RU2418069C2 - Method of constructing recombinant bacteria belonging to genus pantoea and method of l-amino acids production with application of bacteria belonging to genus pantoea - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention describes method of constructing recombinant bacteria, belonging to genus Pantoea, method of constructing recombinant bacterium belonging to genus Pantoea - L-amino acid producent, bacterium belonging to genus Pantoea, obtained by said method, as well as method of obtaining L-amino acid with application of said bacterium.

EFFECT: invention makes it possible to increase production of L-amino acids.

12 cl, 7 dwg, 5 tbl, 6 ex

Description

Technical field

The present invention relates to a method for constructing a recombinant bacterium belonging to the genus Pantoea, and to a method for producing L-amino acids using this bacterium.

Description of the Related Art

Typically, increasing the activity of gene products involved in the biosynthesis of L-amino acids or nucleic acids is a common method used to increase the production of L-amino acids or nucleic acids. This increase was often achieved by creating mutant strains that are resistant to target substances or their analogues, by enhancing the expression of biosynthesis genes, by eliminating the sensitivity of biosynthesis enzymes to inhibition of feedback by end products or intermediate biosynthesis products, and by creating bacterial strains that are deficient in genes using precursors of the target substance for other pathways, or by creating bacterial strains deficient in genes responsible for degradation left substance.

These manipulations usually result in strains that are unable to grow, grow only at a significantly reduced rate, or require additional nutrients, such as amino acids, for their growth. For example, increased expression of certain genes can become excessive and can lead to significant inhibition of bacterial growth and, as a result, to a decrease in the ability of bacteria to produce the target substance.

One approach to avoiding the above difficulties is to optimize the expression of genes that encode proteins involved in the distribution of carbon, nitrogen or phosphorus streams or in the removal of the target product from a bacterial cell.

A library of synthetic promoters of various strengths has been described for Lactococcus lactis (Jensen PR, and Hammer K., Appl. Environ. Microbiol., 1998, 64, No.1. 82-87 Biotechnol. Bioeng., 1998, 58, 2-3 , 191-5). The library consists of 38 chemically synthesized promoter DNA fragments that have randomized spacers between consensus sequences at positions from the 35th to the 15th nucleotides. To evaluate the strength of the obtained promoters, oligonucleotides from the library were cloned into a pAK80 expression vector that contains β-galactosidase. It was found that most artificial promoters are very weak (less than 500) and only three of them had a strength of about 2000 relative units. However, the practical application for this library of synthetic promoters has not been disclosed.

A method for producing coryneform bacteria having improved amino acid or nucleic acid productivity by introducing a mutation into the promoter sequence of the amino acid or nucleic acid biosynthesis genes is disclosed in European Patent Application EP 1033407 A1. For each of the following genes, up to 8 different mutant promoter variants were used: glutamate dehydrogenase gene (gdh), citrate synthase gene (gltA), isocitrate dehydrogenase gene (icd), pyruvate dehydrogenase gene (pdhA) and arginine succinate synthase (argG) gene. The failure of this technique, described in European patent application EP 1033407 A1, lies in the fact that each of the described mutant strains was prepared separately, that is, one after the other. Also, an increase in the productivity of the L-amino acid in all cases disclosed in this European patent application was achieved by increasing the activity of certain enzymes using a limited number of different promoters for the genes encoding these enzymes. This approach is common in the art of preparing bacteria producing amino acids and bacteria producing nucleic acids and is not intended to optimize or fine-tune the promoter activity of genes necessary for the production of L-amino acids or nucleic acids.

A method of creating a library of artificial promoters was disclosed in PCT application WO 03089605. This method includes: a) obtaining a DNA cassette for insertion, which includes a first recombinase site, a second recombinase site and a selective marker gene located between these sites; c) obtaining a first oligonucleotide that includes: i) a first nucleic acid fragment that is homologous to the region preceding the direction of transcription of the gene of interest on the chromosome (upstream region), and ii) a second nucleic acid fragment that is homologous to the 5'-end of the inserted DNA - cassettes; c) obtaining a second oligonucleotide, which includes: i) a third nucleic acid fragment homologous to the 3 ′ end of said insertion DNA cassette, ii) a promoter precursor that comprises a consensus region of 35 nucleotides (from -35 to -30 nucleotides), a linker sequence, and a consensus region of 10 nucleotides (from 12 to 7 nucleotides), and the linker sequence includes from 14 to 20 nucleotides and is flanked by a region of 35 and a region of 10 nucleotides, wherein said promoter precursor the ora is modified in such a way that it includes at least one altered nucleotide position of the promoter precursor, with a region of 35 and 10 nucleotides each comprising 4 to 6 conserved nucleotides of the promoter, and iii) a fourth nucleic acid fragment which is homologous to the region further downstream of the transcription initiation point of the promoter (downstream region); and d) mixing the first and second oligonucleotides in the amplification reaction with an inserted DNA cassette to obtain a library of double-stranded amplified products containing artificial promoters.

A temperature-sensitive plasmid or Mu integration method (Microbiol Res. 2006 Mar 29, J Bacteriol. 1981 Jan; 145 (1): 358-68) is already known as a method for embedding DNA fragments into the chromosome of bacteria of the Enterobacteriaceae family.

However, there are currently no reports describing the use of Red-dependent integration for embedding target DNA fragments in bacteria belonging to the genus Pantoea. There are also currently no reports describing the optimization of gene expression for the production of a useful metabolite, for example, for the production of an L-amino acid, by fermentation of a bacterium belonging to the genus Pantoea, which is modified in such a way that expression of the target gene is optimized in it.

Description of the invention

An object of the present invention is to provide a method for constructing a recombinant bacterium, comprising:

a) obtaining an exogenous DNA fragment that contains at least 2 sites, at least 30 bp in length each, identical to sites located in the target gene on the chromosome of a bacterium belonging to the genus Pantoea,

b) introducing said linear DNA fragment into a bacterium belonging to the genus Pantoea, said bacterium being modified in such a way that it is resistant to the products of the gam, bet and exo genes from lambda phage and

c) selecting a recombinant bacterium in which said target gene is replaced with said linear DNA fragment or a linear DNA fragment is inserted into the target gene.

It is also an object of the present invention to provide a method as described above, wherein said bacterium is a Pantoea ananatis SC17 (0) bacterium.

It is also an object of the present invention to provide a method for constructing a library of DNA fragments in a bacterial chromosome, comprising:

a) introducing a set of DNA fragments that are partially identical to the target gene on the chromosome of a bacterium belonging to the genus Pantoea, in the specified bacterium, and the specified bacterium is modified so that it is resistant to the products of the gam, bet and exo genes of the phage lambda and

b) selecting a bacterium in which said target gene is replaced with said DNA fragments.

It is also an object of the present invention to provide the method described above, said DNA fragments containing a regulatory element for gene expression selected from the group consisting of their promoter, terminator, ribosome binding site (RBS), operator, and combinations thereof.

It is also an object of the present invention to provide the method described above, wherein said regulatory element comprises a randomized sequence.

Another objective of the present invention is the provision of a method for constructing a bacterium belonging to the genus Pantoea, which has an optimized expression level of a gene encoding a protein involved in the distribution of carbon, nitrogen or phosphorus streams or in the removal of the target substance from the specified bacterium, including:

1) the replacement of natural regulatory elements with a library of DNA fragments obtained by the method described above on the chromosome of the bacteria and

2) selection of bacteria with the specified optimized expression level of the specified gene.

It is also an object of the present invention to provide a method as described above, wherein said bacterium is an L-amino acid producing bacterium.

It is also an object of the present invention to provide the method described above, wherein said L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L- glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

It is also an object of the present invention to provide a method as described above, wherein said bacterium is a nucleic acid producing bacterium.

It is also an object of the present invention to provide the method described above, wherein said nucleic acid is selected from the group consisting of inosine, guanosine, xanthosine and adenosine.

It is also an object of the present invention to provide a bacterium belonging to the genus Pantoea and obtained by the method described above.

It is also an object of the present invention to provide the bacterium described above, wherein an optimized level of gene expression is achieved by modifying the promoter region of the gene, consisting of 35 base pairs.

It is also an object of the present invention to provide a bacterium as described above, said gene being the yhfK gene.

It is also an object of the present invention to provide a bacterium as described above, wherein a region of 35 base pairs is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO : 28 and combinations thereof.

