RU2458982C2 - Method of producing l-cysteine, l-cystine, s-sulphocysteine or l-cysteine thiazolidine derivative, or mixture thereof using bacteria of enterobacteriaceae family - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is bacteria of the Enterobacteriaceae family - a producer of L-cysteine, which is modified such that expression of the following is intensified therein: one or more genes of the cysDNC cluster participating in sulphate activation; or one or more genes of the cysDNC cluster participating in activation of sulphate and a cysQ gene participating in decomposition of adenosine-3'-phosphate-5'-phosphosulphate (PAPS). Disclosed are methods of producing L-cysteine, L-cystine, S-sulphocysteine and L-cysteine thiazolidine derivative, involving: growing said bacteria in a culture medium which contains sulphate; and respectively separating L-cysteine, L-cystine, S-sulphocysteine or L-cysteine thiazolidine derivative from the culture fluid.

EFFECT: invention increases output of said products.

20 cl, 2 tbl, 5 dwg, 6 ex

Description

Description of the invention

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing L-cysteine, L-cystine, a derivative or precursor thereof, or a mixture thereof using a bacterium of the Enterobacteriaceae family, modified in such a way that the expression of genes involved in the process of sulfur assimilation is enhanced .

State of the art

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

Many methods have been described to increase the production of L-amino acids, for example, by transforming a microorganism of recombinant DNA (see, for example, US patent 4,278,765). Other methods are based on increasing the activity of enzymes involved in amino acid biosynthesis and / or reducing the sensitivity of the target enzyme to inhibition by the L-amino acid produced by the feedback principle (see, for example, WO 95/16042 or US patents 5,661,012 and 6,040,160).

The synthesis of L-cysteine from inorganic sulfur is the main mechanism by which reduced sulfur is incorporated into the organic compounds of microorganisms. In this process, inorganic sulfate, the most common source of utilized sulfur in the aerobic biosphere, enters the cell and is reduced to sulfide, which, in turn, is incorporated into L-cysteine using a mechanism similar to the fixation of ammonium to glutamine or glutamate. Many genes are known to be involved in the process of sulfur assimilation, including genes involved in the activation of sulfur (cysD, cysN, cysC) and degradation (cysQ) of adenosine-3'-phosphate-5'-phosphosulfate (PAPS).

Inorganic sulfate is reduced to sulfide due to the sequence of enzymatic reactions carried out by ATP sulforylase (EC 2.7.7.4), adenylyl sulfate kinase (APS kinase) (EC 2.7.1.25), phosphoadenylyl sulfate reductase (PAPS reductase) and sulfite reductase (EC 1.8H.2.1.2. -dependent or EU 1.8.7.1. ferredoxin-dependent). O-Acetyl-L-serine (thiol) lyase (EC 4.2.99.8) incorporates sulfide to form the cysteine amino acid (Krone F.A. et al., Mol.Gen.Genet, 225 (2): 314-9 (1991)).

The DNA sequence of the sulfate-activated locus of E. coli K-12 is established. This sequence includes structural genes encoding ATP sulforylase (cysD and cysN) and APS kinase (cysC), which catalyzes the synthesis of adenylyl sulfate. To date, only these genes that make up the sulfate-activated operon are known. Consensus elements of the operon promoter have been identified, start codons and open reading frames of Cys proteins have been established. ATP sulforylase activity is stimulated by its own GTPase. Comparison of the primary sequences of CysN and Ef-Tu showed that many of the residues necessary for the formation of a correct three-dimensional structure and important for the binding of Ef-Tu and RAS proteins to guanine residues are conserved in CysN. The nodP and nodQ genes from Rhizobium meliloti are important for nodule formation in legumes. Proteins Cys and Nod are very similar to each other. NodP was the smallest subunit of ATP sulforylase. NodQ encodes homologues of both CysN and CysC; thus, these enzymes can be covalently linked in R. meliloti. The fact that the GTP binding sequences of NodQ and CysN are identical suggests that NodQ encodes a regulator of GTPase (Leyh TS et al., J. Biol. Chem., 267 (15): 10405-10 (1992)) .

The initial stages of sulphate assimilation during cysteine biosynthesis lead to sulphate uptake and its activation due to the formation of adenosine 5'-phosphosulphate, conversion to 3'-phosphatadenosine-5'-phosphosulphate and reduction to sulphite. Mutations in the cysQ gene from Escherichia coli resulting in the bacterium need for sulfite or cysteine were obtained in vivo by insertion of the Tn5tac1 and Tn5supF transposons, and in vitro insertion of resistance gene cassettes. The cysQ gene is located at position 95.7 min (from 4517 to 4518 kb) and is transcribed in the opposite direction compared to the neighboring cpdB gene. The insertion into the 3'end of the cysQ gene of the transposon Tn5tac1 with a promoter induced by isopropyl-beta-O-thiogalactopyranoside and oriented in the opposite direction to the cysQ promoter led to auxotrophy only when isopropyl-beta-O-thiogalactopyranoside was added, this conditional phenotype converging RNA polymerases or the interaction between complementary antisense and sense cysQ mRNA. Auxotrophy due to complete loss of cysQ function was a leaky type auxotrophy in some, but not all E. coli strains, and could be compensated by mutations in non-conjugated genes. CysQ mutants were prototrophic during anaerobic growth. Mutations in the cysQ gene did not affect the rate of sulfate uptake or the activity of ATP sulforylase and its protein activators, which together catalyze the synthesis of adenosine 5'-phosphosulfate. Some mutations that compensated for the cysQ null alleles resulted in impaired sulfate transport. The cysQ gene is identical to the amtA gene, which was thought to be necessary for ammonium transport. Computer analysis revealed a significant homology of amino acid sequences between the products of the cysQ and suhB genes from E. coli and the mammalian inositol monophosphatase gene. Previous studies have suggested that 3'-phosphoadenoside-5'-phosphosulfate becomes toxic when accumulated and that CysQ helps control the pool of 3'-phosphoadenoside-5'-phosphosulfate or use it in sulfite synthesis (Neuwald AF et al, J. Bacteriol 174 (2): 415-25 (1992)).

A method has been published for the production of L-threonine by fermentation of microorganisms belonging to the Enterobacteriaceae family, in which the expression of at least one or more genes of the cysteine biosynthesis pathway selected from the group consisting of cysG, cysB, cysZ, cysK, cysM, cysA, cysW, cysU, cysP, cysD, cysN, cysC, cysJ, cysI, cysH, cysE and sbp, enhanced (WO 03006666A2).

A known method of producing L-cysteine using a bacterium belonging to the genus Escherichia, in which the productivity of the L-amino acid by the aforementioned bacterium is increased due to increased expression of cysPTWAM cluster genes (US 2005124049 A1).

But at present, there is no data on the sequences of genes involved in the process of sulfur assimilation by bacteria of the Enterobacteriaceae family, and genes whose enhanced expression is necessary for the production of L-cysteine by a bacterium of the Enterobacteriaceae family.

Description of the invention

The aim of the present invention is to increase the productivity of producer strains and to provide a method for producing L-cysteine, L-cystine, their derivatives or precursors, or mixtures thereof using these strains.

This goal was achieved by discovering the fact that increased expression of genes involved in the process of sulfur assimilation leads to an increase in the production of L-cysteine.

The present invention provides a bacterium belonging to the Enterobacteriaceae family with increased ability to produce L-cysteine.

The aim of the present invention is the provision of bacteria of the Enterobacteriaceae family, a producer of L-cysteine, wherein said bacterium is modified in such a way that expression of one or more genes involved in the process of sulfur assimilation is enhanced in it.

Another objective of the present invention is the provision of the bacteria described above, in which the genes involved in the process of sulfur assimilation include genes involved in the activation of sulfate and the degradation of adenosine-3'-phosphate-5'-phosphosulfate (PAPS).

It is also an object of the present invention to provide a bacterium as described above, in which genes involved in sulfate activation include one or more cysDNC cluster genes.

Another objective of the present invention is the provision of the bacteria described above, in which the genes involved in the degradation of adenosine 3'-phosphate-5'-phosphosulfate (PAPS) include the cysQ gene.

It is also an object of the present invention to provide the bacterium described above, which is modified in such a way that expression of the cysQ gene, one or more genes of the cysDNC cluster, or both is enhanced in it.

It is also an object of the present invention to provide a bacterium as described above, in which the expression of the aforementioned gene (s) is enhanced by modifying the expression control sequence so that the expression of the indicated gene (s) is enhanced.

It is also an object of the present invention to provide a bacterium as described above in which the natural promoter (s) of said gene (s) are replaced by (s) a stronger promoter (s).

Another objective of the present invention is the provision of the above bacteria, which belongs to the genus Pantoea.

It is also an object of the present invention to provide the bacterium described above, which is a Pantoea ananatis bacterium.

Another objective of the present invention is the provision of the above bacteria, which belongs to the genus Escherichia.

It is also an object of the present invention to provide a bacterium as described above, which is an Escherichia coli bacterium.

It is also an object of the present invention to provide a process for the preparation of a compound selected from the group consisting of L-cysteine, L-cystine, derivatives or precursors thereof, which comprises growing the bacterium described above in a culture medium containing sulfate and isolating said compound from the culture fluid.

