RU2303635C2 - Genes of corynebacterium glutamicum encoding resistance and tolerance proteins to stress - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, microbiology.

SUBSTANCE: invention represents nucleic acid molecules isolated from Corynebacterium glutamicum that encode polypeptide with the protein activity providing the resistance to lincomycin. Also, invention relates to recombinant expressing vectors comprising such nucleic acids molecules, and to host-cells wherein these expressing vectors have been inserted. This invention relates to a method for preparing amino acids by culturing above indicated cells.

EFFECT: expanded assortment of enzymes, enhanced effectiveness in preparing amino acids.

36 cl, 4 tbl, 14 ex

Description

Related Applications

This application claims the priority of a previously filed provisional application for US Patent Reg. No. 60/141031, filed June 25, 1999, provisional application for US Patent Reg. No. 60/142692, filed July 1, 1999, as well as provisional patent application US reg. No. 60/151214, filed August 27, 1999. This application also claims the priority of German patent application No. 19930429.7, filed July 1, 1999, German patent application No. 199311413.6, filed July 8, 1999, German patent application No. 19931457.8, filed July 8 1999, German patent application No. 19931541.8, filed July 8, 1999, German patent application No. 199932209.0, filed July 9, 1999, German patent application No. 199932230.9, filed July 9, 1999, German patent application No. 199932914.1, filed July 14, 1999, German patent application No. 19940764.9, filed on August 27, 1999, and German patent application No. 19941382.7, filed August 31 th, 1999. The entire contents of all of the above claims in their entirety incorporated herein by reference.

State of the art

Some products and by-products of the natural processes of metabolism in cells are widely used in various industries, including food, animal feed, cosmetic and pharmaceutical industries. Such molecules, collectively called “fine chemicals”, are organic acids, proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their preparation is usually carried out by large-scale cultivation of bacteria grown for the production and secretion of large quantities of a particular desired molecule. One of the valuable microorganisms used for this purpose is Corynebactecium glutamicum, a gram-positive non-pathogenic bacterium. By selecting strains, a number of mutant strains were obtained that produced a number of desired compounds. However, the selection of strains with increased production of a particular molecule is a long and laborious process.

SUMMARY OF THE INVENTION

The present invention relates to new bacterial nucleic acid molecules having widespread use. Such applications are the identification of microorganisms that can be used to produce fine chemicals, modulating the production of fine chemicals in C. glutamicum or related bacteria, typing or identification of C. glutamicum or related bacteria for use as reference plots for mapping C. glutamicum genome, or as markers for transformation. These new nucleic acid molecules encode proteins, referred to herein as resistance and stress tolerance (SRT) proteins.

C. glutamicum is a gram-positive aerobic bacterium that is widely used in industry for the large-scale production of a number of fine chemicals, as well as for the degradation of hydrocarbons (for example, oil spills) and for the oxidation of terpenoids. Therefore, the nucleic acid molecules for the SRT of the present invention can be used to identify microorganisms that can be used to produce fine chemicals, for example, by fermentation. Modulation of the expression of nucleic acids encoding the SRT of the present invention, or modification of a given sequence of nucleic acid molecules encoding the SRT of the present invention, can be performed to modulate the production of one or more fine chemicals from a microorganism (for example, to increase the yield or production of one or several fine chemicals from bacteria of the species Corynebacterium glutamicum or Brevibacterium).

The SRT nucleic acids of the present invention can also be used to identify a Corynebacterium glutamicum microorganism or a closely related microorganism, or to detect the presence of C. glutamicum or its related bacterium in a mixed population of microorganisms. The present invention relates to the nucleotide sequences of a number of C. glutamicum genes; however, the presence of this microorganism can be established by probing the extracted genomic DNA culture of a single or mixed population of microorganisms under stringent conditions with a probe covering the region of the C. glutamicum gene, which is unique to this microorganism. Although Corynebacterium glutamicum itself is non-pathogenic, however, it is akin to a pathogenic species in humans, such as Corynebacterium diphtheriae (a pathogen that causes diphtheria); moreover, the detection of such microorganisms is of great importance in medicine.