Another objective of the present invention is the provision of a method for producing L-amino acids, including:

a) growing the above bacteria in a culture medium, and

b) isolation of the L-amino acid from the culture fluid. It is also an object of the present invention to provide the method described above, wherein said L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L- glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

Detailed Description of the Best Mode for Carrying Out the Invention

1. The method according to the present invention

The present invention provides a method for constructing a recombinant bacterium belonging to the genus Pantoea. The construction of such a recombinant bacterium is usually achieved by using a bacterium that is modified in such a way that it is resistant to the products of the gam, bet and exo genes from phage lambda. It was in the process of this construction that it was found that the products of the gam, bet, and exo phage lambda genes of the Red-dependent integration system are toxic to bacteria of the genus Pantoea. This problem was solved by isolating a mutant strain that is resistant to the products of the gam, bet and exo phage lambda genes. This obtained mutant strain was used as a recipient strain for integration of DNA fragments using the Red integration system (see Example 1 below).

The present invention also provides a simple and direct one-step method for producing a bacterium-producer of L-amino acid or bacteria-producer of nucleic acid, which has an optimized expression level of a gene involved in the metabolism and biosynthesis of the target substance or in the removal of the target substance from the specified bacterium, and, accordingly, affects the production of L-amino acids and nucleic acids.

The present invention also provides an L-amino acid producing bacterium with an optimized level of gene expression that is involved in the metabolism and biosynthesis of the target substance or in the process of removing the target substance from the specified bacterium, and also provides a method for inactivating the target gene on a chromosome that is not included in the open framework reading (ORF), genes or alleles that are necessary for bacterial growth and L-amino acid production, nucleic acid production.

The present invention also provides a method for producing L-amino acids such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine; and a method for producing a nucleic acid.

The present invention has been made possible through the use of a strain that has been modified to be resistant to the product of the gam, bet and exo genes from lambda phage (hereinafter referred to as “λ-Red genes”) as a strain recipient of DNA fragments.

The strain, which is modified in such a way that is resistant to the product of λ-Red genes (hereinafter referred to as λ-Red-resistant strain), can be obtained in the following way. To obtain the λ-Red-resistant strain, cells of the strain P. ananatis SC17 (US Pat. No. 6,596,517) were electroporated with a plasmid carrying the λ-Red genes. After culturing for 18 hours, approximately 10 ⁵ -10 ⁶ transformants were obtained, among which about 10 clones were large, while the other clones were very small. After 18 hours of cultivation, large clones were approximately 2 mm in diameter, small colonies were approximately 0.2 mm in diameter. During prolonged cultivation (up to 24 hours), small colonies no longer grew, while large colonies continued to grow. Strains of P. ananatis with such a large colony size are resistant to the expression of all three Red genes from lambda phage (gam, bet and exo). A preferred λ-Red-resistant strain for the present invention is Pantoea ananatis strain SC17 (0) (VKPM B-9246).

The λ-Red-resistant strain can be used in many different methods for genetic recombination of a target gene, for example, for inactivation of a gene, for gene integration.

To obtain a recombinant strain, the following steps can be performed:

a) preparation of an exogenous linear DNA fragment containing at least 2 sites at least 30 bp long. each identical to sites located in the target gene on the chromosome of a bacterium belonging to the genus Pantoea,

b) introducing said linear DNA fragment into a bacterium belonging to the genus Pantoea, said bacterium being modified to be resistant to the products of the gam, bet and exo genes from the lambda phage, and

c) selecting a recombinant bacterium in which said target gene is replaced with said linear DNA fragment or a linear DNA fragment is inserted into the target gene.

The term "target gene" means a locus on the chromosome of the specified λ-Red-resistant strain of bacteria of the genus Pantoea, which must be modified. The source of the foreign DNA fragment may be an organism other than a λ-Red-resistant strain, or it may be a λ-Red-resistant strain.

The term "recombinant bacterium" means a bacterium of the genus Pantoea, in the gene of which an integrative cassette is inserted or part of the gene of which is replaced by an integrative cassette.

The term "recombinant bacterium" means a bacterium of the genus Pantoea, in which the specified linear DNA fragment is inserted into the target gene on the chromosome or replaces some part of the target gene.

A DNA fragment according to the present invention means a DNA fragment having a free 5'-end and 3'-end and means that it is not circular DNA. A linear DNA fragment, which is partially identical to the target region, can be obtained by cloning a linear DNA fragment on the chromosome of a bacterium of the Pantoea genus or another bacterium of the Enterobacteriaceae family. A linear DNA fragment can, for example, be cloned once into a plasmid from chromosome DNA to produce a recombinant plasmid and then extracted from the recombinant plasmid using restriction enzymes, or it can be obtained by direct amplification of the target gene from a genomic DNA molecule by PCR.

The embedding gene may be a fused gene consisting of two or more genes or a complex of genes containing two or more genes.

In order to select the required recombinant bacterium, genetic markers for positive (e.g., antibiotic resistance genes) or for negative (e.g. sacB gene) selection can be included in the linear DNA fragment. According to the red-managed integration method, the integrated antibiotic resistance gene can be removed by introducing attL and attR sites, which are the attachment sites of the lambda phage and the PCR product, and by combining the excision system derived from the lambda phage with the red-controlled integration method. To remove the selective marker, in particular, the int-xis system can be used (PCT application WO 05/010175).

To embed a linear DNA fragment containing the required gene, a gene can be used that is involved in the metabolism and biosynthesis of the target substance or in the process of removing the target substance from bacteria of the Pantoea genus. Examples of such genes include, but are not limited to, nitrogen glycolysis or assimilation genes, pentose cycle genes, Krebs cycle genes, and so on. More specifically, examples include, but are not limited to glutamate, glutamate, glutamate synthetase, isocitrate, aconitate, citrate, fosofenolpiruvatkarboksilazoy, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructose, phosphofructokinase, glyukozofosfatizomerazoy, glutamine-oxoglutarate aminotransferase, isopropyl malate synth Zoy and so on. Bacterial genes included in the biosynthesis of L-amino acids, nucleic acids and their precursors can also be used. The phrase "a gene encoding a protein involved in the removal of a target substance from a bacterium" means a gene encoding a membrane protein that directly excretes a target substance from a bacterial cell, or a gene encoding a protein that activates a membrane protein that excretes a target substance.

This basic approach was used to fine tune the expression of the yfhK gene in the Pantoea ananatis strain to increase the level of L-glutamic acid production. The achieved optimization, rather than maximizing the expression of a gene or operon, is an urgent problem in the construction of various bacterial strains capable of producing biologically active metabolites, especially in cases where low levels of expression of the target gene are insufficient to achieve the final goal. On the other hand, overexpression of the target gene can lead not only to a neutral, but also to a negative effect. In addition, the desired level of this key gene remains unknown. Fine tuning is highly desirable to ensure optimization, and therefore a huge number of options should be tested. The usual approach for carrying out this kind of fine tuning involves molecular cloning of the target gene in recombinant plasmids and placing them under the control of various promoters, followed by evaluation of the resulting recombinant strains. After selecting the best options, you need to do a lot of extra work if an improved producer strain is desired.

The method according to the present invention involves constructing a library of DNA fragments in the chromosome of a bacterium belonging to the genus Pantoea by inserting into the chromosome of the indicated bacterium a set of DNA fragments synthesized in vitro, the mutant bacterium belonging to the genus Pantoea and is resistant to the products of the gam, bet and exo genes phage lambda, and is used as a recipient bacterium. The preferred mutant bacterium used in this method is the bacterium Pantoea ananatis SC17 (0). More specifically, the method of the present invention includes constructing a library of DNA fragments that contains one or more regulatory elements for gene expression, including promoters, ribosome binding sites (RBS), and operators. More specifically, the method according to the present invention includes constructing a library of DNA fragments that contain one or more regulatory elements that include a randomized sequence.

The term "bacterium belonging to the genus Pantoea" means that the bacterium belongs to the genus Pantoea in accordance with the classification known to the specialist in the field of microbiology. Some strains of Enterobacter agglomerans have recently been reclassified to Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like based on analysis of the 16S rRNA nucleotide sequence, etc.

The phrase “set of DNA fragments” means a mixture of newly synthesized DNA fragments or a mixture of known DNA fragments obtained from natural or mutated microorganisms, a DNA library, GenBank, etc. A set of DNA fragments can be formed by directly mixing newly synthesized DNA fragments with known sequences, DNA fragments obtained from various sources indicated above, or DNA fragments obtained by chemical synthesis, DNA fragments that have a region containing a randomized sequence.

The phrase “the bacterium is modified in such a way that it is resistant to the gene product” means that the bacterium is modified in such a way that it is able to grow and form colonies in the environment, under the conditions when the gene is expressed in the bacterium.

The phrase "regulatory elements" refers to nucleotide sequences located before, inside and / or after the coding region that control the transcription and / or expression of the coding region together with the protein biosynthesis apparatus. This phrase is commonly used to describe promoters, ribosome binding sites (RBS), operators or other elements of the genome and those that affect the level of gene expression.

