RU2549708C2 - SELF-INDUCED EXPRESSION SYSTEM AND ITS APPLICATION FOR OBTAINING USEFUL METABOLITES BY MEANS OF BACTERIA OF Enterobacteriaceae FAMILY - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents a method of obtaining useful metabolites with the application of bacteria of the Enterobacteriaceae family, in particular bacteria, belonging to the genus Escherichia, which is modified in such a way that it contains a genetic expression system, including a transcription apparatus, regulated by a protein of the LysR type, and modified in such a way that the self-induced positive regulation of the feedback type in the said system is mediated by a coinducer.

EFFECT: method is suitable for the production of L-amino acids with a branched chain, in particular L-valine, L-isoleucine and L-leucine, higher alcohols and D-pantothenic acid; invention makes it possible to obtain L-amino acids with the high degree of efficiency.

23 cl, 2 dwg, 5 tbl, 6 ex

Description

FIELD OF THE INVENTION

The present invention relates to the microbiological industry and, in particular, to a method for producing beneficial metabolites by fermentation of a bacterium of the Enterobacteriaceae family, characterized in that the genetic expression system of the bacterium regulated by a LysR protein is modified in such a way that the functionality of said expression system is mediated by a co-inductor, and, as result, increased expression of the gene regulated by the specified expression system. More specifically, the expression system and method may be useful for enhancing the production of metabolites of the biosynthetic pathway of L-amino acids, such as branched chain L-amino acids (branched L-amino acids).

State of the art

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

Many methods have been described to increase the production of L-amino acids, including, for example, transformation of a recombinant DNA microorganism (see, for example, US Pat. No. 4,278,765) and changing regulatory regions, such as a promoter, leader sequence and / or attenuators, or others known to those skilled in the art areas (see, for example, US Patent Application No. 2006216796 A1 and WO 9615246 A1). Other yield enhancing methods include increasing the activity of enzymes involved in amino acid biosynthesis and / or removing feedback inhibition of key enzymes produced by the L-amino acid (see, for example, Japanese Patent Publication No. 56-18596 (1981), WO 95 / 16042 or US Pat. Nos. 5,661,012 and 6,040,160). For example, a mutant bacterial acetohydroxy acid synthetase I (also referred to as acetolactate synthase I, hereinafter AHAS I), which is resistant to feedback inhibition by L-valine, was used to increase the yield of the branched L-amino acid in the corresponding strains producing L-amino acid (Patent RF №2355763).

The biosynthesis of branched L-amino acids (branched-chain L-amino acids, BCAAs) such as L-valine, L-leucine and L-isoleucine is carried out in a branched biosynthetic pathway. Acetolactate synthase (enzyme classification number (EC) 2.2.1.6) catalyzes the reaction in the first step of the biosynthetic pathway, which is common to all three amino acids. The reaction involves the condensation of activated acetaldehyde (2- (α-hydroxyethyl) thiamine diphosphate), a pyruvate derivative, with pyruvate or 2-oxobutanoate to form 2-acetolactate (AΛ) or 2-aceto-2-hydroxybutanoate (AGB), respectively. AΛ is the precursor of L-valine and L-leucine, and AHB is L-isoleucine. In Escherichia coli (hereinafter E. coli), for example, reactions between pyruvate molecules as well as between pyruvate and 2-oxobutanoate are catalyzed by AHAS isoenzymes such as AHAS I, AHAS II and AHAS III, encoded by the ilvBN, ilvGM and ilvIH genes, respectively. AHAS I and AHAS III are inhibited by the end product (a mechanism is referred to as feedback inhibition or retro-inhibition) - L-valine. Feedback inhibition by the target product plays an important role in the physiological control of biosynthetic pathways in bacteria.

The reaction products catalyzed by AHAS, AΛ, or AHB are substrates for IlvC 2-acetohydroxy acid isomeric reductase (EC 1.1.1.86) encoded by the ilvC gene belonging to the ilvYC operon. The E. coli ilvYC operon is an integral part of the expression system regulated by a LysR-type protein, which is the most widespread positively regulated expression system of bacteria and can be found in bacterial families of prokaryotes from Enterobacteriaceae to Rhizobiaceae (Rhee KY et al., Proc. Nat. Acad. Sci. USA, 1999, 96: 14294-14299). The ilvY gene encodes an IlvY regulatory protein of type LysR (transcriptional regulator), which binds in a highly cooperative manner with two tandem operator sites in the ilvYC region with multidirectional overlapping promoters (Figure 1). Upon binding to the first operator site, the IlvY regulator negatively self-regulates transcription from the ilvY promoter, thus weakening its own synthesis. In addition, IlvY plays a major role in the activation of transcription of the ilvC gene. Activation of ilvC transcription requires the binding of the IlvY regulator to the second operator site and subsequent binding of a co-inductor such as 2-acetolactate (AΛ) or 2-aceto-2-hydroxybutanoate (AGB) to the previously formed IlvY / DNA complex. Upon binding of the co-inductor, a conformational change occurs in the protein / DNA complex, which causes a change in the structure of the ilvC promoter in the -35 region and significantly increases the binding strength of the PHK polymerase (Rhee KY et al., J. Biol. Chem., 1998, 273: 11257- 11266).

In the biosynthesis of L-valine and L-leucine, 2-acetolactate (AΛ) is converted by the action of the IlvC protein to 2,3-dihydroxy-3-methylbutanoate (another name for 2,3-dihydroxyisovalerate, DHIV) (Figure 2). In the biosynthesis of L-isoleucine, 2-aceto-2-hydroxybutanoate (AHB) is converted under the action of the IlvC protein into 2,3-dihydroxy-3-methylpentanoate (another name for 2,3-dihydroxy-3-methylvalerate, DHMV).

Recently, self-induced genetic expression systems have become very attractive for enhancing the expression of a desired gene compared to conventional genetic approaches. For example, the artificially created gene expression system is known from the literature and is regulated by the type of positive feedback, which can function as an amplifier of the genetic signal, which increases the sensitivity to the inducer protein and also increases the expression level in the absence of an external cofactor, acyl homoserinlactone (AGΛ, also referred to as homoserinlactone-HSΛ) (Nistala GJ et al., J. Biol. Eng., 2010, 4: 4). The developed system uses the constantly active version of the LuxR protein [lux operon], a quorum sensing regulator (CS) from Vibrio fischeri, which is independent of the self-inductor (AGΛ) due to the point mutation Ala221Val (Sayut DJ et al., Biochem. Biophys. Res. Commun. 2007, 363: 667-673; Poellinger KA et al., FEMS Microbiol Lett., 1995, 129: 97-101). A genetic expression system, similar, with the exception of small changes to the above, was used to change the kinetic parameters of the expression of the model membrane protein cytochrome bd quinol oxidase in E. coli (K. Bansal et al., J. Biol. Eng., 2010, 4: 6) .

A self-induced positive feedback regulated activation system based on a quorum sensing apparatus from V. fischery (lux bioluminescent genes) and using an intracellular self-inductor pool (GSΛ) was used to express recombinant proteins such as antigens for the preparation of pharmaceutical compositions (WO 2010136897 A2).

However, there is currently no data describing a genetic expression system regulated by a protein of the LysR type, modified in such a way that the functionality of said expression system is mediated by a co-inductor; and the use of this system to obtain beneficial metabolites of the L-amino acid biosynthesis pathway, such as branched L-amino acids, and / or its side path.

Disclosure of the invention

The purpose of the present invention is the provision of a genetic expression system comprising elements of a transcriptional apparatus regulated by a protein of the LysR type, modified in such a way that the regulation of this system, which operates on the principle of self-induction with positive feedback, is mediated by a co-inductor.

Another objective of this study is the provision of a bacterium of the Enterobacteriaceae family, which may belong to the genus Escherichia and, more specifically, to the species Escherichia coli, which is modified to contain the indicated expression system.

Another objective of the present invention is the provision of a method for producing beneficial metabolites, for example, L-amino acids, such as L-amino acid branched chain slots, or their salts, in particular, L-valine, L-leucine and L-isoleucine or their salts. This goal was achieved due to the discovery of the fact that the modification of genes that colorate mutant aceto lactate synthase resistant to feedback inhibition by L-valine in such a way that expression of these genes is regulated by a transcriptional apparatus regulated by a LysR protein and expression of which is mediated by a co-inducer obtained A reaction involving acetolactate synthase leads to an increase in the production of branched L-amino acids.

The purpose of the present invention is the provision of a genetic expression system comprising a transcriptional apparatus regulated by a LysR type protein, which includes a promoter and operator, the expression of which is positively regulated by a LysR type regulatory protein and co-inductor, and the target gene (s) with which the transcriptional apparatus is functionally linked, and the target gene (s) encodes (s) the protein (s) involved in the biosynthesis of the co-inductor, substrate or precursor of the co-inductor, and self-induced positive regulation of type o inverse relation of such expression system mediated by a co-inducer.

It is also an object of the present invention to provide an expression system as described above, characterized in that said system is from a bacterium belonging to the Enterobacteriaceae or Pseudomonadaceae family.

It is also an object of the present invention to provide an expression system as described above, characterized in that said system is from a bacterium belonging to the Enterobacteriaceae family.

It is also an object of the present invention to provide the above-described expression system, characterized in that said system is from a bacterium belonging to the genus Escherichia.

Another objective of the present invention is the provision of the above expression system, characterized in that the bacterium belongs to the species Escherichia coli.

It is also an object of the present invention to provide an expression system as described above, characterized in that said system is from a biosynthetic pathway of an L-amino acid selected from the group consisting of branched L-amino acids, L-lysine, L-cysteine, L-methionine and L-tryptophan.

It is also an object of the present invention to provide an expression system as described above, characterized in that said system is from a branched chain L-amino acid biosynthetic pathway.

It is also an object of the present invention to provide an expression system as described above, characterized in that the promoter is the P _ilvC promoter, the regulatory protein of the LysR type is the IlvY protein, and the co-inductor is 2-aceto-lactic acid, 2-aceto-2-hydroxybutyric acid or their salts.

It is also an object of the present invention to provide an expression system as described above, characterized in that the co-inductor is 2-acetolactic acid or a salt thereof.

It is also an object of the present invention to provide the above expression system, characterized in that the target gene (s) encodes (s) acetohydroxy acid synthetase.

It is also an object of the present invention to provide an expression system as described above, characterized in that said target genes encode proteins selected from the group consisting of:

(A) combinations of items A1 and A2:

(A1) a protein containing the amino acid sequence of SEQ ID NO: 2, or a protein containing the amino acid sequence of SEQ ID NO: 2, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein A2; and

(A2) a protein containing the amino acid sequence of SEQ ID NO: 4, or a protein containing the amino acid sequence of SEQ ID NO: 4, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein A1;

(B) combinations of items B1 and B2:

(B1) a protein containing the amino acid sequence of SEQ ID NO: 6, or a protein containing the amino acid sequence of SEQ ID NO: 6, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein B2; and

(B2) a protein containing the amino acid sequence of SEQ ID NO: 8, or a protein containing the amino acid sequence of SEQ ID NO: 8, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein B1; and

(C) combinations of items C1 and C2:

(C1) a protein containing the amino acid sequence of SEQ ID NO: 32, or a protein containing the amino acid sequence of SEQ ID NO: 32, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein C2; and

(C2) a protein comprising the amino acid sequence of SEQ ID NO: 34, or a protein comprising the amino acid sequence of SEQ ID NO: 34, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein C1.

It is also an object of the present invention to provide the above-described expression system, characterized in that the acetohydroxy acid synthetase is a mutant acetolactate synthase I resistant to feedback inhibition by L-valine.

It is also an object of the present invention to provide the above-described expression system, characterized in that the operator includes a region to which the above-described regulatory protein of the LysR type binds.

