RU2458981C2 - Method of producing l-cysteine using bacteria of enterobacteriaceae family - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is L-cysteine producing bacteria, which relates to the Pantoea or Escherichia genus of the Enterobacteriaceae family, which is modified such that it has stronger expression of one or more genes selected from a group consisting of glutaredoxin coding genes grxC and grxB, and a glutathione reductase coding gene (gorA). Disclosed is a method of producing L-cysteine, involving growing said bacteria in a culture medium containing thiosulphate, and separating L-cysteine from the culture fluid.

EFFECT: invention increases output of L-cysteine.

9 cl, 4 tbl, 4 dwg, 5 ex

Description

State of the art

Technical field

The present invention relates to the microbiological industry, in particular to a method for producing L-cysteine using a bacterium of the Enterobacteriaceae family, which is modified in such a way that expression of one or more genes encoding glutaredoxins and glutathione reductase is enhanced in it.

State of the art

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

Many methods have been described to increase the production of L-amino acids, for example, by transforming a microorganism of recombinant DNA (see, for example, US patent 4,278,765). Other methods are based on increasing the activity of enzymes involved in the biosynthesis of amino acids and / or reducing the sensitivity of the target enzyme to inhibition by the L-amino acid produced by feedback (see, for example, PCT application WO95 / 16042 or US patent No. 4346170; 5661012; and 6040160 )

S^+VI) in vivo восстанавливается до сульфита (

S^+IV) с помощью фосфоаденилилсульфатредуктазы (PAPS-редуктаза). Мутант Escherichia coli, лишенный глутатионредуктазы и глутаредоксинов (gor^-grxA^-grxB^-grxC^-), практически не растет на сульфате. С помощью метода, основанного на тиолспецифичном флуоресцентном алкилировании и гель-электрофорезе, было показано, что глутатионилированная PAPS-редуктаза восстанавливается до глутаредоксинов через монотиольный механизм. Обратимое глутатионилирование может регулировать активность PAPS-редуктазы in vivo (Lillig С.Н. et al., J Biol Chem.; 278(25):22325-30 (2003)).Inorganic Sulfate (

S ^{+ VI} ) in vivo is reduced to sulfite (

S ^{+ IV} ) using phosphoadenylyl sulfate reductase (PAPS reductase). The mutant Escherichia coli, devoid of glutathione reductase and glutaredoxins (gor ^- grxA ^- grxB ^- grxC ^- ), practically does not grow on sulfate. Using a method based on thiolspecific fluorescent alkylation and gel electrophoresis, it was shown that glutathioneylated PAPS reductase is reduced to glutaredoxins via a monothiol mechanism. Reversible glutathionylation can regulate PAPS reductase activity in vivo (Lillig, C.N. et al., J Biol Chem .; 278 (25): 22325-30 (2003)).

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides (SS proteins) or mixed disulfides formed between proteins and glutathione (GSH) (protein-S-SG) in the conjugated system with GSH, NADPH and GSH reductase. Glutaredoxins can thus be considered as disulfide reducing agents via GSH. The bacterium E. coli has three glutaredoxins (Grx1, Grx2 and Grx3) and two thioredoxins (Trx1, Trx2). Grx3 (9 kDa, encoded by the grxC gene) was inserted from an E. coli mutant that was completely defective in Grx1 and Trx1. Grx1 and Grx3 are 33% identical in sequence and have a similar structure (thioredoxin / glutaredoxin fold). Grx3 can recover RR1a (class 1a ribonucleotide reductase from E. coli) with 5% catalytic efficiency of Grx1. Grx2 and Grx3, apparently, are not involved in the reduction of sulfate to sulfite, which is catalyzed in E. coli by PAPS reductase. It was shown that the wild-type strain and the trxB strain ^- grxC ^- show the same growth rates on rich LB medium, which suggests that E. coli can compensate for the main disorders of the thioredoxin / glutaredoxin pathway when fully provided with nutrients. On the M9 minimal medium, the wild type and mutant trxB ^- grxC ^- also grew at the same rate, while trxA ^- grxC ^- grew very slowly and reached a lower final growth level (Vlamis-Gardikas A. et al., J Biol Chem .; 277 (13 ): 10861-8 (2002)).

Grx3 associated with cellular GSH is believed to be the third hydrogen donor system in the absence of thioredoxin and classical glutaredoxin (Grx1). Under ordinary conditions, it is obvious that Grx3 must have other functions in the cell (Aslund F. et al., PNAS, 91, 9813-7 (1994)).

Gor transcription is induced by H ₂ O ₂ , the putative OxyR binding site and the start of the OxyR-regulated transcript before the gorA gene from E. coli are mapped (Toledano M.V. et al., Cell, 78, 897-909 (1994)).

The gor gene has been found to be regulated not only through OxyR, but also via rpoS. It was found that in the stationary phase, rpoS regulates not only the enzymatic activity of gor, but also transcriptional activity. The absence of the above protein increases the resistance of E. coli to certain oxidative stress substances, such as H ₂ O ₂ and NEM (N-ethyl maleimide). Therefore, it is likely that gor does not function as a typical antioxidant (Becker-Hapak M. and Eisenstark A., FEMS Microbiology Letters 134 (1), 39-44 (1995)).

Co-expression of several genes in E. coli was measured using reverse transcription-multiplex fluorescence PCR. GorA transcription has been shown to activate as wild-type bacteria grow. GorA was activated during the exponential growth phase. The maximum induction was 4.6. Treatment with H ₂ O ₂ stimulated the expression of the gorA gene previously identified as a component of the OxyR regulon. Induction by means of H ₂ O ₂ was much faster and was a reversible reaction, and it was the case with the OxyR-dependent and σS-independent type. NaCl induced the expression of genes controlled by OxyR (C. Michán et al., J Bacteriol .; 181 (9): 2759-64 (1999)).

The expression of up to 16 E. coli genes encoding the components of both the glutaredoxin and thioredoxin systems and the components of OxyR and SoxRS regulons was evaluated. An inverse relationship between nrdAB expression has been demonstrated.

and expression of genes encoding the components of both the glutaredoxin (grxA, gorA) and thioredoxin (trxB, trxC) systems (Prieto-Alamo M.J. et al., J Biol Chem .; 275 (18): 13398-405 (2000)).