Nucleic acid molecules encoding the SRT of the present invention can also serve as starting sites for mapping the C. glutamicum genome or genomes of related microorganisms. Similarly, these molecules or their variants or parts can serve as markers for genetically engineered species of Corynebacterium or Brevibacterium.

SRT proteins encoded by the new nucleic acid molecules of the present invention, for example, allow C. glutamicum to survive under chemical stress or environmental hazards that are harmful to the microorganism. Considering the availability of cloning vectors for use in Corynebacterium glutamicum, such as the vectors described by Sinskey et al. In US Pat. ., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984) & Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 ( 1984)), the nucleic acid molecules of the present invention can be used for the genetic construction of this microorganism in order to obtain from it a better or more efficient producer nta of one or more fine chemicals because of the ability of these proteins to stimulate the growth and reproduction of C. glutamicum (as well as the continuous production of one or more fine chemicals) under conditions that usually inhibit the growth of a given microorganism, such as common conditions in the process of large-scale enzymatic growth. So, for example, by overexpressing or constructing a molecule induced by heat shock of a protease to optimize its activity, the ability of this bacterium to decompose improperly laid proteins can be increased when this bacterium is exposed to high temperatures. With a decrease in the number of improperly laid (and possibly incorrectly regulated or non-functional) proteins that impede the implementation of normal reaction mechanisms in a cell, the ability of this cell to function normally in such a culture increases, which, in turn, should increase its viability. This overall increase in the number of cells with greater viability and activity in a given culture should also lead to an increase in the yield, production and / or production efficiency of one or more desired chemicals of fine organic synthesis, which is caused by at least a relatively large number of cells, producing these chemicals in a given culture.

The present invention also relates to new nucleic acid molecules encoding SRT proteins, which allow, for example, C. glutamicum to survive under chemical stress or environmental exposure, dangerous for this microorganism. Nucleic acid molecules encoding an SRT protein are referred to herein as SRT nucleic acid molecules. In a preferred embodiment, the SRT protein is involved in metabolic pathways that allow C. glutamicum to survive under chemical stress or environmental hazards that are harmful to the microorganism. Examples of such proteins are proteins encoded by the genes shown in Table 1.

In accordance with one of its aspects, the present invention relates to isolated nucleic acid molecules (eg, cDNA, DNA or RNA) containing the nucleotide sequence encoding the SRT protein or its biologically active parts, as well as to nucleic acid fragments used as primers or probes for hybridization to detect or amplify an SRT-coding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, an isolated nucleic acid molecule contains one of the nucleotide sequences represented by the oddly numbered SEQ ID NOs in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 5 : 7 ...) or the coding region or complement of one of these nucleotide sequences. In other particularly preferred embodiments of the present invention, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence that hybridizes with that molecule or that is at least about 50%, and preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more, is homologous to the nucleotide sequence the sequence represented by the oddly numbered SEQ ID Nos in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 ...) or parts thereof. In other preferred embodiments of the present invention, an isolated nucleic acid molecule encodes one of the amino acid sequences represented by evenly numbered SEQ ID NO: in the Sequence List (e.g., SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 ...). Preferred SRT proteins of the present invention are also proteins that preferably have at least one of the SRT activities described in this application.

In another embodiment, an isolated nucleic acid molecule encodes a protein or part thereof, wherein said protein or part thereof comprises an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (for example, a sequence having the evenly numbered SEQ ID NO: in the Sequence Listing) , for example, a sufficiently homologous amino acid sequence of the present invention, such that the protein or part thereof retains SRT activity. Preferably, this protein or a part thereof encoded by this nucleic acid molecule retains the ability to increase the survival of C. glutamicum under chemical stress or environmental exposure hazardous to the microorganism. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, and preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and most preferably at least about 95%, 96%, 97%, 98% or 99% or more homologous to the amino acid sequence of the present invention (for example, the entire amino acid sequence selected from evenly numbered SEQ sequences ID NO: in the List after sequences). In another preferred embodiment, said protein is a full-length C. glutamicum protein that is substantially homologous to the entire amino acid sequence of the present invention (encoded by an open reading frame shown by the corresponding oddly numbered SEQ ID NO: in the Sequence List (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 ...).