It is also an object of the present invention to provide the method described above, wherein said bacterium has enhanced expression of genes involved in the biosynthesis of L-cysteine.

It is also an object of the present invention to provide the method described above, wherein said bacterium has enhanced expression of genes involved in the biosynthesis of L-methionine.

The present invention is described in more detail below.

Detailed Description of the Best Mode for Carrying Out the Invention

1. Bacteria

The bacterium according to the present invention is a bacterium belonging to the Enterobacteriaceae family, a producer of L-cysteine, modified in such a way that the expression of genes involved in the process of sulfur assimilation by this bacterium is enhanced.

“Bacteria producing L-cysteine” means a bacterium capable of producing and storing L-cysteine in the medium when such a bacterium is cultured in a nutrient medium.

The term “bacterium producing L-cysteine” also means a bacterium capable of producing and accumulating in the culture medium L-cysteine in quantities greater than the wild-type strain, unmodified or parental strains, for example Pantoea ananatis, such as Pantoea ananatis (Enter obacter agglomerans ) strains AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (US patent 7,090,998), or Escherichia coli, such as E. coli K-12. Strain SC17 was selected as a strain with low mucus production from strain AJ13355 isolated from the soil of Iwata-shi, Shitsuoka-ken, Japan (Iwata-shi, Shizuoka-ken, Japan) as a strain that can grow in a low pH medium containing L-glutamic acid and a carbon source (U.S. Patent No. 6,596,517). Strain SC17 was deposited with the National Institute of Advanced Industrial Science and Technology, the International Depository of Organizations for Patenting Purposes, Tsukuba 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 4, 2009 and received accession number FERM BP-11091. The term “bacterium-producer of L-cysteine” also means that the microorganism is capable of producing and storing L-cysteine in the medium in concentrations of not less than 0.5 g / l and more preferably not less than 1.0 g / l.

L-cysteine produced by this bacterium can be converted in the medium to L-cystine due to the formation of disulfide bonds. Mainly, as described below, it is believed that if L-cysteine is produced in large quantities by increasing the activity of DsbA, then the formation of L-cystine from L-cysteine with the participation of the oxidative system DsbA-DsbB-UQ is triggered. In addition, S-sulfocysteine can be obtained by the reaction of L-cysteine and thiosulfuric acid in a medium (Szczepkowski T.W., Nature, vol. 182 (1958)). In addition, L-cysteine produced in bacterial cells can be condensed with ketone, aldehyde or, for example, pyruvic acid, which is present in the cells, with the aim of producing a thiazolidine derivative via a semi-ketal intermediate (see Japanese Patent No. 2992010). Such a thiazolidine derivative and a semi-ketal may be present as an equilibrium mixture. Consequently, the ability to produce L-cysteine is not limited to the ability to accumulate only L-cysteine in the medium or cells, but also includes the ability to accumulate L-cystine or their derivatives, or precursors, or a mixture thereof in the medium.

Examples of the aforementioned L-cysteine or L-cystine derivative include, for example, S-sulfocysteine, thiazolidine derivatives, polythioketal, L-methionine, S-adenosylmethionine and others. L-cysteine is a precursor of L-methionine. L-cysteine is involved in the biosynthesis of L-methionine in the conversion of O-succinyl-L-homoserine to L-cystathionine. Thus, elevated levels of L-cysteine can lead to the accumulation of L-methionine. In turn, L-methionine is the starting compound for the synthesis of S-adenosylmethionine and others. In addition, if the bacterium has, in addition to the ability to produce L-cysteine, the ability to produce L-methionine or adenosylmethionine, the production of compounds such as L-methionine or adenosylmethionine , can be increased by enhancing the expression of genes involved in the process of sulfur assimilation.

Examples of L-methionine producing bacteria and parent strains that can be used to produce L-methionine producing bacteria include, but are not limited to, Escherichia bacteria strains such as AJ11539 (NRRL B-12399), AJ11540 (NRRL B- 12400), AJ11541 (NRRL B-12401), AJ11542 (NRRL B-12402) (GB Patent GB 2075055); strains 218 (VKPM B-8125) (patent of the Russian Federation RU 2209248) and 73 (VKPM B-8126) (patent of the Russian Federation RU 2215782), resistant to L-methionine analogue norleucine, or the like.

Examples of L-cysteine or L-cystine precursors include, for example, O-acetylserine, which is a precursor of L-cysteine. Precursors of L-cysteine or L-cystine also include, for example, N-acetylserin, which is a precursor of O-acetylserine, and others.

O-acetylserine (OAS) is a precursor to the biosynthesis of L-cysteine. OAS is a metabolite of bacteria and plants and is synthesized by acetylation of L-serine in an enzymatic reaction catalyzed by serine acetyltransferase (SAT). Next, OAS is converted to L-cysteine in cells.

An L-cysteine producing bacterium can basically have the ability to produce L-cysteine, or it can be given this ability by modifying a microorganism such as described below using mutagenesis or recombinant DNA technology. Unless otherwise specified, the term L-cysteine refers to reduced L-cysteine, L-cystine, a derivative or precursor thereof, as described above, or mixtures thereof.

The concept of a bacterium is not limited in any way, provided that the bacterium according to the present invention belongs to the Enterobacteriaceae family and is capable of producing L-cysteine. The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia and so on. Particularly preferred are bacteria belonging to the Enterobacteriaceae family according to the classification of the NCBI database (National Center for Biotechnology Information, http://www.ncbi. Nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347).

The phrase "a bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea, according to the classification system known to a specialist in the field of microbiology. Some Enterobacter agglomerans species have recently been reclassified to Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or other similar ones, based on nucleotide sequence analysis of 16S RNA, and others (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).

The phrase "a bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to a specialist in the field of microbiology, but the list of bacteria is not limited in any way. Among examples of bacteria belonging to the genus Escherichia, but not limited to, the bacterium Escherichia coli (E. coli) may be mentioned.

Examples of bacteria producing L-cysteine and parent strains that can be used to produce bacteria producing L-cysteine include, but are not limited to, strains of Escherichia bacteria, such as E. coli JM15, transformed with various alleles of the cysE gene encoding serine acetyltransferase resistant to reverse regulation (US patent No. 6,218,168, patent application of Russia 2003121601), E. coli W3110, which overexpresses genes encoding proteins that regulate the secretion of cell-toxic substances (US patent No. 5,972,663), E. coli strains with reduced sulfohydrase activity (Japanese patent application 11155571 A2), E. coli W3110 with increased activity of the positive transcriptional regulator of the cysteine regulon encoded by the cysB gene (WO 01/27307 A1), etc.

The term “sulfur assimilation process” means a process in which sulfur from the environment, such as sulfate, is converted to organic sulfur for use in cellular metabolism. The two main end products of this process are the most important amino acids - L-cysteine and L-methionine. The key in this process is to increase the level of available organic sulfur for the biosynthesis of L-cysteine and L-methionine.

Examples of "one or more genes involved in the process of sulfur assimilation and degradation of adenosine 3'-phosphate 5'-phosphosulfate (PAPS)" include the cysG, cysD, cysN, cysC, cysQ genes, and combinations thereof. The cysG, cysD, cysN, cysC, cysQ genes are involved in sulfur assimilation, and the cysQ gene is involved in PAPS degradation. Examples of “one or more genes involved in sulfur assimilation and PAPS degradation” include the cysD, cysN and cysC or cysQ genes individually, the combination of cysD and cysN genes, the combination of cysD, cysN and cysC genes, the combination of cysD, cysN and cysQ and a combination of cysD, cysN, cysC and cysQ genes.

A known system of sulfate activation in E. coli. This system includes structural genes encoding the enzymes ATP sulforylase (cysD and cysN) and APS kinase (cysC). The initial stages of sulphate assimilation during cysteine biosynthesis lead to sulphate uptake and its activation due to the formation of adenosine 5'-phosphosulphate, conversion to 3'-phosphoadenosine-5'-phosphosulphate and reduction to sulphite. The cysQ gene is involved in this process. The cysG gene from E. coli encodes a CysG protein that is a subunit of uroporphyrin-IIIC-methyltransferase / precorrin-2-dehydrogenesis / syrohydrochlorinferrochelatase. The cysD gene from E. coli encodes the CysD protein component of the sulfatadenylyl transferase. The cysN gene from E. coli encodes the CysN protein component of the sulfatadenylyl transferase. The cysC gene from E. coli encodes a CysC protein subunit of adenylyl sulfate kinase. The cysQ gene from E. coli encodes a CysQ protein that supposedly helps control the pool of 3'-phosphoadenosine-5'-phosphosulfate or its use in sulfite synthesis. In the Escherihica coli bacteria, the cysD, cysN and cysC genes form the cysDNC operon.

The genes of P. ananatis are known and cloned, homologous to the genes of the E. coli sulfur utilization system. The nucleotide sequence of the cysG gene from P. ananatis is presented in SEQ ID NO: 1. The nucleotide sequence of the cysD gene from P. ananatis is presented in SEQ ID NO: 2. The nucleotide sequence of the cysN gene from P. ananatis is presented in SEQ ID NO: 3. The nucleotide sequence of the cysC gene from P. ananatis is presented in SEQ ID NO: 4. The nucleotide sequence of the cysQ gene from P. ananatis is presented in SEQ ID NO: 5.