To study the role in protection against oxidative stress and the redox-dependent formation of photosynthetic complexes, mutants with disorders in the components of the glutathione-glutaredoxin (GSH / Grx) system of the Rhodobacter capsulatus were constructed. The absence of the glutaredoxin 3 gene (grxC) or glutathione synthetase B (gshB) gene led to a slowdown in growth under aerobic conditions and increased sensitivity to oxidative stress, confirming the role of the GSH / Grx system in protecting against oxidative stress. Both mutants were highly sensitive to disulfide stress, which shows a significant contribution of the GSH / Grx system to the thiol disulfide redox buffer system of the cytoplasm. Like mutations in the thioredoxin system, mutations in the GSH / Grx system influenced the formation of redox-dependent photosynthetic complexes in R. capsulatus. The expression of the glutaredoxin1 genes grxC, gshB, grxA and glutathione reductase gorA, encoding the components of the GSH / Grx system, was not induced by oxidative stress. Mutations in the genes grxC and / or gshB, and / or trxC (thioredoxin 2) influenced the expression of these genes, thereby demonstrating the interaction of various defense systems against oxidative stress. OxyR and SoxRS regulons control the expression of many genes involved in protection against oxidative stress in E. coli in response to H ₂ O ₂ and superoxide, respectively. The data obtained and the known genomic sequence of R.capsulatus suggest that R.capsulatus SoxRS does not have a system, but there is another superoxide-sensitive regulator. At the same time, the expression of gorA and grxA is regulated by H ₂ O ₂ in E. coli, but not in R. capsulatus, so the OxyR regulons of these two species are completely different (Li K. et al., J Bacteriol .; 186 ( 20): 6800-8 (2004)).

Mutants having abnormalities in cysteine synthase A or B, or both, were isolated from Salmonella typhimurium LT2. Parent strains were able to grow on minimal medium containing sulfate, sulfite, sulfide or thiosulfate as a source of sulfur. Mutants without cysteine synthase B were not able to grow on thiosulfate, while mutants without cysteine synthase A grew with slight differences in growth rate at the four above-mentioned sources of inorganic sulfur. Mutants without both cysteine synthases did not grow on a medium containing any of the analyzed sources of inorganic sulfur. Isolation of cysteine synthase B was accompanied by the isolation of S-sulfocysteine synthase. In addition, both of these activities were also transduced together. The indicated activities are probably associated with the cysM gene and can be co-transduced with the cysK gene with high frequency. Based on these results, it can be concluded that thiosulfate is assimilated only through S-sulfocysteine only using S-sulfocysteine synthase (Nakamura T. et al., J. Bacteriology, 156 (2), 656-62 (1983)).

The metabolism of S-sulfocysteine was studied in S. typhimurium. From an experiment with feeding mutants requiring sulfite and sulfide, it was found that this amino acid is converted to cysteine and sulfite, and serves as the sole source of sulfur. In the primary extracts, two different NADPH-dependent systems were identified that are able to participate in the metabolism of S-sulfocysteine. Both systems were purified and identified as glutathione-glutathione reductase and thioredoxin-thioredoxin reductase (Funane K. et al., Agric. Biol. Chem. 51 (5), 1247-56 (1987)).

But there is currently no evidence of increased expression of one or more genes encoding glutaredoxins and glutathione reductase 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 strains producing L-cysteine, and the provision of a method for producing L-cysteine using these strains.

This goal was achieved by discovering the fact that increased expression of one or more genes among genes encoding glutaredoxins and glutathione reductase can increase L-cysteine production.

The present invention provides a bacterium belonging to the Enterobacteriaceae family, in particular a bacterium belonging to the genus Pantoea or Escherichia, 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, characterized in that said bacterium is modified in such a way that expression of one or more genes among genes encoding glutaredoxins and glutathione reductase is enhanced.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Pantoea.

It is also an object of the present invention to provide a bacterium as described above, characterized in that said bacterium belongs to the genus Escherichia.

It is also an object of the present invention to provide a bacterium as described above, characterized in that the genes encoding glutaredoxins are selected from the group comprising the grxC gene from Pantoea ananatis, the grxB gene from Escherichia coli and the grxC gene from Escherichia coli.

It is also an object of the present invention to provide a bacterium as described above, characterized in that the gene encoding glutathione reductase is the gorA gene.

It is also an object of the present invention to provide a bacterium as described above, characterized in that the expression of said gene (s) is enhanced by increasing the number of copies of said gene (s).

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

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

It is also an object of the present invention to provide a method for producing L-cysteine, which comprises growing the bacterium in a thiosulfate-containing culture medium and isolating L-cysteine from the culture fluid in order to produce and store it.

It is also an object of the present invention to provide a method for producing L-cysteine as described above, characterized in that the expression of genes involved in the biosynthesis of L-cysteine is enhanced in said bacterium.

The present invention is described in more detail below.

Detailed Description of the Best Mode for Carrying Out the Invention

1. The bacterium according to the present invention

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 one or more genes among the genes encoding glutaredoxins and glutathione reductase indicated by the bacterium is enhanced.

According to the present invention, the term “bacterium producing L-cysteine” means a bacterium capable of producing and accumulating L-cysteine in a 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 natural or parental strains, for example Pantoea, such as Pantoea ananatis {Enterobacter agglomerans) strains AJ13355 (FERM BP -6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (US Pat. No. 7,090,998), or E. coli, such as E. coli K-12. The term "bacterium-producer of L-cysteine" also means that the microorganism is able to produce and accumulate in the medium L-cysteine in concentrations of not less than 0.5 g / l, and, more preferably, not less than 1.0 g / l

The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia and others. Particularly preferred are bacteria belonging to the Enterobacteriaceae family according to the classification of the NCBI database (National Center for Biotechnology Information, ttp: //www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi? Id = 91347). Bacteria belonging to the genus Escherichia or Pantoea are preferred.

