pMW118-attL-Cm-attR used for PCR amplification of the cat gene.

Figure 3 shows the construction of a chromosomal DNA fragment containing the inactivated ybiV gene.

Examples

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

Example 1. Construction of PCR matrices and helper plasmids

Plasmid pMW118-attL-Cm-attR, used as a template for PCR, and helper plasmid pMW-intxis-ts were obtained as described below.

(1) pMW118-attL-Cm-attR

Plasmid pMW118-attL-Cm-attR was constructed on the basis of plasmid pMW118-attL-Tc-attR obtained by crosslinking the following four DNA fragments:

1) a BglII-EcoRI fragment (114 bp) carrying attL (SEQ ID NO: 3), which was obtained by PCR amplification of the corresponding chromosome region of E. coli strain W3350 (containing prophage λ) using oligonucleotides P1 and P2 (SEQ ID NOS: 4 and 5) as primers (these primers contained additional recognition sites for BglII and EcoRI restriction enzymes);

2) a PstI-HindIII fragment (182 bp) carrying attR (SEQ ID NO: 6), which was obtained by PCR amplification of the corresponding chromosome region of E. coli strain W3350 (containing prophage λ) using P3 and P4 oligonucleotides (SEQ ID NOS: 7 and 8) as primers (these primers contained additional recognition sites for the restriction enzymes PstI and HindIII);

3) a large fragment of BglII-HindIII (3916 bp) of the plasmid pMW118-ter_rrnB. Plasmid pMW118-ter_rrnB was obtained by crosslinking the following three DNA fragments:

- a large DNA fragment (2359 bp) carrying the AatII-EcoRI fragment of plasmid pMW118 obtained by the following method: pMW118 was digested with restriction enzyme EcoRI, processed using a Klenow fragment of DNA polymerase I), and then digested with restriction enzyme AatII;

- a small fragment of AatII-BglII (1194 bp) of pUC19 plasmid carrying the bla ampicillin resistance gene (Ap ^R ), which was obtained by PCR amplification of the corresponding region of pUC19 plasmid using P5 and P6 oligonucleotides (SEQ ID NOS: 9 and 10) as primers (these primers contained additional recognition sites for the restriction enzymes AatII and BglII);

- a small fragment of BglII-PstIpol (363 bp) of the ter_rrnB transcription terminator obtained by PCR amplification of the corresponding chromosome region of E. coli strain MG1655 using oligonucleotides P7 and P8 (SEQ ID NOS: 11 and 12) as primers (data primers contained additional recognition sites for the restriction enzymes BglII and PstI);

4) a small fragment of EcoRI-PstI (1388 bp) (SEQ ID NO: 13) of the plasmid pML-Tc-tet_thrL carrying the tetracycline resistance gene and ter_thrL transcription terminator; plasmid pML-Tc-ter_thrL was obtained in two stages:

- the plasmid pML-ter_thrL was obtained by cleavage of the plasmid pML-MCS (Mashko, SV et al., Biotekhnologiya (in Russian), 2001, no.5, 3-20) using restriction enzymes Xbal and BamHl and subsequent crosslinking of a large fragment (3342 bp) and an XbaI-BamHI fragment (68 bp) carrying the ter_thrL terminator and obtained by PCR amplification of the corresponding chromosome region of E. coli strain MG1655 using oligonucleotides P9 and P10 (SEQ ID NOS: 14 and 15) as primers (these primers contained additional recognition sites for the restriction enzymes XbaI and BamHI);

- plasmid pML-Tc-ter_thrL was obtained by cleavage of the plasmid pML-ter_thrL using restriction enzymes KpnI and XbaI and subsequent processing using a Klenow fragment of DNA polymerase I (Klenow fragment of DNA polymerase I) and cross-linking with a small fragment of EcoRI-Van91I (1317 bp) of the plasmid pBR322 carrying the tetracycline resistance gene (pBR322 was digested with restriction enzymes EcoRI and Van91I and then treated with the Klenov fragment of DNA polymerase I).