The phrase “randomized sequence” means that during the normal chemical synthesis of a DNA fragment, random nucleotides (usually denoted as N, where N is adenine, guanine, cytosine or thymine) are inserted at certain positions of the DNA fragment or in some area of the DNA fragment having random nucleotide sequences, respectively. DNA fragments contain sequences called "regulatory elements".

The method according to the present invention also includes obtaining an L-amino acid producing bacterium with an optimized expression level of the target gene, which encodes a protein involved in the metabolism and biosynthesis of the target substance, or in the removal of the target substance from the specified bacterium and, as a result, affects the production of L- amino acids. The method according to the present invention is divided into the following steps:

1) introducing a library of artificially created DNA fragments that contain regulatory elements for the expression of the specified target gene, in the chromosome of the specified bacteria instead of the natural regulatory elements of the specified target gene, and

2) selection of bacteria with the desired phenotype.

The term “expression”, as used herein, means the production of a protein product encoded by a gene.

The phrase "optimized expression level" means the expression of the target gene or several genes, the result of which is a bacterium with the desired phenotype.

The phrase “desired phenotype” includes one or more characteristics of a bacterium that are objects for improvement. Preferably, this may be the ability of the bacterium to produce L-amino acids in greater quantities than the parent strain; it may also be the ability to grow on a minimal medium that does not contain the additives that are commonly used to complement auxotrophy or other factors necessary for growth or a combination thereof.

The phrase “gene encoding a protein involved in the metabolism or biosynthesis of the target substance” means a gene encoding a protein that is involved in the metabolic pathways of carbon, nitrogen or phosphorus. Examples of such genes include, but are not limited to, nitrogen glycolysis or assimilation genes, pentose cycle genes, Krebs cycle genes, etc. More specifically, examples include, but are not limited to glutamate, glutamine synthetase, glutamate synthase, isocitrate dehydrogenase, aconitate, sitratsintazoy, phosphoenolpyruvate carboxylase, pyruvate dehydrogenase, pyruvate kinase, phosphoenolpyruvate synthase, enolase, phosphoglyceromutase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, fructose, phosphofructokinase, glyukozofosfatizomerazoy, glutamine-oxoglutarate aminotransferase, isopropyl malate synth zoy etc. Bacterial genes involved in the biosynthesis of L-amino acids, nucleic acids and their precursors may also be included.

The phrase "a gene encoding a protein involved in the removal of a target substance from a bacterium" means a gene encoding a membrane protein that directly removes a target substance from a bacterial cell, or a gene encoding a protein that activates a membrane protein that excretes a target substance.

A mixture of DNA fragments containing regulatory elements is inserted into the bacterial chromosome instead of the natural regulatory element, resulting in a population of bacterial cells with different levels of expression of the genes of interest. The selection of bacteria with the desired phenotype can be carried out by directly assessing the amount of accumulated L-amino acids in a standard minimal medium or by other methods suitable for determining the characteristics necessary for the desired phenotype.

The term “L-amino acid producing bacterium,” as used herein, means a bacterium that is capable of producing the target L-amino acid and causes the accumulation of L-amino acid in the fermentation medium in large quantities, compared to the natural or parent strain , and preferably means that the microorganism is capable of accumulating in the medium the target L-amino acid in an amount of not less than 0.5 g / l, more preferably not less than 1.0 g / l.

The determination of the level of enzymatic activity and the selection of regulatory elements of sequences that are optimal for certain conditions require a specialist in-depth knowledge in this technical field; selection of regulatory elements of sequences based on scientific assumption or intuition may not lead to the desired or optimal result. In addition, the preparation of even a limited number of mutants with different levels of enzymatic activity is time-consuming and time-consuming, since such mutants are usually prepared one after another. However, the method according to the present invention advantageously offers a simple and direct one-step procedure for integrating a natural or artificial DNA fragment into a chromosome, generating a population of bacterial cells with a wide range of expression levels of the target gene. For example, the randomization of four nucleotides in a certain region of a chemically synthesized regulatory element provides 4 ⁴ , or 256 theoretically possible variants of this region.

In addition, it is common to increase the activity of the target enzyme by inserting a copy of the gene encoding the enzyme into the bacterial plasmid.

However, the expression level of a gene integrated into the bacterial chromosome and the expression level of the same gene integrated into the plasmid populating the bacterium may not be the same. Therefore, another advantage of the method according to the present invention is that the method allows to evaluate and select the bacterium under conditions optimal for expression of the gene of interest, followed by direct use of the bacterium for the production of L-amino acids without additional manipulations.

Methods for producing plasmid DNA, cutting and ligation of DNA, transformation, selection of oligonucleotides as primers and the like can be conventional methods well known to those skilled in the art. These methods are described, for example, in Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

The bacterium according to the present invention can be a producing bacterium of L-amino acid and nucleic acid with an optimal expression level of a gene that encodes a protein involved either in the distribution of carbon, nitrogen and phosphorus, or in the removal of the target substance from the specified bacterium, while the optimization of expression leads to increased production of L-amino acids. Such a bacterium can be obtained by the method according to the present invention.

More specifically, the bacterium of the present invention produces an L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glycine, L-histidine, L- isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. Also, the bacterium of the present invention includes an L-glutamic acid producing bacterium having an optimized gene expression level that affects the production of L-glutamic acid. In addition, L-glutamic acid plays a significant role in the biosynthesis of L-leucine, L-lysine, L-isoleucine, L-valine, L-histidine, L-aspartate, L-alanine, L-tyrosine and L-phenylalanine as a donor amino groups. And the bacterium of the present invention is an L-leucine producing bacterium with an optimized gene expression level that affects the production of L-leucine.

Therefore, an improved nitrogen supply for the gene encoding aminotransferase is useful for the production of other amino acids such as L-leucine, L-lysine, L-isoleucine, L-valine, L-histidine, L-aspartate, L-alanine, L-tyrosine and L-phenylalanine.

Examples of bacteria producing L-glutamic acid include mutant strains belonging to the genus Pantoea that are deficient in the activity of α-ketoglutarate dehydrogenase or have a reduced activity of α-ketoglutarate dehydrogenase, and can be obtained as described above. Such strains include Pantoea ananatis strain AJ13356 (US Pat. No. 6,331,419). The strain Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry) at the National Institute of Biological Sciences and Human Technology (currently called the National Institute of Advanced Industrial Science and Technology, International Depository of Organizations for Patenting Goals, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan - National Institute of Advanced Industrial science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, February 19, 1998 and received accession number FERM P-16645). Then, the strain was internationally deposited in accordance with the conditions of the Budapest Treaty of January 11, 1999, and the strain received accession number FERM BP-6615. The strain Pantoea ananatis AJ13356 does not have α-KGDH activity as a result of inactivation of the αKGDH-E1 gene subunit (sucA). The above strain, when isolated, was identified as Enterobacter agglomerans and deposited as Enterobacter agglomerans AJ13356 strain. However, it was later classified as Pantoea ananatis on the basis of the 16S pPHK nucleotide sequence and other evidence. Although strain AJ13356 was deposited with Enterobacter agglomerans at the above depository, for the purposes of this description it will be referred to as Pantoea ananatis.

The strategy described above for producing L-glutamic acid can be used to produce bacteria that produce other L-amino acids.

The bacterium of the present invention is a nucleic acid producing bacterium having an optimized expression level of a gene encoding a protein involved in the distribution of carbon, nitrogen or phosphorus streams, or in removing the target substance from said bacterium, which leads to an increase in nucleic acid production. The nucleic acid is selected from the group consisting of inosine, xanthosine, guanosine and adenosine. Such a bacterium can be obtained by the method according to the present invention.

2. The method of obtaining L-amino acids

A method for producing an L-amino acid is a method comprising the steps of growing a bacterium according to the present invention in a culture medium for the purpose of producing and accumulating an L-amino acid in a culture fluid and isolating the L-amino acid from the culture fluid. More specifically, a method for producing L-amino acid includes a method for producing L-glutamic acid, which includes the steps of growing a bacterium according to the present invention in a nutrient medium for the production and accumulation of L-glutamic acid in the culture fluid and the isolation and purification of L-glutamic acid from the culture fluid .

According to the present invention, the cultivation, isolation and purification of an L-amino acid from a culture or similar liquid may be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a bacterium.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation of the microorganism used, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. As vitamins, thiamine, yeast extract and the like can be used.

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

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the L-amino acid can be isolated and purified by ion exchange chromatography, concentration and / or crystallization.

Brief Description of the Drawings

Figure 1 shows the genetic maps of auxiliary ("helper") plasmids that induce the expression of λ-Red genes.