The term “bacterium belonging to the genus Pantoea)) means that the bacterium belongs to the genus Pantoea according to the classification system known to the person skilled 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 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. Among examples of bacteria belonging to the genus Escherichia, but not limited to, the bacterium Escherichia coli (E. coli) may be mentioned.

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

The 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) in terms of a cell or an increase in the expression level of 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 a variety of methods, including Northern hybridization, quantitative RT-PCR (RT-PCR) and the like. Wild type strains, for example, Pantoea ananatis FERM 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 commonly used to refer to promoters, ribosome binding sites (RBS), operators and / or other genome elements involved in the regulation of gene expression level.

Enhanced gene expression can be achieved by introducing several copies of the gene into the bacterial chromosome, for example, using the method of homologous recombination, Mu integration, or other similar methods. For example, one act of Mu integration allows up to 3 copies of a gene to be introduced into the bacterial chromosome.

An increase in the number of copies of the gene can also be achieved by introducing several copies of the gene into the chromosomal DNA of the bacterium. In order to introduce several copies of a gene into a bacterial chromosome, homologous recombination can be carried out using a sequence with several copies of which are present as a target in the chromosomal DNA. Sequences that have multiple copies in chromosomal DNA include, but are not limited to, repeating DNA, or inverted repeats at the ends of motile elements. It is also possible to introduce a gene into a transposon and ensure its movement to introduce several copies of the gene into chromosomal DNA.

Enhanced gene expression, according to the present invention, can be achieved by placing a DNA fragment under the control of a stronger than natural promoter. For example, the lac promoter, trp promoter, trc promoter, P _R , or P _L phage λ promoters are known as strong promoters. A strong promoter can be used in conjunction with an increase in the number of copies of a gene. 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 / cluster of genes and facilitating the expression of this gene / cluster of genes. The strength of the promoter is determined by the frequency of the act of initiation of RNA synthesis.

Also, the action of the promoter can be enhanced, for example, by introducing mutations into the promoter to increase the level of transcription of the gene located after the specified promoter. In addition, it is known that the translational ability of RNA is significantly influenced by the replacement of certain nucleotides between the ribosome binding site (RBS ribosome binding site) and the start codon, and mainly sequences located immediately before the start codon. For example, depending on the nature of the three nucleotides preceding the start codon, there is a 20-fold difference in expression level (Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984).

Moreover, it is possible to introduce a nucleotide substitution into the promoter region of a gene in the bacterial chromosome, leading to increased functioning of the promoter. Changing the expression control sequence can be carried out, for example, in the same way as the gene replacement procedure using the thermosensitive plasmid described in PCT application WO 00/18935 and Japanese application JP 1-215280 A.

The grxB gene from E. coli encodes a glutaredoxin 2 protein. The grxC gene from E. coli encodes a glutaredoxin 3 protein. The gorA gene from E. coli encodes a glutathione reductase protein. Glutathione reductase catalyzes the reduction reaction of disulfide glutathione: disulfide glutathione + NADPH + H ⁺ <=> 2 glutathione + NADP ⁺ .

The nucleotide sequence of the gorA, grxB and grxC genes from E. coli has already been determined. The gorA gene (synonyms: ECK3485, b3500; nucleotides 3644322 to 3645674 in the sequence presented in the GenBank database with inventory number NC_000913.2; gi: 16131372) is located between the yhiR and dinQ genes on the chromosome of the E. coli K12 bacterium. The grxB gene (synonyms: ECK1049, b1064; nucleotides complementary to nucleotides at positions 1122630 to 1123277; in the sequence presented in the GenBank database with inventory number NC_000913.2; gi: 16129027) is located between the yceB and mdtH genes on the chromosome of bacterium E. coli K12. The grxC gene (synonyms: ECK3600, b3610, yibM; nucleotides complementary to nucleotides at positions 3782214 to 3782465; in the sequence presented in the GenBank database with inventory number NC_000913,2; gi: 16131481) is located between the secB and yibN genes on the bacterial chromosome E. coli K12.

The nucleotide sequences of the gorA and grxC genes from P. ananatis have not yet been known. The nucleotide sequence of the grxC gene from P. ananatis is presented in SEQ ID NO: 1. The nucleotide sequence of remgorA from P. ananatis is presented in SEQ ID NO: 2.

Due to the fact that DNA sequences can differ among species and strains of the genus Pantoea or Escherichia, the gorA, grxB and grxC genes are not limited to the sequences presented in SEQ ID NO: I, SEQ ID NO: 2 or in the GenBank database, and can include genes homologous to the above genes.

Thus, the gorA, grxB and grxC genes can be variants that hybridize under stringent conditions with the nucleotide sequences presented in SEQ ID NO: I, SEQ ID NO: 2 or in GenBank, or with probes that can be synthesized based on these nucleotide sequences. "Stringent conditions" include those conditions under which specific hybrids, such as hybrids with a degree of homology of at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably , at least 95%, are formed, and non-specific hybrids, for example, hybrids with a degree of homology lower than the above, are not formed. A practical example of harsh conditions is a single or multiple washing, preferably two or three times, at a salt concentration corresponding to standard washing conditions for Southern hybridization, for example, I × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, at 60 ° C.

The duration of washing depends on the type of membrane used for blotting and, as a rule, is recommended by the manufacturer. For example, the recommended wash time for Hybond ™ N + nylon membrane (Amersham) under severe conditions is 15 minutes. Two or three times washing is preferred. The length of the probe can be selected depending on the hybridization conditions, usually it is from 100 bp up to 1 kb

Methods for obtaining plasmid DNA, DNA cleavage and donation, 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.

A method for producing L-cysteine is a method comprising the steps of cultivating a bacterium according to the present invention in a nutrient medium containing thiosulfate as a sulfur source in order to accumulate L-cysteine in the nutrient medium and isolate L-cysteine from the culture fluid.