In the Pantoea ananatis bacterium, the cysG, cysD, cysN and cysC genes form the cysGDNC operon. Enhanced cysG expression is not necessary. However, cysG expression can be enhanced.

Due to the fact that DNA sequences can vary among species and strains of the genus Pantoea, the cysG, cysD, cysN, cysC and cysQ genes are not limited to the sequences presented in the Sequence Listing under the numbers SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and may include genes homologous to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

In this regard, the cysG, cysD, cysN, cysC and cysQ genes can be variants that hybridize under stringent conditions with nucleotide sequences complementary to the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4 or SEQ ID NO: 5, or a sample that can be prepared from any of these nucleotide sequences. "Stringent conditions" are the conditions under which specific hybrids are formed, for example hybrids having a homology of at least 60%, 70%, 80%, 90%, 95%, 98% or 99% in various cases, and non-specific hybrids, having a degree of homology less than indicated above, are not formed. An example of harsh conditions can be a single or multiple washing, or, for example, double or triple washing in a solution with a salt concentration of 1 × SSC 0.1% SDS or 0.1 × SSC 0.1% SDS at 60 ° C. The duration of washing depends on the type of membrane used for hybridization and is generally recommended by the manufacturer. For example, the recommended washing time for Hybond ™ N ⁺ Nylon Membrane (Amersham) under severe conditions is 15 minutes. Washing can be done 2-3 times. The sample length may vary depending on hybridization conditions, but usually ranges from 100 bp. up to 1 kb

The phrase “the bacterium has been modified so that gene expression is enhanced” means that the level of gene expression in the modified bacterium is higher than in an unmodified strain, for example, a wild-type strain. Examples of such modifications include an increase in the number of copies of the expressed gene (s) per cell or an increase in the expression level of the gene (s) and others. The number of copies of an expressed gene can be measured, for example, by restriction of chromosomal DNA and subsequent Southern hybridization, using a probe constructed on the basis of the nucleotide sequence of the gene, fluorescence in situ hybridization (FISH) and similar methods. The level of gene expression can be measured using various methods, including Northern hybridization, quantitative reverse transcription - PCR (RT-PCR) and the like. Wild type strains, for example, Pantoea ananatis PERM BP-6614, can serve as a control.

The term “expression” means the formation of a protein product encoded by a gene.

The phrase “expression control sequence” refers to nucleotide sequences localized before, inside, or after the coding region that controls the transcription and / or expression of the coding region in association with the cell protein biosynthesis apparatus. This phrase is usually used to refer to promoters, ribosome-binding sites (RBS), operators and / or other elements of the genome involved in the regulation of gene expression level.

In addition, increased gene expression can be achieved by placing the DNA fragment of the present invention under the control of a stronger than natural promoter. The term "natural promoter" means a region of DNA existing in a natural organism located in front of an open reading frame (ORF - opened reading frame) of a gene or cluster of genes and facilitating the expression of this gene or cluster of genes. The strength of the promoter is determined by the frequency of the act of initiation of RNA synthesis. For example, the promoters P _lac , P _trp , P _trc and the promoters P _R or P _L phage lambda are known as strong promoters. Examples of methods for evaluating promoter strength and strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)) and others. Enhanced gene expression can be achieved by placing a strong terminator after the DNA fragment of the present invention.

In addition, in addition to the above methods, gene expression can be enhanced by increasing the number of copies of the gene, for example, using the technique of recombinant genes (molecules). For example, recombinant DNA can be obtained by ligation of a gene fragment containing a target gene with a vector for functioning in a host bacterium, such as a multi-copy vector, and then introduced into the bacterium for transformation. Examples of said vector include vectors that autonomously replicate in the cells of a host bacterium.

In order to introduce such recombinant DNA into the bacterium, any transformation methods known to date can be used. For example, suitable methods are the treatment of recipient cells with calcium chloride, as well as the increase in cell permeability for DNA described for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970) ), and the preparation of competent cells from cells in the growth phase, followed by the introduction of the DNA described in Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, Gene, 1, 153 (1977)). In addition, a suitable method is the preparation of protoplates or spheroplasts from DNA recipient cells, which can easily absorb recombinant DNA, followed by the introduction of recombinant DNA into these cells, this method is applicable to Bacillus subtilis, actinomycetes and yeast (Chang, S. and Choen , SN, Mol. Gen. Genet., 168, 111 (1979); Bibb, MJ, Ward, JM and Hopwood, OA, Nature, 274, 398 (1978); Hinnen, A., Hicks, JB and Fink, GR , Proc. Natl. Sci. USA, 75, 1929 (1978)). In addition, microorganisms can be transformed by electroporation (Japanese Patent Application Laid-Open No. 2-207791).

An increase in the number of copies of a gene can also be achieved by introducing multiple copies of the specified gene into the genomic DNA of the bacterium. To introduce multiple copies of a gene into the genomic DNA of a bacterium, homologous recombination is performed using the sequence, multiple copies of which are present in the genomic DNA as targets. As sequences, multiple copies of which are represented in genomic DNA, repeating DNA, inverted repeats present at the ends of mobile elements can be used. Another target gene may be introduced at a distance from the target gene present in the genome in tandem, or may be introduced instead of the unnecessary gene in the genome in multiple copies. The transfer of such a gene can be achieved using a temperature-dependent or integrative vector.

Alternatively, as described in Japanese Patent Laid-open No. 2-109985, it is also possible to include a gene in a transposon, the movement of which will lead to the introduction of multiple copies into genomic DNA. The movement of the indicated gene into the genome can be confirmed by Southern hybridization using a portion of the gene as a sample.

Genes involved in the biosynthesis of L-cysteine include various alleles of the cysE gene encoding reverse regulation resistant serine acetyltransferases (US Pat. No. 6,218,168, Russian Patent Application 2003121601); genes encoding proteins that regulate the secretion of cell-toxic substances (US Pat. No. 5,972,663), the cysB gene encoding a positive transcriptional regulator of cysteine regulon (WO 01/27307 A1). Another example of L-cysteine production is a decrease in the activity of cysteine desulfohydrase (Japanese Patent Application 11155571 A2).

Genes involved in the biosynthesis of L-methionine may include genes for methionine regulon. The methionine regulon may contain mutant genes encoding proteins with reduced activity of repression of amino acid biosynthesis. An example of such genes is the modified type of the metJ gene, which encodes a protein for repressing the biosynthesis of L-methionine from E. coli, whose activity during repression of methionine biosynthesis is reduced (JP patent application JP 2000157267 A2).

Methods for obtaining plasmid DNA, DNA cleavage and ligation, transformation, selection of oligonucleotides as primers and other similar methods are conventional methods well known to those skilled in the art. These methods are described in Sambrook, J., and Russell D., "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (1989).

2. The method according to the present invention

The method according to the present invention is a method for the production of compounds selected from the group consisting of L-cysteine, L-cystine, their derivatives or precursors, or a mixture thereof, comprising the steps of growing the bacteria of the present invention in a nutrient medium in order to produce and accumulate said compound in a nutrient medium and the allocation of the specified compounds from the culture fluid. Examples of a derivative or precursor of L-cysteine include S-sulfocysteine, a thiazolidine derivative, a semi-ketal corresponding to the aforementioned thiazolidine derivative, O-acetylserine, N-acetylserine, etc.

The cultivation of bacteria, isolation from the culture fluid and purification of the compounds can be carried out by traditional fermentation methods used for amino acids produced by the bacterium.

The growth medium can be either synthetic or natural, provided that it contains sources of carbon, nitrogen, mineral compounds and, if necessary, nutritional supplements in the amount necessary for the growth of bacteria. Carbon sources include various carbohydrates, such as glucose and sucrose, and various organic acids. Depending on the method of assimilation of the bacteria used, alcohols such as ethanol and glycerin may be used. Ammonia, various ammonium salts such as ammonium sulfate, other nitrogen compounds such as amines, natural nitrogen sources such as peptone, soybean hydrolyzate, and microbial fermentolizate are used as nitrogen sources. As sources of sulfur according to the present invention, ammonium sulfate, magnesium sulfate, iron sulfate, manganese sulfate and the like are used. As mineral compounds, monosubstituted potassium phosphate, sodium chloride, calcium chloride, magnesium salts, iron salts, manganese salts and the like are used. As vitamins, thiamine, yeast extract and the like are used.

The cultivation is preferably carried out under aerobic conditions, such as shaking and aeration with stirring, at a temperature of from 20 to 40 ° C, preferably from 30 to 38 ° C. The pH is usually in the range of 5 to 9, preferably in the range of 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffers. Usually, growing for 1 to 5 days leads to the accumulation of the target compound in the culture fluid.

After cultivation, insoluble components, such as cells, can be removed from the nutrient medium by centrifugation or filtration on a membrane, after which the target compound can be isolated and purified by ion exchange, concentration or crystallization.

L-cysteine obtained as described above can be used to produce derivatives of L-cysteine. Cysteine derivatives include methylcysteine, ethylcysteine, carbocysteine, sulfocysteine, acetylcysteine, etc.

In addition, when the thiazolidine derivative of L-cysteine is produced in the medium, L-cysteine can be produced by selecting said thiazolidine derivative from the medium in order to upset the equilibrium reaction between the indicated thiazolidine derivative and L-cysteine, which leads to excessive production of L-cysteine. In addition, when S-sulfocysteine is produced in the medium, it can be converted to L-cysteine by reduction with a reducing agent such as dithiotriethol.