Figure 2 shows the construction of an in vitro promoter library.

Figure 3 shows the integration of PCR of DNA fragments into the chromosome of P. ananatis.

Figure 4 shows 2 variants of the modification of the natural yhfK gene. A) A fragment of the chromosome of a natural strain of P. ananatis carrying the yhfK gene. B) The nucleotide sequence of a fragment of a chromosome containing the upstream region of the yhfK gene. C) The first modification option: embedding a cassette containing the terminator T _thr , cut-out marker Km ^R and the promoter P _tac immediately after the stop codon of the crp gene. D) Second modification option: replacing the upstream region of the yhfK gene with an integrative cassette containing the terminator T _thr , a marker Km ^R cut out, a randomized promoter P _tacN from the obtained library of promoters, and an AGGAGG consensus SD sequence.

Figure 5 shows the structure of the chromosome in front of the dadX (A) and gntK (B) genes containing replaced regions with promoters.

Figure 6 shows the structure of the plasmid RSFPlacsacB.

Figure 7 shows the design scheme of plasmids RSF-Red-TER and RSF-Red-Kan.

Examples

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

Example 1. The selection of the recipient strain for highly efficient λ-Red-mediated recombinant construction of the strain P. ananatis.

λ-Red-dependent recombination was used to integrate DNA fragments obtained by PCR with 40 bp flanking regions that are homologous to regions in the P. ananatis genome. For this purpose, two new helper plasmids, RSF-Red-TER, RSF-Red-Kan, which express the λ gam, bet and exo genes (hereinafter referred to as “λ-Red genes") were constructed (Figure 1). A detailed construction of these plasmids is described below in the Reference Example. Both plasmids can be used for a wide range of host bacteria having different genotypes because: 1) they have a replicon of plasmid RSF1010 for a wide range of hosts that can be stably retained in many gram-negative and gram-positive bacteria and even in plants (Scholz, et al., 1989; Buchanan-Wollaston et al ,, 1987); 2) The λ-gam, bet, and exo λ-genes are controlled by the promoter P _lacUV5 , which is recognized by many bacterial RNA polymerases (see, for example, Brunschwig, E. and Darzins, A., Gene, 111, 1, 35- 41 (1992); Dehio, M. et al., Gene, 215, 2, 223-229 (1998)); 3) the automatically regulated element P _lacUV5- lacI and ρ-independent terminator of transcription of the rrnB operon from E. coli (T _rrnB ) provides a low level of basal expression of λ-Red genes (Skorokhodova, A.Yu et al., Biotekhnologiya (Rus), 5, 3-21 (2004)). In addition, the RSF-Red-TER plasmid contains the levansaccharose gene (sacB), which allows the plasmid to be extracted from cells in a medium containing sucrose. In E. coli cells, the frequency of integration of DNA fragments obtained by PCR with a short flanking region provided by the plasmids RSF-Red-TER and RSF-Red-Kan was as high as for the helper plasmid pKD46 (Datsenko, KA, Wanner, BL , Proc. Natl. Acad. Sci. USA, 97, 6640-6645, (2000)). However, the expression of λ-Red genes was toxic to P. ananatis cells. Cells transformed with helper plasmids RSF-Red-TER or RSF-Red-Kan had a very low growth rate on LB medium containing IPTG (1 mM) and the corresponding antibiotic (chloramphenicol, 25 mg / ml, or kanamycin, 40 mg / ml), and the extremely low frequency of λ-Red-dependent recombination (10 ^-8 ), in the case when it generally occurred.

Mutant P. ananatis strains resistant to the expression of all three Red genes from lambda phage (gam, bet and exo) were selected. For this, cells of strain P. ananatis SC17 (US Pat. No. 6,596,517) were electroporated with the plasmid RSP-Red-TER. Approximately 10 ⁶ transformants were obtained after culturing for 18 hours and up to 10 clones were large, while all other clones were very small. Large colonies were approximately 2 mm in diameter, small colonies were approximately 0.2 mm after 18 hours of cultivation. During continued cultivation (up to 24 hours), small colonies no longer grew, while large colonies continued to grow. One of the large-sized mutant colonies of the P. ananatis strain, which was resistant to the expression of all three Red genes from lambda phage (gam, bet and exo), was selected for further analysis.

RSF-Red-TER plasmid DNA from this large colony and from several small colonies was isolated and retransformed into E. coli MG1655 to test the plasmid's ability to synthesize active products of Red system gene genes. A control experiment of Red-dependent integration in the obtained transformants showed that only the plasmid purified from a large colony provides the expression of λ-Red genes necessary for Red-dependent integration.

To check whether there is a Red-dependent integration in the selected large clone, electroporation of a linear DNA fragment was carried out by PCR, containing a Km ^R marker and a 40 bp flanking region, which is homologous to hisD gene, and synthesized in this way to be integrated into the SmaI recognition site of hisD gene from P. ananatis; two small clones were used as a control. The nucleotide sequence of hisD gene from P. ananatis is shown in SEQ ID NO: 42. The oligonucleotides shown in SEQ ID NO: 43 and 44 were used as primers, and plasmid pMW118- (λattL-Km ^r -λattR) was used as a template in PCR; two small clones that were not resistant to λ-Red genes were used as controls. The construction of the plasmid pMW118- (λattL-Km ^r -λattR) is described below in Example link 2. Plasmid RSF-Red-TER is capable of inducibly expressing Red genes, due to the presence of the lacI gene on the plasmid. Two induction conditions were checked: in the first group, IPTG (1 mM) was added one hour before electroporation, in the second group, IPTG was added at the time of cultivation to prepare an electrocompetent culture. The growth rate of cells bearing plasmid RSF-Red-TER, originating from a large colony, was not significantly lower than the growth rate of the strain without plasmid SC17. The addition of IPTG caused only a slight inhibition of the growth rate of these crops. At the same time, the offspring of small colonies grew very slowly without IPTG and induction practically stopped their growth. After electroporation of cultures of progeny cells of a large colony, many Km ^R clones (18 clones with a short induction time and about 100 clones with a prolonged induction time) grew. All 100 colonies tested had His ^- phenotype; PCR verification of 20 clones confirmed the expected chromosome structure of these cells. On the other hand, after electroporation of progeny cells of small clones, no integrants were obtained.

The large clone thus obtained was grown on plates containing 7% sucrose to eliminate the plasmid and retransformed with the plasmid RSF-Red-TER. The non-plasmid strain was named SC17 (0) and was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (1st Road passage, 1, Moscow, 117545, RF) on September 21, 2005 under accession number VKPM B-9246. All clones that grew after this retransformation were large, as was the parent clone SC17 (0). An experiment was conducted on Red-mediated integration into strain SC17 (0), which was retransformed with the plasmid RSF-Red-TER. Three independently obtained transformants were tested; used the same DNA fragment that was used in the previous experiment. A short induction time was used (1 hour before electroporation). In each triplet, more than 100 Km ^R clones grew. All tested clones had His ^- phenotype. Therefore, it was concluded that a mutant strain called SC17 (0), which is resistant to the expression of λ-Red genes, was selected. This strain can be used as a recipient for Red-dependent integration into the P. ananatis chromosome.

A similar experiment was performed with the helper plasmid RSF-Red-Kan. After electrotransformation of SC17 by this plasmid, all the clones that grew on solid LB medium were the same size as the parent strain SC17, however, a significant growth retardation was observed after induction. In order to reproduce the selection of the λ-Red-resistant strain using the helper plasmid RSF-Red-Kan, the cells after electroporation were placed on solid LB medium supplemented with IPTG in various concentrations. Lack of growth was observed on a plate with 1 mM IPTG; only a few large clones grew on a plate with 50 μM IPTG; and many small and several large clones grew on a plate containing 20 μM IPTG. One of the large colonies that grew on medium with 20 μM IPTG was found to be a recipient for λ-Red-dependent integration. In this experiment, the integration frequency for this clone was the same as for strain SC17 (0). The procedure developed in the last experiment can be successfully applied to select λ-Red-resistant derivatives of other bacterial strains or strains for which the expression of λ-Red genes is toxic.

Example 2. Design and characterization of a library of promoters suitable for optimizing the expression level of a gene of interest.

To develop the design of a group of promoters with different strengths in P. ananatis, the approach previously implemented for E. coli was applied (PCT application WO 2004080386, WO 2005/010175).

Preparation of a promoter library generated by PCR.