Another objective of the present invention is the provision of the above expression system, characterized in that the regulatory protein of the LysR type is selected from the group consisting of:

(D) a protein containing the amino acid sequence of SEQ ID NO: 10; and

(E) a variant of a protein containing the amino acid sequence of SEQ ID NO: 10, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having an activity of a regulatory protein of the LysR type.

Also the aim of the present invention is the provision of the above expression system, characterized in that the promoter includes:

(F) DNA containing the nucleotide sequence of SEQ ID NO: 30; or

(G) a DNA variant containing the nucleotide sequence of SEQ ID NO: 30, which contains the substitution, deletion, insertion or addition of one or more nucleotide residues, and having the activity of the nucleotide sequence of SEQ ID NO: 30.

It is also an object of the present invention to provide an L-amino acid producing bacterium belonging to the Enterobacteriaceae family, characterized in that the bacterium is modified so that it contains an expression system as described above.

It is also an object of the present invention to provide a bacterium as described above, characterized in that the bacterium contains a gene encoding a regulatory protein of the LysR type.

Also the purpose of the present invention is the provision of the above bacteria, characterized in that the bacterium belongs to the genus Escherichia.

Also the aim of the present invention is the provision of the above bacteria, characterized in that the bacterium belongs to the species Escherichia coli.

It is also an object of the present invention to provide the above bacteria, characterized in that the L-amino acid is a branched chain L-amino acid.

It is also an object of the present invention to provide the above bacteria, characterized in that the branched chain L-amino acid is selected from the group consisting of L-valine, L-leucine and L-isoleucine.

The purpose of the present invention is the provision of a method for producing a branched chain L-amino acid, including:

(i) growing the above bacteria in a culture medium such that said branched chain L-amino acid accumulates in the culture fluid; and

(ii) isolating a branched chain L-amino acid from the culture fluid.

It is also an object of the present invention to provide a method for producing the above branched L-amino acid with a branched chain, characterized in that said L-amino acids of the branched chain slot are selected from the group consisting of L-valine, L-leucine and L-isoleucine.

Brief Description of the Figures

Figure 1 shows a transcriptional regulation scheme for the ilvY and ilvC genes. IlvY means a transcriptional regulator of the type LysR, AΛ or AGB - co-inductor 2-acetolactate or 2-aceto-2-hydroxybutanoate, O1 and O2 - operators 1 and 2, TS _ilvC - start transcription of the ilvC gene, TS _ilvY - start transcription of the ilvY gene, signs minus (-) and plus (+) are the negative and positive effects on gene transcription, respectively.

Figure 2 shows a diagram of the biosynthesis of L-valine from pyruvate. AΛ means 2-acetolactate, DHIV - 2,3-dihydroxyisovalerate, AHAS-acetolactate synthase, IlvC - isomereductase

Description of sequences

SEQ ID NO: I is the nucleotide sequence of the ilvB gene.

SEQ ID NO: 2 is the amino acid sequence of the large wild-type acetolactate synthase I subunit.

SEQ ID NO; 3 - nucleotide sequence of the ilvN gene.

SEQ ID NO: 4 is the amino acid sequence of the small wild-type acetolactate synthase I subunit.

SEQ ID NO: 5 is the nucleotide sequence of the ilvI gene.

SEQ ID NO: 6 is the amino acid sequence of the large wild-type acetolactate synthase III subunit.

SEQ ID NO: 7 is the nucleotide sequence of the ilvH gene.

SEQ ID NO: 8 is the amino acid sequence of the small subunit of wild-type acetolactate synthase III.

SEQ ID NO: 9 is the nucleotide sequence of the ilvY gene.

SEQ ID NO: 10 is the amino acid sequence of the IlvY protein.

SEQ ID NOs: 11-29 — nucleotide sequences of primers P1-P19.

SEQ ID NO: 30 is the nucleotide sequence of the PilvC promoter.

SEQ ID NO: 31 is the nucleotide sequence of the ilvG gene.

SEQ ID NO: 32 is the amino acid sequence of the large wild-type acetolactate synthase II subunit.

SEQ ID NO: 33 is the nucleotide sequence of the ilvM gene.

SEQ ID NO: 34 is the amino acid sequence of the small wild-type acetolactate synthase II subunit.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Bacteria

The term “beneficial metabolite” as used in the present invention is not limited in any way, provided that this metabolite can be obtained by enzymatic reaction or biosynthetically and may include L-amino acids, higher alcohols and D-pantothenic acid.

The term "bacterium-producer of L-amino acids" may mean a bacterium of the Enterobacteriaceae family, which is capable of producing and causing the accumulation of L-amino acids in the culture fluid when the bacterium is grown (cultivated) in a nutrient medium. The ability of a bacterium to produce an L-amino acid can mean the ability of a bacterium to produce an L-amino acid in a nutrient medium or in bacterial cells, which leads to the accumulation of an L-amino acid in quantities sufficient to isolate it from the culture fluid or from bacterial cells when the bacterium is grown (cultivated) ) in a nutrient medium.

The term "L-amino acid" may mean L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine L-glutamic acid, L-glutamine, glycine L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

The term “branched L-amino acid” may include L-valine, L-leucine and L-isoleucine.

A bacterium may be capable of producing a useful metabolite, such as an L-amino acid, initially, in accordance with its natural characteristics, or may be modified by mutations (mutagenesis) or recombinant DNA technologies so that it becomes capable of production.

A bacterium belonging to the Enterobacteriaceae family can be selected from bacteria belonging to the genera of this family, such as Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, etc., and capable of producing L-amino acid. More specifically, bacteria classified as Enterobacteriaceae can be used according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/ wwwtax.cgi? id = 543). Examples of bacteria that can be modified according to the present invention include bacteria belonging to the genus Escherichia, Enterobacter or Pantoea.

The selection of bacterial strains belonging to the genus Escherichia that can be modified in the present invention is not limited in any way, however, as examples of bacteria of the genus Escherichia described in Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In FC Neidhardt et al. (Ed.), E. coli and Salmonella: cellular and molecular biology, 2 ^nd ed. ASM Press, Washington, DC, 1996), may be included among the bacteria of the present invention. As a specific example, E. coli. Strains, such as E. coli W3110 (ATTC 27325), E. coli MG1655 (ATCC 47076), etc., which originate from the original wild-type strain, i.e. E. coli K-12 strain. This strain can be obtained, in particular, from the American Type Culture Collection, “American Type Culture Collection, (ATCC)” (PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned an individual registration number, and strains can be ordered according to the registration number (see link www.atec.org). The strain registration numbers are on the American Type Culture Collection, ATCC catalog directory.

Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, etc. Examples of Pantoea bacteria include Pantoea ananatis, etc. Recently, some Enterobacter agglomerans species have been reclassified as Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii based on analysis of the nucleotide sequence of 168 rRNA and other evidence. Bacteria belonging to the genus Enterobacter or Pantoea can be used in accordance with the present invention, as they belong to the Enterobacteriaceae family. Genetic engineering strains of Pantoea ananatis, such as strain Pantoea ananatis AJ 3355 (PERM BP-6614), strain AJ 3356 (FERM BP-6615), strain AJ 3601 (PERM BP-7207) and their derivatives can be used in in accordance with the present invention. These strains were classified as Enterobacter agglomerans upon isolation, and they were deposited as Enterobacter agglomerans. However, they were later reclassified as Pantoea ananatis based on analysis of the 16S pPHK nucleotide sequence and other evidence.

Examples of bacteria belonging to the Pseudomonadaceae family may be from the genus Pseudomonas. Examples of bacteria of the genus Pseudomonas include Pseudomonas putida, P. aeruginosa, and P. syringae.

The term “bacterium that is modified to contain a genetic expression system” may mean a bacterium, for example, an L-amino acid producing bacterium belonging to the Enterobacteriaceae family, in which a genetic expression system regulated by a LysR protein is modified in such a way that the functionality of said a self-induced system with positive reverse regulation is mediated by a co-inductor. For example, the term “bacterium that is modified to contain a genetic expression system” may mean a bacterium of the Enterobacteriaceae family that has the ability to produce an L-amino acid, such as a branched L-amino acid, that is modified so that it has one or more genes the specified expression system.

Branched Chain L-Amino Acid Bacteria The term “branched chain L-amino acid producing bacteria” may mean a bacterium that is capable of producing and causing the accumulation of a branched L-amino acid in a culture fluid, such as L-valine, L-leucine and L -isoleucine, when the specified bacterium is grown (cultivated) in a nutrient medium.

Branched L-amino acids, in addition to L-valine, L-leucine and L-isoleucine, may also include unnatural branched L-amino acids, such as L-homoleucine and L-homoisoleucine. The ability of a bacterium to produce a branched L-amino acid can be imparted to the bacterium or increased by selecting the bacterium during breeding. The term “branched chain L-amino acid producing bacterium” can also mean a bacterium that is capable of producing and causing the accumulation of the branched L-amino acid slots in the culture fluid in a larger amount than an unmodified strain, for example, a wild-type strain or a parent strain, and also may mean a bacterium that is capable of producing and causing the accumulation of a branched L-amino acid in the culture fluid in an amount of not less than 0.5 g / l or even not less than 1.0 g / l.

A branched chain L-amino acid producing bacterium may be a bacterium of the Enterobacteriaceae family that contains a regulatory region that positively affects the expression of acetolactate synthase genes by a complex that includes a transcriptional regulatory protein and a co-inductor that is a reaction product catalyzed by acetolactate synthase. Moreover, the bacterium may be a bacterium of the Enterobacteriaceae family, in which the activity of mutant acetolactate synthase is increased. More precisely, the bacterium can be a bacterium producing a branched L-amino acid of the Enterobacteriaceae family, characterized in that the production of a branched L-amino acid is increased due to the increased activity of mutant acetolactate synthase by introducing a regulatory site into the bacterium. The bacterium is a producing bacterium of a branched L-amino acid belonging to the genus Escherichia, characterized in that the production of a branched L-amino acid bacterium is increased due to the increased activity of mutant acetolactate synthase resistant to feedback inhibition by L-valine.

A branched L-amino acid producing bacterium of the Enterobacteriaceae family is not limited to the bacterium described above. More specifically, a branched L-amino acid producing bacterium can also be a bacterium producing L-valine, L-leucine and / or L-isoleucine.

L-valine producing bacterium

Examples of parental strains that can be used to produce L-valine producing bacteria in accordance with the present invention include, but are not limited to, strains having modifications leading to overexpression of ilvGMEDA operon genes (US Patent No. 5,998,178). It is desirable to remove the fragment of the ilvGMEDA operon responsible for the weakening of the expression so that the expression of the operon is not attenuated by the resulting L-valine. Furthermore, the ilvA gene can be destroyed in the operon in order to reduce the activity of threonine deaminase.

Examples of parental strains that can be used to produce L-valine producing bacteria also include mutant strains having a mutation in aminoacyl-tRNA synthetase (US Patent No. 5,658,766). For example, an E. coli strain VL1970 can be used which has a mutation in the ileS gene encoding isoleucine-tRNA synthetase. The strain E. coli VL1970 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1st Dorozhniy proezd, 1) on June 24, 1988 with accession number VKPM B-4411. Further, mutant strains that require lipoic acid and / or with an insufficient amount of H ⁺ -ATPase (WO 96/06926) can also be used as parent strains.

As the parent strain, L-valine producing bacteria belonging to the genus Escherichia can be used, such as H-81 (VKPM B-8066), NRRL B-12287 and NRRL B-12288 (US Patent No. 4,391,907), VKPM B-4411 (US Patent No. 5,658,766), VKPM B-7707 (European Patent Application EP 1016710 A2), NS1610 (reference to Patent EP1942183, Example 7 or examples mentioned therein) and the like.