Brief Description of Drawings

The Figure 1 shows the sequence of the natural promoter P _nlpD (SEQ ID NO: 65) and the modified promoter P _nlp8 (SEQ ID NO: 66).

The Figure 2 shows the construction of plasmid pM12.

Figure 3 shows the construction of the plasmid pM12-ter (thr).

Figure 4 shows the design of an integJ cassette intJS.

Figure 5 shows the construction of plasmid pMIV-5JS.

Examples

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

Example 1. Construction of strains with enhanced gene expression of the cysGDNC cluster.

1. Construction of strain P. ananatis EYPSG8

The DNA fragment carrying the promoter of the nlpD gene of the bacterium E. coli was obtained by PCR. The chromosomal DNA of strain E. coli MG1655 was used as a template, and oligonucleotides P1 (SEQ ID No: 6) and P2 (SEQ ID No: 7) served as primers. Strain MG1655 (ATCC 47076) is available from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 55 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 0.2 kb in length, and it was purified using agarose gel electrophoresis. Then the purified fragment was treated with endonucleases PaeI and SalI. The resulting DNA fragment was ligated with the plasmid pMIV-5JS (the construction of the plasmid pMIV-5JS is described in Reference example 1), pretreated with endonucleases PaeI and SalI. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on plates with LB agar containing ampicillin (50 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinct individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. The resulting plasmids contained the promoter of the E. coli nlpD gene and were named pMIV-Pnlp0.

A DNA fragment containing the terminator of the rrnB gene of the bacterium E. coli was obtained by PCR. The chromosomal DNA of E. coli strain MG1655 was used as a template; oligonucleotides P3 (SEQ ID No: 8) and P4 (SEQ ID No: 9) were used as primers. PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 59 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 0.3 kb in length, and it was purified using agarose gel electrophoresis. Then, the purified fragment was treated with BamHI and XbaI endonucleases. The resulting DNA fragment was ligated with the plasmid pMIV-Pnlp0, pretreated with BamHI and XbaI endonucleases. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar containing ampicillin (50 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinguishable individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. The resulting plasmid contained the terminator of the rrnB gene of E. coli and was named pMIV-Pnlp0-ter.

A DNA fragment containing the yeaS gene of the bacterium E. coli was obtained by PCR. Chromosomal DNA of E. coli strain MG1655 was used as a template, and oligonucleotides P5 (SEQ ID No: 10) and P6 (SEQ ID No: 11) served as primers. PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 55 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 0.7 kb in length, and it was purified using agarose gel electrophoresis. The purified fragment was then treated with SalI and XbaI endonucleases. The resulting DNA fragment was ligated with the plasmid pMIV-Pnlp0-ter, pretreated with SalI and XbaI endonucleases. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar containing ampicillin (50 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinguishable individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. The resulting plasmids contained the E. coli yeaS gene and were named pMIV-Pnlp0-yeaS3.

Then, randomization of the −10 region of the P _nlpD promoter and selection of the P _nlp8 promoter were _performed . The 3 ′ end of the P _nlpD promoter was obtained by PCR amplification. Plasmid pMIV-Pnlp0 was used as a template, and oligonucleotides P1 (SEQ ID No: 6) and P7 (SEQ ID No: 12) served as primers. Primer P7 contains random nucleotides indicated in SEQ ID No: 12 by the designation "n" (for A or G, or C, or T). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The 5'-end of the P _nlpD promoter was obtained by PCR amplification. Plasmid pMIV-Pnlp0 was used as a template, and oligonucleotides P2 (SEQ ID No: 7) and P8 (SEQ ID No: 13) were used as primers. Primer P8 contains random nucleotides indicated in SEQ ID No: 13, the designation "n" (for A or G, or C, or T). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. Both amplified DNA fragments were purified by agarose gel electrophoresis. Then the purified fragments were treated with BglII endonuclease followed by ligation of the fragments in an equimolar ratio. The ligation mixture was incubated overnight at 4 ° C and then used as a template for the next PCR reaction; oligonucleotides P1 (SEQ ID No: 6) and P2 (SEQ ID No: 7) were used as primers. PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 12 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C.

The amplified DNA fragment was about 0.2 kb in length, and it was purified using agarose gel electrophoresis. Then the purified fragment was treated with endonucleases PaeI and SalI. Then, the obtained DNA fragment was ligated with the plasmid pMIV-Pnlp0-yeaS3, pretreated with endonucleases PaeI and SalI. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar containing ampicillin (50 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinguishable individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. The resulting plasmids contained the P _nlp8 promoter (Figure 1) and were named pMIV-Pnlp8-yeaS7.

The plasmid pMIV-Pnlp8-yeaS7 was then treated with HindIII endonuclease, followed by purification and addition of a large fragment of DNA polymerase I (Klenov fragment). The resulting DNA fragment was purified and treated with NcoI endonuclease. Next, the obtained DNA fragment was purified and ligated in an equimolar ratio with the plasmid pMW-Pomp-cysE5 (WO 2005007841), pre-treated with SmaI and NcoI endonucleases. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar containing ampicillin (50 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinguishable individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. The resulting plasmids contained the cysE5 gene and were named pMIV-EY2. To confirm the operability of the cysE5 allele in the obtained transformants, the enzymatic activity of serine acetyltransferase was measured.

The next step was the integration of the cysE5 and yeaS genes into the chromosome of P. ananatis strain SC17 (US Pat. No. 6,596,517). Plasmid pMH10 (D. Zimenkov et al., Biotechnology, 6, 1-22 (2004)) was used to transform P. ananatis strain SC17 by electroporation. The obtained transformants were plated on LB agar containing kanamycin (20 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies and reseeded twice. Then, using the electroporation method, P. ananatis bacteria were transformed with the obtained strain SC17 / pMH10 (this strain grows at 30 ° C) with plasmid pMIV-EY2. Next, the transformants were subjected to heat shock at high temperature (42 ° C, 20 minutes) and plated on LB agar containing chloramphenicol (20 mg / L), the plates were incubated overnight at 39 ° C to form distinct individual colonies. About 50 clones were reseeded at 39 ° C, and each of them was seeded in 1 ml of LB medium and incubated for 48 hours at 39 ° C. After incubation, all 50 variants were tested for the loss of plasmids pMH10 and pMIV-EY2, and variants resistant to chloramphenicol (20 mg / L) but sensitive to kanamycin (20 mg / L) and ampicillin (50 mg / L) were selected. Desired integrants were identified by PCR analysis using primers P1 and P6. The resulting strain lines were named EY01-EY50, all of which were tested for their ability to produce cysteine in fermentation tubes. For further experiments, the best producer, strain EY19, was selected.

In order to get rid of the resistance of P. ananatis EY19 strain to chloramphenicol, this strain was transformed with the plasmid pMT-Int-Xis2 (WO 2005010175) by electroporation. The obtained transformants were plated on LB agar with tetracycline (10 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies. Desired transformants were identified among the selected chloramphenicol-sensitive variants (20 mg / L). Such a “cured" strain was named EY19 (s).

The next step was the replacement of the cysPTWA gene promoter region in the EY19 (s) strain with the P _nlp8 promoter region. PCR was performed using plasmid pMIV-Pnlp8-yeaS7 as the template and primers P1 (SEQ ID No: 6) and P2 (SEQ ID No: 7). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 20 cycles: 20 seconds at 94 ° C, 20 seconds at 59 ° C, 15 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 0.2 kb in length, and it was purified using agarose gel electrophoresis. Then the purified DNA fragment was processed with a Klenov fragment. The resulting DNA fragment was ligated in an equimolar ratio with the plasmid pMW118- (λattL-Km ^r -λattR) (see Reference Example 2), pretreated with XbaI endonuclease and Klenov fragment. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar containing kanamycin (20 mg / L), the plates were incubated overnight at 37 ° C until the formation of distinct individual colonies. Plasmids were isolated from the obtained transformants and analyzed using restriction analysis. Plasmids containing the P _nlp8 promoter were named pMW-Km-Pnlp8. Then, PCR was performed using the plasmid as the template and primers (SEQ ID No: 14) and P10 (SEQ ID No: 15). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 30 cycles: 20 seconds at 94 ° C, 20 seconds at 54 ° C, 90 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The resulting DNA fragment was about 1.6 kb in length, purified by agarose gel electrophoresis and used to transform P. ananatis strain SC17 by electroporation. The obtained transformants were plated on LB agar containing kanamycin (20 mg / L), plates were incubated overnight at 34 ° C until distinctive individual colonies were formed. The desired transformants were identified by PCR with primers P11 (SEQ ID No: 16) and P12 (SEQ ID No: 17). The resulting strain was named SC17-Pnlp8-PTWA. Chromosomal DNA was isolated from bacteria of the SC17-Pnlp8-PTWA strain, 10 μg of which was used for transformation of P. ananatis EY19 (s) by electroporation. The obtained transformants were seeded with LB agar containing kanamycin (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinguishable individual colonies. The desired transformants were identified by PCR with primers P11 and P12. The resulting strain was named EYP197. To get rid of the resistance of P. ananatis EY19 strain to kanamycin, this strain was transformed with plasmid pMT-Int-Xis2 by electroporation. The obtained transformants were plated on LB agar with tetracycline (10 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies. Desired transformants were identified by selection of kanamycin-sensitive variants (20 mg / L). Such a “cured" strain was named EY197 (s).