According to the present invention, the cultivation of bacteria, isolation from the culture fluid and purification of L-cysteine can be carried out by enzymatic methods traditionally used for the amino acid that is 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 ° C to 40 ° C, preferably from 30 ° C to 38 ° C. The pH value usually ranges from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted with ammonia, calcium carbonate, various acids, bases and buffers. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

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

Brief Description of Drawings

Figure 1 depicts the sequence of the natural Pnlp promoter and the Pnlp8 promoter.

Figure 2 shows the construction of plasmid pM12-ter (thr).

Figure 3 shows the design of an integJ cassette intJS.

Figure 4 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 a strain with enhanced cysM gene expression

1. Construction of the strain P.ananatis EYPSG8

The DNA fragment containing the promoter of the nlpD gene from E. coli was obtained by PCR. The chromosomal DNA of E. coli strain MG1655 (ATCC 700926) was used as a template; P1 (SEQ ID No: 6) and P2 (SEQ ID No: 7) were used as primers. Primer P1 (SEQ ID No: 6) contains a SalI restriction enzyme site at the 5 ′ end. Primer P2 (SEQ ID No: 7) contains a PaeI restriction enzyme site at the 5'-end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 55 ° C, 15 s at 72 ° C; final elongation: 5 min 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 endonuclease 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 with ampicillin (50 mg / L), plates were incubated overnight at 37 ° C until distinctive individual colonies were formed. Plasmids were isolated from the obtained transformants and analyzed by restriction analysis. The resulting plasmid contained the promoter of the nlpD gene from E. coli, and was named pMIV-Pnlp0.

A DNA fragment containing the terminator of the rrnB gene from 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. Primer P3 (SEQ ID NO: 8) contains a XbaI restriction enzyme site at the 5 ′ end. Primer P4 (SEQ ID No: 8) contains a BamHI restriction enzyme site at the 5'-end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 59 ° C, 15 s at 72 ° C; final elongation: 5 min at 72 ° C. The amplified DNA fragment was about 0.3 kb in length, and it was purified using agarose gel electrophoresis. The purified fragment was then 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 with 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 by restriction analysis. The resulting plasmid contained the E. coli rrnB gene terminator and was named pMIV-Pnlp0-ter.

A DNA fragment containing the yeaS gene from E. coli was obtained by PCR. The chromosomal DNA of E. coli strain MG1655 was used as a template; oligonucleotides P5 (SEQ ID No: 10) and P6 (SEQ ID No: 11) were used as primers. Primer P5 (SEQ ID No: 10) contains a SalI restriction enzyme site at the 5'-end. Primer P6 (SEQ ID No: 11) contains a XbaI restriction enzyme site at the 5'-end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 55 ° C, 15 s at 72 ° C; final elongation: 5 min at 72 ° C. The amplified DNA fragment was about 0.7 kb in length, and it was purified using agarose gel electrophoresis. Then, the purified fragment was 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 with 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 by restriction analysis. The resulting plasmid contained the yeaS gene from E. coli and was 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) were used as primers. Primer P7 contains random nucleotides indicated in the sequence SEQ ID NO: 12 by the designation “n” (for A or G, or C or T), and a BglII restriction enzyme site at the 5 ′ end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 15 s at 72 ° C; final elongation: 5 min 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 the sequence SEQ ID NO: 13 by the designation "n" (for A or G, or C, or T), and a BglII restriction enzyme site at the 5'-end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 15 s at 72 ° C; final elongation: 5 min at 72 ° C. Both amplified DNA fragments were purified by agarose gel electrophoresis. Then, the obtained 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 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 12 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 15 s at 72 ° C; final elongation: 5 min 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 PaeI and SalI endonucleases. Next, 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 with 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 by restriction analysis. The resulting plasmid contained the P _nlp8 promoter (Figure 1), and was 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 (pMT-Pomp-cysE5 was obtained from pMIV-Pomp-cysE5 by cloning the XbaI-Eco88I fragment treated with the Klenov fragment from pACYC-184 (tet R) at the PvuI site of plasmid pMIV-Pomp-cysE5; plasmid pMIV-Pomp-cysE5 was obtained by subcloning a PaeI + SacI fragment from pMW-Pomp-cysE5 (see PCT application WO 2005007841), at the same sites of plasmid pMIV-JS) pretreated 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 with 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 by 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 cysE5 and yeaS genes into the chromosome of P.ananatis strain SC17 (US patent 6596517). The 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 with kanamycin (20 mg / L), plates were incubated overnight at 30 ° C until distinctive individual colonies were formed. The resulting strain SC17 / pMH10 was reseeded twice. Then, using the electroporation method, P.ananatis bacteria were transformed with the obtained strain SC17 / pMH10 (this strain grows at 30 ° С) with plasmid pMIV-EY2. The obtained transformants were heat shocked at a 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 seeded at 39 ° C, and each of them was seeded in 1 ml of LA 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 with primers P1 and P6. The resulting strain line was 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.

To relieve the P.ananatis EY19 strain of chloramphenicol resistance, EY19 was transformed with the plasmid pMT-Int-Xis2 (WO 2005/010175) 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 selecting variants sensitive to chloramphenicol (20 mg / L). Such a “cured" strain was named EY19 (s).