A library of promoters suitable for optimizing the expression level of a gene of interest was constructed by randomizing four internal nucleotides from a 35-nucleotide region (“-35”) of the P _tac initiating promoter. The library was integrated before the lacZ gene on the chromosome of P. ananatis strain SC17 (0) and the LacZ activity in each transform was determined. The structure of the chromosome region of P. ananatis in front of the lacZ gene is shown in Figure 3. The nucleotide sequences of the yghU, scrK, lacZ genes from P. ananatis are shown in SEQ ID NO: 45, 46 and 47, respectively.

The “-35” region of the P _tac promoter is shown in SEQ ID NO: 16. To obtain promoters of different strengths, a DNA fragment with a group of P _tac-like promoters other than the “-35” promoter was constructed by PCR using plasmid pDR540 (Pharmacia ", Sweden) as a matrix. For this purpose, either the “I” primer (SEQ ID NO: 1), which fully corresponds to the P _tac sequence, or the “1 '” primer, a “randomized” primer (SEQ ID NO: 2), was used as 5'-primers. Both contained a BglII recognition site at the 5'-end. Four of the six positions in primer “1” from the consensus region “-35” were randomized during chemical synthesis and they contained a combination of any four nucleotides designated as “n” in SEQ ID NO: 2. Primer “2” (SEQ ID NO: 3), which was used as a primer at the 3'-end, contains 46 nucleotides from the 3'-end of P _tac , the SD sequence and the beginning of the coding region of the lacZ gene. DNA fragments obtained by PCR, which contained either the original promoter P _tac , or a group of randomized tac-like promoters, were used later for design.

At the same time, the DNA fragment was amplified with the Km resistance gene cut out, flanked by the attL and attR sites of the lambda phage, using plasmid pMW118- (λattL-Km ^r -λattR) as the template and primers “3” (SEQ ID NO: 4) and “4 "(SEQ ID NO: 5). The resulting DNA fragment had a recognition site at one end for BglII restriction enzyme, which is useful for ligation with fragments of tac-like promoters, and a 42 bp site homologous to the P. ananatis chromosome and located in front of the scrK gene in order to provide subsequent integration into the bacterial genome (see Figure 3), from the other end. Two PCR generated fragments were treated with BglII and ligated in vitro using T4 DNA ligase.

Integration of PCR-generated promoter library into P. ananatis chromosome

The resulting ligase mixtures were used for λ-Red-dependent integration into the chromosome of P. ananatis. The helper plasmid was used as a carrier of λ-Red phage genes. To obtain electrocompetent P. ananatis cells, strain SC17 (0), transformed with the RSF-Red-TER plasmid, was grown overnight at 34 ° C in LB medium with the addition of chloramphenicol to a concentration of 50 μg / ml. The culture was then diluted 100 times with fresh LB medium containing 50 μg / ml chloramphenicol, and grown until it reached an optical density of OD _{600 of} approximately 0.3 at 34 ° C with aeration. After that, IPTG was added to a concentration of 1 mM and cultivation was continued until an optical density of OD ₆₀₀ = 0.7 was reached. Cell samples with a volume of 10 ml were washed 3 times with equal amounts of deionized ice water and resuspended in 40 μl of 10% ice glycerol. Immediately prior to electroporation, 100-200 ng of amplified in vitro DNA fragments were dissolved in 5 μl of deionized water and added to the cell suspension. The procedure was carried out using a device for electrotransformation of bacteria ("BioRad", USA, catalog number 165-2089, version 2-89). The applied ripple parameters were as follows: the electric field intensity was 20 kV / cm, the ripple time was 5 ms. After electroporation, 1 ml of glucose-enriched LB medium (0.5%) was immediately added to the cell suspension. Then the cells were grown with aeration at a temperature of 34 ° C for 2 hours and scattered on plates with solid LB medium containing 40 μg / ml kanamycin, followed by night incubation at a temperature of 34 ° C. Selected Km ^R integrants were scattered on plates with LB medium supplemented with IPTG (1 mM) and sucrose (5 g / l) at 34 ° C and grown to form single colonies. Km ^R , Cm ^S variants were isolated in such a way that the helper plasmid RSF-Red-TER was removed from the integrants.

PCR verification.

In order to confirm the identity of the obtained chromosome structure of the selected KmR, CmS colonies, PCR was performed using primers “5” (SEQ ID NO: 6), which is the homologue of the attR site at the 5'-end and “6” (SEQ ID NO: 7 ), whose annealing site is localized in the structural part of the lacZ gene.

DNA sequencing of the obtained tac-like promoters.

Initially, PCR amplification of genomic DNA fragments isolated from selected clones was performed using a genomic DNA isolation kit (Sigma, USA). For sequencing of the obtained DNA fragments, the Sanger method and primers “5” (SEQ ID NO: 6) and “7” (SEQ ID NO: 8) were used, both for PCR and for sequencing.

Test for β-galactosidase.

All cell cultures were grown overnight on LB medium supplemented with 0.5 × M9 saline, glucose (5 g / l) and 40 μg / ml kanamycin, then diluted 100 times with fresh medium and grown at 34 ° C with aeration for 3 hours. Β-galactosidase activity was measured by the usual method (Miller, 1972). The data presented (Table 1) are the average of three independently grown cultures of each clone. The standard error did not exceed 30%.

As can be seen from Table 1, the activity values in representative clones of the library with P _tas-like promoters differ by a factor of two. You can also see that the selected procedure allows you to construct a library of mutant promoters only with a randomized region of "-35".

Example 3. Increasing the production of glutamic acid by fine-tuning the level of expression of the yhfK gene.

The yhfK gene encodes presumably the Glu transporter. It has been disclosed that its amplification on a plasmid increases the productivity of the producer strain of L-glutamic acid (US patent application 2005/0196846). To fine-tune the level of expression of the yhfK gene, Red-dependent integration was used, as described above, to modify the 5'-terminal region of the gene in its natural location. The structure of the 5'-terminal region of the yhfK gene on the P. ananatis chromosome is shown in Fig. 4A. The nucleotide sequence of the region is shown in SEQ ID NO: 48.

On the P. ananatis chromosome, the yhfK gene is located after 42 bp after the crp gene (see Fig. 4A). No regulatory elements (terminator, promoter, or SD sequence) were found in the 5'-terminal region of the yhfK gene (Fig. 4B). Therefore, it was suggested that the introduction of a strong promoter and, possibly, an SD sequence in front of this gene can enhance its expression level significantly. At the same time, to ensure the stability of the mRNA of the crp gene in strains with various modifications of the region adjacent to the 3'-end of the crp gene without violating its expression, immediately after the stop codon of this gene, the leader peptide of the threonine operon of E. coli T was introduced _thr (see Example Link 2). The structures of the modifications obtained are shown in FIGS. 4C and D.

To construct integrative cassettes, DNA fragments with a cut out Km ^R marker and the natural promoter _Ptac or randomized promoters were amplified by PCR using genomic DNA fragments that were isolated from clones of the promoter library using primers: direct primer "9" (SEQ ID NO: 11 ) and reverse primer “8” (SEQ ID NO: 9) or “8 '” (SEQ ID NO: 10). Reverse primer “8” allows amplification of the natural upstream region of the yhfK gene; primer “8 '” allows amplification of the 5'-terminal region of the yhfK gene with an additional SD sequence. The obtained DNA fragments contained the XbaI recognition site at the 5'-ends and were homologous for 40 a or 42 bp desired integration site at 3'-ends. In parallel, the DNA fragment containing T _thr was amplified by PCR using the plasmid pML-ter_thrL (see Example link 2) as a matrix and primers "10" (SEQ ID NO: 12) and "II" (SEQ ID NO: 13 ) The obtained second fragment contained the XbaI recognition site at the 3'-end and was homologous for 43 bp desired integration site at the 5'-end. All PCR-generated DNA fragments were digested with XbaI and fragments containing various promoters were ligated with a fragment containing the terminator _{Th thr} .

The resulting ligation mixtures were precipitated with alcohol, dissolved in deionized water, and used for Red-dependent integration into strain SC17 (0) / RSF-Red-TER using the same procedure as that used to integrate the promoter library. The fact of integration was confirmed by PCR using primers "12" (SEQ ID NO: 14) and "13" (SEQ ID NO: 15).

To transfer these modifications to the L-glutamic acid producing strain AJ 13601 (European Patent Application 1078989), genomic DNA was isolated from the obtained strains using the Genomic DNA Isolation Kit (Sigma, United States). After electroporation of these DNA fragments into strain AJ13601, strains were obtained having the yhfK gene under the control of the promoter P _tac or the consensus sequence SD and the promoter P _tacN, respectively. The promoter P _tacN generally means a promoter with a randomized region. The procedure was carried out using a device for electrotransformation of bacteria ("BioRad", USA, catalog number 165-2089, version 2-89). The pulsation parameters used were as follows: electric field intensity 12.5 kV / cm, pulse time 10 ms. The promoter fragments used in these experiments contained only the first 11 bp (from 21 bp) of the lactose operator O ₁ , so they were not repressed by LacI.