Mutation N348A was introduced using site-specific mutagenesis. For this, the 3'-end of the serA gene (with mutation), which encodes phosphoglycerate dehydrogenase, was obtained by PCR amplification of chromosomal DNA of strain SC17 with primers P13 (SEQ ID No: 18) and P14 (SEQ ID No: 19), and 5 The 'end of the serA gene was obtained by PCR amplification of chromosomal DNA of strain SC17 with primers P15 (SEQ ID No: 20) and P16 (SEQ ID No: 21). The conditions for the first PCR were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 25 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 60 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The conditions for the second PCR were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 20 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 20 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. Both amplified DNA fragments were purified by agarose gel electrophoresis and processed with SmaI exonuclease. Next, the obtained DNA fragments were ligated in an equimolar ratio. The ligation mixture was incubated overnight at 4 ° C and used as a template for subsequent PCR (with primers P13 and P15 (SEQ ID No: 20)). PCR conditions were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 15 cycles: 20 seconds at 94 ° C, 20 seconds at 60 ° C, 75 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 1.3 kb in length, and it was purified using agarose gel electrophoresis. The purified fragment was treated with SalI and XbaI endonucleases. After restriction, the DNA fragment was ligated in an equimolar ratio with the plasmid pMIV-Pnlp8-ter, pretreated with the same endonucleases. The ligation mixture was incubated overnight at 4 ° C and then used to transform E. coli strain MG1655 by electroporation. The obtained transformants were plated on LB agar with ampicillin (50 mg / L), plates were incubated overnight at 37 ° C until the formation of distinguishable individual colonies. Plasmids were isolated from the obtained transformants and analyzed by sequencing. The resulting plasmids containing the serA gene with the N348A mutation were named pMIV-Pnlp8-serA348.

The next step was the integration of the serA348 allele into the chromosome of P. ananatis SC17 strain. The plasmid DNA pMIV-Pnlp8-serA348 was used to transform P. ananatis strain SC17 / pMH10 (this strain grows at 30 ° C) by electroporation. The obtained transformants were subjected to heat shock (20 minutes at 42 ° C) and plated on LB agar with chloramphenicol (20 mg / L), plates were incubated overnight at 39 ° C to form distinct individual colonies. About 50 clones were reseeded at 39 ° C, and each of them was seeded in 1 ml of LB medium and incubated for 48 hours at 39 ° C. After incubation, all 50 variants were tested for the absence of plasmids pMH10 and pMIV-Pnlp8-serA348, by selection of variants resistant to chloramphenicol (20 mg / L), but sensitive to kanamycin (20 mg / L) and ampicillin (50 mg / L). Desired integrants were identified by PCR analysis using primers P1 and P15. In all the obtained variants, the activity of phosphoglycerate dehydrogenase (PGD) was measured, and the SC17int-serA348 variant with the highest activity was selected for subsequent experiments.

The next step was the transfer of an integrated copy of serA348 to strain EYP197 (s).

Chromosomal DNA was isolated from SC17int-serA348 strain, 10 μg of which was used to transform P. ananatis strain EYP197 (s) by electroporation. The obtained transformants were plated on LB agar with chloramphenicol (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinct individual colonies. The desired transformants were identified by PCR analysis with primers P1 and P15. The resulting strains were named EYPS1976.

The next step was the introduction of a gcd deletion in strain EYPS1976. To get rid of the resistance of P. ananatis strain EYPS1976 to chloramphenicol, strain EYPS1976 was transformed with plasmid pMT-Int-Xis2 by electroporation. The obtained transformants were plated on LB agar with tetracycline (10 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies. Desired transformants were identified by selection of chloramphenicol-sensitive variants (20 mg / L). Such a cured strain was named EY1976 (s).

The strain P. ananatis SC17 (0), in which the gcd gene is delegated, was constructed using a method first developed by Datsenko, KA and Wanner, BL (Proc. Natl. Acad. Sci. USA, 2000, 97 (12), 6640- 6645) and called "Red-Dependent Integration". The DNA fragment containing the kanamycin resistance cassette Km ^R was obtained by PCR using primers P17 (SEQ ID No: 28) and P18 (SEQ ID NO: 29) and plasmid pMW118-attL-Km-attR-ter_rrnB (Additional Example 2 ) as a matrix. The obtained PCR fragment was purified using agarose gel electrophoresis and used for electroporation into P. ananatis strain SC17 (0) containing plasmid pKD46 with temperature-dependent replication. Plasmid pKD46 (Datsenko, KA and Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) carries a 2154 nucleotide phage λ fragment (chromosome position: 31088-33241, accession number in GenBank: J02459) and contains the genes for Red-dependent homologous phage recombination (γ, β and exo genes) under the control of the arabinose-induced P _araB promoter. Plasmid pKD46 is required for integration of the PCR product into the chromosome of strain SC17 (0). Mutants with a delegated gcd gene and labeled with a Km resistance gene were tested by PCR with locus-specific primers P37 (SEQ ID No: 54) and P38 (SEQ ID No: 28). The PCR product obtained in the reaction with cells of the parental SC17 (0) gcd ⁺ strain as a matrix was 2560 bp in length. The PCR product obtained in the reaction with the cells of the mutant strain as a matrix had a length of 1541 bp The mutant strain was named SC17 (0) Δgcd :: Km.

Chromosomal DNA was isolated from SC17Δgcd :: Km strain, 10 μg of extracted DNA was used to transform P. ananatis strain EYPS1976 (s) by electroporation. The obtained transformants were plated on LB agar with kanamycin (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinguishable individual colonies. The desired transformants were identified by PCR analysis with primers P17 (SEQ ID No: 28) and P18 (SEQ ID No: 29). The resulting strain was named EYPSG8.

To get rid of the resistance of P. ananatis EYPSG8 strain to kanamycin, the EYPSG8 strain was transformed with the plasmid pMT-Int-Xis2 by electroporation. The obtained transformants were plated on LB agar with tetracycline (10 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies. Desired transformants were identified by selection of kanamycin-sensitive variants (20 mg / L). Such a cured strain was named EYPSG8 (s).

2. Construction of strain EYPSG-Pnlp8-cysGDNC

The promoter region of the cysGDNC genes of strain EYPSG8 (s) was replaced by the P _nlp8 promoter. PCR was performed using the plasmid DNA pMW-Km-Pnlp8 as the template and primers P19 (SEQ ID No: 24) and P20 (SEQ ID No: 25). The conditions for PCR were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 30 cycles: 20 seconds at 94 ° C, 20 seconds at 54 ° C, 90 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The resulting DNA fragment was about 1.6 kb in length. and purified by agarose gel electrophoresis. The purified fragment was used to transform strain A ananatis SC17 by electroporation. The obtained transformants were plated on LB agar containing kanamycin (20 mg / L), plates were incubated overnight at 34 ° C until distinctive individual colonies were formed. The desired transformants were identified by PCR with primers P21 (SEQ ID No: 26) and P22 (SEQ ID No: 27). The resulting strain was named SC17-Pnlp8-cysGDNC. In order to transfer cysGDNC under the control of the P _nlp8 promoter in the EYPSG8 (s) strain, the chromosomal DNA of the SC17-Pnlp8-cysGDNC strain was isolated, 10 μg of which was used to transform P. ananatis EYPSG8 (s) by electroporation. The obtained transformants were seeded with LB agar containing kanamycin (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinguishable individual colonies. The desired transformants were identified by PCR with primers P21 and P22, the resulting strain was named EYPSG-Pnlp8-cysGDNC.

To get rid of the resistance of P. ananatis EYPSG-Pnlp8-cysGDNC strain to kanamycin, the EYPSG-Pnlp8-cysGDNC strain was transformed with the plasmid pMT-Int-Xis2 by electroporation. The obtained transformants were plated on LB-arap with tetracycline (10 mg / L), the plates were incubated overnight at 30 ° C until the formation of distinguishable individual colonies. Desired transformants were identified by selection of kanamycin-sensitive variants (20 mg / L). Such a cured strain was named EYPSG-Pnlp8-cysGDNC (s).

Example 2. Construction of a strain with enhanced gene expression of cysGDNC cluster and cysQ gene

First, the promoter region of the cysQ gene in strain EYPSG-Pnlp8-cysGDNC (s) was replaced by the promoter region P _cysK . PCR was performed using the plasmid DNA pMW-Km-PcysK as the template and primers P10 (SEQ ID No: 15) and P24 (SEQ ID No: 29). Plasmid pMW-Km-PcysK was obtained by inserting a DNA fragment containing the promoter region of the cysK gene from P. ananatis obtained by PCR using chromosomal DNA of strain SC17 as a template and primers (SEQ ID No: 30) and P26 (SEQ ID No: 31), into the plasmid pMW118-attK-Km-attR-ter_rrnB, pre-ligated and sequentially treated with XbaI restriction enzyme and Klenov fragment. The conditions for PCR were as follows: 3 minutes denaturation at 95 ° C; conditions for the first two cycles: 1 minute at 95 ° C, 30 seconds at 50 ° C, 40 seconds at 72 ° C; conditions for the next 30 cycles: 20 seconds at 94 ° C, 20 seconds at 54 ° C, 90 seconds at 72 ° C; final stage: 5 minutes at 72 ° C. The amplified DNA fragment was about 1.6 kb in length. and purified by agarose gel electrophoresis. The purified fragment was used to transform P. ananatis strain SC17 by electroporation. The obtained transformants were plated on LB-arap containing kanamycin (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinguishable individual colonies. The desired transformants were identified by PCR with primers P25 and P26. The resulting strain was named SC17-PcysK-cysQ.