The next step was the replacement of the cysPTWA gene promoter region with the Pnlp8 promoter region in strain EY19 (s). PCR was performed using the plasmid pMIV-Pnlp8 -yeaS7 as template and primers P1 and P2. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 20 cycles: 20 s at 94 ° C, 20 s at 59 ° C, 15 s at 72 ° C; final elongation: 5 min 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 a Klenov fragment. After that, the obtained DNA fragment was ligated in an equimolar ratio with the plasmid pMW118- (λattL-Km ^r -λattR) (Reference example 2), pre-treated 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 with kanamycin (20 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 by restriction analysis. The resulting plasmid containing the Pnlp promoter was named pMW-Km-Pnlp8. Then, PCR was performed using the plasmid pMW-Km-Pnlp8 as template and primers P9 (SEQ ID No: 14) and P10 (SEQ ID No: 15). PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 30 cycles: 20 s at 94 ° C, 20 s at 54 ° C, 90 s at 72 ° C; final elongation: 5 min at 72 ° C. The resulting DNA fragment with a length of about 1.6 TPN purified by agarose gel electrophoresis and used to transform the strain P.ananatis SC17 (0) by electroporation. Transformants were plated on LB agar with kanamycin (20 mg / L), plates were incubated overnight at 34 ° C until distinct 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 SC17-Pnlp8-PTWA strain, 10 μg of which was used to transform P.ananatis EY19 (s) strain by electroporation. The obtained transformants were seeded on LB agar with 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 and P12. The resulting strain was named EYP197. To get rid of the resistance of the strain P.ananatis EY19 to kanamycin, strain EY19 was transformed with the plasmid pMT-Int-Xis2 by electroporation. The obtained transformants were plated on LB agar with tetracycline (10 mg / L), plates were incubated at 30 ° C overnight until distinct individual colonies were formed. 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) 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 the 5'-end of the gene serA was obtained by PCR amplification of the chromosomal DNA of strain SC17 with primers P15 (SEQ ID No: 20) and P16 (SEQ ID No: 21). Both primers P14 (SEQ ID No: 19) and P16 (SEQ ID No: 21) contain a Smal restriction enzyme site at the 5'-end. The conditions for the first PCR were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 25 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 60 s at 72 ° C; final elongation: 5 min at 72 ° C. The conditions for the second PCR were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 20 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 20 s at 72 ° C; final elongation: 5 min at 72 ° C. Both amplified DNA fragments were purified by agarose gel electrophoresis and treated with SmaI endonuclease. 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). Primer P13 (SEQ ID No: 18) contains a SalI restriction enzyme site at the 5'-end. Primer P15 (SEQ ID No: 20) contains a XbaI restriction enzyme site at the 5'-end. PCR conditions were as follows: 3 min denaturation at 95 ° C; conditions for the first two cycles: 1 min at 95 ° C, 30 s at 50 ° C, 40 s at 72 ° C; conditions for the next 15 cycles: 20 s at 94 ° C, 20 s at 60 ° C, 75 s at 72 ° C; final elongation: 5 min at 72 ° C. The amplified DNA fragment was about 1.3 kb in length, and it was purified using agarose gel electrophoresis. The resulting 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 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 with 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 by sequencing. The resulting plasmid containing the serA gene with the N348A mutation was 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 seeded at 39 ° C, and each of them was seeded in 1 ml of LA 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, and variants resistant to chloramphenicol (20 mg / L) but sensitive to kanamycin (20 mg / L) and ampicillin (50 mg / L) were selected. The desired integrants were identified by PCR analysis with primers P1 and P15. In all the obtained variants, the specific 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 distinguishable individual colonies. The desired transformants were identified by PCR analysis with primers P1 and P15. The resulting strain was named EYPS1976.

The next step was the introduction of a gcd deletion in strain EYPS1976. To get rid of the resistance of the strain P.ananatis EYPS1976 to chloramphenicol, the strain EYPS1976 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 chloramphenicol-sensitive variants (20 mg / L). Such a cured strain was named EY1976 (s).

The strain P.ananatis SC17 (0) (Russian patent application RU 2006134574), in which the gcd gene was deleted, was constructed using the method first developed by Datsenko K.A., Wanner BL (Proc. Natl. Acad. Sci. USA, 2000 97 (12), 6640-6645), called "Red-Dependent Integration". The DNA fragment containing the kanamycin resistance cassette Km ^R was obtained by PCR using primers P17 (SEQ ID NO: 22) and P18 (SEQ ID NO: 23) and plasmid pMW118-attL-Km-attR-ter_rrnB (Reference example 2 ) as a matrix. The resulting PCR product was purified by agarose gel electrophoresis and used for electroporation of P.ananatis SC17 (0) strain containing plasmid pKD46 with temperature-dependent replication. Plasmid pKD46 (Datsenko K.A. and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 12: 6640-45) carries a 2154 nucleotide phage λ fragment (chromosomal position: 31088 - 33241 sequences with inventory number J02459 in the GenBank database), and contains the genes of Red-dependent homologous phage recombination (genes γ, β and exo-genes), which are controlled by 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 deleted gcd gene and labeled with a Km resistance gene were verified by PCR. For testing, locus-specific primers P19 (SEQ ID NO: 3) and P20 (SEQ ID NO: 4) were used. 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 seeded on LB agar with 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 P17 (SEQ ID No: 22) and PI8 (SEQ ID No: 23). The resulting strain was named EYPSG8.

To get rid of the resistance of the 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), plates were incubated at 30 ° C overnight until distinctive individual colonies were formed. 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 EYPSGint1M2

The EYPSGint1M2 strain was obtained by integrating the cysM gene from E. coli into the EYPSG8 (s) chromosome. In the first step, cysM integration was achieved by mini-integration of the plasmid pMIV-Pnlp1-cysM (Reference Example 3) into the chromosome of strain SC17 using the μ transposase contained in plasmid pMH10 (as described above). The resulting construct (int (Pnlp1-cysM)) was transferred from strain SC17 to strain EYPSG8 by chromosome transformation using a Cm ^R marker for selection. The obtained strain SC17 int (PompC-cysE5-Pnlp8-yeaS) Pnlp8-cysPTWAint (Pnlp8-serA348) Agcdint (Pnlp1-cysM)) was named EYPSGint1M2 and was used for further experiments after curing from the antibiotic resistance marker.

Example 3. The activity of S-sulfocysteine reductase and glutathione reductase strains SC17 / pMIV-grxC and SC17 / pMIV-gorA

To evaluate the ability of the grxC and gorA genes from P.ananatis to increase the activity of S-sulfocysteine reductase and glutathione reductase, strain SC17 was transformed with the plasmids pMIV-grxC (Reference Example 4) and pMIV-gorA (Reference Example 4), resulting in strains of SC17 / pMIV -grxC and SC17 / pMIV-gorA, respectively.

