Recombinant plasmid pt7Δpcka::loxpcat, providing synthesis of l-threonine in cells of escherichia coli, and recombinant strain escherichia coli ftr2717 (kccm-10475)-producer of l-threonine

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns biotechnology, in particular genetic engineering. The recombinant plasmid pT7ApckA::loxpcat is designed by cloning of an incomplete pckA-gene in pT7B1ie-vector. The plasmid contains a fragment of a pckA-gene which includes a gene steady against Chloramfenicolum and loxP-sites. By transformation of cells E.coli with a plasmide DNA pT7ApckA::loxpcat strain E.coli FTR2717-producer L-threonine is obtained.

EFFECT: increase of L-threonine output in presence of high concentration of glucose.

2 cl, 3 dwg, 2 tbl, 3 ex

The invention

The technical field to which the invention relates

The present invention relates to a microorganism which produces L-threonine, and to a method for producing L-threonine using this microorganism. In particular, the present invention relates to a microorganism which contains the chromosome inactivated genestdcBCandpckAand significantly improves the productivity of L-threonine, which is caused by the inactivation of these two genes, as well as to a method for producing L-threonine using such a microorganism.

Description of the prior art

It is known that L-threonine is an essential amino acid, which is widely used as fodder, feed additives and animal growth promoter, but also as a component of drug in aqueous solutions and additional raw materials in medical products. Currently, in developed countries, L-threonine is produced only five companies, including Ajinomoto Company in Japan, and L-threonine in two or three times the price of lysine, which is known that it is of high value in the international market due to its high price, 5000-6000 dollars per ton. Therefore, for the production of L-threonine there is the possibility of rapid growth in the global market.

Currently, L-threonine receive only using methodologically fermentation, mainly using mutants derived from wild-type microorganisms, includingEscherichia colirepresentatives of the genusCorynebacterium,representatives of the genusBrevibacterium,representatives of the genusSerratiaand representatives of the genusProvidencia. Examples of such mutants include mutants having resistance to amino acid analogs or drugs, and their auxotrophs on diaminopimelic acid, methionine, lysine and isoleucine (Japanese patent publication No. Heisei 2-219582; Korean patent publication No. 1998-32951;Appl. Environ. Biotechnol., 29:550-553, 1988). However, these mutant strains for their auxotrophic properties in relation diaminopimelic acid or isoleucine unprofitable in the sense that they have low productivity of L-threonine and grow only in the environment in which it is added expensive diaminopimelic acid or isoleucine. In other words, when using a mutant that requires for its growth in diaminopimelic acid, this enzymatic production of L-threonine requires high costs. In the case of auxotroph by isoleucine fermentation medium for this auxotroph should also contain additive expensive isoleucine, which leads to increased costs in the production of L-threonine.

These problems can be overcome by the use of leaky mutant by isoleucine, which is described in Korean patent publication No. 92-8365, excluding the presence in the environment of isoleucine and producing large amounts of L-threonine as compared with the known strains. However, this classical mutation method for selection of new bacterial strains able to produce large amounts of L-threonine, is time-consuming and inefficient, and its biggest drawback is that it only partially improves the productivity of L-threonine.

In this regard, instead of using data auxotroph using the methods of metabolic engineering developed other methods for mass production of L-threonine using a recombinant microorganism producing L-threonine, and which have increased activity of enzymes involved in the biosynthesis of L-threonine. Namely, using the methods of genetic recombination, there are the genes corresponding to enzymes involved in the metabolism of L-threonine, clone selected genes in appropriate gene carriers and embed them in microbial mutants for improved productivity of L-threonine in these mutants.

The authors of the present invention previously developed a method of creating a strain producing L-threonine, using such methods of metabolic engineering, which is disclosed in Korean patent request is e No. 2001-6976. In detail, the high yield of L-threonine can be achieved by using a recombinant microorganism, which has one or multiple chromosomal copies of the gene encoding fosfoenolpiruvatcarboksilaza (hereinafter referred to as simply "ppc"), which catalyzes the formation of oxalacetate (OAA) as a precursor in the biosynthesis of L-threonine from phosphoenolpyruvate (PEP), and also contains an operon containing genes encoding enzymes involved in the biosynthesis of L-threonine from aspartate, aspartokinase 1-homoerythromycin (thrA), homoserine (thrBand trionychinae (thrC).