L-Leucine Producer Bacteria

Examples of parental strains that can be used to produce L-leucine producing bacteria include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (e.g., strain 57 (VKPM B- 7386, US Patent No. 6,124,121)) or leucine analogs including p-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397B and JP 8-70879A); E. coli strains obtained using genetic engineering methods and described in WO 96/06926; E. coli H-9068 (JP 8-70879 A), and the like.

The bacterium can be improved by enhancing the expression of one or more genes involved in the biosynthesis of L-leucine. Examples of such genes include the leuABCD operon genes, which can be represented by the mutant leuA gene encoding isopropylmalate synthase with retroactivated L-leucine depleted (US Patent No. 6,403,342). In addition, the bacterium of the present invention can be improved by enhancing the expression of one or more genes encoding proteins that export L-amino acids to a slot from a bacterial cell. Examples of such genes include the b2682 and b2b83 genes (ud zH genes) (European Patent Application EP 1239041 A2).

As the parent strain can be used bacteria-producers of L-leucine belonging to the genus Escherichia, such as H-9070 (FERM BP-4704) and H-9072 (FERM BP-4706) (US Patent No. 5744331), VKPM B-7386 and VKPM B-7388 (RF Patent No. 2140450), W1485atpA401 / pMWdAR6, W14851ip2 / pMWdAR6, AJ12631 / pMWdAR6 (European Patent Application EP 0872547) and the like.

L-isoleucine producing bacterium

Examples of parent strains that can be used to produce L-isoleucine-producing bacteria include, but are not limited to, 6-dimethylaminopurine resistant mutants (Japanese Patent Application 5-304969A), mutant strains resistant to isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutant strains additionally resistant to DL-ethionine and / or arginine hydroxamate (Japanese application 5-130882A). In addition, recombinant strains transformed with genes encoding proteins involved in the biosynthesis of L-isoleucine, such as threonine deaminase and acetohydroxate synthase (Japanese Patent Application 2-458A, European Patent Application EP 0356739 and US Patent No. 5,998,178) can also be used as parent strains.

Bacteria producing higher alcohols and D-pantothenic acid

The bacterium producing L-amino acids of the Enterobacteriaceae family and, more precisely, the bacterium producing L-branched amino acids of the Enterobacteriaceae family can be used to produce higher alcohols and organic acids, or their derivatives. For example, higher alcohols such as isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol, and an organic acid such as D-pantothenic acid (vitamin B5) can be obtained using a similar bacterium.

It is known in the art that in the bacteria of the Enterobacteriaceae family, the biosynthetic pathway of L-valine, L-leucine and L-isoleucine passes through keto-acid intermediates. More specifically, 2-oxovalerate (2-ketovalerate, 2-KIV) is a precursor for L-valine and L-leucine; and 2-oxobutanoate is a precursor for L-isoleucine. It is also known from the prior art that keto-acid intermediates or their derivatives can be precursors for higher alcohols with a carbon chain length of more than 2 carbon atoms due to the Ehrlich degradation pathway (Yan Y. and Liao JCJ Ind. Microbiol. Biotechnol., 2009 36: 471-479). For example, 2-oxoisovalerate is a precursor for isobutanol, and its derivative 4-methyl-2-oxopentanoate is a precursor for 3-methyl-1-butanol; 2-oxobutanoate is a precursor for propan-1-ol, and its derivative 3-methyl-2-oxopentanoate is a precursor for 2-methyl-1-butanol.

The ability to produce higher alcohols can be imparted to E. coli strains that are initially unable to produce higher alcohols by introducing the kivd and adh2 genes from donor microorganisms encoding, respectively, ketoacid decarboxylase (CDC) with broad substrate specificity and alcohol dehydrogenase (ADH). For example, the kivd gene from Bacillus subtilis and the adh2 gene from Lactococcus lactis can be used. In this aspect, reference can be made to Connor M.R. and Liao J.C., Appl. Env. Mocrobiol., 2008, 74: 5769-5775; and Patent Application WO 2009046370 A2.

Thus, the ability of a bacterium to produce higher alcohols, such as isobutanol, 3-methyl-1-butanol and 2-methyl-1-butanol, can be imparted to the bacteria by introducing a genetic expression system and suitable heterologous bacteria into the bacterium producing the L-amino acid of the Enterobacteriaceae family genes, for example, kivd and adh2 or their variants from donor microorganisms.

It is also known in the art that in bacteria of the Enterobacteriaceae family, 2-oxovalerate is a precursor of D-pantothenic acid. Thus, the ability to produce D-pantothenic acid can be imparted to bacteria by modifying the Enterobacteriaceae family of bacteria with a genetic expression system.

2. Genetic expression system

The expression system may be a regulatory transcription system for one or more target genes, which includes elements of the transcriptional apparatus regulated by a protein of the LysR type. Elements of the transcriptional apparatus regulated by a LysR-type protein may include a promoter, an operator, the expression of which is positively regulated by a LysR-type regulatory protein and a co-inductor.

The expression system may also include one or more target genes that are functionally linked to the aforementioned transcriptional apparatus. The term "operably linked" may mean that the target gene is associated with regulatory sequence (s), such as a promoter or operator in such a way that expression of this gene is possible.

One or more target genes may encode proteins involved in the biosynthesis of a co-inductor, substrate, or precursor of said co-inductor.

In accordance with the above-described scheme of the expression system, the expression of the system can be self-induced and positively regulated by the type of feedback mediated by the co-inductor obtained with the participation of the product (s) of expression of the target gene (s). Such an expression system may be referred to as a “self-induced feedback-regulated expression system,” but for the sake of simplicity, may also be referred to herein as an “expression system” or “expression system”. The expression system can self-regulate on the principle of positive feedback by the co-inductor, which can be a substrate or precursor for a useful metabolite in the biosynthetic pathway, more precisely, in the biosynthetic pathway of branched L-amino acids and / or its side path.

A “regulatory protein of the LysR type” may also be called a “transcriptional regulator of the LysR type (LTTR)”, “a transcriptional regulator of the LysR family”, or simply “regulator”. A regulatory protein of the LysR type may belong to a diverse family of oligomeric bacterial transcription factors that regulate a wide range of transcription elements in response to a wide range of external signals. Representatives of this family can act as transcriptional activators and / or transcriptional repressors and have several common structural characteristics, such as i) a DNA-binding domain having a helix-rotate-helix motif (residues can be from positions 1 to 65 with N- end of LTTR), (ii) the domains involved in the recognition of the co-inductor and / or response (residues can be from positions 100 to 173 and from 196 to 206), and (iii) the domain necessary for DNA and response binding (residues can be from provisions 227 to 253), as described in Schell MA, Ann . Rev. Microbiol., 1993, 47: 597-626. In the absence of a co-inductor, LTTR can bind to regulated promoters via a double double stranded 15 bp region with a common structure and a DNA region at about -65, as described by Schell M.A., Ann. Rev. Microbiol., 1993, 47: 597-626. In the presence of a co-inductor, additional LTTR interactions with RNA polymerase binding sites (about -35) and / or DNA bending can occur, resulting in transcription activation (Schell M.A., Ann. Rev. Microbiol., 1993, 47: 597-626). Moreover, some representatives of the LysR-type regulatory protein family may have four other functional characteristics, such as: (i) be a co-inductor-dependent transcriptional regulator of various sizes from 276 to 324 amino acid residues, (ii) bind to the operator sites of DNA regardless of the presence of the co-inductor having a similar position and structural motive, (iii) multidirectionally transcribed from a promoter that is located close to or even overlaps with the promoter of the regulated gene, and (iv) suppress ennuyu transcription in varying degrees, e.g., from 3 to 10 times, i.e., be negatively self-regulatory, as described by Schell M.A., Ann. Rev. Microbiol., 1993, 47: 597-626.

By way of example, but not limited to, the expression system may include a transcriptional apparatus regulated by a LysR-type protein that contains a self-inducing upregulated promoter, an operator, and a target gene (s). The expression system is a system with positive regulation by a regulatory protein of the LysR type and a co-inductor. An example of a self- _{inducing upregulated} promoter may include the P _ilvC promoter. An example of a regulatory protein of the LysR type may include an IlvY protein. Examples of a co-inductor may include a representative of 2-aceto-2-hydroxycarboxylic acids, such as 2-acetolactic acid (2-acetolactic acid, AL, AΛ), 2-aceto-2-hydroxybutyric acid (2-aceto-2-hydroxybutyric acid, AHB , AGB) or their salts. Examples of target genes may include genes encoding acetolactate synthase I, II and / or III, or mutant variants thereof. An example expression system can be described schematically without limiting the type, amount, and arrangement of elements of the expression system.

The ilvY and ilvC genes are located next to each other on opposite sides of the chain as shown in Figure 1. Transcription of the ilvY and ilvC genes is initiated from divergent overlapping promoters ilvY and ilvC, respectively. However, the terms “ilvC promoter” or “P _ilvC promoter” may mean promoters of the ilvY and ilvC genes. In the promoter region are two tandem operators (Figure 1).

One or more target genes are functionally linked to a transcriptional apparatus, more specifically, to a promoter. For example, if the target genes are ilvBN genes encoding acetolactate synthase I, and when the promoter is the ilvC promoter, then the native portion of the promoter upstream of the ilvBN genes is replaced by a DNA fragment that includes the inducible ilvC gene promoter and the second operator in this way that the expression of these genes is controlled by this promoter. For example, the ilvBN4 operon genes encoding AHAS I, which is resistant to feedback inhibition by L-valine, are placed under the ilvC promoter, which becomes transcriptionally activated only when a complex is formed between AΛ or AHB, the IlvY transcriptional regulator, and the second operator site. The expression cassette, which includes the P _ilvC promoter and the operon of the ilvBN4 genes, allows the ilvBN4 genes to be transcribed for the production of AHAS I. AHAS I converts two moles of pyruvate to one mole of 2-acetolactate (AΛ) or one mole of pyruvate and one mole of 2-oxobutanoate to one mole 2-aceto-2-hydroxybutanoate (AHB) depending on the presence of 2-oxobutanoate.

An advantage of the present invention is that part of the reaction product (AΛ or AHB) catalyzed by AHAS I acts as a co-inductor and binds to IlvY / DNA complex, inducing the transcription of ilvBN4 operon genes and, thus, providing AΛ or AHB synthesis. Another part of AΛ or AHB can be converted to the target product - a branched L-amino acid (L-valine, L-leucine or L-isoleucine) using the IlvC keto-acid reductisomerase and other enzymes from the biosynthetic pathway of the branched L-amino acids as described above.

Continuous supply of the expression system with 2-acetolactate or 2-aceto-2-hydroxybutanoate can give the system the property of self-inducibility; and cyclic co-inductor flow through a self-inducing expression system regulated by the principle of positive feedback can lead to a continuous supply of biosynthesis pathways of branched L-amino acids with precursors such as 3-hydroxy-3-methyl-2-oxobutanoate for L-valine and L-leucine or 3-hydroxy-3-methyl-2-oxopentanoate for L-isoleucine.

The proposed approach for gene expression can be called as a self-induced genetic expression system that has positive regulation by the type of feedback and is mediated by the co-inductor AΛ or AGB.

An expression system that operates on the principle of self-induction with positive feedback is not limited to the expression system mentioned above, including the transcriptional regulator ilvY. The expression system may also include other expression systems using regulatory proteins of the LysR type, which are described in Table 1.

The term "acetolactate synthase" can mean an enzyme found in bacteria, such as bacteria of the Enterobacteriaceae family, bacteria of the genus Corynebacterium, bacteria of the genus Bacillus, etc. Examples of bacteria of the Enterobacteriaceae family can be bacteria belonging to the genus Escherichia, Pantoea, Erwinia, Providencia and Serratia, such as E. coli, Pantoea ananatis (P. ananatis) and the like. An example of a coryneform bacterium may be a bacterium belonging to the genus Corynebacterium, such as Corynebacterium glutamicum. Examples of bacteria belonging to the genus Bacillus may be Bacillus subtilis. Bacillus amyloliquefaciens FZB42 and Bacillus amyloliquefaciens DSM7. The term "acetolactate synthase" may also mean an enzyme having acetolactate synthase activity.