Cells of strains SC17, SC17 / pMIV-grxC and SC17 / pMIV-gorA from night cultures grown in M9 medium with the appropriate additives were diluted with fresh medium at a ratio of 1:20 and grown at 34 ° C. Cells were harvested at the late exponential phase (approximately 6 hours). The collected cells were washed once with saline, resuspended in 50 mM Tris-HCl, pH 7.0.2 mM DTT, and destroyed by ultrasound. The soluble protein fraction was used as a protein preparation for measuring the activity of S-sulfocysteine reductase and glutathione reductase.

S-sulfocysteine reductase, together with its cofactor, glutathione (GSH), catalyzes the reduction of S-sulfocysteine to cysteine and sulfite to give the oxidized form of glutathione (GSSG). Methods for measuring activity were based on coupling this reaction with glutathione reductase, which, together with its cofactor, NADPH, catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). The method used was a spectrophotometric measurement in which the oxidation of NADPH to NADP ^{+ was} monitored to reduce absorption at 340 nm. The unit of measurement of the activity of S-sulfocysteine reductase can be expressed in terms of oxidation of NADPH or reduction of GSSG, since their molar ratio is 1: 1.

The reaction mixture (1 ml) contained: 100 mm Tris-HCl, pH 7.5, 0.125 mm NADPH, 0.1 mm glutathione (GSH, reduced form), 1 unit glutathione reductase (Sigma G3664), 2 mM S-sulfocysteine and an enzyme preparation. The reaction was initiated by the addition of S-sulfocysteine and carried out at 20 ° C. One unit of S-sulfocysteine reductase was determined as the amount of enzyme required for catalysis of the reduction of one micromole of NADPH per minute at pH 7.5 and 20 ° C. The reaction product (cysteine) was determined by HPLC.

Glutathione reductase was measured spectrophotometrically by the Carsberg method (Carlberg I., Mannervik B., Glutathione reductase. Meth. Enzymol. 113, 485-490 (1985)).

The measurement results are presented in Table 1. As can be seen from Table 1, strain SC17 / pMIV-grxC showed greater activity of S-sulfocysteine reductase than SC17 and SC17 / pMIV-gorA; SC17 / pMIV-gorA showed greater glutathione reductase activity compared to SC17 and SC17 / pMIV-grxC.

Example 4. Production of L-cysteine by P.ananatis strains 1. Production of L-cysteine by P.ananatis strains transformed with plasmids carrying the grxC and gorA genes from P.ananatis, and the grxB and grxC genes from E. coli.

To assess the effect of increased expression of the grxC and gorA genes from P.ananatis, and the grxB and grxC genes from E. coli, on the production of L-cysteine, P.ananatis strain EYPSGint1M2 was transformed with plasmids pMIV-grxC, pMIV-gorA and pMIV-AC ( Reference example 4), and pMIV-grxB (Ec), and pMIV-grxC (Ec) (Reference example 6), with obtaining strains EYPSGint1M2 / pMIV-grxC, EYPSGint1M2 / pMIV-gorA, EYPSGint1M2 / pMIV-AC, EYPSGint1M2 -grxB (Ec) and EYPSGint1M2 / pMIV-grxC (Ec), respectively.

The strains P.ananatis EYPSGint1M2, EYPSGint1M2 / pMIV-grxC, EYPSGint1M2 / pMIV-gorA, EYPSGint1M2 / pMIV-AC, EYPSGint1M2 / pMIV-grxB (Ec) and EYPSGint1M2 / p for 34 h (° C) at 17 ° C pM on L-agar. Then, cells from approximately 30 cm ^{2 of the} surface of the plate were plated in a 500 ml L-medium flask (50 ml) and cultured with aeration for 5 hours at 32 ° C. After this, the cultures were subcultured in 450 ml of fermentation medium in Jar fermenters (Marubisi) and cultivated at 30 ° С, pH 7.4 (with control of aqueous NH3 (~ 4.66 M)), D _O2 > 25% (with stirring at 900 rpm).

After cultivation, the amount of L-cysteine accumulated in the medium was determined by the method described by Gaitonde M.K. (Biochem J .; 104 (2): 627-33 (1967)), with the following modifications: 150 μl of the sample was mixed with 150 μl of 1M H ₂ SO ₄ , incubated for 5 min at 20 ° C, then 700 μl of N was added to the mixture ₂ O, 150 μl of the resulting mixture was transferred to a new tube and 800 μl of solution A was added (1M Tris pH 8.0, 5 mM DTT), the resulting mixture was incubated for 5 min at 20 ° C, centrifuged for 10 min at 13000 rpm then 100 μl of the mixture was transferred to 20 × 200 mm tubes, 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 in for 10 min in a water bath, then 4.5 ml of ethanol was added and OD _{560 was} measured. The cysteine concentration was calculated by the formula C (cis g / l) = 11.3 * OD ₅₆₀ . The measurement results are shown in Table 2. As can be seen from Table 2, the strains EYPSGint1M2 / pMIV-grxC, EYPSGint1M2 / pMIV-gorA, EYPSGint1M2 / pMIV-AC, EYPSGint1M2 / pMIV-grxB (Ec) and EYPSGint1Mx / pMIV / pMIV / pMIV / grMB / Ec2) more accumulated L-cysteine compared to EYPSGint1M2. In addition, the use of the gorA, grxB or grxC genes led to a significant reduction in the accumulation of a by-product, S-sulfocysteine. The most significant effect of reducing the accumulation of S-sulfocysteine was observed in a strain transformed with two gorA and grxC genes simultaneously.

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

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 (D) Na ₂ S ₂ O ₃ 5H ₂ O 188.4 g / l

Sterilization of groups (A, B, C and D) was carried out separately to prevent adverse interactions during sterilization.

Thiosulfate was added via a separate pump, activated in conjunction with an alkaline pump (thiosulfate was added in an amount equivalent to the added 4.6 N NH ₄ OH).

2. Production of L-cysteine by P.ananatis strains with integrated copies of the grxC and gorA genes

To evaluate the effect of increased expression of the grxC and gorA genes on the production of L-cysteine, P. ananatis strains EYPSGint1M2intAC and EYPSGint1M2Pnlp8-grxC were also constructed.