In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an SRT fusion protein) comprising a biologically active domain that is at least about 50% or more homologous to one of the amino acid sequences of the present invention (for example, one of the evenly numbered sequences of SEQ ID NO: in the List of Sequences) and has the ability to increase the survival of C. glutamicum under chemical stress or environmental exposure, dangerous for the microorganism, or has one or more activities listed in Table 1, and the specified isolated molecule also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory region.

In another embodiment, said isolated nucleic acid molecule has a length of at least 15 nucleodites and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of the present invention (e.g., the oddly numbered SEQ ID NO sequence in the Sequence List). Preferably, said isolated nucleic acid molecule corresponds to a natural nucleic acid molecule. More preferably, the isolated nucleic acid encodes the naturally occurring C. glutamicum SRT protein or a biologically active portion thereof.

In another aspect, the present invention relates to vectors, for example, to recombinant expression vectors containing the nucleic acid molecule of the present invention, and to host cells into which these vectors have been introduced. In one embodiment of the invention, said host cell is used to produce the SRT protein by culturing it in a suitable medium. Then, said SRT protein can be isolated from a given medium or host cell.

In yet another aspect, the present invention relates to a genetically modified microorganism into which the SRT gene has been introduced or in which it has been modified. In one embodiment, the genome of said microorganism has been modified by introducing a nucleic acid molecule of the present invention encoding a wild-type SRT sequence or a mutated SRT sequence as a transgene. In another embodiment, the endogenous SRT gene in the genome of the microorganism has been modified, for example, functionally destroyed by homologous recombination with the modified SRT gene. In another embodiment, an endogenous or introduced SRT gene in a microorganism has been modified using one or more point mutations, deletions or inversions, but it encodes a functional SRT protein. In yet another embodiment, one or more regulatory regions (e.g., a promoter, repressor, or inducer) of an SRT gene in a microorganism has been modified (e.g., by deletions, truncations, inversions, or point mutations) so that expression of the SRT gene is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Cocynebacterium glutamicum being particularly preferred. In a preferred embodiment of the invention, said microorganism is also used to produce the desired compound, such as an amino acid, with lysine being particularly preferred.

In another aspect, the present invention relates to a method for identifying the presence or activity of Corynebacterium diphtheriae in an individual. This method comprises detecting in an individual one or more nucleotide or amino acid sequences of the present invention (for example, the sequences shown in SEQ ID NO: 1-304), and thereby detecting the presence or activity of Corynebacterium diphtheriae in that individual.

In another aspect, the present invention relates to an isolated SRT protein or part thereof, for example, to its biologically active part. In a preferred embodiment of the invention, an isolated SRT protein or part thereof has the ability to increase the survival of C. glutamicum under chemical stress or environmental exposure hazardous to the microorganism. In another preferred embodiment of the invention, the isolated SRT protein or part thereof is sufficiently homologous to the amino acid sequence of the present invention (for example, the evenly numbered sequence SEQ ID NO: in the Sequence List), while this protein or part thereof retains the ability to increase the survival of C. glutamicum under the influence of chemicals or the environment, dangerous for this microorganism.