The term "activity of acetolactate synthase" may mean the ability of the enzyme to catalyze the reaction of formation of i) 2-acetolactate and CO ₂ from two pyruvate and / or ii) 2-aceto-2-hydroxybutanoate and CO ₂ molecules from pyruvate and 2-oxobutanoate under appropriate conditions, such as temperature, ionic strength, acidity (pH), concentration of cofactors and substrates, etc. The activity of acetolactate synthase can be measured using the method of Stormer FC and Umbarger HE, Biochem. Biophys. Res. Commun., 1964, 17 (5): 587-592.

Acetolactate synthase I, II or III (AHAS I, AHAS II or AHAS III) (EC 2.2.1.6) may also be referred to as acetolactate synthase. Acetolactate synthase is a heterotetrameric protein with an α ₂ β ₂ type structure consisting of two catalytic and two regulatory domains (Weinstock, O. et al., J. Bacterial., 1992, 174 (17): 5560-55bb). It is believed that the large subunit (approximately 60-kDa) is catalytic, while the small subunit (approximately 11-kDa) is regulatory.

AHAS I is encoded by the ilvB and ilvN genes forming the ilvBN operon. AHAS II is encoded by the ilvG and ilvM genes forming the ilvGMEDA operon. Genes encoding AHAS II are usually not expressed in E. coli K-12 cells (Guardiola J. et al., Mol. Gen. Genet, 1977, 156: 17-25). AHAS III is encoded by the ilvI and ilvH genes forming the ilvIH operon.

AHAS I from E. coli, for example, may have amino acid residue substitutions, such as N17K and / or A30P, which report feedback inhibition resistance by L-valine (RF Patent No. 2355763, US Patent Application No. 2009197309 A1). Also, substitution of alanine at position 33 for any amino acid or several amino acids, such as replacing it with 12 amino acids containing a translation termination site, leads to the IlvN33 protein shortened by 45 amino acid residues (RF Patent No. 2355763, US Patent Application No. 2009197309 A1) . This mutant protein is also resistant to feedback inhibition by L-valine.

The small subunit of the mutant AHAS I containing the substitution N17K (Asn at position 17 is replaced by Lys, i.e., the corresponding AAC codon is replaced by AAG) in the wild type AHAS I, can be encoded by the mutant ilvN gene, which can be designated as the ilvN4 gene or the ilvN ValR4 gene, as described in EP 1942183. AHAS I, having the amino acid sequence shown in SEQ ID NOs: 2 and 4, may be referred to as "wild-type acetolactate synthase I".

Based on the model of the valine-binding region of the regulatory (small) subunit of AHAS III from E. coli, shortening of the carboxyl end of the small subunit was made. Such a modification led to the fact that the shortened AHAS III enzyme lost its sensitivity to valine (Mendel S. et al., J. Mol. Biol., 2003, 325 (2): 275-284).

Acetolactate synthase III, which is shortened from the carboxyl end of the small subunit by 35, 48, 80 or 95 amino acid residues, may be referred to as “mutant acetolactate synthase III”. DNA encoding the small subunit of the mutant AHAS III may be referred to as the “ilvH mutant gene”. The ilvIH operon containing the mutant ilvH gene may be referred to as the "mutant ilvIH operon". AHAS III, having the amino acid sequence shown in SEQ ID NOs: 6 and 8, may be referred to as "wild-type acetolactate synthase III".

Mutant AHAS I may contain a deletion, substitution, insertion or addition of one or more amino acid residues at one or more positions in addition to 17, 30 and / or 33 in the original amino acid sequence, provided that the activity of acetolactate synthase is maintained. In addition, the mutant AHAS III may also contain a deletion, substitution, insertion or addition of one or more amino acid residues at one or more positions in the original amino acid sequence, provided that the activity of acetolactate synthase is maintained. In addition, the carboxyl end of the mutant AHAS III can be shortened by one or more amino acid residues, not limited to 35, 48, 80 or 95 amino acid residues, provided that the activity of acetolactate synthase has not decreased.

The number of “several” amino acid residues varies depending on the position in the tertiary structure of the protein or the type of amino acid residue. This is possible because some amino acids are similar to each other in structure and function within the protein, and the mutual rearrangements of such amino acids do not greatly affect the tertiary structure or function of the protein. AHAS III can be shortened by a sequence of amino acid residues of various lengths until the truncated enzyme is able to bind and activate the catalytic (large) subunit, so that the mutant AHAS III remains active. Thus, mutant AHAS I, II and III can have a homology of at least 65%, at least 80%, at least 90%, at least 95%, at least 97% or at least 99% compared with the initial amino acid sequence of acetolactate synthase, provided that the activity of acetolactate synthase is maintained. In this regard, in the present description, the term "homology" may mean "identity".

Mutant AHAS I can be obtained by introducing mutations into the wild-type ilvN gene using known methods. For example, the mutant operon ilvBN4, which contains the ilvB gene and the mutant ilvN4 gene, can be obtained by PCR (polymerase chain reaction), as described in White T.J. et al., Trends Genet., 1989, 5: 185-189, using primers selected based on the nucleotide sequence of the UvN gene (SEQ ID NO: 3). Genes encoding acetolactate synthase from other microorganisms can be obtained in a similar way.

A mutant AHAS III with a truncated carboxyl end of the small subunit can be obtained by site-directed mutagenesis using PCR, which allows DNA fragments with overlapping regions to be obtained by introducing stop codons at the desired position of the UvH gene (But SN et al., Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene, 1989, 77: 51-59). PCR primers suitable for the synthesis of ilvIH operon genes can be selected based on the nucleotide sequence of the ilvH gene (SEQ ID NO: 7).

The ilvB gene encodes the large subunit of acetolactate synthase I (KEGG, Kyoto Encyclopedia of Genes and Genomes, Kyoto Encyclopedia of Genes and Genomes, No.b3671). The ilvB gene (GenBank accession number NC_000913.2; nucleotides complementary to nucleotides at position 3849119 to 3850807; Gene ID: 948182) is located between the ilvN and ivbL genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvB gene and the amino acid sequence of the large subunit of acetolactate synthase I encoded by the ilvB gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The ilvN gene encodes a small subunit of acetolactate synthase I (KEGG, No.b3670). The ilvN gene (GenBank accession number NC_000913.2; nucleotides complementary to nucleotides at position 3848825 through 3849115; Gene ID: 948183) is located between the uhpA and ilvB genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvN gene and the amino acid sequence of the small subunit of acetolactate synthase I encoded by the ilvN gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The ilvI gene encodes a large subunit of acetolactate synthase III (KEGG, No.b0077). The ilvI gene (accession number in GenBank NC_000913.2; nucleotide positions 85630 to 87354; Gene ID: 948793) is located between the leoO and ilvH genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvI gene and the amino acid sequence of the large subunit of acetolactate synthase III encoded by the ilvI gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The ilvH gene encodes the small subunit of acetolactate synthase III (KEGG, No.b0078). The ilvI gene (accession number in GenBank NC_000913.2; nucleotide positions 87357 to 87848 Gene ID: 947267) is located between the ilvI and fruR genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvH gene and the amino acid sequence of the small subunit of acetolactate synthase III encoded by the ilvH gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The ilvY gene encodes the DNA-binding double transcriptional regulator ilvY (transcriptional regulator of the LysR family, positive regulator for ilvC) (KEGG, No.B3773). The ilvY gene (accession number in GenBank NC_000913.2; nucleotides complementary to nucleotides at position 3954950 through 3955843; Gene ID: 948284) is located between the ilvA and ilvC genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvI gene and the amino acid sequence of the IlvY protein encoded by the ilvY gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

The ilvG gene is a pseudogen (KEGG, No.b4488). The ilvG gene (accession number in GenBank NC_000913.2; nucleotide positions 3948583 through 3950227; Gene ID: 2847699) is located between the ilvX and ilvM genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvG gene is shown in SEQ ID NO: 31. The ilvG gene can encode the large subunit of the acetolactate synthase II, provided that it contains the replacement of the TGA codon with the AAT codon at position 982 through 984 from the start of the gene, or a mutation as described in Lawther R.P. et al., J. Bacteriol., 1982, 159: 294-298. The amino acid sequence of the large subunit of acetolactate synthase II encoded by the ilvG gene, in which the TGA codon is replaced by the AAT codon at positions from 982 to 984, is shown in SEQ ID No: 32.

The ilvM gene encodes the small subunit of acetolactate synthase II (KEGG, No.b3769). The ilvM gene (GenBank accession No. NC_000913.2; nucleotide positions 3950224 through 3950487; Gene ID: 948279) is located between the ilvG and ilvE genes on the E. coli K-12 chromosome. The nucleotide sequence of the ilvM gene and the amino acid sequence of the small subunit of acetolactate synthase II encoded by the ilvM gene are shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.

Since there may be some differences in DNA sequences between genera or strains of bacteria of the Enterobacteriaceae family, the ilvB, ilvN, ilvI, ilvH, ilvG and ilvM genes encoding acetolactate synthase and the ilvY gene encoding a transcriptional regulator are not limited to the genes represented in SEQ ID NOs : 1, 3, 5, 7, 31, 33, and 9, but may include genes that are variants or homologues of the sequences shown in SEQ ID NOs: I, 3, 5, 7, 31, 33, and 9, and which encode protein variants IlvB, IlvN, IlvI, IlvH, IlvG, IlvM and IlvY.

The term "protein variant" used in the present invention may mean a protein that contains one or more changes in the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 32, 34 and 10, which may be substitutions, division, insertion and / or addition of one or more amino acid residues, provided that the activity of such a protein is the same as that of the proteins IlvB, IlvN, Ilvl, IlvH, IlvG, IlvM and IlvY, respectively. The number of changes in protein variants depends on the type or position of the amino acid residue in the tertiary structure of the protein. It can be from 1 to 30, or from 1 to 15, or from 1 to 5 in SEQ ID NO: 2, 4, 6, 8, 32, 34 and 10

Substitution, division, insertion and / or addition of one or more amino acid residues can be conservative mutations, provided that the activity and / or function of the protein variant is preserved or similar to the activity of the IlvB, IlvN, IlvI, IlvH, IlvG, IlvM and IlvY proteins. An example of a conservative mutation may be a conservative substitution. Conservative substitutions may be reciprocal substitutions between Ser or Thr for Ala, substitutions of Gln, His or Lys for Arg; replacement of Glu, Gln, Lys, His or Asp with Asn; replacement of Glu or Gln with Asp; replacement of Ser or Ala with Cys; replacement of Asn, Glu, Lys, His, Asp or Arg with Gln; replacing Gly, Asn, Gln, Lys, or Asp with Glu; replacing Pro with Gly; replacing Asn, Lys, Gln, Arg, or Trp with His; replacing Leu, Met, Val or Phe with Ile; replacing Ile, Met, Val or Phe with Leu; replacement of Asn, Glu, Gln, His or Arg with Lys; replacing Ile, Leu, Val or Phe with Met; replacing Trp, Tyr, Met, Ile or Leu with Phe; replacement of Thr or Ala with Ser; replacement of Ser or Ala with Thr; replacing Phe or Tyr with Trp; replacing His, Phe or Trp with Tyr; and replacing Met, Ile, or Leu with Val.