L-threonine is synthesized from aspartate by multistage, in which the aspartate is formed from JAA converted to PPC from PEP. The biosynthesis of L-threonine inhibited in the presence in the environment of glucose in relatively high concentrations compared with the rate of bacterial growth and the total speed of the tricarboxylic acid cycle. In this situation, the expressionppc-gene cupressinum, and expression of the gene encoding PEP-carboxykinase (hereinafter referred to as simply "pckA"), which catalyzes the conversion of OAA to PEP increases. Elevated levels ofpckAlead to the formation as a precursor of the biosynthesis of amino acids PEP from OAA, where this PEP are synthesized by-products (Goldie H. Medina V.,Mol Gen. Genet., 220(2):191-196, 1990; Dang et al.,E. coli and Salmonella, 1:191-102, 1996). For this reason,pckAgene needs to substantially inactivate the purpose of obtaining large quantities of L-threonine by increasing the intensity of the metabolic pathways responsible for the biosynthesis of L-threonine.

On the other hand, there are several ways degradation of L-threonine, which include the following three ways. The first includes the path initiated by trainingsgerate, producing α-aminoβ-ketobutyrate. Received α-aminoβ-ketobutyrate is converted to acetyl-COA and glycine or spontaneously breaks up aminooctane, which is converted into pyruvate. The second way is connected with trainingexercises producing α-ketobutyrate, which subsequently catabolized to propionyl COA and, ultimately, to the intermediate of the tricarboxylic acid cycle - succinyl-COA. The third way uses trivialities (F.C. Neidhardt et al.Escherichia coli and Salmonella: cellular and molecular biology, 2^nded. ASM press. Washington DC, pp.369-370). From them trainingsgerate is an operon, which is expressed during hypoxia and high levels of threonine.

In the present invention using the method of genetic recombination (Korean patent application No. 2002-015380) created the microorganism with improved productivity of L-threonine in the specific is aktivacii this gene operon ( tdcBC).

On the other hand, in International patent publication no WO 02/29080 A2 disclosed is a method of obtaining L-threonine using defective bypckA-the gene of the microorganism, which is obtained by introducing into a microorganism strain wild-type recombinant vector carrying a partially deleteriouslypckA-the gene. However, when using the specified microorganism remains an open question about the formation of L-threonine as the path of degradation and intracellular receipt synthesized L-threonine is still activated in this organism.

When carrying out the present invention the authors with the purpose of solving the problems existing in the prototype, completed an intensive and thorough study of methods for obtaining microorganisms capable of producing a large amount of L-threonine even when they are grown in a medium with high glucose concentration and without degradation of the produced L-threonine, and found that when chromosomepckA-gene of this organism is inactivated by the method of genetic recombination, the microorganism is destroyedtdcBC-the operon, created by the authors of the present invention, has a high productivity of L-threonine as compared with traditionally used by microorganisms producing L-threonine.

For this reason, the objective of the present invented the I is to create a microorganism, able to effectively produce a large amount of L-threonine.

Summary of the invention

In order to achieve the above objective, the present invention has created a new strain ofE. colithat contains the chromosome inactivated genestdcBCandpckA.

In strainE. coliwith inactivatedtdcBC/pckA-genespckA-inactivate gene by introducing a fragment of alienpckAgene, containing the gene for resistance to the antibiotic and the binding site of the site-specific recombinase at each of its ends, in a strain ofE. colicontaining operontdcBCassociated with the degradation of L-threonine, which is inactivated, and then spent homologous recombination between the fragment alienpckAgene andpckA-genome chromosome for inactivation of chromosomalpckA-gene.

In addition, in the present invention, a method of obtaining L-threonine, using strains ofE. coliwith inaktivirovannyetdcBC/pckA-the genome.

Brief description of drawings

The above and other objectives, features and other advantages of the present invention will become clearer from the following detailed description together with illustrated drawings, in which

figure 1 schematically depicts the cloning technologypckA-gene;

in figure 2 schemati the Eski depicted technology for producing recombinant microorganism, in which you entered the codepckAgene, containing the gene for resistance to chloramphenicol (catand loxP sites, ΔpckA::loxpcat; and

figure 3 presents a photograph showing the results of southern blotting, which identified the gene for resistance to chloramphenicol (cat), built-inpckA-gene chromosome of strainE. coliproducing L-threonine (lane 1: recombinant strain selected in the presence of chloramphenicol in accordance with the present invention; track 2: the parent strain TRN212 and lane 3: molecular mass marker).