The present invention also relates to an isolated SRT protein preparation. In preferred embodiments, the SRT protein includes the amino acid sequence of the present invention (for example, one of the evenly numbered sequences of SEQ ID NO in the Sequence List). In another preferred embodiment, the present invention relates to an isolated full-length protein that is substantially homologous to the complete amino acid sequence of the present invention (e.g., SEQ ID NO sequence having an even number in the Sequence List) (encoded with an open reading frame in the corresponding SEQ ID NO having an odd number on the Sequence List). In yet another embodiment, the protein is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98% or 99% or more homologous to the complete amino acid sequence of the present invention (for example, the sequence SEQ ID NO having an even number in the Sequence List). In other embodiments, an isolated SRT protein contains an amino acid sequence that is at least about 50% or more homologous to one of the amino acid sequences of the present invention (for example, SEQ ID NO having an even number in the Sequence List), and which capable of increasing the degree of survival of C. glutamicum under conditions of exposure to chemicals or the environment that are dangerous for a given microorganism, or it has one or more activities indicated and in Table 1.

Alternatively, the isolated SRT protein comprises an amino acid sequence encoded by a nucleotide sequence that hybridizes, for example, under stringent conditions, to one of the SEQ ID NO sequences numbered by even numbers in the Sequence List, or which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and most preferably at least about 95%, 96%, 97%, 98% or 99 % or more homologous to the specified sequence. It is also preferred that the preferred forms of the SRT proteins also have one or more biological SRT activities described herein.

The SRT polypeptide or a biologically active portion thereof may be operably linked to a non-SRT polypeptide to form a fusion protein. In preferred embodiments of the invention, said fusion protein has an activity that is different from the activity inherent in only one SRT protein. In other preferred embodiments of the invention, this fusion protein enhances the yields, production and / or production efficiency of the desired fine chemicals from C. glutamicum. In particularly preferred embodiments of the invention, integration of said fusion protein into a host cell helps modulate the production of a desired compound from that cell.

In another aspect, the present invention relates to methods for screening molecules that modulate the activity of an SRT protein, either by interacting with the protein itself, or with its substrate, or by binding to a partner of the SRT protein, or by modulating the transcription or translation of the SRT nucleic acid molecule of the present inventions.

In another aspect, the present invention relates to a method for the production of fine chemicals. This method involves culturing a cell containing a vector directing the expression of the SRT nucleic acid molecule of the present invention so that fine chemicals are produced. In a preferred embodiment of the invention, said method further comprises the step of producing a cell containing said vector, wherein the cell is transfected with a vector directing the expression of SRT nucleic acid. In another preferred embodiment of the invention, said method further comprises the step of isolating a fine chemical product from a given culture. In a particularly preferred embodiment of the invention, said cell is obtained from bacteria of the genus Corynebacterium or Brevibacterium, or is selected from the strains shown in Table 3.

In another aspect, the present invention relates to methods for modulating the production of a molecule from a microorganism. Such methods include contacting the cell with an agent that modulates the activity of the SRT protein or expression of the SRT-coding nucleic acid so that the cell-associated activity is changed compared to the same activity, but in the absence of this agent. In a preferred embodiment of the invention, the resistance of the indicated cell to one or more toxic chemical products or the resistance to one or more environmental influences is modulated, so that this leads to an increase in the yield or degree of production of the desired chemical product of fine organic synthesis by this microorganism. The specified agent that modulates the activity of the SRT protein may be an agent that stimulates the activity of the SRT protein or expression of SRT nucleic acid. Examples of agents that stimulate SRT protein activity or expression of a nucleic acid encoding SRT are small molecules, active SRT proteins, and nucleic acids encoding SRT proteins that have been introduced into the cell. Examples of agents that inhibit the activity or expression of SRT are small molecules and antisense SRT nucleic acid molecules.