Plasmid pMW118-attL-Cm-attR was constructed by crosslinking a large BamHI-XbaI fragment (4413 bp) of the plasmid pMW118-attL-Tc-attR and a synthetic BglII-XbaI DNA fragment (1162 bp) containing the promoter P _A2 (early T7 phage promoter), cat chloramphenicol resistance gene (Cm ^R ), ter_thrL transcription terminator and attR region. A synthetic DNA fragment (SEQ ID NO: 16) was obtained as follows:

1. The plasmid pML-MCS was digested with restriction enzymes KpnI and XbaI and crosslinked with a small fragment of KpnI-XbaI (120 bp) containing the P _A2 promoter (early promoter of phage T7) and obtained by PCR amplification of the corresponding DNA fragment of phage T7 using oligonucleotides P11 and P12 (SEQ ID NOS: 17 and 18, respectively) as primers (these primers contained additional recognition sites for KpnI and XbaI restriction enzymes). As a result, the plasmid pML-P _A2 -MCS was obtained.

2. The XbaI site was deleted from the plasmid pML-P _A2 -MCS, whereby the plasmid pML-P _A2 -MCS (XbaI ^- ) was obtained.

3. A small fragment of BglII-HindIII (928 bp) of the plasmid pML-P _A2 -MCS (XbaI ^- ) containing the P _A2 promoter (early phage T7 promoter) and the chloramphenicol resistance gene cat (Cm ^R ) was crosslinked with a small fragment of HindIII-HindIII (234 bp) of the plasmid pMW118-attL-Tc-attR containing the transcription terminator ter_thrL and the attR region.

4. A synthetic DNA fragment (1156 bp) was obtained by PCR amplification of the ligation mixture using oligonucleotides P9 and P4 (SEQ ID NOS: 14 and 8) as primers (these primers contained additional recognition sites for HindIII and XbaI).

(2) pMW-intxis-ts

The recombinant plasmid pMW-intxis-ts containing the cI repressor gene and int-xis genes of phage λ under the control of the P _R promoter was constructed on the basis of the pMWP _lac lacI-ts vector. The pMWP _lac lacI-ts vector was obtained by replacing the AatII-EcoRV fragment of the plasmid pMWP _lac lacI (Skorokhodova, A. Yu. Et al., Biotekhnologiya (in Russian), 2004, no.5, 3-21) with the AatII-EcoRV fragment plasmids pMAN997 (Tanaka, K. et al., J. BacterioL, 2001, 183 (22): 6538-6542) carrying par and ori loci, as well as repA ^ts replicon gene pSC101.

Two DNA fragments were amplified using phage λ DNA ("Fermentas") as a template. The first DNA fragment contained a DNA sequence from 37168 to 38046 nucleotides, a repressor gene cI, promoters P _RM and P _R and a leader sequence of its gene. This DNA fragment was amplified by PCR using oligonucleotides P13 and P14 (SEQ ID NOS: 19 and 20) as primers. The second DNA fragment containing the xis-int phage λ genes and the DNA sequence from 27801 to 29100 nucleotides was amplified by PCR using oligonucleotides P15 and P16 (SEQ ID NOS: 21 and 22) as primers. All primers contained the corresponding restriction sites.

The first DNA fragment obtained by PCR amplification and carrying the cI repressor gene was digested with the restriction enzyme ClaI, processed with the Klenow fragment of DNA polymerase I, and then digested with the restriction enzyme EcoRI. The second DNA fragment obtained by PCR amplification was digested with EcoRI and PstI. Plasmid pMWP _lac lacI-ts was digested with the restriction enzyme BglII, processed with the Klenow fragment of DNA polymerase I, and then digested with the restriction enzyme PstI. The vector fragment of the plasmid pMWP _lac lacI-ts was eluted from the agarose gel and crosslinked with the two aforementioned DNA fragments, resulting in the recombinant plasmid pMW-intxis-ts.

Example 2. Construction of a strain with an inactivated ybiV gene

1. Deletion of the ybiV gene

The strain containing the ybiV gene deletion was constructed using the technique developed by Datsenko, KA and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640-6645), known as "Red-dependent integration". The DNA fragment containing the Cm ^R marker encoded by the cat gene was obtained by PCR using primers P17 (SEQ ID NO: 23) and P18 (SEQ ID NO: 24) and plasmid pMW118-attL-Cm-attR as a matrix ( the construction of the plasmid is described in Example 1). Primer P17 contains a 36 N DNA region complementary to the DNA region located at the 5'-end of the ybiV gene, as well as a DNA region complementary to the attL region. Primer P18 contains a 35 N DNA region complementary to the DNA region located at the 3 ′ end of the ybiV gene, as well as a DNA region complementary to the attR region. The following temperature profile for PCR was used: denaturation at 95 ° C for 3 min; the first two cycles: 1 min at 95 ° C, 30 sec at 50 ° C, 40 sec at 72 ° C; subsequent 25 cycles: 30 sec at 95 ° C, 30 sec at 54 ° C, 40 sec at 72 ° C; and final polymerization: 5 min at 72 ° C.