Detailed description of the invention

The strain ofE. colithat contains associated with the degradation of L-threonine operon, specifically inactivating using genetic recombination and having high productivity of L-threonine due to inactivation of the operon, can be used as the parent strain in the present invention. Preferred parent strain is a strain ofE. coliTRN212 (inventory number: KCCM-10353; Korean patent application No. 2002-015380), which is created in the present invention.

The present invention is characterized by obtaining a new strain ofE. coliproducing a large number and a high yield of L-threonine in the result of inaktivirovaniepckA-the gene involved in the inhibition of the synthesis of L-threonine is in the parent strain of E.coli containing associated with the degradation of L-threonine inactivated operon (tdcBC). Inactivation of both genestdcBCandpckAleads to prevent degradation and intracellular receipt of L-threonine, mediated by products broadcasttdcBC-operon, and the inhibition of the synthesis of L-threonine, mediated translational productpckA-gene, and leads to the production of large quantities of L-threonine.

Therefore, in the present invention created a strain ofE. coliwith inaktivirovannye genetdcBC/pckA, which is obtained by introducing a fragment of alienpckAgene, including the gene of resistance to the antibiotic that has a binding site site-specific recombinase at each of its ends to the strain ofE. colicontaining associated with the degradation of L-threonine operontdcBCthat is inactivated, and then allowing homologous recombination between the fragment alien and is contained in the chromosomepckAgene for inactivation of chromosomalpckA-the gene.

In addition,pckAgene on the chromosome of the parent strain ofE. coliinactivate when removing built-in chromosomal pckA gene resistance gene to an antibiotic by activating the site-specific recombinase expressed in this bacterial strain, and the presence of one copy of the linking site-specific the recombinase in chromosome pckA-gene.

InaktivirovaniepckAgene bacterial chromosome is carried out by homologous recombination with a fragment of alienpckA-gene. A fragment of alienpckAgene inactivate by embedding in it a gene of resistance to the antibiotic. This vaccine fragment alienpckAgene is introduced into the parent strainE. colithen there is a double crossover recombination betweenpckA-genome bacterial chromosome and a fragment of alienpckAgene for inaktivirovaniepckAgene bacterial chromosome. The presence of the resistance gene to an antibiotic in alien inactivatingpckA-gene facilitates selection of cells with inactivatingpckA-gene.

Non-limiting examples of the gene of resistance to the antibiotic used for inactivation ofpckA-gene include a gene for resistance to chloramphenicol, the gene of resistance to kanamycin gene of resistance to gentamicin and the gene for resistance to ampicillin.

On the other hand, after the selection of a strain ofE. coliwith inactivatingpckA-genome provide an opportunity to be expressed site-specific recombinases to remove resistant to the antibiotic gene, integrated in the bacterial chromosome. That is, the gene of resistance to this antibiotic is inserted into thepckA-gene bacterial chromosome connect with the surrounding sites site-specific recombinases and removed by activation of site-specific recombinases, expressed in the bacterial strain. Examples of such site-specific recombinases include, without limitation, the binding sites FLP, Cre and XerC/d Deletion of the gene of resistance to this antibiotic allows you to re-use the same gene of resistance to this antibiotic as a selective marker, if you want to inactivate the other gene is identical to the bacterial strain.

In order to inactivate chromosomalpckAgene, in the present invention using the fragment containing the gene for resistance to chloramphenicol, each end of which is connected to the loxP site. This loxP site is recognized by a site-specific recombinase Cre. As a result of activation of the Cre recombinase expressed in the strain ofE. coli, the gene of resistance to the antibiotic, located between the two loxP sites, is removed from the bacterial chromosome.

The expression of Cre recombinase in the strain ofE. coliyou can implement known in the art by the way. In the present invention plasmid pJW168 bearingcre-gene is inserted into a strain ofE. colifor expression in it Cre enzyme.