The integration of the grxC and gorA genes was carried out as a result of mini-µu integration (using the µu transposase contained in plasmid pMH10) of plasmid pMIV-AC into the chromosome of strain SC17. The resulting construct (intAC) was transferred from strain SC17 to strain EYPSGint1M2 by chromosome transformation. The resulting strain was named EYPSGint1M2intAC.

The pomotor region of the grxC gene in the chromosome of strain EYPSGint1M2 was replaced by the Pn1p8 promoter region using the method of Red-dependent integration. The DNA fragment for Red-dependent integration was obtained by PCR with primers P31 (SEQ ID No: 42) and P32 (SEQ ID No: 43) and plasmid DNA pMW-Km-Pnlp8-cysE (Reference Example 5) as a template. The resulting strain was named EYPSGint1M2Pnlp8-grxC.

Strains P.ananatis EYPSGint1M2, EYPSGint1M2intAC and EYPSGint1M2Pnlp8-grxC were cultured as described above, the amount of L-cysteine accumulated in the medium was determined using the method described above. The measurement results are presented in Table 3. As can be seen from Table 3, EYPSGint1M2intAC and EYPSGint1M2Pnlp8-grxC accumulated a larger amount of L-cysteine compared to EYPSGint1M2.

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

In order to evaluate the effect of increased expression of grxC and grxC genes from E. coli on L-cysteine production, E. coli strains MT / pACYC-DES / pMIV-grxB and MT / pACYC-DES / pMIV-grxC were constructed.

For this, the E. coli strain MT / pACYC-DES (European patent EP 1528108 B1) was transformed with the plasmids pMIV-grxB and pMIV-grxC (see Reference Example 6) to obtain strains MT / pACYC-DES / pMIV-grxB and MT / pACYC-DES / pMIV-grxC, respectively.

Strains MT / pACYC-DES, MT / pACYC-DES / pMIV-grxB and MT / pACYC-DES / pMIV-grxC were cultured overnight with shaking at 34 ° C in 2 ml of nutrient broth with the addition of 100 mg / l ampicillin and 25 μg / ml streptomycin. 0.2 ml of the obtained cultures were sown in 2 ml of fermentation medium containing chloramphenicol (30 mg / l) and ampicillin (100 mg / l) in 20 × 200 mm tubes, and grown at 34 ° C for 42 hours on a rotary shaker with 250 rpm 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, 6% glucose, 100 mg / L L-methionine and 3 g / L Na ₂ S ₂ O ₃ .

After cultivation, the amount of L-cysteine accumulated in the medium was determined by the method described by Gaitonde, M.K. (Biochem. J., 104: 2, 627-33 (1967)). The results obtained are presented in Table 4. As can be seen from Table 4, strains MT / pACYC-DES / pMIV-grxB and MT / pACYC-DES / pMIV-grxC accumulated a greater amount of L-cysteine compared to MT / pACYC-DES. Strain MT / pACYC-DES / pMIV-grxC also accumulated less S-sulfocysteine than MT / pACYC-DES.

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 Mu-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 directions (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) in pM12, pre-treated with Hindlll and Mph1103I restriction enzymes, the plasmid pM12-ter (thr) was constructed (Figure 2).

The integj cassette intJS was designed as follows (Figure 3):

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) CmR fragment 1.03 kbp in length was obtained by PCR amplification using a phosphorylated forward primer (SEQ ID NO: 36) and a reverse primer (SEQ ID NO: 37) (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) (a restriction enzyme recognition site of PstI is isolated), and a phosphorylated reverse primer (SEQ ID NO: 39) (a restriction enzyme recognition site of SacI is isolated). The plasmid pMW118-attL-tet-attR-ter_rrnB was used as a matrix;

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

e) LattL-CmR and LattR fragments were digested with PstI restriction enzyme, ligated, and the resulting LattL-CmR-LattR fragment 1.31 kbp in length. has been cleaned;

f) a double-stranded DNA fragment with a length of 70 bp 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 SacI, ligated, and the resulting 1.38 kb LattL-Cm ^R -LattR-MCS cassette was purified.

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 4).

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 pMWl 18-attL-Tc-attR (WO 2005/010175) by replacing the tetracycline resistance marker with the kanamycin resistance gene taken from pUC4K plasmid (Vieira J. and Messing J., Gene, 19 (3): 259-68 (1982)).

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

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

1) a BglII-EcoRI fragment (114 bp) containing the attL site (SEQ ID NO: 44) was obtained by PCR amplification of the corresponding chromosome region (containing prophage λ) of E. coli W3350 using primers P33 and P34 (SEQ ID NOS: 45 and 46) (these primers contained additional recognition sites for BglII and EcoRI endonucleases);

2) a PstI-HindIII fragment (182 bp) containing the attR site (SEQ ID NO: 47) was obtained by PCR amplification of the corresponding chromosome region (containing prophage λ) of E. coli W3350 using primers P35 and P36 (SEQ ID NOS: 48 and 49) (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 obtained as follows: pMW118 was digested with EcoRI restriction enzyme, processed with a Maple fragment of DNA polymerase I, and then with AatII restriction enzyme;

2 - a small fragment of AatII-BglII (1194 bp) of the pUC19 vector carrying the bla ampicillin resistance gene (Ap ^R ) was obtained by PCR amplification of the corresponding region of the pUC19 vector using primers P37 and P38 (SEQ ID NOS: 50 and 51) (these primers contained additional recognition sites for the endonucleases AatII and BglII);

3 - a small fragment of BglII-PstlIol (363 bp) of the ter_rrnB transcriptional terminator was obtained by PCR amplification of the corresponding region of E. coli chromosome MG1655 using primers P39 and P40 (SEQ ID NOS: 52 and 53) (these primers contained additional recognition sites for endonucleases BglII and PstI);

4) a small fragment of EcoRI-PstI (1388 bp) (SEQ ID NO: 54) 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 MG1655 using P41 and P42 oligonucleotides (SEQ ID NOS: 55 and 56) 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).