Next, the cysQ gene, under the control of the P _cysK promoter, was transferred to the EYPSG-Pnlp8-cysGDNC (s) strain. 10 μg of chromosomal DNA isolated from strain SC17-PcysK-cysQ was used to transform P. ananatis EYPSG-Pnlp8-cysGDNC (s) by electroporation. The obtained transformants were seeded with LB-arap containing kanamycin (20 mg / L), the plates were incubated overnight at 34 ° C until the formation of distinguishable individual colonies. The desired transformants were identified by PCR with primers P21 and P22, the resulting strain was named EYPSG-Pnlp8-cysGDNC-PcysK-cysQ.

Example 3. Construction of a strain with enhanced cysQ gene expression

The cysQ gene, under the control of the P _cysK promoter, was transferred to strain EYPSG8 (s). Chromosomal DNA was isolated from strain SC17-PcysK-cysQ. 10 μg of isolated DNA was used for electroporation transformation of P. ananatis EYPSG8 (s). The obtained transformants were plated on LB agar containing kanamycin (20 mg / L), plates were incubated overnight at 34 ° C until distinctive individual colonies were formed. The desired transformants were identified by PCR analysis with primers P25 and P26. The resulting strain was named EYPSG-PcysK-cysQ.

Example 4. Production of L-cysteine by P. ananatis strains

To test the effect of increased expression of genes involved in the process of sulfur assimilation in the production of L-cysteine, the productivity of P. ananatis strains EYPSG8, EYPSG8-Pnlp-cysGDNC, EYPSG8-Pnlp-cysGDNC-PcysK-cysQ and EYPSG8-Pcysys was compared.

The P. ananatis strains EYPSG8, EYPSG8-Pnlp-cysGDNC, EYPSG8-Pnlp-cysGDNC-PcysK-cysQ and EYPSG8-PcysK-cysQ were grown on L-agar plates for 17 hours at 34 ° C. Then, cells collected from approximately 30 cm ^{2 of the} surface of the plate were incubated in a 500 ml flask with L medium (50 ml) and grown with aeration for 5 hours at 32 ° C. After this, the cultures were transferred into 450 ml of fermentation medium in Jar-fermenters (Marubishi).

After cultivation, the amount of L-cysteine accumulated in the medium was determined using the method described by Gaitonde MK (Biochem J .; 104 (2): 627-33 (1967)), with changes: 150 μl of the sample was mixed with 150 μl of 1M H ₂ SO ₄ , incubated for 5 minutes at 20 ° C, then 700 μl of H ₂ O was added to the mixture, 150 μl of the resulting mixture was transferred to a new tube and 800 μl of solution A was added (1M Tris pH8.0, 5 μM DTT). The resulting mixture was incubated for 5 minutes at 20 ° C, centrifuged for 10 minutes at 13000 rpm and then 100 μl of the mixture was transferred into 20 × 200 mm tubes. Then, 400 μl of H ₂ O, 500 μl of glacial acetic acid and 500 μl of solution B (0.63 g of ninhydrin; 10 ml of glacial acetic acid; 10 ml of 36% HCl) were added, the mixture was incubated for 10 minutes in a boiling water bath. Then added 4.5 ml of ethanol and measured the optical density of OD ₅₆₀ . The cysteine concentration was calculated by the formula C (cis g / l) = 11.3 * OD ₅₆₀ . The results of three independent jar fermentations are presented in Table 1. As can be seen from Table 1, the strains EYPSG8-Pnlp8-cysGDNC, EYPSG8-Pnlp8-cysQ and EYPSG8-Pnlp8-cysGDNC-PcysK-cysQ had a larger amount of L-cysteine accumulated in the comparison with strain EYPSG8. The composition of the medium for fermentation (g / l):

Concentration Unit (A) Glucose fifty g / l MgSO ₄ 7H ₂ O 0.3 g / l (B) Trypton Difco 2 g / l Difco Yeast Extract one g / l (NH ₄ ) ₂ SO ₄ fifteen g / l KH ₂ PO ₄ 1,5 g / l NaCl 0.5 g / l L-histidine HCl 0-0.1 g / l L-methionine 0-0.35 g / l FeSO ₄ 7H ₂ O 2 mg / l CaCl ₂ fifteen mg / l Sodium Citrate 5H ₂ O one g / l Na ₂ MoO ₄ H ₂ O 0.15 mg / l CoCl ₂ 6H ₂ O 0.7 mg / l MnCl ₂ 4H ₂ O 1,6 mg / l ZnSO ₄ 7H ₂ O 0.3 mg / l Gd 113 0,03 mg / l (C) Pyridoxine (sodium salt) 2 mg / l

Example 5. Production of L-cysteine by E. coli strains

To evaluate the effect of increased expression of the cysQ gene from E. coli on the production of L-cysteine, an E. coli strain MT / pACYC-DES / pMIV-PompC-cysQ was constructed. For this, the E. coli strain MT / pACYC-DES (European patent EP 1528108 B1) was transformed with the plasmid pMIV-PompC-cysQ. Plasmid pMIV-PompC-cysQ was constructed using the method below.

First, the promoter region of the E. coli ompC gene was amplified from the chromosomal DNA of E. coli strain MG1655 by PCR using primers PrOMPCF (SEQ ID No: 55) and PrOMPCR (SEQ ID No: 56). Fragment 0.3 kb was isolated, digested with restriction enzymes with PaeI and SalI, and ligated with plasmid pMIV-5JS pretreated with the same restriction enzymes.

Next, the cysQ gene from the chromosome of strain MG1655 was amplified by PCR using primers mz017 (SEQ ID No: 57) and mz018 (SEQ ID No: 58). 0.76 kb PCR fragment was isolated, digested with restriction enzymes SalI and XbaI and cloned at the SalI / XbaI sites of the previously obtained plasmid. The resulting plasmid was named pMIV-PompC-cysQ.

The strain MT was obtained from strain MG1655 as follows. First, a mutation in the metC gene was induced by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) followed by a multiple ampicillin enrichment procedure. Mutants capable of growing on cystathionine, but not on homoserine, were selected. Strains lacking metC can also be obtained using recombinant DNA technology according to the procedure described in US Pat. No. 6,946,268.

Next, the destroyed tnaA gene from strain CGSC7152 (tnaA300 :: Tn10 (TcR), E. coli Genetic Stock Center, USA) was transferred to the obtained metC ^- strain using the standard P1 transduction procedure (Sambrook et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)). The pACYC-DES plasmid was constructed by insertion of the ydeD gene, cysE mutant gene and serA5 mutant gene into the pACYC184 vector under the control of the P _ompA promoter. The ydeD gene, which encodes a membrane protein that is not involved in the biosynthesis of any amino acid, can enhance L-cysteine production when additional copies of the gene are introduced into the cells of the corresponding producer strain (US Pat. No. 5,972,663). The serA5 gene encodes feedback resistance phosphoglycerate dehydrogenase (US Pat. No. 6,180,373).

Strains MT / pACYC-DES and MT / pACYC-DES / pMIV-PompC-cysQ were grown overnight at 34 ° C in 2 ml of nutrient broth containing 100 mg / l ampicillin and 25 mg / l streptomycin. 0.2 ml of the obtained culture was seeded in 2 ml of fermentation medium containing tetracycline (20 mg / l) and ampicillin (100 mg / l) in 20 × 200 mm tubes and grown at 34 ° C for 42 hours on a shaker at 250 rpm / min The following composition of the fermentation medium was used: 15.0 g / l (NH ₄ ) ₂ SO ₄ , 1.5 g / l KH ₂ PO ₄ , 1.0 g / l MgSO ₄ , 20.0 g / l CaCO ₃ , 0.1 mg / l thiamine, 1% LB environments, 4% glucose, 300 mg / l of L-methionine.

After cultivation, the amount of L-cysteine accumulated in the medium was determined using the method described by Gaitonde M.K. (Biochem J .; 104 (2): 627-33 (1967)). The results are presented in Table 2. As can be seen from Table 2, the strain MT / pACYC-DES / pMIV-PompC-cysQ had a greater amount of L-cysteine accumulated in the medium compared to the strain MT / pACYC-DES.

In order to evaluate the effect of enhancing the expression of the cysDNC operon, cysDN operon or cysC gene, and the effect of jointly enhancing the expression of the cysDNC operon and cysQ gene from E. coli on the production of L-cysteine, E. coli strains MT-intPcysK-cysDNC / pACYC- were constructed DES and MT-intPcysK-cysDNC / pACYC-DES / pMIV-PompC-cysQ.