In another aspect, the present invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing a wild-type SRT gene or mutant SRT gene into a cell, either located on a separate plasmid or integrated into the genome of the host cell. If the gene is integrated into the genome, such integration can occur randomly, or it can be carried out by homologous recombination, so that the native gene is replaced by the introduced copy, which leads to the production of the desired compound from the modulated cell. In a preferred embodiment, said yields are elevated. In another preferred embodiment, the specified chemical substance is a chemical product of fine organic synthesis. In a particularly preferred embodiment of the invention, said fine fusion chemical is an amino acid. In particularly preferred embodiments of the invention, said amino acid is L-lysine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleic acid and SRT protein molecules that contribute to the survival of C. glutamicum when exposed to chemicals or environmental conditions that are harmful to the microorganism. The molecules of the present invention can be used to modulate the production of fine chemicals from microorganisms, since these SRT proteins provide continuous growth and propagation of C. glutamicum in the presence of toxic chemical conditions or adverse environmental conditions such as may arise during large-scale enzymatic cultivation . By increasing the growth rate, or at least maintaining normal growth in poor, if not toxic, conditions, the yield, production and / or production efficiency of one or more fine chemicals from such a culture can be increased, which is due, at least at least a relatively higher number of cells producing the specified chemical product of fine organic synthesis in a given culture. Aspects of the present invention will be described in more detail below.

I. Fine chemicals

The term "fine chemicals" is known to those skilled in the art and means molecules produced by a microorganism used in various industries, such as, but not limited to, the pharmaceutical industry, agriculture, and the cosmetic industry. Such compounds are organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., Eds. VCH: Weinheim and its references), lipids, saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propanediol and butanediol) carbohydrates (e.g. hyaluronic acid and trehalose ), aromatic compounds (for example, aromatic amines, vanillin and indigo), vitamins and cofactors (described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, Vitamins, p.443-613 (1996) VCH: Weinheim and available therein links; and Ong, AS, Niki E. & Packer, L. (1995) Nutrition, Lipids, Health and Disease, Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68) and other chemical compounds described by Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the references therein. The metabolism and use of some of these fine chemicals will be described in more detail below.

A. Metabolism and use of amino acids

Amino acids include the basic structural units of all proteins and, thus, are necessary for the normal functioning of all organisms. The term "amino acid" is well known in the art. Proteinogenic amino acids, a total of 20 species, serve as structural units of the protein in which they are joined by peptide bonds, and non-proteinogenic amino acids (several hundred of which are known) are not usually found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids can have an optical D- or L-configuration, although L-amino acids are usually the only type found in natural proteins. The pathways of biosynthesis and degradation of each of these 20 proteinogenic amino acids have been characterized in prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3 ^rd edition, pages 578-590 (1988)). “Essential” amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), so-called because they mainly satisfy the nutritional requirements, due to the complexity of their biosynthesis, easily turn into during metabolism by simple biosynthesis into the remaining 11 “interchangeable” amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, series, and tyrosine). Higher animals retain the ability to synthesize some of these amino acids, but essential amino acids must come from food for normal protein synthesis in the body.

In addition to their function in protein biosynthesis, these amino acids themselves are of interest as chemical compounds, and many of them are known to find their application in the food, feed, chemical, cosmetic, agricultural and pharmaceutical industries. Lysine is an important amino acid for the nutrition of not only humans, but also animals with a single-chamber stomach, such as poultry and pigs. Glutamate is the most widely used compound as flavoring agents (sodium mono-glutamate, MGN) and is widely used in the food industry as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in both the pharmaceutical and cosmetic industries. Threonine, tryptophan and D / L-methionine are common dietary supplements (Leuchtenberger, W. (1996) Amino aids-technical production and use, p. 466-502, Rehm et al. (Eds.) Biotechnology vol. 6, chapter 14a, VCH: Weimheim). In addition, it was found that these amino acids can be used as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim, 1985.