The overnight culture was grown at 34 ° C on LB medium supplemented with MES (0.1 M, pH 7.0), glucose (5 g / l), tetracycline (10 μg / ml) and chloramphenicol (25 μg / ml). Then the culture was diluted 100 times with fresh medium containing glucose (40 g / l), MgSO ₄ · 7H ₂ O (0.5 g / l), (NH ₄ ) ₂ SO ₄ (16 g / l), KH ₂ PO ₄ ( 0.3 g / l), KCl (1 g / l), MES (10 g / l), betaine (1 g / l), FeSO ₄ · 7H ₂ O (10 mg / l), MnSO ₄ · 5H ₂ O ( 10 mg / l), lysine (100 mg / l), methionine (100 mg / l), DAP (100 mg / l), calcium pantothenate (10 mg / l), CaCO ₃ (30 g / l), 10 μg / ml tetracycline and 50 μg / ml chloramphenicol, and were grown for 26 hours at 34 ° C under aeration. Glutamate production and accumulation were measured by thin layer chromatography (TLC). The averaged data from the results of growing three independent crops are presented.

The selection was carried out on the production of L-glutamic acid. One of the best clones having a high level of production of L-glutamic acid (No. 11), and one of the clones having a reduced growth rate and production of L-glutamic acid (No. 52), the 5'-terminal region of the yhfK gene was selected and sequenced. LacZ activity expressed under the control of these P _tac-like promoters was measured as described above (data are presented in Table 1).

As can be seen from table 2, in these experimental conditions, the best results were obtained by combining a high-performance consensus SD sequence (SD sequence, AGGAGG, is included in the primer "8 '" (SEQ ID NO: 10) as its component) and relative weak promoter P _tac11 (strain AJ13601 P _tac11 SDyhfK). The yield of glutamate produced by this strain was higher ((Δ1% in yield) compared to the original AJ13601 strain. Using the more efficient promoter P _tac52 in combination with the natural 5'-terminal region of the yhfK gene led, as a rule, to worse results.

The results described above prove that the use of a promoter library, as described above (in combination with other regulatory elements, such as the SD sequence, repressor and activator binding sites, and so on), provide working tools for fine-tuning the expression level of the gene of interest .

Reference Example 1. Construction of helper plasmids RSF-Red-TER and RSF-Red-Kan.

The design scheme of helper plasmids is presented in FIG. 5.

At the first stage of constructing helper plasmids RSF-Red-TER and RSF-Red-Kan, the RSFsacBPlacMCS vector was constructed. For this purpose, DNA fragments containing the cat gene from plasmid pACYC184 and the structural part of the sacB gene from B. subtillis were amplified by PCR using oligonucleotides SEQ ID NO: 49, 50, 51 and 52, respectively. These oligonucleotides contain the following suitable restriction sites at the 5'-end, which are necessary for subsequent cloning: BglII, SacI, XbaI and BamHII, respectively. The resulting fragment of the sacB gene size of 1.5 thousand p. cloned into the XbaI-BamHI sites of the previously obtained vector pMW119-P _lac lacI. This vector was constructed in the same manner as described for the vector pMW118-P _lac lacI (Skorokhodova, A. Yu et al., Biotekhnologiya (Rus), 5, 3-21 (2004)), but it does contain a polylinker site from plasmid pMW219. instead of plasmid pMW218. After that, the previously described fragment of the cat gene size of 1.0 thousand p. treated with restriction enzymes BglII and SacI and cloned into the BamHI-SacI sites of the plasmid RSF-P _lac lacIsacB, which was obtained in the previous step. The resulting plasmid pMW-P _lac lacIsacBcat contained a fragment of P _lacUV5- lacI-sacB-cat. To subclone this fragment into the RSF1010 vector, plasmid pMWP _lac lacIsacBcat was digested with restriction enzyme BglII, treated with a Klenow fragment of DNA polymerase I to form a sticky end, and then SacI was cut. Fragment of BglII-SacI 3.8 thousand bp in size plasmids pMWP _lac lacIsacBcat were eluted from a 1% agarose gel and ligated with RSF1010, which contained the vector treated with the restriction enzyme PstI, a Klenow fragment of DNA polymerase I and SacI. The ligation mixture was transformed into E. coli TG1 strain and plated on LB agar containing chloramphenicol (50 mg / L). After restriction analysis of plasmids isolated from grown clones, the plasmid RSFsacB was obtained. To construct the RSFsacBP _lac MCS vector, the DNA fragment containing the P _lacUV5 promoter was PCR amplified using oligonucleotides SEQ ID NOS: 53 and 54 as primers and plasmid pMW119-P _lac lacI as a template. The resulting fragment is 146 bp in size. digested with restriction enzymes SacI and NotI and ligated with a large fragment of SacI-NotI from the plasmid RSFsacB.

After this, a 2.3 thousand bp DNA fragment containing λ-Red-αβγ genes and tL3 transcription terminator was amplified by PCR using oligonucleotides SEQ ID NO: 55 and 56 as primers and plasmid pKD46 as a template (Datsenko, KA, Wanner, BL, Proc. Natl. Acad. Sci. USA, 97, 6640-6645, (2000)). The resulting fragment was cloned into the PvuI-NotI sites of the RSFsacBP _lac MCS vector. Thus, the RSFRed plasmid was constructed. In order to prevent end-to-end reading of Red genes from the cat gene promoter, a ρ-independent transcription terminator of the rrnB operon from E. coli was inserted between the cat gene and the P _lacUV5 promoter. For this purpose, DNA fragments containing the P _lacUV5 promoter and the T _rrnB terminator were amplified by PCR using oligonucleotides SEQ ID NO: 57 and 54 as primers and plasmid pMW119-P _lac lacI as the matrix and oligonucleotides SEQ ID NO: 58 and 59 as primers and chromosomes of strain E. coli BW3350 as a matrix, respectively. The resulting fragments were treated with restriction enzyme KpnI and ligated. After this, a 0.5 kb DNA fragment containing both P _lacUV5 and d T _rrnB was amplified by PCR using oligonucleotides SEQ ID NOS: 54 and 59 as primers. The resulting DNA fragment was digested with EcoRI restriction enzyme, treated with a Klenow fragment of DNA polymerase I to form sticky ends, cut with BamHI restriction enzyme and ligated with a large Ecl136II-BamHI fragment of RSFsacBPlacMCS vector. The resulting plasmid was named RSF-Red-TER.

To obtain the RSF-Red-Kan plasmid, a 1.3 kb DNA fragment containing the kan gene and T _rrnB terminator was amplified by PCR using the previously obtained plasmid pMW118-SceKm as a matrix and oligonucleotides SEQ ID NO: 58 and 60 as primers. To construct the plasmid pMW-SceKm, a DNA fragment containing the kan gene flanked by the desired restriction endonuclease recognition sites (including the I-SceI meganuclease recognition site) was generated by PCR using oligonucleotides SEQ ID NO: 60 and 61 as primers and plasmids pUC4K in as a matrix. The resulting fragment was treated with XbaI and BamHI and inserted into XbaI-BamHI sites on plasmid pMW118-P _lac lacI.

The resulting fragment size of 1.3 thousand BP cut with restriction endonuclease BamHI, treated with a Klenow fragment of DNA polymerase I to form a sticky end, and cleaved with KpnI. In parallel, the RSF-Red-TER plasmid was sequentially treated with the restriction enzyme HindIII, the Klenow fragment of DNA polymerase I and the restriction enzyme KpnI, and the large fragment was eluted from 1% agarose gel. After ligation of the obtained fragments with the reaction mixture obtained after ligation, the TG1 strain was transformed and the cells were plated on LB-arap containing 50 mg / L kanamycin. Plasmid RSF-Red-Kan was selected after restriction analysis of plasmids isolated from growing clones.

Example reference 2. Construction of the plasmid pMW118- (λattL-Km ^r -λattR).