Homology between proteins or DNA can be determined using several well-known approaches, for example, BLAST and FASTA computer algorithms and the ClustalW method. The BLAST algorithm (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/), which allows for hierarchical search, is embedded in the programs blastp, blastn, blastx, megablast, tbiastn and tbiastx; these programs assign significance to found objects using the statistical methods described in Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-2268; "Applications and statistics for multiple high-scoring segments in molecular sequences" (Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5877). The BLAST algorithm computes three parameters: the number of amino acid residues, identity, and similarity. The FASTA search algorithm is described in Pearson W.R. (“Rapid and sensitive sequence comparison with FASTP and FASTA”, Methods Enzymol., 1990, 183: 63-98). The ClustalW method is described in Thompson J.D. et al. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.” Nucleic Acids Res., 1994, 22: 4673-4680).

Moreover, the genes ilvB, ilvN, ilvI, ilvH, ilvG, ilvM and ilvY can be variants of nucleotide sequences.

The term "nucleotide sequence variant" may mean a nucleotide sequence encoding a "protein variant". The term "variant nucleotide sequence" can also mean a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequences shown in the Sequence Listing under the numbers SEQ ID NOs: 1, 3, 5, 7, 31, 33 or 9, or probe, which can be synthesized based on the indicated nucleotide sequences under stringent conditions, provided that it encodes a functional acetolactate synthase or regulatory protein before inactivation. "Harsh conditions" means those conditions in which a specific hybrid is formed, for example, a hybrid having a homology of not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96% , not less than 97% or not less than 99%, and in which a non-specific hybrid is formed, for example, a hybrid having a homology lower than that indicated above. A practical example of harsh conditions is a one-time, two- or three-time washing at a salt concentration of 1 × SSC, 0.1% SDS or 0.1 × SSC, 0.1% SDS, at 60 ° C or 65 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for a Hybond ™ -N + positive charge nylon membrane (GE Healthcare) under severe conditions is 15 minutes. Rinsing can be done two or three times. As a probe, a portion of the sequence complementary to the sequence shown in SEQ ID NOs: 1, 3, 5, 7, 31, 33 or 9 can be used. A similar probe can be obtained by PCR using oligonucleotide primers selected based on the sequences shown in SEQ ID NOs: I, 3, 5, 7, 31, 33 or 9, and a DNA fragment containing the nucleotide sequence as a template. The recommended probe length can be selected depending on hybridization conditions and is> 50 bp, usually about 100-1000 bp For example, when using a DNA fragment of about 300 bp in length as a probe washing conditions can be as follows: 2 × SSC, 0.1% SDS at 50 ° C, 60 ° C or 65 ° C.

Since the genes encoding the proteins IlvB, IlvN, IlvI, IlvH, IlvG, IlvM and IlvY from E. coli are known (see above), the nucleotide sequences encoding the proteins IlvB, IlvN, IlvI, IlvH, IlvG, IlvM and IlvY, can be obtained by PCR (polymerase chain reaction), as described in White TJ et al., Trends Genet., 1989, 5: 185-189 using primers selected based on the nucleotide sequence of the ilvB, ilvN, ilvI, ilvH, ilvG, ilvM and ilvY genes. Genes encoding the IlvB, IlvN, IlvI, IlvH, IlvG, IlvM and IlvY proteins or variants thereof can be obtained in a similar manner.

The term "self-induced positively regulated promoter" may mean a promoter known to a person skilled in the art. A traditionally self-induced, positively regulated promoter may mean a promoter that has: a) increased activity if the level of the transcription factor or activator is increased, i.e. positive regulation takes place; b) the absence or low activity in the absence of a transcription factor or activator, however, there is a promoter activation if it is associated with a transcription factor or activator, i.e. induction takes place; and c) can be induced by the expression product of a coding gene controlled by this promoter, including any post-translationally modified expression product, as well as its analogues and derivatives or complexes formed by it, including products such as individual substances or their complexes resulting from activity coding gene expression product, i.e. self-induction takes place.

An example of a self-induced positively regulated promoter is the Puvc promoter (SEQ ID NO: 30) (Wek RC and Hatfield GW, J. Biol. Chem., 1986, 261 (5): 2441-2450; Opel ML and Hatfield GW, Mol Microbiol, 2001, 39 (1): 191-198), located in front of the structural part of the ilvC gene, the P _cysP promoter, located in front of the cysPUWAM transcription unit, the P _cysK promoter, located in front of the cysK gene, and the P _metR promoter structural part of the metE gene. The P _ilvC promoter can be regulated by the IlvY / AΛ or IlvY / AGB induction complex. The promoters P _cysP and P _cysK can be regulated by the CysB / O-acetyl-L-serine self-induction complex. The P _metR promoter can be regulated by a MetR / L-homocysteine self-induction complex. Self-induced positively regulated promoters are not limited, and may include the substitution, deletion, insertion or addition of one or more nucleotide residues, provided that the functionality of the promoter is preserved.

The term "self-induced negatively regulated promoter" may mean a promoter known to a person skilled in the art. A traditionally self-induced negatively regulated promoter may mean a promoter that has: a) reduced activity if the level of the transcription factor or activator is lowered, i.e. negative regulation takes place; b) the absence or low activity in the presence of a transcription factor or activator, however, there is an activation of the promoter if the boundary concentration of the transcription factor or activator is reached, i.e. induction takes place; and c) can be deregulated by the expression product of a coding gene controlled by this promoter, including any post-translationally modified expression product, as well as its analogues and derivatives or complexes formed by it, including products such as individual substances or their complexes resulting from activity coding gene expression product, i.e. self-induction takes place.

The term “self-inducing promoter” can also mean “self-regulating promoter”, “self-regulating promoter” or the like, regardless of the positive or negative type of regulation.

The term "expression system is modified in such a way that the regulation of the indicated system, which operates on the principle of self-induction with positive feedback, is mediated by the co-inductor" may mean that the expression system is responsible for the synthesis of the co-inductor by regulating the expression of the gene encoding the enzyme, which is responsible for the synthesis of the co-inductor. The specified co-inductor can bind to the regulatory protein / DNA complex to activate transcription of the gene encoding the protein, which is responsible for the synthesis of the co-inductor. Thus, it is obvious that the functionality or regulation of said expression system can be mediated by a co-inductor.

The term "inducible expression system" may mean an expression system in which the level of transcription can be changed at least 1.5 times, or at least 2 times, or at least 3 times, or at least 5 times or not less than 10 times, or not less than 15 times, or not less than 20 times, or not less than 30 times, or not less than 100 times, or more. The term “inducible expression system” can include any expression system that can be induced, for example, by a co-inductor, such as 2-acetolactate, 2-acetohydroxy-2-butanoate, arabinose, lactose, IPTG, etc .; an inducer, such as a regulatory protein, belonging to a protein of the LysR type family; external influences, for example, heating, cooling, etc .; or growth conditions, such as cell density, acidity (pH), etc. The term "inducible expression system" may also include any expression system that can be repressed, for example, an expression system that can be repressed by the addition of chemical compounds, proteins, under the influence of external factors, etc.

The term “target gene” may mean a gene whose expression level is to be deregulated using an expression system. As examples of target genes, the gene (s) encoding (e) the protein (s) involved (e) in the biosynthesis of a co-inductor, or substrate or precursor of a co-inductor, or co-inductor synthase can be cited. More specifically, examples of target genes can be genes encoding the large and / or small subunit (s) of AHAS I, II and / or III, or mutant variants thereof. An example of target genes is the ilvBN4 operon encoding a mutant AHAS I resistant to feedback inhibition by L-valine.

The term "deregulation of the expression level" of the target gene (s) may mean weakening, inactivation or increased expression of the specified gene (s); increased expression is one example.

The term "operator site" may mean a DNA fragment that is functionally linked to the promoter site and affects the transcription of a gene located under the promoter. An example of an operator site may be a DNA fragment operably linked to PilvC or PilvY promoters and to which an IlvY transcriptional regulator can bind.

The term “regulator of the gene (s) of the expression system”, which may also be called “regulator of the self-induced system with positive reverse regulation” or “regulator,” can mean a protein belonging to the family of regulatory proteins of the LysR type, capable of directly or indirectly regulating gene transcription . Examples of regulatory proteins are shown in Table 1.

The term "gene inactivation" may mean that the modified gene encodes a completely inactive or non-functional protein. It is also possible that the modified portion of the DNA is not capable of naturally expressing the gene due to deletion of part of the gene or the whole gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s) or modification of the adjacent portion of the gene, inclusion of sequences that control gene expression, such as a promoter (s), enhancer (s), attenuator (s), ribosome binding site (s) (RBS, ribosome binding site), etc. Gene inactivation can also be carried out by conventional methods, such as mutagenesis by treating microorganisms, for example, UV irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, disruption of the gene structure using homologous recombination and / or mutagenesis by insertion deletion (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 5978-83; Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97 (12): 6640-45), also referred to as “λRed dependent integration”.

The term “increased gene expression” or “gene expression increased” may mean that due to genetic modification, the expression level of the mutant gene is higher than the expression level of the gene in an unmodified strain, for example, a wild-type strain or a parent strain, such as E. coli MG1655 strain , E. coli K-12 (VKPM B-7) or E. coli B7 ΔilvGM ΔilvIH P _L -ilvBN4. Examples of such modifications include an increase in the number of copies of expressed genes in a cell and / or an increase in gene expression by modification of a region adjacent to the gene, including sequences that control gene expression, such as promoters, enhancers, attenuators, ribosome binding sites, etc., and other examples. Methods that can be used to increase gene expression may also include introducing the gene into a vector that is capable of increasing the copy number of the gene in Enterobacteriaceae family bacteria. Examples of such vectors include, but are not limited to, vectors with a wide range of hosts (broad-host-range vectors), for example, pCM110, pRK310, pVK101, pBBR122, pBHR1 and the like. Increased gene expression can also be achieved by introducing multiple copies of the gene into the bacterial chromosomal DNA, for example, by homologous recombination, Mu integration, or similar methods.

Enhanced expression of the gene (s) and / or operon genes can also be achieved by substituting a DNA fragment under the control of a strong promoter. Examples of strong promoters are the lac promoter, trp promoter, trc promoter, tac promoter, P _R or P _L phage λ promoters. Strong promoters providing a high level of gene expression in bacteria belonging to the Enterobacteriaceae family can be used in this invention. On the other hand, the action of the promoter can be enhanced, for example, by introducing a mutation into the promoter region of a gene on the bacterial chromosome, which leads to an increase in the level of transcription of the gene under the promoter. In addition, it is known that the replacement of several nucleotides in the region between the ribosome binding site and the start codon, especially in the sequence immediately before the start codon, significantly affects the translational ability of mRNA. For example, a 20-fold variation in the level of gene expression was found depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol., 1981, 35: 365-403; Hui A. et al. , EMBO J., 1984, 3: 623-629). The use of a strong promoter can be combined with an increase in the number of copies of the gene.

The number of copies, the presence or absence of the gene and / or operon genes can be measured, for example, by restriction of chromosomal DNA followed by Southern blotting using a probe selected based on the gene sequence, in situ fluorescence hybridization (fluorescence in situ hybridization , FISH) and similar methods. The level of expression of the gene and / or operon genes can be determined using various known methods, including measuring the amount of transcribed mRNA using well-known methods, for example, Northern blotting, quantitative RT-PCR (RT-PCR), etc. The amount of protein encoded by the gene can be determined by known methods, including SDS-PAGE (SDS-PAGE) electrophoresis followed by Western blotting (Western blotting analysis), and the like.

Methods for preparing plasmid DNA, DNA restriction and ligation, transformation, selection of nucleotides as a primer, etc. may be conventional methods known to a person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual, 2 ^nd ed.". Cold Spring Harbor Laboratory Press (1989). Molecular cloning and expression of heterologous genes are described in Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, Molecular Biotechnology: principles and applications of recombinant DNA, 4 ^th ed., Washington, DC: ASM Press (2009) ; Evans Jr., TC and Xu M. - Q., “Heterologous gene expression in E. coli”, 1 ^st ed., Humana Press (2011).