In one of the embodiments of the present invention incompletepckAgene amplified by PCR using as template genomic DNA isolated from producing L-threonine strainE. colithat includes inaktivirovannye the tdcBC-operon. Amplificatory incompletepckA-gene clone in pT7Blue vector (Novagen Co.), thus obtaining the recombinant vector pT7Blue/pckA containing incompletepckA-gene. In addition, ploxpcat2-plasmids (Betriz Palmeros et al.,Gene, 247:255-264, 2000) obtain a DNA fragment loxpcat2, containing the gene for resistance to chloramphenicol and loxP sites and are ligated with NruI-processed pT7Blue/pckA, thus creating a recombinant plasmid pT7ΔpckA::loxpcat containing fragmentpckA-the gene includes a gene for resistance to chloramphenicol and loxP sites. Therefore, in the present invention is made, as shown above, the recombinant plasmid pT7ΔpckA::loxpcat.

In another embodiment of the present invention the fragmentpckAgene, containing the gene for resistance to chloramphenicol, each end of which is connected to the loxP site is inserted into a strain ofE. coliTRN212 containingtdcBC-operon, which inactivate by homologous recombination using resistant to kanamycin gene having at each of its two ends loxP site. Then carry out homologous recombination betweenpckA-genome bacterial chromosome and a fragment of alienpckAgene, containing the gene for resistance to chloramphenicol and loxP sites, thus creating a recombinant strain ofE. colicontainingtdcBC-gene and an inactivatedpckA-gene chromos who we are. Specified recombinant strain ofE. coliwas marked "FTR2717" and deposited in the Korean Center of Cultures of Microorganisms (KCCM) on March 20, 2003 under inventory number KCCM-10475.

Recombinant strain ofE. coliFTR2717 detects the following characteristics:

(1) compared with the wild-type strain he has resistance to threonine analogues, analogues of lysine, isoleucine analogues and analogues of methionine;

(2) its chromosome contains endogenousppc-gene and endogenous threonine operon, containing genesthrA, thrBandthrCand one or multiple copies of the exogenousppc-gene and an exogenous genesthrA, thrBandthrC;

(3) it includes an inactivated gene operontdcBCinvolved in the degradation of L-threonine; and

(4) it includes an inactivatedpckA-the gene involved in the inhibition of the synthesis of L-threonine, and therefore it produces a large amount of L-threonine in high concentrations of glucose in the environment.

A better understanding of the present invention can be achieved by using the following examples, which are provided for illustration and should not be construed as limiting the present invention.

EXAMPLE 1: CloningpckA-gene

The obtained recombinant vector carrying thepckA-gene (see figure 1). At the beginning of the strainE. coliTRN212 (inventory number: KCCM-10353), includingtdcBC-/i> operon and producing L-threonine (QIAGEN Co.), allocate using QIAGEN Genomic-tip system bacterial genomic DNA. Using extracted genomic DNA as template, perform PCR amplification incomplete, about 1.5 TPN, thepckA-the gene. In this PCR using a set of primers consisting of a forward and reverse primer, represented respectively in SEQ ID NO: 1 and 2. The PCR conditions include 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 1 min 30 sec.

The resulting PCR products are subjected to electrophoresis in 0.8% of agarose gel, then cut out from the gel strip 1.5 TPN From this cut strip by cleaning allocate 1.5 TPN DNA fragment using a set of DNA Gel Purification Kit (QIAGEN Co.) and clone it into EcoRV-processed pT7Blue vector (Novagen Co.) by ligating on blunt ends at 16°C, thus creating a recombinant vector pT7Blue/pckA containing incompletepckA-gene. Then using the vector pT7Blue/pckA strain ofE. coliNM522 transform and put a smear on a solid medium (LB: 1% NaCl, 1% tryptone, 0,5% yeast extract)containing ampicillin (100 mg/l), followed by incubation overnight at 37°C. Grown on solid medium colony inoculant in 3 ml of liquid medium containing ampicillin, followed by incubation overnight at 37°C is a set of QIAGEN mini prep kit (QIAGEN Co.) from cultivated bacteria produce plasmid DNA and examine its size. In addition, the orientation ofpckA-gene is determined using Nrul and Stulrestriction mapping. After that plasmid DNA is treated with restriction enzyme Nrul and subjected to electrophoresis in a 0.7% agarose gel. This gel cut strip size approximately 4.3 TPN, and cut from the strip by cleaning allocate fragment of 4.3 TPN

EXAMPLE 2: Construction of recombinant vector carrying inactivatingpckA-gene and strain ofE. coliwith inaktivirovannyepckA-the genome.

2-1) Construction of recombinant vector carrying inactivatingpckA-the gene.