The resulting PCR product with a length of 1699 bp (Figure 2), purified by agarose gel, was used for electroporation into E. coli strain MG1655 (ATCC 700926) containing the plasmid pKD46 with a heat-sensitive replicon. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 6640-6645) contains a 2154 bp DNA fragment of phage λ. (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, the 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 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 ybiV gene by PCR

YbiV deleted mutants containing the Cm resistance gene were verified by PCR. Locus-specific primers P19 (SEQ ID NO: 25) and P20 (SEQ ID NO: 26) 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 ybiV ⁺ MG1655 as a matrix of cells is 945 bp The length of the PCR product obtained by the reaction using the mutant strain MG1655 ΔybiV :: cat as a matrix of cells is 1941 bp (Figure 3).

Example 3. Production of L-threonine by E. coli strain B-3996-ΔybiV.

To assess the effect of inactivation of the ybiV gene on the production of threonine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: 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).

Both strains of E. coli, B-3996 and B-3996-ΔybiV, were grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). 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% sucrose. 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. A stain 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 ten independent in vitro fermentations are shown in Table 1. As follows from Table 1, strain B-3996-ΔybiV accumulated a larger amount of L-threonine compared to strain B-3996.

The fermentation medium of the following composition was used (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.

Example 4. Production of L-lysine by E. coli strain AJ11442-ΔybiV

To assess the effect of inactivation of the ybiV gene, lysine production DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: cat described above can be transferred to the 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, WC196 (pCABD2) and WC196 (pCABD2) -ΔybiV, can be grown in L medium containing 50 mg / l chloramphenicol and 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.

Can be used in a fermentation medium of the following composition (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. CaCO _{3 is} also added to a concentration of 30 g / l, previously sterilized by dry heat at 180 ° C for 2 hours.

Example 5. Production of L-cysteine by E. coli strain JM15 (ydeD) -ΔybiV

To assess the effect of inactivation of the ybiV gene on L-cysteine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: 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) -ΔybiV 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 6. Production of L-leucine by E. coli strain 57-ΔybiV

To assess the effect of inactivation of the ybiV gene on L-leucine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: 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 strain 57-pMW-ΔybiV can be obtained.

Both strains of E. coli, 57 and 57-ΔybiV, can be grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). 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 by paper chromatography (mobile phase composition: butanol-acetic acid-water = 4: 1: 1).

A fermentation medium (pH 7.2) of the following composition (g / l) 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 are sterilized separately.

Example 7. Production of L-histidine by E. coli strain 80-ΔybiV

To assess the effect of inactivation of the ybiV gene on L-histidine production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: cat 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 B-7270.

Both strains of E. coli, 80 and 80-ΔybiV, can be grown on L-broth containing chloramphenicol (30 mg / ml) at 29 ° C for 6 hours. Then, 0.1 ml of the obtained cultures can be introduced into 2 ml of fermentation medium in 20 × 200 mm test tubes, and the cultures can be grown at 29 ° С 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.

A fermentation medium (pH 6.0) of the following composition (g / l) can be used:

Glucose 100.0 Memeno 0.2 total nitrogen L-proline 1.0 (NH ₄ ) ₂ SO ₄ 25.0 KN 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 8. The production of L-glutamic acid strain E. coli VL334thrC ⁺ -ΔybiV

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

Both strains, VL334thrC ⁺ and VL334thrC ⁺ -ΔybiV, can be grown on L-agar plates containing chloramphenicol (30 μg / ml) at 37 ° C for 18-24 hours. Then one loop of cells can be transferred to tubes containing 2 ml of fermentation medium. The fermentation medium may 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 are 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 by 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 9. Production of L-phenylalanine by E. coli strain AJ12739-ΔybiV

To assess the effect of inactivation of the ybiV gene on the production of L-phenylalanine, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: 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 Road passage, 1) November 6, 2001 with accession number VKPM B-8197.

Both strains, AJ12739 and AJ12739-ΔybiV, can be grown at 37 ° C for 18 hours in a nutrient broth containing chloramphenicol (30 μg / ml); 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 0.11 mm coated with a 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.