The term “gene encoded enzyme activity is increased” may mean that the total activity of the enzyme per cell or the specific activity per molecule of the enzyme is higher than the total and / or specific activity of the enzyme in an unmodified strain, for example, a wild-type strain or parent strain. To compare the activity of the enzyme, a strain of E. coli K-12 containing wild-type acetolactate synthase can be taken as an unmodified strain.

The term “biosynthetic pathway,” which can also be called a “metabolic pathway” or “biochemical pathway,” can mean a set of anabolic or catabolic (bio) chemical reactions to convert a biomolecule of one species into a biomolecule of another species, for example, a more complex species towards target end product or beneficial metabolite. The term "biosynthetic pathway" is usually understood by a person skilled in the art. The desired end product may be an L-amino acid, nucleoside, nucleotide, cofactor, lower or higher alcohol, organic acid, their derivatives, protein, etc.

The term "substrate" can mean a chemical or biochemical compound (substance) of any kind that can be converted or is supposed to be converted into a substance or compound of another type by the action of an enzyme. The term “substrate” can mean not only a single substance, but also a combination of compounds, such as solutions, mixtures, and other substances that contain at least one substrate or its derivative (s). The term “substrate” can also mean compounds that act as an acceptable carbon source for the synthesis of chemical and biochemical substances, which may be, for example, sugar derived from biomass, intermediates or end metabolites that are used in the biosynthetic pathway of a modified metabolism microorganism. Various types of chemical or biochemical substances and compounds can be converted into the desired end product as a result of a single enzyme-catalyzed reaction. Examples of substrates include pyruvate and 2-oxobutanoate.

The term "precursor" can mean a chemical or biochemical compound (substance) of any kind from which another, more complex chemical or biochemical compound (substance) can be obtained in the direction of the target end product or beneficial metabolite. In the biosynthesis of L-valine, examples of precursors include pyruvate, 2-acetolactate, 3-hydroxy-3-methyl-2-oxobutanoate, 2,3-dihydroxy-3-methylbutanoate and 2-oxoisovalerate. In the biosynthesis of L-leucine, examples of precursors can be pyruvate, 2-acetolactate, 3-hydroxy-3-methyl-2-oxobutanoate, 2,3-dihydroxy-3-methylbutanoate and 2-oxoisovalerate, 2-isopropyl malate, etc. In the biosynthesis of L-isoleucine, examples of precursors can be L-threonine, pyruvate, 2-aceto-2-hydroxybutanoate, 3-hydroxy-3-methyl-2-oxopentanoate, 2,3-dihydroxy-3-methylpentanoate, etc.

The terms “substrate” and “predecessor” may be used interchangeably.

The term "biomolecule" can mean a chemical or biochemical compound (substance) of any kind, a product obtained using a microorganism. Biomolecules can be proteins, polysaccharides, lipids, nucleic acids and small molecules, such as primary metabolites, secondary metabolites and natural substances.

The term "beneficial metabolite" can mean a chemical or biochemical compound (substance) of any kind, obtained with the help of a microorganism for industrial, feed, food, pharmacological or other purposes. Branched L-amino acids such as L-valine, L-isoleucine and L-leucine can be mentioned as useful metabolites; higher alcohols such as isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol; and an organic acid such as D-pantothenic acid.

The expression system can be introduced into the bacterium as described above to enhance gene expression, for example, using a vector containing the expression system. The expression system can also be introduced into the bacterium by substituting the native promoter of the target gene (s) with a self-induced promoter. The bacterium may contain a gene encoding a regulatory protein of the LysR type, which positively regulates the promoter. Expression of a gene encoding a regulatory protein of the LysR type can be enhanced, however, this amplification is not significant. In the case where the promoter is the ilvC promoter, the regulatory protein of the LysR type is the ilvY protein, endogenous expression of the ilvY gene is usually sufficient.

3. A method for producing beneficial metabolites, such as L-amino acids, higher alcohols and D-pantothenic acid. A method for producing beneficial metabolites, more specifically, L-amino acids, especially branched L-amino acids, such as L-valine, L-leucine and L -isoleucine, higher alcohols such as isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol and organic acids such as D-pantothenic acid may include the steps of growing the bacteria in a nutrient medium for the purpose of formation, excretion and accumulation beneficial metabolite in a nutrient medium and L-extraction acids, higher alcohol and / or organic acids from the culture fluid.

Cultivation (cultivation), isolation and purification of metabolites from the culture fluid can be carried out by methods similar to traditional methods of fermentation, in which an amino acid, higher alcohol or organic acid is produced by a microorganism. The nutrient medium used to produce beneficial metabolites may be a normal nutrient medium that contains sources of carbon, nitrogen, inorganic ions and other necessary organic components. Various carbohydrates can be used as a carbon source, such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and starch hydrolysates; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid and succinic acid; and the like. As a nitrogen source, various inorganic ammonium salts can be used, such as ammonium sulfate, ammonium chloride and ammonium phosphate; organic nitrogen such as soybean hydrolysates; aqueous ammonia; and the like. It is desirable that vitamins such as vitamin B ₁ , essential substances, for example, organic additives, such as nucleic acids, adenine and RNA, or yeast extract and the like. could be present in appropriate or even minimal amounts. In addition, if necessary, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions and the like can be added.

Cultivation can be performed under aerobic conditions for 16 to 72 hours at a temperature in the range of 30-45 ° C or in the range of 30-37 ° C with an acidity (pH) of the nutrient medium in the range of 5-8 or 6.5-7, 2. The pH can be adjusted using inorganic or organic acids or bases, such as, for example, gaseous ammonia. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid residues such as cells and cell debris can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the target L-amino acid or organic acid can be isolated and purified using any combination of traditional methods, such as ion exchange chromatography, concentration and crystallization. Higher alcohols can be isolated from the culture fluid, for example, by distillation followed by purification by repeated distillation or chromatography.

Examples

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

Example 1. Construction of an expression cassette regulated by acetohydroxy acid and analysis of its expression using the lacZ reporter gene in various genetic environments

IlvYC gene transcription is well known (K. Rhee et al., Proa. Natl. Acad. Sci. USA, 1999, 96 (25): 14294-14299; Opel ML and Hatfield GW Mol. Microbiol., 2001, 39 (1) : 191-198). The authors of the present invention have studied the possibility of using the ilvC gene promoter for a metabolically regulated expression system. For this purpose, the cat gene was first introduced after the ilvY gene on the chromosome of E. coli strain MG1655 (ATCC 47076) using λRed-dependent integration. The DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR (polymerase chain reaction) using oligonucleotide primers P1 (SEQ ID NO: 11) for plot ilvY-attL and P2 (SEQ ID NO: 12) for plot attR-ilvYn plasmids pMW118-λattL-cat-λattR (Katashkina Zh.I. et al., Mol. Biol. (Mosk.), 2005, 39 (5): 823-831) as a matrix. The obtained DNA fragment was introduced into the E. coli strain MG1655 / pKD46 by electrotransformation using a Bio-Rad electroporator (USA) (No. 165-2098, version 2-89) in accordance with the manufacturer's instructions. As a result, the E. coli transformant MG1655cat-ilvY was obtained, which contained a chloramphenicol resistance marker (λattL-cat-λattR, Cm ^R ) on the chromosome above the ilvY gene. The recombinant plasmid pKD46 (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 6640-6645) with a temperature-sensitive replicon was used as a donor of phage λ genes responsible for ARed-dependent recombination the system. The E. coli strain MG1655 containing the recombinant plasmid pKD46 can be obtained from E. coli Genetic Stock Center, Yale University, New Haven, USA, accession number CGSC7669. After integration of the plasmid pKD46 into the E. coli strain MG1655, the E. coli strain MG1655 / pKD46 was obtained.

The cat-ilvY-Puvc fragment containing λattL-cat-λattR, the ilvY gene and the ilvY-ilvC intergenic region with the P _ilvC promoter were obtained by PCR using oligonucleotide primers P3 (SEQ ID NO: 13) for the attL-lacZ region and P4 primer (SEQ ID NO: 14) for the plot of ilvCp-lacZ and chromosomal DNA of strain E. coli MG1655 cat-ilvY as a matrix. The resulting DNA fragment was inserted into the chromosome of E. coli strain MG1655 / pKD46 above the lacZ gene using λRed-dependent integration. As a result, the E. coli strain MG1655 cat-ilvY-P _ilvC - _{lacZ was} obtained. Chloramphenicol resistant (Cm ^R) colonies were selected on plates containing lysogenic medium (lysogenic broth, LB) (Sambrook , J. and Russell, DW ( 2001) Molecular Cloning: A Laboratory Manual (3 ^{rd ed) Cold Spring Harbor Laboratory.} . Press), agar 1.5% and chloramphenicol 40 mg / L.

The insertion of the fragment was checked by PCR. For this purpose, colonies that were grown for 24 h were checked for the presence of a cat-ilvY-P ilvC- _lacZ fragment instead of the native lacZ gene by PCR using oligonucleotide primers P13 (SEQ ID NO: 23) and P14 (SEQ ID NO: 24 ) For this, a fresh isolated colony was suspended in 20 μl of water, and 1 μl of the resulting suspension was used for PCR. The temperature profile was as follows: initial denaturation of DNA for 5 min at 94 ° C; then 30 cycles: denaturation for 30 sec at 94 ° C, annealing for 30 sec at 53 ° C, elongation for 3 min at 72 ° C; and final elongation for 7 min at 72 ° C. Several analyzed Cm ^R colonies should contain the required DNA fragment 3053 bp long, which confirms the presence of the cat-ilvY-P _ilvC - _lacZ fragment instead of the native lacZ gene 288 bp long. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by culturing at 37 ° C. Thus, E. coli strain MG1655 cat-ilvY-P _ilvC - _{lacZ was} obtained.

In order to obtain data on the expression level of the ilvBN and ilvBNA genes encoding AHAS I, the expression level of the lacZ reporter gene was evaluated under the control of the P _ilvC promoter under various genetic environments (Table 2). Strains additionally containing a deletion of ilvAYC genes were also analyzed. The ΔilvAYC-Km ^R modification was introduced using P1 transduction (Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor).

All modifications were combined using P1 transduction and the resulting strains were cured of “cut out” markers as described in EP 1942183.

The ilvAYC deletion was constructed in two steps using ARed-dependent integration. First, a PCR fragment was prepared using oligonucleotide primers P5 (SEQ ID NO: 15) and P6 (SEQ ID NO: 16) and plasmids pMW118-λattL-kan-λattR (Katashkina Zh.I. (2002) Development of the methods for targeted modification of E. coli genetic loci for the construction of amino-acids-producing strains. PhD Thesis. Moscow) as a matrix containing a kanamycin resistance marker (λattL-kan-λattR, Km ^R ). Then, the resulting fragment was introduced into E. coli MG1655 / pKD46 by electrotransformation, as described above. Kanamycin-resistant ilvAYC gene clones were selected on kanamycin-containing plates as described above. Thus, the E. coli strain MO1655ΔilvAYC :: Km ^R was obtained.

The insertion of the fragment was checked by PCR. For this purpose, the colonies that were grown for 24 h were checked for the presence of the ΔilvAYC :: Km ^R fragment instead of native ilvAYC genes by PCR using primers P15 (SEQ ID NO: 25) and P16 (SEQ ID NO: 26). For this purpose, a fresh isolated colony was suspended in 20 μl of water, and 1 μl of the resulting suspension was used for PCR. The temperature profile was as follows: initial denaturation of DNA for 5 min at 94 ° C; then 30 cycles: denaturation for 30 sec at 94 ° C, annealing for 30 sec at 57 ° C, elongation for 2 min at 72 ° C; and final elongation for 7 min at 72 ° C. Several analyzed Km ^R colonies should contain the required 1761 bp DNA fragment, which confirms the presence of the ΔilvAYC :: Km ^R fragment instead of native ilvAYC genes 2926 bp long. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by culturing at 37 ° C. Thus, the E. coli strain MG1655 ΔilvAYC :: Km ^R was obtained.