1,2 TPN loxpcat-fragment, which contains resistant to chloramphenicol gene with loxP-site at each end, is treated with restriction enzyme HincII plasmids ploxpcat2 (plasmids carrying resistant to chloramphenicol gene having at their ends loxP sites; Beatriz Palmeros et al.,Gene,247:255-264, 2000, Professor G. Gosset, University of Mexico). The obtained DNA fragment of 1.2 TPN are ligated with Nrul-processed pT7Blue/pckA obtained in example 1 by ligating on blunt ends, thus creating a recombinant vector pT7ΔpckA::loxpcat length of almost 5.7 TPN and containing an inactivatedpckA-gene (see figure 2).

2-2) Obtaining strainE. coliwith inaktivirovannyepckA-the genome.

Recombinant vector pT7ΔpckA::loxpcat obtained in example 2-1)is inserted into a strain ofE. coliNM522. This tra is formed strain NM522 put a smear on a solid medium (LB: 1% NaCl, 1% tryptone, 0,5% yeast extract)containing ampicillin and chloramphenicol, followed by incubation overnight at 37°C. Grown on this medium, colonies inoculant in 3 ml of liquid medium containing ampicillin and chloramphenicol, followed by incubation overnight at 37°C. From this bacterial culture using a set of QIAGEN mini prep kit allocate plasmid DNA and examine its size and orientation of the built-inpckA-the gene. After that plasmid DNA is subjected to double processing with > PST and Cloned and electrophoresis in a 0.7% agarose gel. From the gel, cut out a strip 2.7 TPN and from this band by cleaning allocate fragment of 2.7 TPN(ΔpckA::loxpcat).

The resulting fragmentpckA-gene ΔpckA::loxpcat containing resistant to chloramphenicol gene having at their ends loxP sites inserted by electroporation in producing L-threonine strain ofE. coliTRN212 (inventory number: KCCM-10353). Then the transformed strain TRN212 put a smear on a solid medium containing chloramphenicol to select only resistant to chloramphenicol cells, resulting in the selection of cells in which chromosomepckA-gene replaced by a fragment of alienpckA-gene (ΔpckA::loxpcat). Using southern blot analysis in accordance with the same method as in the following experimental example 1, predelay, whether in the selected clones specifically destroyed chromosomepckA-the gene.

The selected clones identified using southern blot analysis for the presence of specific destroyed in chromosomepckA-gene, transforming pJW168-plasmid (a gift from Prof. Guillermo Gosset from the University in Mexico city), which contains thecre-the gene encoding the site-specific recombinases that recognizes loxP sites. These transformed cells were cultured overnight in culture medium containing 10 mm L-arabinose removal resistant to chloramphenicol gene embedded in the desired bacterial chromosome. Then the culture fluid 10⁷-fold diluted and applied brushstroke on solid LB medium with the addition of ampicillin (100 mg/l) followed by incubation overnight at 30°C. Each of the 100 colonies grown on solid medium, inoculant in two 3 ml portions of liquid LB medium containing or not containing ampicillin, followed by incubation overnight at 30°C. Determine the colony, who died in a medium containing chloramphenicol, but survived in an environment that does not contain chloramphenicol. In making this selection select only those clones that have a deletion in resistant to chloramphenicol gene.

EXPERIMENTAL EXAMPLE 1: Assessment of fracture in chromosomepckA-gene using southern blotting

Strain TRN212 as the parent strain and one of the chloramphenicol-resistant clones selected in example 2-2), cultured overnight in 3 ml of liquid medium containing chloramphenicol (15 mg/l). Then using a set of QIAGEN genomic kit 20 of the cell culture distinguish genomic DNA and process it during the night with EcoRV. The resulting DNA fragments are separated by size in a 0.7% agarose gel. After electrophoresis, the separated DNA fragments are transferred to a nylon membrane (Biodyne B membrane, Young Sci.) overnight by capillary transfer (Molecular Cloning, Vol. 1, pp.6.31-6.38). The membrane is dried and then exhibit in UV light (120 mJ/cm²SpectroLinker™) for immobilization of DNA fragments in the membrane (Molecular Cloning, Vol. 1, pp.6.45). The resulting membrane is incubated in prehybridization solution I (Roche #1093657) at 55°C for 2 h and hybridized denatured DNA probe in thermostat for hybridization (BAMBINO 230300) at 55°C.