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

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

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

1) a BglII-EcoRI fragment (114 bp) carrying the attL site (SEQ ID NO: 29), which was obtained by PCR amplification of the corresponding chromosome region of E. coli strain W3350 (containing prophage λ) using primers P1 and P2 ( SEQ ID NOS: 30 and 31). These primers contained additional recognition sites for BglII and EcoRI endonucleases;

2) a PstI-HindIII fragment (182 bp) carrying the attR site (SEQ ID NO: 32), which was obtained by PCR amplification of the corresponding region of the E. coli W3350 chromosome (containing prophage λ) using primers P3 and P4 ( SEQ ID NOS: 33 and 34). These primers contained additional recognition sites for the endonucleases PstI and HindIII;

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

a large DNA fragment (2359 bp) carrying the AatII-EcoRI fragment of plasmid pMW118, which was prepared as follows: plasmid pMW118 was digested with EcoRI restriction endonuclease, treated with Klenow fragment of DNA polymerase I and digested with AatII restriction endonuclease;

- a small fragment of AatII-BglII (1194 bp) of pUC19 plasmid carrying the bla gene for ampicillin resistance (Ap ^R ) was obtained by PCR amplification of the corresponding region of pUC19 plasmid using primers P5 and P6 (SEQ ID NOS: 35 and 36 ) These primers contained additional recognition sites with AatII and BglII endonucleases;

- a small fragment of BglII-PstIpol (363 bp) of the ter_rrnB transcription terminator was obtained by PCR amplification of the corresponding region of E. coli chromosome MG1655 using primers P7 and P8 (SEQ ID NOS: 37 and 38). These primers contained additional recognition sites for BglII and PstI endonucleases;

4) a small fragment of EcoRI-PstI (1388 bp) (SEQ ID NO: 39) of the plasmid pML-Tc-ter_thrL carrying the tetracycline resistance gene and transcription terminator; plasmid pML-Tc-ter_thrL was obtained in two stages:

- the plasmid pML-ter_thrL was obtained by digestion of the plasmid pML-MCS (Mashko, SV et al., Biotekhnologiya (in Russian), 2001, no. 5, 3-20) with restriction endonucleases XbaI and BamHI, followed by ligation of a large fragment (3342 p. o.) with an XbaI-BamHI fragment (68 bp) that contains the ter_thrL terminator obtained by PCR amplification of the corresponding region of E. coli chromosome MG1655 using primers P9 and P10 (SEQ ID NOS: 40 and 41), These the primers contained additional recognition sites for endonucleases XbaI and BamHI;

- plasmid pML-Tc-ter_thrL was obtained by digestion of the plasmid pML-ter_thrL with restriction endonucleases KpnI and XbaI, followed by treatment with a Klenow fragment of DNA polymerase I and ligation with a small fragment of EcoRI-Van91I (1317 bp) of the resistance plasmid pBR322 carrying the plasmid tetracycline (pBR322 was digested with restriction endonucleases EcoRI and Van91I and then treated with a Klenow fragment of DNA polymerase I).

Example 4. The use of a library of randomized promoters.

Using the procedure described in Example 2, it is possible to obtain in one stage a library of clones with different levels of expression of genes of interest.

On the other hand, a library of created promoters can be used to construct a set of strains with different expression levels of genes of interest, for which the ratio between the indicated expression levels can be approximately calculated a priori.

To confirm this statement, several promoters from the library of created promoters were integrated into the chromosome of strain P. ananatis SC17 (0) before the dadX gene (SEQ ID NO: 64) encoding alanine racemase. For this purpose, genomic DNA was isolated from the AJ13601P _tac11 SDyhfK, AJ13601P _tac52 SDyhfK and AJ13601P _tac SDyhfK strains (see Example 3) using the Genomic DNA Isolation Kit (Sigma, USA). The obtained DNA samples were used as PCR matrices with primers PdadA (SEQ ID NO: 65) and PdadX (SEQ ID NO: 66) to obtain an in vitro integrative cassette containing a P _tac-like promoter linked to a cut-out marker of resistance to kanamycin and transcription terminator T _thr . In addition, the gntK gene (SEQ ID NO: 67) encoding gluconate kinase was modified in a similar manner. In this case, chromosomal DNAs isolated from clones containing the P _tac , P _tac49, and P _{tac11 promoters} , from the promoter library, and attL-gntK primers (SEQ ID NO: 68) and PtacN-gntK (SEQ ID NO: 69) to obtain an in vitro integrative cassette containing a P _tac-like promoter coupled to a cut-out marker of resistance to kanamycin. These oligonucleotides contain the regions necessary for integration that are homologous to the desired regions of the dadX or gntK gene at their 5'-ends, and regions that are homologous to the terminal regions of the amplified DNA fragment at their 3'-ends. The integrative cassettes thus obtained were integrated into the chromosome of strain SC17 (0) / RSFRedTER using the procedure described above.

The structures obtained on the chromosome are shown in FIG. 5. The validation of the design in the strains was carried out by PCR using dadX-t1 primers (SEQ ID NO: 70), dadX-t2 (SEQ ID NO: 71) and gntR (SEQ ID NO: 72), gntK (SEQ ID NO: 73) respectively. In the designed strains, the specific activities of alanine racemase or gluconate kinase were evaluated. Alanine racemase activity was evaluated by resistance to D-cycloserine on LB agar plates (Peter, A., Bron, Appl. Environ. Microbiol., Vol. 68, No. 11, Nov. 2002, p. 563-5670).

The specific activity of gluconate kinase was evaluated spectrophotometrically at 340 nm by the appearance of NADP-H in a coupled reaction with 6-phosphogluconate dehydrogenase. For this, the strains were grown on M9 minimal medium at pH = 6.0 with the addition of glucose (5 g / l) to a final optical density of A ₅₉₅ = 1.0. Cells were washed once with 1 ml of 50 mM Tris-HCl buffer (pH 7.5) and resuspended in a solution containing 50 mM Tris-HCl buffer (pH 7.5), 2 mM DTT, 2 mM MgCl ₂ . Cells were destroyed by ultrasound. The activity test solution contained 50 mM Tris-HCl buffer (pH 8.0), 4 mM NADP, 2 mM MgCl ₂ , 4 mM ATP, 4 mM gluconate, and 2 units / ml 6-phosphogluconate dehydrogenase as an enzyme for the coupled reaction. Specific activity was determined based on the molar extinction coefficient of NADP-H equal to 6.2 · 10 ³ .

As shown in Table 3, in the constructed strains there was a clear correlation between the relative activities of the enzymes, defined as the ratio of the specific activity that a strain containing one of the P _tac-like promoters possessed to the specific activity that the strain containing the P _tac promoter had before the corresponding genome.

Example 5. The use of a combined recombination system λRed-λInt / Xis for the introduction of multiple modifications into the chromosome.

The possibility of reintegration of various cassettes flanked by attL and attR sites of phage λ into the P. ananatis chromosome using the Red-dependent recombination of linear fragments with homology arms of about 40 bp in length was tested. For this purpose, several genes on the chromosome were replaced with a tetracycline resistance gene. The primers used in the experiments are shown in Table 4.

The results presented in Table 5 indicate that under non-selective conditions, the frequency of reintegration at the same point (when att sites of 120 and 150 bp serve as homology shoulders) is 10-30 times higher than at the desired integration point ( using shoulders homology 36-40 bp). But it’s still possible to select clones containing two modifications. In the case of electroporation of the chromosome (the method is described in Example 3), all reintegrations of the cassette flanked by att sites of phage λ took place at the desired point.

Example 6. The procedure of two-stage λ Red-dependent recombination for the introduction of unlabeled point mutations in the chromosome of P. ananatis.

A two-stage λ Red-dependent integration procedure was developed to introduce an unlabeled point mutation into the P. ananatis chromosome. The procedure includes the following stages: a) introducing into a bacterium resistant to the products of the phage genes λ gam, bet and exo, an exogenous linear DNA fragment that includes a double selective / counter-selective marker flanked by 2 sites at least 30 bp in length identical to the sites localized near the desired point on the chromosome of the bacterium; b) replacement using λ Red-dependent integration of the marker with a short DNA fragment containing the desired point mutation, flanked by sites identical to sites located near the desired point on the chromosome; and c) selection of bacteria with unlabeled point mutation using counter-selection.

Any suitable antibiotic resistance marker may be used as a selective marker in the method of the present invention. For example, markers of resistance to chloramphenicol, ampicillin, kanamycin, tetracycline, streptomycin and the like.

The sacB gene from B. subtilis, encoding levansucrase, the expression of which is lethal for bacteria containing this gene and growing on nutrient medium containing sucrose, can be used as a counter-selective marker (marker of negative selection) (Gay, P. et al., J . Bacteriol., 164, 918-921 (1985)); the tetA gene, which allows for counter-selection in the presence of fusaric acid (Bochner, B.R. et al., J. Bacteriol., 143, 926-933 (1980)); a rpsL gene conferring sensitivity to streptomycin (Russel, C. B. and Dahlquist, F.W., J. Bacteriol., 171, 2614-2618 (1989)) and the like.

To illustrate the procedure for introducing a point mutation, the native SmaI recognition site located inside the hisD gene of the bacterium P. ananatis was replaced with the XhoI recognition site.

Construction of a double selective / contraselective marker.