Modifications cat-ilvY-P _ilvC- lacZ, ΔilvAYC :: Km ^R , ΔilvGM, ΔilvIH and ΔilvBN, P _L -ilvBN and P _L -ilvBN4 were introduced into E. coli strain K-12 (VKPM B-7) using P1- transduction. The construction of ΔilvBN, ΔilvGM, ΔilvH and P _L -ilvBN is described in EP 1942183. The VKPM B-7 strain (also known as B7) can be replaced by another K-12 sub-strain, such as K-12 MG1655, which is available from the American Type Culture Collection " American Type Culture Collection, (ATCC) ”(PO Box 1549, Manassas, VA 20108, United States of America). The resulting strains are shown in Table 2.

To measure β-galactosidase activity, the strains were grown to the log average growth phase on M9: LB medium (9: 1, v / v) containing glucose (0.4%, weight / v). The medium for strains having a deletion of ilvAYC and strains with insufficient AHAS additionally contained Ile (25 mg / L) and Val (25 mg / L). Β-galactosidase activity was measured according to Miller's method (Miller J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor). The data presented in Table 2 indicate that the expression level of the lacZ reporter gene under the Pave promoter varies within more than two orders of magnitude depending on the genetic environment. The maximum expression level is achieved in E. coli strains in which ilvBN4 genes are overexpressed, encoding AHAS I resistant to L-valine inhibition, or containing inactivated IlvC isomereductase encoded by ilvC gene.

Example 2. Construction of a mutant strain of E. coli B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4

The strain E. coli B7 ΔilvGM ΔilvIH P _L -ilvBN4 was modified so that the strain contained the cat-ilvY-P _{ilvC fragment} obtained as described in Example 1. The phage promoter P _L located in front of the ilvBN4 genes was replaced by the cat-ilvY- regulatory region P _ilvC using λRed-dependent integration in E. coli strain B7 ΔilvIH ΔilvGM P _L -ilvBN4 (the construction of this strain is described in EP 1942183). For this purpose, a DNA fragment containing a cat-ilvY-P _ilvC expression cassette flanked by short regions adjacent to the ilvB gene was obtained by PCR using oligonucleotide primers P7 (SEQ ID NO: 17) and P8 (SEQ ID NO: 18) and chromosomal DNA of E. coli strain MG1655 cat-ilvY-P _ilvC- lacZ as a template. The obtained PCR fragment was introduced into the E. coli strain B7 ΔilvGM ΔilvIH P _L -ilvBN4 / pKD46 by electrotransformation as described above. Thus, E. coli strain B7 ΔilvGM ΔilvIH cat-ilvY-P _{ilvC -ilvBN4 was} obtained in which the λ phage promoter P _L located above the ilvBN4 operon genes encoding feedback resistance AHAS I is replaced by a self-induced P promoter _ilvC .

Replacement was checked by PCR. To this end, a fresh, isolated colony was suspended in 20 μl of water, and 1 μl of the resulting suspension was used for PCR. The temperature profile was as follows: initial denaturation of DNA for 5 min at 94 ° C; then 30 cycles: denaturation for 30 sec at 94 ° C, annealing for 30 sec at 59 ° C, elongation for 2 min at 72 ° C; and final elongation for 7 min at 72 ° C. Several analyzed Cm ^R colonies should contain the required 2865 bp DNA fragment, which confirms the presence of the cat-ilvY-P _ilvC - _ilvBN4 fragment instead of the original 382 bp P _L -ilvBN4 fragment. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by culturing at 37 ° C. Thus, E. coli strain B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC - _{ilvBN4 was} obtained.

Example 3. Properties of the mutant strain of E. coli B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4

Cells were grown to the mid-logarithmic growth phase on M9: LB medium (9: 1, v / v) containing glucose (0.4%, weight / v). AHAS I activity was measured in crude cell extracts with or without 10 mM L-Val as described in Stormer F. and Umbarger N., Biochem. Biophys. Res. Commun., 1964, 17 (5): 587-592. The average results of the three experiments are presented in Table 3. As follows from Table 3, the cat-ilvY-PilvC regulatory region positively affects the expression level of genes encoding AHAS I.

Example 4. Production of L-valine by E. coli strain B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4

The modified E. coli strain B7 ΔlvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4 and the control strain B7 ΔlvGM ΔluIH P _L -ilvBN4 were separately grown at 32 ° C for 18 hours in Luria-Bertani medium (also called lysogenic medium, as described in Sambrook, J. and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (3 ^{rd ed.) Cold Spring Harbor Laboratory} Press).. Then 0.2 ml of the obtained medium was introduced into 2 ml of fermentation medium in 20 × 200 mm test tubes and grown at 30 ° C for 60 hours on a rotary shaker at 250 rpm.

The composition of the enzymatic medium (g / l):

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 15.0 KH ₂ PO ₄ 1,5 MgSO ₄ × 7H ₂ O 1,0 Thiamine HCl 0.1 CaCO ₃ 25,

with the addition of LB medium: 10% (v / v).

The fermentation medium was sterilized at 116 ° C for 30 minutes, glucose and CaCO _{3 were} sterilized separately under the following conditions: glucose at 110 ° C for 30 minutes, CaCO ₃ at 116 ° C for 30 minutes, the pH was maintained at 7.0 KOH solution.

After growing, the accumulated L-valine was measured by thin layer chromatography (TLC). TLC plates (10 × 20 cm) were coated with a layer (0.11 mm) of Sorbfil silica gel containing a non-fluorescent indicator (Sorbpolymer, Krasnodar, Russian Federation). Samples were applied to the plates using a Camag Linomat 5 applicator. The iso-propanol: ethyl acetate: 25% aqueous ammonia: water mixture (16: 16: 5: 10, v / v) was used as the mobile phase. For visualization, a solution of ninhydrin (2%) in acetone was used. After staining, the plates were dried and scanned using Camag TLC Scanner 3 in the absorption mode with detection at 520 nm, the data were processed using winCATS software (version 1.4.2).

The average results of four independent in vitro fermentations are shown in Table 4. As follows from Table 4, the modified E. coli strain B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC- ilvBN4 caused a greater accumulation of L-valine compared to the original E. coli strain B7 ΔilvGM ΔilvIH P _L -ilvBN4.

Example 5. Construction of a strain of E. coli B7 ΔilvGM ΔilvIH ilvY ^inactive -P ^ilvC -ilvBN4

The expression cassette ilvY-P _ilvC -ilvBN4 ilvY comprises a gene encoding a regulatory protein type LysR. Thus, the strain containing this cassette has two copies of the ilvY gene: one copy is contained in the native locus on the chromosome, and the other copy is located in front of the ilvBN4 operon. To find out whether the positive effect of the ilvY-P _ilvC- ilvBN4 _{cassette is} associated with duplication of the ilvY gene, the ilvY gene was inactivated in the ilvY-P _ilvC- ilvBN4 _expression cassette. The nucleotide sequence of the ilvY gene was modified so that upon inactivation of the ilvY gene, the regulatory region of the ilvBN4 operon genes was not affected. More precisely, a 31 nucleotide DNA fragment was inserted into the ilvY gene of the ilvY-P _ilvC- ilvBN4 _expression cassette to replace the fifth codon (GAT) of the structural part of the ilvY stop gene with the TGA codon.

Inactivation of the ilvY gene was carried out as follows. First, a PCR fragment containing a λattL-cat-λattR cassette with regions adjacent to the interior of the ilvY gene was obtained using oligonucleotide primers P9 (SEQ ID NO: 19) and P10 (SEQ ID NO: 20) and plasmid pMIV5-JS as template . Plasmid pMIV5-JS was constructed as described in EP 1942183. E. coli MG1655 / pKD46 cells were electrotransformed with the obtained PCR fragment. Chloramphenicol resistant transformants were selected as described above. Thus, the E. coli strain MG1655 ilvY :: cat was obtained containing a chloramphenicol resistance marker (Cm ^R ) in the structural part of the ilvY gene. The insertion of the fragment was checked by PCR.

Colonies grown for 24 h were checked for the presence of the ilvY :: cat fragment, integrated instead of the native ilvY gene, by PCR using oligonucleotide primers P11 (SEQ ID NO: 21) and P12 (SEQ ID NO: 22). To this end, a fresh, isolated colony was suspended in 20 μl of water, and 1 μl of the resulting suspension was used for PCR. The temperature profile was as follows: initial denaturation of DNA for 5 min at 94 ° C; then 30 cycles: denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, and elongation at 72 ° C for 1 min 30 sec; final elongation for 7 min at 72 ° C. Several analyzed Cm ^R colonies should contain a 1603 bp DNA fragment, which confirms the presence of the ilvY :: cat fragment instead of the 297 bp native ilvY fragment. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by culturing at 37 ° C. Thus, the E. coli strain MG1655 ilvY :: cat was obtained.

The strain E. coli B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC- ilvBM (see Example 2} was removed from the Cm ^R marker (cat) by introducing the transition plasmid pMWts-λInt / Xis (Katashkina Zh.I. et al., Mol. Biol. (Mosk.), 2005, 39 (5): 823-831) Thus, an unlabelled strain of E. coli B7 ΔilvGM ΔilvIH ilvY-P _ilvC - _{ilvBN4 was} obtained. IlvAYC genes were removed from strain E.coli B7 ΔilvGM ΔilvIH ilvY-P _ilvC- ilvBN4 by P1 transduction using E. coli MG1655 ΔilvAYC :: Km (see Example 1) as a donor as described above After cloning the λRed genes onto plasmid pKD46, E. coli strain B7 ΔilvGM ΔilvIH ilvY-P _ilvC- ilvBN4 ΔilvAYC :: Km ^{R was} electrotransformed with a PCR fragment containing a λattL-cat-λattR cassette with regions adjacent to the inner part of the ilvY gene.This fragment was obtained by PCR using oligonucleotide primers P11 (SEQ ID NO: 21) and P12 (SEQ ID NO: 22) and the chromosomal DNA of E. coli strain MG1655 ilvY :: cat as a matrix. the E. coli strain B7 ΔilvIH ΔilvIH ΔilvAYC :: Km ^R ilvY :: cat-P _ilvC - _{ilvBN4 was} obtained, which was further used as a donor strain for transduction of the cassette ilvY :: cat-P _ilvC- ilvBN4 into E.coli B7 ΔilvGM strain ΔilvIH ilvY-P _ilvC -ilvBN4. P1 transduction was performed as described above.

The insert insert test ilvY :: cat-P _ilvC- ilvBN4 was tested by PCR. To this end, colonies grown for 24 hours were checked for the presence of a DNA fragment integrated instead of the ilvY-P ilvC- _ilvBN4 cassette using PCR using primers P11 (SEQ ID NO: 21) and P19 (SEQ ID NO: 29) . To this end, a fresh, isolated colony was suspended in 20 μl of water, and 1 μl of the resulting suspension was used for PCR. The temperature profile was as follows: initial denaturation of DNA for 5 min at 94 ° C; then 30 cycles: denaturation at 94 ° C for 30 sec, annealing at 58 ° C for 30 sec, and elongation at 72 ° C for 2 min; final elongation for 7 min at 72 ° C. Several analyzed Cm ^R colonies should contain a 1654 bp DNA fragment, which confirms the presence of the ilvY :: cat-P _ilvC - _ilvBN4 fragment instead of the original 339 bp ilvY-P _ilvC - _ilvBN4 construct. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by culturing at 37 ° C. Thus, E. coli strain B7 ΔilvGM ΔilvIH ΔilvAYC :: Km ^R ilvY :: cat-P _ilvC -ilvBN4 was obtained. This strain was further used as a donor for transduction of the ilvY :: cat-P _ilvC - _iluBN4 cassette into the E. coli B7 strain ΔilvGM ΔilvIH ilvY-P _ilvC- ilvBN4 as described above. Thus, the E. coli strain B7 ΔilvGM ΔilvIH ilvY :: cat-P _{ilvC -ilvBN4 was} obtained, possessing only one copy of the ilvY gene located at its native locus due to inactivation of the copy of the ilvY gene in the cassette ilvY :: cat-P _ilvC -ilvBN4. The cat gene was removed by introducing the transition plasmid pMWts-λInt / Xis. Thus, an unmarked strain of E. coli B7 ΔilvGM ΔilvIH ilvY ^inactive -P _ilvC -ilvBN4 was obtained.