Can be used in a fermentation medium of the following composition (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 10. The production of L-tryptophan strain E. coli SV164 (pGH5) -ΔybiV

To assess the effect of inactivation of the ybiV gene on L-tryptophan production, DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: cat described above 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, SV164 (pGH5) and SV164 (pGH5) -ΔybiV, 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) and chloramphenicol (30 μg / 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 that can be used are shown in Table 3; the groups of components A, B, C, D, E, F and H are sterilized separately, as shown in Table 3, in order to avoid unwanted interactions during sterilization.

Example 11. Production of L-Proline by E. coli strain 702ilvA-ΔybiV

To assess the effect of inactivation of the ybiV gene on the production of L-proline, DNA fragments from the chromosome of the E. coli MG1655 ΔybiV :: 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 Road passage, 1) with accession number VKPM V-8012.

Both strains of E. coli, 702ilvA and 702ilvA-ΔybiV, can be grown for 18-24 hours at 37 ° C on plates with L-agar containing chloramphenicol (30 μg / ml). Then fermentation using these strains can be carried out under the same conditions as described in Example 8.

Example 12. The production of L-arginine strain E. coli 382-ΔybiV

To evaluate the effect of inactivation of the ybiV gene, L-arginine production DNA fragments of the chromosome of the E. coli strain MG1655 ΔybiV :: cat described above were 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, 382 and 382-ΔybiV, were grown with stirring at 37 ° C for 18 hours in 3 ml of nutrient broth containing chloramphenicol (30 mg / ml); 0.3 ml of the obtained cultures were introduced into 3 ml of fermentation medium in 20 × 200 mm test tubes, and the cultures were grown at 32 ° C for 48 hours on a rotary shaker.

After growing, the amount of L-arginine accumulated in the medium was determined using paper chromatography, and the following composition of the mobile phase was used: 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 was excised; L-arginine was eluted with a 0.5% aqueous solution of CdCl ₂ , after which the amount of L-arginine was determined spectrophotometrically at a wavelength of 540 nm. The results of ten independent in vitro fermentations are shown in Table 2. As follows from Table 2, strain 382-ΔybiV accumulated a greater amount of L-arginine compared to strain 382.

The fermentation medium of the following composition was used (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 were sterilized separately. CaCO _{3 was} dry heat sterilized at 180 ° C for 2 hours. The pH was adjusted to 7.0.

Example 13. Removal of the resistance gene Cm (cat gene) from the chromosome of strains producing L-amino acids of the bacterium E. coli

The Cm resistance gene (cat gene) can be removed from the chromosome of strains producing L-amino acids by using the int-xis system. To this end, the L-amino acid producer strain into which DNA fragments from the above E. coli MG1655 ΔybiV :: cat strain were transferred using P1 transduction (see Examples 3-12) can be transformed with the plasmid pMWts-Int / Xis. Selection of transformed clones can be performed using LB medium containing ampicillin (100 μg / ml). Cells can be incubated at 30 ° C overnight. The cat gene can be removed from transformed clones by growing individual colonies at 37 ° C (at this temperature, the CIts repressor is inactivated to some extent, while the int / xis genes are activated), followed by selection of Cm ^S Ap ^R variants. Removing the cat gene from the strain chromosome can be confirmed by PCR. Locus-specific primers P21 (SEQ ID NO: 27) and P22 (SEQ ID NO: 28) can be used for this confirmation by PCR. PCR conditions may be the same as described above. The PCR product obtained in the case of cells with the deleted cat gene should be 0.2 kb in length. Thus, an L-amino acid producing strain can be obtained in which the ybiV gene is inactivated and from which the cat gene is deleted.

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 32.1 ± 0.6 21.3 ± 0.2 B-3996-ΔybiV 30.9 ± 0.8 21.9 ± 0.3

table 2 Strain OD ₅₄₀ L-arginine, g / l 382 12.4 ± 0.8 11.6 ± 0.8 382-ΔybiV 13.1 ± 0.9 12.6 ± 0.8

Table 3 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. The bacterium producing L-threonine or L-arginine, belonging to the genus Escherichia, modified in such a way that the ybiV gene is inactivated in this bacterium. 2. The bacterium according to claim 1, characterized in that said ybiV gene is inactivated due to deletion of the ybiV gene in the bacterial chromosome. 3. A method for producing L-threonine or L-arginine, comprising growing a bacterium according to any one of claims 1 and 2 in a nutrient medium, causing production and accumulation of said L-amino acid in the culture fluid; and isolating said L-amino acid from the culture fluid.

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