The plasmid RSFsacB described in Example link 1 was digested with XbaI restriction endonuclease and ligated at a low DNA concentration (1 ng / μl). Then, the E. coli TG1 strain was transformed with the mixture obtained after ligation. After isolation and restriction analysis of plasmid DNA from Cm ^R clones grown after transformation, the RSFPlacsacB plasmid with the missing lacI gene was selected (Figure 6).

Introduction of the marker P _lacUV5 -sacB-cat into the P. ananatis chromosome.

Plasmid RSFPlacsacB was used as template for PCR amplification of the P _lacUV5 -sacB-cat cassette. The primers HisD-Plac-5 (SEQ ID NO: 90) and HisD-cat-3 (SEQ ID NO: 91) were used for this reaction. The primers contained sites homologous to regions of the chromosome flanking the SmaI recognition site located within the hisD gene of P. ananalis bacterium at its 5'-end.

The DNA fragment obtained in this reaction was used for electroporation into strain SC17 (0) / RSFRedkan as described above (for conditions, see Example 1). After electroporation, cells were plated on plates in LB agar supplemented with chloramphenicol (20 mg / L). To verify the grown integrants, PCR was performed using HisD-L5 primers (SEQ ID NO: 62) and HisD-R3 (SEQ ID NO: 63) (for conditions, see Example 1).

Replacing the marker with a short DNA fragment containing the desired mutation using λ Red-dependent integration.

A short PCR DNA fragment containing the desired mutation was prepared as follows. First, two complementary primers were hybridized, XhoI-F (SEQ ID NO: 92) and XhoI-Rev (SEQ ID NO: 93). The result was a short double-stranded DNA fragment containing the desired mutation in the center (replacing the SmaI recognition site with XhoI). In order to increase the flank regions homologous to the chromosome, PCR amplification of the obtained fragment was carried out using His-SL (SEQ ID NO: 94) and His-SR (SEQ ID NO: 95) primers. The obtained DNA fragment contained the XhoI recognition site in the center, flanked by 83 bp sites homologous to the corresponding sites on the P. ananatis chromosome.

The indicated DNA fragment was used for electroporation into the strain SC17 (0) hisD :: P _lacUV5 -sacB-cat described above with the auxiliary plasmid RSFRedkan. Electroporation cells were cultured in 10 ml of LB medium supplemented with 40 mg / L kanamycin and 1 mM IPTG until OD ₅₉₅ = 0.5 was achieved. Cells were collected using centrifugation, washed three times with ice water and once with 10% glycerol and resuspended in 50 μl of 10% glycerol. After adding 100 ng of the integrative DNA fragment, electroporation was performed (E = 20 kV / cm, pulse time 5 ms). After electroporation, the cells were plated on plates with LB agar containing 20% sucrose. Checked 100 grown sucrose resistant clones. About 25% of the tested clones had the Suc ^R Cn ^S His ⁺ phenotype. Genomic DNA was isolated from these clones and the chromosome fragment containing the expected mutation in the center was amplified by PCR using HisD-L5 and HisD-R3 primers. Restriction analysis of amplified fragments showed the presence of the desired mutation (replacing the SmaI recognition site with XhoI) in all selected clones.

Although the invention has been described in detail with reference to the best mode of carrying out the invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention.

Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1 Clone "-35" area 5 '-> 3' LacZ activity, Miller units SEQ ID NO: P _tac TTGACA 6000 16 84 TTTGCA 2000 17 65 TTGTGA 1250 eighteen 49 TTGTAA 1200 19 47 TTGAGA 1000 twenty 60 TTTCAA 770 21 eighteen TAGTGA 150 22 82 TCGCGA one hundred 23 25 Tccgga 80 24 21 TCTCGA 70 25 8 TGAAGA 47 26 52 GTGCTA 2800 27 eleven TTGGAA 730 28

table 2 Strain OD ₅₄₀ Glutamate, g / l Exit, % Aj13601 13.7 15.6 39.0 AJ13601P _tac yhfK 13.7 14.2 35.5 AJ13601P _tac52 SDyhfK 11.7 10.4 26.0 AJ13601P _tac11 SDyhfK 13,4 16,0 40,0

Table 3 Promoter Gene lacZ dadX gntKV The relative activity of β-galatosidase Relative D-Cycloserine Resistance The relative activity of gluconate kinase P _tac one one one P _tac52 0.47 0.5 P _tac49 0.20 0.41 P _tac11 0.12 0.2 0.025

Table 4 Modification DNA fragment primers for integration Testing Primers ΔrecD SEQ ID NOs: 74 and 75 SEQ ID NOs: 76 and 77 ΔresC SEQ ID NOs: 78 and 79 SEQ ID NOs: 80 and 81 ΔresB SEQ ID NOs: 82 and 83 SEQ ID NOs: 84 and 85 ΔsbcB SEQ ID NOs: 86 and 87 SEQ ID NOs: 88 and 89

Table 5 The strain is the recipient DNA for electroporation The number of colonies per 10 ⁹ electroporated cells grown on a medium containing: Replica Tc ^R Clone Replica tetracycline tetracycline and chloramphenicol The number of tested clones Tc ^R The number of Cm ^R clones among the tested Tc ^R clones Chromosomal Electroporation SC17recD :: attKm ^R Chromosome SC17hisD :: attCm ^R 23 thirty 17 17 Red dependent integration SC17 (0) hisD :: attCm ^R (RSFRedkan) PCR fragment recB :: attTc ^R 46 0 35 one PCR fragment recC :: attTc ^R 271 0 33 one PCR fragment sbcB :: attTc ^R 432 84 34 3

Claims (12)

1. A method of constructing a recombinant bacterium belonging to the genus Pantoea, including:
a) obtaining an exogenous linear DNA fragment, which has at least 2 sections of at least 30 bp each in length identical to the sites located in the target area on the chromosome of the specified bacterium,
b) introducing said linear DNA fragment into said bacterium, wherein said bacterium is modified to be resistant to gam, bet, and exo gene products from lambda phage, and
c) selection of a recombinant bacterium in which the target region is replaced with the indicated linear DNA fragment or the linear DNA fragment is inserted in the target region. 2. The method according to claim 1, characterized in that the bacterium is a bacterium Pantoea ananatis SC17 (0). 3. The method according to claim 1, characterized in that the specified target site in a recombinant bacterium is modified due to inactivation or removal. 4. A method of constructing a bacterium belonging to the genus Pantoea, containing a library of DNA fragments, including:
a) obtaining a set of exogenous DNA fragments, each of which has at least 2 sections of at least 30 bp in its composition. each in length identical to the sites located in the target area on the chromosome of the specified bacterium,
b) introducing the specified set of linear DNA fragments into the specified bacterium, while the specified bacterium is modified in such a way that it is resistant to the products of the gam, bet and exo genes from the phage lambda, and
b) selection of recombinant bacteria in which the target region is replaced with the specified linear DNA fragment or a linear DNA fragment is integrated in the target region. 5. The method according to claim 4, characterized in that said DNA fragments contain a regulatory element for gene expression selected from the group consisting of a promoter, a ribosome binding site (RBS), an operator, and combinations thereof. 6. The method according to claim 5, characterized in that said regulatory element comprises a randomized sequence. 7. A method of constructing a recombinant bacterium belonging to the genus Pantoea, a producer of L-amino acids, including:
a) replacing the natural regulatory elements of the target area with exogenous DNA fragments by the method according to claim 1 and
b) selecting a bacterium with an optimized level of gene expression in the target region of the bacterial chromosome, characterized in that said gene encodes a protein that is involved in the biosynthesis and metabolism of the L-amino acid or in the excretion of the L-amino acid from the specified bacterium. 8. The method according to claim 7, characterized in that said L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L- glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. 9. A bacterium belonging to the genus Pantoea and obtained by the method according to claim 7, is a producer of an L-amino acid selected from the group consisting of L-glutamic acid, L-leucine, L-lysine, L-isoleucine, L-valine, L β-histidine, L-aspartate, L-alanine, L-tyrosine and L-phenylalanine. 10. The bacterium according to claim 9, characterized in that said natural regulatory elements contain in the target region a fragment of 35 base pairs in size from the promoter of a gene selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO; 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and combinations thereof. 11. The bacterium of claim 10, wherein the specified gene is the yhfK gene. 12. A method of obtaining an L-amino acid selected from the group consisting of L-glutamic acid, L-leucine, L-lysine, L-isoleucine, L-valine, L-histidine, L-asparagate, L-alanine, L-tyrosine and L-phenylalanine including:
a) growing bacteria according to claim 9 in a nutrient medium and
b) isolating said L-amino acid from the culture fluid.

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