First, the promoter region of the cysK gene from E. coli was amplified by PCR with the chromosomal DNA of E. coli strain MG1655 and primers N1 (SEQ ID No: 59) and N2 (SEQ ID No: 60). The obtained fragment with P _{cysK was} isolated, treated with restriction enzymes PaeI and SalI, and ligated into plasmid pMIV-5JS, previously treated with the same restriction enzymes. The resulting plasmid was named pMIV-P _cysK .

Then, cysDNC operon was cloned using PCR with DNA of strain MG1655 as a template and primers (SEQ ID No: 61) and N4 (SEQ ID No: 62). The obtained PCR fragment with the cysDNC operon was isolated, treated with restriction enzymes SalI and XbaI and ligated into the plasmid pMIV-P _cysK , pre-treated with the same restriction enzymes. The resulting plasmid was named pMIV-P _cysK -cysDNC.

Then, cysDN operon was cloned using PCR with DNA of strain MG1655 as a template and primers N3 (SEQ ID No: 61) and N5 (SEQ ID No: 63). The obtained PCR fragment with the cysDN operon was isolated, treated with restriction enzymes SalI and XbaI and ligated into the plasmid pMIV-P _cysK pretreated with the same restriction enzymes. The resulting plasmid was named pMIV-P _cysK -cysDN.

Also, cysC gene was cloned using PCR with the DNA of strain MG1655 as template and primers N6 (SEQ ID NO: 64) and N4 (SEQ ID NO: 62). The obtained PCR fragment with the cysDNC operon was isolated, treated with restriction enzymes SalI and XbaI and ligated into the plasmid pMIV-P _cysK , pre-treated with the same restriction enzymes. The resulting plasmid was named pMIV-P _cysK -cysC.

The plasmids pMIV-P _cysK cysDNC, pMIV-P _cysK cysDN and pMIV-P _cysK cysC were used to transform MT / pACYC-DES by electroporation to obtain strains MT / pACYC-DES / pMIV-PcysK-cysDNC, MT / pACYC-DES / pMIV-PcysK-cysDN and MT / pACYC-DES / pMIV-PcysK-cysC, respectively.

The next step was the integration of the P _cysK -cysDNC fragment into the chromosome of E. coli MT strain. To this end, the cysDNC operon under the control of the P _cysK promoter was integrated using mini-μu integration (using the μu transposase contained in plasmid pMH10) plasmid pMIV-P _cysK -cysDNC into the chromosome of strain MG1655. The resulting construct (intP _cysK -cysDNC) was transferred from strain MG1655-int-P _cysK -cysDNC to strain MT using P1 transduction (Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) with the aim of obtaining a strain of E. coli MT-int-P _cysK -cysDNC. The plasmid pACYC-DES was used to transform the strain MT-int-P _cysK -cysDNC by electroporation to obtain the E. coli strain MT-int-P _cysK -cysDNC / pACYC-DES, and both plasmids pACYC-DES and pMIV-PompC-cysQ were used to transform the strain MT-int-P _cysK -cysDNC by electroporation to obtain the strain MT-int-P _cysK -cysDNC / pACYC-DES / pMIV-PompC-cysQ.

Strains MT-intPcysK-cysDNC / pACYC-DES and MT-intPcysK-cysDNC / pACYC-DES / pMIV-PompC-cysQ containing the cysDNC operon integrated into the chromosome and strains MT / pACYC-DES / C, MT / pACYC-DES / pMIV-PcysK-cysDN and MT / pACYC-DES / pMIV-PcysK-cysC containing the cysDNC operons, cysDN and the cysC gene in the pMIV plasmid were grown separately at 34 ° C. in 2 ml of culture medium supplemented with 100 mg / l ampicillin and 20 μg / ml tetracycline. 0.2 ml of the obtained cultures was transferred into 2 ml of fermentation medium containing tetracycline (20 μg / ml) and ampicillin (100 mg / ml) in 20 × 200 mm tubes, and grown at 34 ° C for 42 hours with centrifugation at 250 rpm min The composition of the fermentation medium was as follows: 15.0 g / l (NH ₄ ) ₂ SO ₄ , 1.5 g / l KH ₂ PO ₄ , 1.0 g / l MgSO ₄ , 20.0 g / l CaCO ₃ , 0.1 mg / l thiamine, 1% LB , 4% glucose, 300 mg / l L-methionine.

After cultivation, the amount of L-cysteine accumulated in the medium was determined using the method described by Gaitonde, MK (Biochem. J., 104: 2, 627-33 (1967)). The results are presented in Table 2. As can be seen from Table 2, the strain MT-intPcysK-cysDNC / pACYC-DES had a weak positive effect on the production of L-cysteine compared with the control strain MT / pACYC-DES, the strain MT-intPcysK-cysDNC / pACYC-DES / pMIV-PompC-cysQ showed a significant increase in L-cysteine production compared to MT / pACYC-DES. Also, the strain MT / pACYC-DES / pMIV-PcysK-cysC DES had a weak positive effect on L-cysteine production, the strains MT / pACYC-DES / pMIV-PcysK-cysDNC and MT / pACYC-DES / pMIV-PcysK-cysDN showed Significant increase in L-cysteine accumulation compared to MT / pACYC-DES. The largest amount of accumulated L-cysteine was observed when combining the cysDNC operon under the control of the P _cysK promoter and the cysQ gene under the control of the P _ompC promoter in the strain MT-intPcysK-cysDNC / pACYC-DES / pMIV-PompC-cysQ.

Example 6. Production of L-methionine by E. coli strains

In order to evaluate the effect of enhancing the expression of the cysDNC operon, cysDN operon, cysC gene and cysQ gene from E. coli, or a combination thereof, for production of L-methionine, E. coli strain 73 (VKPM B-8126) containing the indicated operons or genes in various chromosome combinations and / or cloned in plasmids pMIV-P _ompC -cysQ, pMIV-P _cysK -cysDNC, pMIV-P _cysK -cysDN, pMIV-P _cysK -cysC, can be constructed as described in Example 5. E. coli strain 73 (VKPM B B-8126) has been deposited in the Russian National collection of industrial microorganisms (VKPM) (Russia, 117545 Moscow, 1 ^st road trip, 1) May 14, 2001 under accession number VKPM B-812 6, then on February 1, 2002, an international deposit of this strain was made in accordance with the terms of the Budapest Treaty.

The strain E. coli 73 and the resulting strains can be individually grown in 2 ml of minimal medium ((NH ₄ ) ₂ SO ₄ - 18 g / l, K ₂ HPO ₄ - 1.8 g / l, MgSO ₄ - 1.2 g / l, thiamine - 0.1 mg / l, yeast extract - 10 g / l, glucose - 60 g / l, threonine - 400 mg / l, ampicillin - 100 mg / l, if necessary) in 20 ml tubes and incubated overnight with By aeration at 32 ° C, 0.2 ml of each overnight culture can be transferred to 20 ml tubes with 2 ml of fresh fermentation medium with or without IPTG and grown at 32 ° C for 48 hours on a rotary shaker.

The composition of the fermentation medium:

(NH ₄ ) ₂ SO ₄ 18.0 g / l K ₂ HPO ₄ 1.8 g / l MgSO ₄ 1.2 g / l CaCO ₃ 20.0 g / l Thiamine 0.1 mg / l Glucose 60.0 g / l Threonine 400 mg / l Yeast extract 1.0 g / l Ampicillin 100 mg / l, if necessary.

After growth, plasmid stability and optical density of the culture fluid at 540 nm can be determined by conventional methods. The amount of methionine accumulated in the culture fluid can be determined by thin layer chromatography (TLC). The composition of the liquid phase for TLC can be as follows: isopropanol - 80 ml, ethyl acetate - 80 ml, NH ₄ OH (30%) - 15 ml, H ₂ O - 45 ml.

Reference Example 1. Construction of the plasmid pMIV5JS

Plasmid PMIV-5JS was constructed according to the scheme below. First, plasmid pM12 was constructed by integrating an in vivo Mi-dependent integration cassette into plasmid pMW1 derived from pMW119 (Figure 2). Two terminator oligonucleotide sequences complementary to each other were synthesized (SEQ ID No: 32 and SEQ ID No: 33). The thrL terminator was obtained by annealing these synthetic oligonucleotides in the forward (SEQ ID No: 32) and reverse (SEQ ID No: 33). The thrL terminator was flanked by the HindIII and PstI sites. Then, using the insertion of the synthetic terminator sequence Ter (thr) into pM12 pretreated with HindIII and Mph 1103I, the plasmid pM12-ter (thr) was constructed (Figure 3).