Example 6. Production of L-valine by E. coli strain B7 ΔilvGM ΔilvIH ilvY ^inactive -P _ilvC -ilvBN4

The modified E. coli strain B7 ΔilvGM ΔilvIH ilvY ^inactive -P _ilvC- ilvBN4 and the control strain B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC- ilvBN4 were separately grown at 32 ° C for 18 hours in Luria-Bertani medium (also called lys medium as described in Sambrook, J. and Russell, DW (2001) Molecular Cloning: a Laboratory Manual (3 ^{rd ed.) Cold Spring Harbor Laboratory} Press).. Then 0.2 ml of the obtained medium was introduced into 2 ml of fermentation medium in 20 × 200 mm test tubes and grown at 30 ° C for 60 hours on a rotary shaker at 250 rpm.

The composition of the enzymatic medium (g / l):

Glucose 60.0 (NH ₄ ) ₂ SO ₄ 15.0 KH ₂ PO ₄ 1,5 MgSO ₄ × 7H ₂ O 1,0 Thiamine HCl 0.1 CaCO ₃ 25,

with the addition of LB medium: 10% (v / v).

The fermentation medium was sterilized at 116 ° C for 30 minutes, glucose and CaCO _{3 were} sterilized separately under the following conditions: glucose at 110 ° C for 30 minutes, CaCO ₃ at 116 ° C for 30 minutes. The pH was maintained in the region of 7.0 with a KOH solution.

After growing, the accumulated L-valine was measured by thin layer chromatography (TLC). TLC plates (10 × 20 cm) were coated with a layer (0.11 mm) of Sorbfil silica gel containing a non-fluorescent indicator (Sorbpolymer, Krasnodar, Russian Federation). Samples were applied to the plates using a Camag Linomat 5 applicator. The iso-propanol: ethyl acetate: 25% aqueous ammonia: water mixture (16: 16: 5: 10, v / v) was used as the mobile phase. For visualization, a solution of ninhydrin (2%) in acetone was used. After staining, the plates were dried and scanned using Camag TLC Scanner 3 in the absorption mode with detection at 520 nm, the data were processed using winCATS software (version 1.4.2).

The average results of four independent in vitro fermentations are shown in Table 5. As can be seen from Table 5, the modified E. coli strain B7 ΔilvGM ΔilvIH ilvY ^inactive -P _ilvC- ilvBN4 caused a greater accumulation of L-valine compared to the original E. coli strain B7 ΔilvGM ΔilvIH P _L -ilvBN4. In addition, inactivation of the ilvY gene in E. coli strain B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC- ilvBN4 did not adversely affect the production of L-valine strain.

Although the invention has been described in detail with reference to the best methods 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 Regulatory proteins of the LysR type. Organism Regulatory protein Co-inductor KEGG, No. Biosynthetic pathway E.coli IlvY 2-Acetolactate, B3773 The biosynthesis of valine, leucine, isoleucine 2-Aceto-2-hydroxybutanoate E.coli Lysr Diaminopimelic acid B2839 Lysine biosynthesis E.coli, Salmonella typhimurium Cysb O-Acetyl-L-Serine B1275, Cysteine biosynthesis N-Acetyl-L-Serine STM1713 E. coli, S. typhimurium Metr L-homocysteine B3828 Methionine biosynthesis STM3964 Pseudomonas putida, Trpi Indol glycerol phosphate PP_0084, Tryptophan biosynthesis P. aeruginosa, RA0037, P. syringae PSPTC_0157

table 2 The influence of different genetic environments on Puvc-dependent transcription. Activity of β-galactosidase LacZ. Strain The specific activity of β-galactosidase, in Miller units B7 (+ IPTG, 1 mm) 1200 B7 ΔilvBN ΔilvGM ΔilvIH cat-ilvY-P _ilvC -lacZ 10 B7 ΔilvGM ΔilvIH P _L -ilvBN cat-ilvY-P _ilvC -lacZ 940 B7 ΔilvGM ΔilvIH P _L -ilvBN4 cat-ilvY-P _ilvC -lacZ 3700 B7 ΔilvGM ΔilvIH P _L -ilvBN ΔilvAYC-Km ^R cat-ilvY-P _ilvC -lacZ 4400 B7 ΔilvGM ΔilvIH P _L -ilvBN4 ΔilvAYC-Km ^R cat-ilvY-P _ilvC -lacZ 4400

Table 3 AHAS I activity measured in strains containing a cat-ilvY-P _ilvC - _ilvBN4 cassette. Strain The activity of AHAS I, nmol / min * mg without L-Val with L-Val, 10 mM (percentage of value measured without adding L-Val) B7 ΔilvGM ΔilvIH P _L -ilvBN4 (control) 59 51 (86%) B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -BN4, clone 1 91 80 (88%) B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4, clone 4 121 106 (88%)

Table 4 Production of L-valine using a modified E. coli strain. Values are given as mean ± SD, where SD is the standard deviation. Strain OD _540nm L-Val, g / l B7 ΔilvGM ΔilvIH P _L -ilvBN4 (control) 59 4.2 ± 0.4 B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4 53 5.7 ± 0.5

Table 5 L-valine production by a modified E. coli strain. Values are given as mean ± SD, where SD is the standard deviation. Strain OD540HM L-Val, g / l B7 ΔilvGM ΔilvIH P _L -ilvBN4 (control) 59 3.7 ± 0.4 B7 ΔilvGM ΔilvIH cat-ilvY-P _ilvC -ilvBN4 (control) 52 5.5 ± 0.2 B7 ΔilvGM ΔilvIH ilvY ^inactive -P _ilvC -ilvBN4 56 5.7 ± 0.5

Claims (23)

1. Genetic expression system, including:
a transcriptional apparatus regulated by a LysR-type protein, which includes a promoter and an operator, the expression of which is positively regulated by a LysR-type regulatory protein and a co-inductor, and
the target gene (s) with which the indicated transcriptional apparatus is operably linked, the target gene (s) encoding (s) the protein (s) involved in the biosynthesis of the indicated co-inductor, substrate or precursor said co-inductor,
characterized in that the self-induced positive regulation by feedback type of the indicated expression system is mediated by a co-inductor. 2. The expression system according to claim 1, characterized in that the said system is from a bacterium belonging to the family Enterobacteriaceae or Pseudomonadaceae. 3. The expression system according to claim 1, characterized in that said system is from a bacterium belonging to the Enterobacteriaceae family. 4. The expression system according to claim 3, characterized in that the said system is from a bacterium belonging to the genus Escherichia. 5. The expression system according to claim 4, characterized in that said bacterium belongs to the species Escherichia coli. 6. An expression system according to any one of claims 1 to 4, characterized in that said system is from a biosynthetic pathway of an L-amino acid selected from the group consisting of branched L-amino acids, L-lysine, L-cysteine, L-methionine and L tryptophan. 7. The expression system according to claim 6, characterized in that said system is a branched chain L-amino acid biosynthetic pathway. 8. The expression system according to any one of claims 1 to 5 and 7, characterized in that the promoter is a P _ilvC promoter, said regulatory protein of the LysR type is IlvY protein, and the co-inductor is 2-aceto-lactic acid, 2-aceto-2-hydroxybutyric acid or their salts. 9. The expression system according to claim 8, characterized in that the co-inductor is 2-acetolactic acid or its salt. 10. The expression system according to any one of claims 1 to 5 and 7, characterized in that the target gene (s) encodes (s) acetohydroxyacid synthetase. 11. The expression system according to claim 10, characterized in that said target genes encode proteins selected from the group consisting of:
(A) combinations of items A1 and A2:
(A1) a protein containing the amino acid sequence of SEQ ID NO: 2, or a protein containing the amino acid sequence of SEQ ID NO: 2, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein A2; and
(A2) a protein containing the amino acid sequence of SEQ ID NO: 4, or a protein containing the amino acid sequence of SEQ ID NO: 4, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein A1;
(B) combinations of items B1 and B2:
(B1) a protein containing the amino acid sequence of SEQ ID NO: 6, or a protein containing the amino acid sequence of SEQ ID NO: 6, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein B2; and
(B2) a protein containing the amino acid sequence of SEQ ID NO: 8, or a protein containing the amino acid sequence of SEQ ID NO: 8, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein B1; and
(C) combinations of items C1 and C2:
(C1) a protein containing the amino acid sequence of SEQ ID NO: 32, or a protein containing the amino acid sequence of SEQ ID NO: 32, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein C2; and
(C2) a protein comprising the amino acid sequence of SEQ ID NO: 34, or a protein comprising the amino acid sequence of SEQ ID NO: 34, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having the activity of acetolactate synthase with protein C1. 12. The expression system according to claim 11, characterized in that said acetohydroxy acid synthetase is a mutant acetolactate synthase I, which is resistant to feedback inhibition by L-valine. 13. The expression system according to any one of claims 1 to 5 and 7, characterized in that said operator includes a site to which said regulatory protein of the LysR type binds. 14. The expression system according to item 13, wherein the specified regulatory protein of the LysR type is selected from the group consisting of:
(D) a protein containing the amino acid sequence of SEQ ID NO: 10; and
(E) a variant of a protein containing the amino acid sequence of SEQ ID NO: 10, which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having an activity of a regulatory protein of the LysR type. 15. The expression system according to any one of claims 1 to 5 and 7, characterized in that said promoter includes:
(F) DNA containing the nucleotide sequence of SEQ ID NO: 30; or
(G) a DNA variant containing the nucleotide sequence of SEQ ID NO: 30, which contains the substitution, deletion, insertion or addition of one or more nucleotide residues, and having the activity of the nucleotide sequence of SEQ ID NO: 30. 16. A bacterium-producer of L-amino acids belonging to the family Enterobacteriaceae, characterized in that the bacterium is modified so that it contains an expression system according to any one of claims 1 to 15. 17. The bacterium according to clause 16, wherein said bacterium contains a gene encoding a regulatory protein of the LysR type. 18. The bacterium according to clause 16, wherein said bacterium belongs to the genus Escherichia. 19. The bacterium according to p. 18, characterized in that the bacterium belongs to the species Escherichia coli. 20. A bacterium according to any one of claims 16-19, characterized in that said L-amino acid is a branched chain L-amino acid and said bacterium is modified so that it contains an expression system according to claim 7. 21. The bacterium according to claim 20, characterized in that the branched chain L-amino acid is selected from the group consisting of L-valine, L-leucine and L-isoleucine. 22. A method of producing a branched chain L-amino acid, comprising:
(i) growing the bacterium according to claim 20 in a nutrient medium such that said branched chain L-amino acid accumulates in the culture fluid; and
(ii) isolating said branched chain L-amino acid from the culture fluid. 23. The method for producing the branched-chain L-amino acid according to claim 22, wherein said branched-chain L-amino acid is selected from the group consisting of L-valine, L-leucine and L-isoleucine.

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