Reference Example 3. Construction of the plasmid pMIV-Pnlp1-cysM

The promoter region of the nlpD gene from P. ananatis was amplified by PCR with primers P21 (SEQ ID NO: 5) and P22 (SEQ ID NO: 24) and chromosomal DNA of strain SC17 as a template. The resulting DNA fragment with a length of about 0.2 TPN kb was purified by agarose gel electrophoresis and treated with PaeI and SalI endonucleases, followed by ligation with plasmid pMIV-5JS, previously treated with the same endonucleases.

The termination region of the rrnB gene from E. coli was amplified by PCR with examples of P23 (SEQ ID NO: 25) and P24 (SEQ ID NO: 26) and chromosomal DNA of strain MG1655 as a template. The resulting DNA fragment with a length of about 0.25 kb was purified using agarose gel electrophoresis and processed with XbaI and BamHI endonucleases, followed by ligation with the plasmid obtained in the previous step, previously treated with the same endonucleases.

The cysM gene from E. coli was amplified by PCR with examples of P25 (SEQ ID NO: 27) and P26 (SEQ ID NO: 28) and the chromosomal DNA of strain MG1655 as a matrice. The resulting DNA fragment with a length of about 1.1 TPN was purified using agarose gel electrophoresis and processed with SalI and XbaI endonucleases, followed by ligation with the plasmid obtained in the previous step, previously treated with the same endonucleases. Thus, the plasmid pMIV-Pnlp1-cysM was obtained.

Reference example 4. Construction of plasmids pMIV-grxC, pMIV-gorA and pMIV-AC

The grxC gene from P. ananatis was amplified by PCR with primers P27 (SEQ ID NO: 29) and P28 (SEQ ID NO: 30) and chromosomal DNA of strain SC17 as a template. The gorA gene from P.ananatis was amplified by PCR with primers P29 (SEQ ID NO: 31) and P30 (SEQ ID NO: 41) and chromosomal DNA of strain SC17 as a template. Each of the obtained genes was cloned into pMIV-5JS (at SalI and XbaI sites). Thus, plasmids pMIV-grxC and pMIV-gorA, respectively, were obtained.

Plasmids carrying both the grxC and gorA genes were constructed as follows. Plasmid pMIV-grxC was digested with HindIII endonuclease, then digested with a Klenov fragment of DNA polymerase I followed by endonuclease NcoI. The resulting 1.3 kb The grxC gene DNA fragment was purified and ligated with the plasmid pMIV-gorA pretreated with SmaI and NcoI endonucleases. A plasmid containing both the grxC and gorA genes was named pMIV-AC.

Reference Example 5. Construction of the plasmid pMW-Km-Pnlp8-cvsE

The cysE gene from P.ananatis was amplified by PCR from the chromosome of strain SC17 using primers P43 (SEQ ID NO: 57) and P44 (SEQ ID NO: 58). Primer P43 (SEQ ID No: 57) contains a SalI restriction enzyme site at the 5'-end. 0.9 kb the fragment was isolated and treated with SalI endonucdease.

The Pnlp8 promoter was amplified by PCR from plasmid pMIV-Pnlp8-yeas7 using primers P45 (SEQ ID NO: 59) and P46 (SEQ ID NO: 60). Primer P45 (SEQ ID No: 59) contains a SalI restriction enzyme site at the 5'-end. 0.3 kb the fragment was isolated and processed with SalI endonuclease and ligated with the cysE gene fragment described above. The ligation mixture was used as a template for PCR with primers P44 (SEQ ID NO: 58) and P46 (SEQ ID NO: 60).

The resulting 1.2 kb the fragment was isolated and processed by the Klenov fragment and cloned into the plasmid vector pMW118- (λattL-Km ^r -λattR) (Reference example 2), pretreated with restriction enzyme XbaI and the Klenov fragment. The resulting plasmid was named pMW-Km-Pnlp8-cysE.

Reference example 6. Construction of plasmids pMIV-grxB (Ec) and pMIV-grxC (Ec).

The grxB gene from E. coli was amplified by PCR with primers P47 (SEQ ID NO: 61) and P48 (SEQ ID NO: 62) and the chromosomal DNA of E. coli strain MG1655 as a template. The resulting gene was cloned into pMIV-5JS (at XhoI and XbaI sites). Thus, the plasmid pMIV-grxB (Ec) was obtained.

The grxC gene from E. coli was amplified by PCR with primers P49 (SEQ ID NO: 63) and P50 (SEQ ID NO: 64) and the chromosomal DNA of E. coli strain MG1655 as a template. The resulting gene was cloned into pMIV-5JS (at XhoI and XbaI sites). Thus, the plasmid pMIV-grxC (Ec) was obtained.

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.

Claims (9)

1. A bacterium is a producer of L-cysteine belonging to the genus Pantoea or Escherichia of the Enterobacteriaceae family and modified in such a way that expression of one or more genes selected from the group consisting of:
coding for glutaredoxins genes grxC and grxB and a gene coding for glutathione reductase. 2. The bacterium according to claim 1, characterized in that the grxC gene is a gene from Pantoea ananatis or Escherichia coli. 3. The bacterium according to claim 1, characterized in that the grxB gene is a gene from Escherichia coli. 4. The bacterium according to claim 1, characterized in that the gene encoding glutathione reductase is the gorA (gor) gene from Pantoea ananatis. 5. The bacterium according to any one of claims 1 to 4, characterized in that the expression of the specified gene (s) is enhanced by increasing the number of copies of the specified gene (s). 6. The bacterium according to any one of claims 1 to 4, characterized in that the expression of the indicated gene (s) is enhanced by modifying the sequence (s) that controls the expression of the indicated gene (s). 7. The bacterium according to claim 6, characterized in that the natural (s) promoter (s) of the indicated gene (s) is replaced (s) by a stronger (s) promoter (s). 8. A method for producing L-cysteine, which comprises growing a bacterium according to any one of claims 1 to 7 in a nutrient medium containing thiosulfate and isolating L-cysteine from the culture fluid to produce and accumulate L-cysteine. 9. The method according to claim 8, characterized in that in said bacterium the expression of genes involved in the biosynthesis of L-cysteine is enhanced.

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