The design of the integrative cartridge intJS (Figure 4):

a) a 0.12 kb LattL fragment was obtained by PCR amplification using a forward primer (SEQ ID No: 34) (BglII restriction enzyme recognition site is highlighted), and a phosphorylated reverse primer (SEQ ID No: 35). The plasmid pMW118-attL-tet-attR-ter_rrnB (WO 2005/010175) was used as a matrix;

b) Cm ^R fragment of 1.03 kbp was obtained by PCR amplification using a phosphorylated forward primer (SEQ ID No: 36) and a reverse primer (SEQ ID No: 37) (the PstI restriction enzyme recognition site is highlighted). Plasmid pACYC184 was used as a matrix;

c) LattR fragment with a length of 0.16 kb was obtained by PCR amplification using a forward primer (SEQ ID No: 38) (PstI restriction enzyme recognition site isolated) and phosphorylated reverse primer (SEQ ID No: 39) (SacI restriction enzyme recognition site isolated). The plasmid pMW118-attL-tet-attR-ter_rrnB was used as a matrix;

d) LattL and Cm ^R fragments were ligated and the resulting 1.15 kb fragment. has been cleaned;

e) fragments of LattL-Cm ^R and LattR were digested with restriction enzyme PstI, ligated, the resulting fragment of LattL-Cm ^R -LattR 1.31 kb in length has been cleaned;

f) a 70 bp double-stranded DNA fragment containing a multiple cloning site (MCS) was obtained by renaturation of two synthesized oligonucleotides: an oligonucleotide whose sequence is shown in SEQ ID No: 40, and an oligonucleotide having a complementary SEQ ID No: 40 sequence;

g) LattL-Cm ^R -LattR and MCS fragments were digested with Sacl, ligated, and the resulting LattL-Cm ^R -LattR-MCS cassette, 1.38 kbp, has been cleaned;

In the last step, the LattL-Cm ^R -LattR-MCS fragment was digested with restriction enzymes BglII and HindIII and cloned into the plasmid pM12-ter (thr) pretreated with BamHI and HindIII to obtain the plasmid pMIV-5JS (Figure 5).

Reference example 2. Construction of the plasmid pMW118- (λattL-Km ^r -λattR).

Plasmid pMW118- (λattL-Km ^r- λattR) was constructed on the basis of plasmid pMW118-attL-Tc-attR (WO 2005/010175) with the replacement of the tetracycline resistance marker with the kanamycin resistance gene taken from pUC4K plasmid (Vieira, J. and Messing, J., Gene, 19 (3): 259-68 (1982)).

For this purpose, a large fragment of the EcoRI - HindIII plasmid pMW118-attL-Tc-attR was ligated with two fragments of the plasmid pUC4K: HindIII - PstI fragment (676 bp) and EcoRI - HindIII fragment (585 bp).

The base fragment pMW118-attL-Tc-attR was obtained by ligation of the following four DNA fragments:

1) a BglII-EcoRI fragment (114 bp) carrying the attL site (SEQ ID No: 41) was obtained by PCR amplification of the corresponding chromosome region (containing prophage λ) of E. coli W3350 using primers P27 and P28 (SEQ ID NoS: 42 and 43) (these primers contained additional recognition sites for BglII and EcoRI endonucleases);

2) a PstI-HindIII fragment (182 bp) carrying the attR site (SEQ ID No: 44) was obtained by PCR amplification of the corresponding chromosome region (containing prophage λ) of E. coli W3350 using primers P29 and P30 ( SEQ ID NoS: 45 and 46) (these primers contained additional recognition sites for the endonucleases 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 ligation of the following three fragments:

1) a large DNA fragment (2359 bp) carrying the AatII-EcoRI fragment of plasmid pMW118 was prepared as follows: pMW118 was digested with EcoRI restriction enzyme, digested with a Maple fragment of DNA polymerase I, and then with AatII restriction enzyme;

2) a small fragment of AatII-BglII (1194 bp) of pUC 19 vector carrying the bla ampicillin resistance gene (Ap ^R ) was obtained by PCR amplification of the corresponding region of pUC 19 vector using primers P31 and P32 (SEQ ID NoS: 47 and 48) (these primers contained additional recognition sites for the endonucleases AatII and BglII);

3) a small fragment of BglII-PstI (363 bp) of the ter_rrnB transcriptional terminator was obtained by PCR amplification of the corresponding region of E. coli chromosome MG1655 using primers P33 and P34 (SEQ ID NoS: 49 and 50) (these primers contained additional recognition sites for endonucleases BglII and PstI);

4) a small fragment of EcoRI-PstI (1388 bp) (SEQ ID No: 51) of the plasmid pML-Tc-ter_thrL, carrying the tetracycline resistance gene and ter_thrL transcription terminator; plasmid pML-Tc-ter_thrL was obtained in two stages:

- plasmid pML-ter_thrL was obtained by processing the plasmid pML-MCS (Mashko S.V. et al., Biotechnology, 2001, no. 5, 3-20) with restriction enzymes XbaI and BamHI, followed by ligation of a large fragment (3342 bp .) with an XbaI-BamHI fragment (68 bp) carrying the ter_thrL terminator, obtained by PCR amplification of the corresponding region of the E. coli chromosome chromosome MG1655 using oligonucleotides P35 and P36 (SEQ ID NoS: 52 and 53) as primers (these primers contained additional recognition sites for endonucleases XbaI and BamHI);

- the plasmid pML-Tc-ter_thrL was obtained by processing the plasmid pML-ter_thrL with restriction enzymes KpnI and XbaI, the Klenov fragment of DNA polymerase I, and ligation with a small EcoRI-Van91I fragment (1317 bp) of the pBR322 vector carrying the tetracycline resistance gene (pBR322 was treated with restriction enzymes EcoRI and Van91I, and then a Klenov fragment of DNA polymerase I).

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 may be made and equivalent replacements may be 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 Strains The concentration of L-cysteine, g / l EYPSG8-Pnlp8 0.85 ± 0.07 EYPSG8-Pnlp8-cysGDNC 1.37 ± 0.09 EYPSG8-PcysK-cysQ 1.06 ± 0.08 EYPSG8-Pnlp8-cysGDNC-PcysK-cysQ 1.69 ± 0.11

table 2 Strains The concentration of L-cysteine, g / l MT / pACYC-DES 1.05 ± 0.13 MT / pACYC-DES / pMIV-PompC-cysQ 1.46 ± 0.20 MT-intPcysK-cysDNC / pACYC-DES 1.13 ± 0.14 MT-intPcysK-cysDNC / pACYC-DES / pMIV-PompC-cysQ 1.70 ± 0.11 MT / pACYC-DES / pMIV-PcysK-cysDNC 1.67 ± 0.12 MT / pACYC-DES / pMIV-PcysK-cysDN 1.51 ± 0.11 MT / pACYC-DES / pMIV-PcysK-cysC 1.10 ± 0.10

Claims (20)

1. A bacterium of the Enterobacteriaceae family - a producer of L-cysteine, modified in such a way that expression is enhanced in it:
- one or more genes of the cysDNC cluster involved (them) in sulfate activation; or
- one or more genes of the cysDNC cluster involved (them) in the activation of sulfate, and the cysQ gene involved in the degradation of adenosine-3'-phosphate-5'-phosphosulfate (PAPS). 2. The bacterium according to claim 1, characterized in that the expression of the specified (s) gene (s) is enhanced by modifying the sequence (s) that controls (their) expression of the specified (s) gene (s). 3. The bacterium according to claim 2, characterized in that the indicated sequence (s) that control (s) the expression of the specified gene (s) is the natural promoter (s). 4. The bacterium according to claim 3, characterized in that the natural promoter (s) of the indicated gene (s) is replaced by the stronger promoter (s). 5. The bacterium according to claim 1, characterized in that said bacterium belongs to the genus Pantoea. 6. The bacterium according to claim 5, characterized in that said bacterium is a Pantoea ananatis bacterium. 7. The bacterium according to claim 1, characterized in that said bacterium belongs to the genus Escherichia. 8. The bacterium according to claim 7, characterized in that said bacterium is a bacterium Escherichia coli. 9. A method for producing L-cysteine, including:
- growing bacteria according to any one of claims 1 to 8 in a nutrient medium containing sulfate; and
- the allocation of L-cysteine from the culture fluid. 10. The method according to claim 9, characterized in that in said bacterium the expression of genes involved in the biosynthesis of L-cysteine is enhanced. 11. The method according to claim 9, characterized in that in said bacterium the expression of genes involved in the biosynthesis of L-methionine is enhanced. 12. A method of producing L-cystine, comprising:
- growing bacteria according to any one of claims 1 to 8 in a nutrient medium containing sulfate;
- the conversion of L-cysteine to L-cystine; and
- the allocation of L-cystine from the culture fluid. 13. The method according to p. 12, characterized in that the specified bacteria enhanced expression of genes involved in the biosynthesis of L-cysteine. 14. The method according to p. 12, characterized in that in the specified bacteria, the expression of genes involved in the biosynthesis of L-methionine is enhanced. 15. A method of producing S-sulfocysteine, including:
- growing bacteria according to any one of claims 1 to 8 in a nutrient medium containing sulfate;
- the conversion of L-cysteine to S-sulfocysteine; and
- the allocation of S-sulfocysteine from the culture fluid. 16. The method according to p. 15, characterized in that in the specified bacteria, the expression of genes involved in the biosynthesis of L-cysteine is enhanced. 17. The method according to clause 15, wherein the specified bacteria enhanced expression of genes involved in the biosynthesis of L-methionine. 18. A method of obtaining a thiazolidine derivative of L-cysteine, including:
- growing bacteria according to any one of claims 1 to 8 in a nutrient medium containing sulfate;
- the conversion of L-cysteine to a thiazolidine derivative of L-cysteine; and
- the selection of a thiazolidine derivative of L-cysteine from the culture fluid. 19. The method according to p. 18, characterized in that the specified bacteria enhanced expression of genes involved in the biosynthesis of L-cysteine. 20. The method according to p. 18, characterized in that the specified bacteria enhanced expression of genes involved in the biosynthesis of L-methionine.

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