The DNA probe was prepared as follows. First, using a set QIAGEN, produce plasmid ploxpcat2 and handle it with HincII to obtain a DNA fragment (about 1.2 TPN)containing resistant to chloramphenicol gene with loxP-site at each of both ends. Dedicated 1,2 TPN fragment boil in water for 5 min and quickly cooled on ice, thus obtaining single-stranded the NC. The obtained single-stranded DNA have been labelled with DIG-UDP, using the set of DIG Labeling and Detection Kit (Roche #1093657)by incubation overnight at 37°C.

After hybridization, the membrane is washed with a wash of solutions I and II (Roche #1093657) to remove non-specific bound peroxidase DNA molecules. This washed the membrane incubated in prehybridization solution II (Roche #1093657) at room temperature for 30 min and then at room temperature for 30 min, subjected to interaction with anti-DIG-antibody specifically binding with DIG-UTP. The membrane is washed with a wash solution III (Roche #1093657) to remove non-specific bound peroxidase anti-DIG antibodies and using a set of Labeling and Detection Kit (Roche #1093657)are at room temperature before the appearance of visible bands. The results are presented in figure 3.

As shown in figure 3, in the case of the parent strain TRN212 band is not detected (lane 2), since the strain TRN212 does not contain resistant to chloramphenicol gene. On the contrary, chloramphenicolbuy clone, selected in accordance with the present invention, detects a band of approximately 3.6 TPN (track 1). The results obtained indicate that the corresponding selective clones contain in their chromosome resistant to chloramphenicol gene.

EXAMPLE 3: Comparison of selective clones producing L-threonine, the ri their cultivation in Erlenmeyer flasks.

Among the finally selected recombinant clonesE. coliexample 2-2), which was removed built-resistant to chloramphenicol gene, thirty clones were productive in relation to L-threonine. Each of them cultivated in Erlenmeyer flask containing culture medium, prepared in accordance with the composition shown below in table 1. Then, for each culture fluid determine the yield of L-threonine. Briefly, after growing on solid LB-medium at 32°C each of the thirty clones of a single colony of each clone inoculant one loop in 25 ml of the indicated culture medium and cultivated at 32°C and 250 rpm for 48 hours After each centrifugation of the culture liquid obtained supernatant was diluted with distilled water in 250 times. The concentration of L-threonine in this diluted supernatant was measured by HPLC. The results are presented below in table 2.

In the parent strain TRN212 yield of L-threonine is 23 g/l As shown in table 2, of the thirty-tested clones twenty-eight find the best productivity of L-threonine than the parent strain TRN212. In particular, nine clones demonstrate the yield of L-threonine in excess of 26 g/l, about 13,04% higher than the output in the parent strain TRN212. Thirty clones were selected one clone with the highest yield of L-threonine (more than 26 g/l) and marked as "FTR2717 (inventory number: KCCM-10475)".

INDUSTRIAL APPLICABILITY

As indicated above, the present invention created a microorganism with inaktivirovannyepckA-the genome, which is obtained by embedding resistant to the antibiotic gene in the chromosome is th DNA of the parent strain of E.coli, producing high levels of L-threonine, namely strainE. colicontaining participates in the degradation of L-threoninetdcBC-operon, which is inactivated when the procedure recombination of DNA.

Since the native chromosomaltdcBC-operon is inactivated, this microorganism in accordance with the present invention has the effect of preventing degradation and intracellular intake of L-threonine. In addition, due to inactivation ofpckA-a gene that is involved in the inhibition of the synthesis of L-threonine, the microorganism according to the invention has more effective ways of biosynthesis of L-threonine. For this reason, the microorganism according to the invention is suitable for mass production of L-threonine, as it may in large quantities and with high yield to produce L-threonine even in the presence of high concentrations of glucose.

1. Recombinant plasmid pT7ΔpckA::loxpcat for synthesis of L-threonine in Escherichia coli cells containing a fragment of the pckA gene, which includes resistant to chloramphenicol gene and loxP sites, which is obtained by cloning incomplete pckA gene in 7Blue-vector with education 7Blue/pckA-plasmids, processing it NruI and ligation with loxpcat-DNA fragment, obtained from plat2-plasmid containing resistant to chloramphenicol gene and loxpcat2 sites.

2. Strain Eschrichia coli FTR2717 (xsm-10475) - producer of L-threonine, transformed with recombinant plasmid according to claim 1.