The biosynthesis of these naturally occurring amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for a review of bacterial amino acid biosynthesis and its regulation see Umbarger, H.E. (1978) Ann.Rev. Biochem. 47: 533-606). Glutamate is synthesized by reductive amination of α-ketoglutarate, an intermediate in the citric acid cycle. Each of glutamine, proline, and arginine is sequentially produced from glutamate. Serine biosynthesis is a three-step process starting with 3-phosphoglycerate (an intermediate in glycolysis) and ending with the formation of this amino acid after the stages of oxidation, transamination and hydrolysis. Cysteine and glycine are produced from serine; in this case, the first one is formed by condensation of homocysteine with serine, and the second one is formed by transferring the β-carbon atom of the side chain to tetrahydrofolate in the reaction catalyzed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the precursors of the glycolytic and pentose-phosphate pathways of metabolism, that is, erythrose-4-phosphate and phosphoenolpyruvate in the 9-step biosynthesis pathway, which differs only in the last two stages after prefenate synthesis. Tryptophan is also produced from these two starting molecules, but its synthesis proceeds in 11 stages. Tyrosine can also be synthesized from phenylalanine in the reaction catalyzed by phenylalanine hydroxylase. Alanine, valine and leucine are biosynthetic products of pyruvate, the end product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate in the citric acid cycle. Asparagine, methionine, threonine and lysine are each produced by converting aspartate. Isoleucine is formed from threonine. A complex 9-stage metabolic pathway leads to the production of histidine from 5-phosphoribosyl-1-pyrophosphate, activated sugar.

When excess of the protein synthesis, amino acids necessary for a given cell can not be stored, and instead they are degraded with formation of intermediates involved in the major pathways of metabolism occurring in the cell (for review see. Stryer L. Biochemistry 3 ^rd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle" p. 495-516 (1988)). Although this cell has the ability to convert unwanted amino acids into the necessary intermediate compounds of metabolism, obtaining amino acids is expensive in terms of energy costs, the cost of obtaining precursor molecules and enzymes necessary for their synthesis. Thus it is not surprising that amino acid biosynthesis is regulated by the inhibition of the feedback type, where the presence of a particular amino acid serves to slow or complete termination of its own production (for a review of feedback mechanisms in amino acid biosynthetic reactions cm. Stryer L. Biochemistry 3 ^rd ed. Ch .24 Biosynthesis of Amino Acid and Heme p.575-600 (1988)). Thus, the yield of any particular amino acid is limited by the amount of amino acid present in the cell.

B. Metabolism of vitamins, cofactors and nutritious pharmaceutical products and their use

Vitamins, cofactors and nutritious pharmaceutical products contain molecules of another group, the ability to synthesize which in higher animals has been lost and which must be supplied with food, although they are easily synthesized by other organisms, such as bacteria. These molecules themselves are either biologically active substances or precursors of biologically active substances that can serve as electron carriers or as intermediates in various metabolic pathways. In addition to their nutritional value, these compounds are also of great industrial importance as coloring agents, antioxidants and catalysts or other processing aids. (For a review of the structure, activity, and industrial uses of these compounds, see, for example, Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, Vitamins, p. 433-613 VCH: Weinheim, 1996). The term "vitamin" is known to experts and means nutrients that the body needs for normal functioning, but which he himself can not synthesize. The vitamin group may include cofactors and nutritional pharmaceutical compounds. The term "cofactor" includes non-protein compounds necessary for normal enzymatic activity. Such compounds may be organic or inorganic compounds; however, the cofactor molecules of the present invention are preferably organic compounds. The term "nutritious pharmaceutical preparation" includes nutritional supplements useful for the life of plants and animals, and especially for human health. Examples of such molecules are vitamins, antioxidants, as well as certain lipids (e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been well characterized (Ullman's Encyclopedia of Industrial Chemistry, vol. A27, Vitamins, p. 433-613 VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, AS, Niki E. & Packer, L. (1995) “Nutrition, Lipids, Health and Disease” Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, IL X, 374 S).

Thiamine (Vitamin B ₁ ) is produced by the chemical combination of pyrimidine and thiazole groups. Riboflavin (vitamin B ₂ ) is synthesized from guanosine 5'-triphosphate (GTP) and ribose-5'-phosphate. Riboflavin, in turn, is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). A family of compounds under the general name “Vitamin B ₆ ” (for example, pyridoxine, pyridoxamine, pyridox-5′-phosphate and the commercially used pyridoxine hydrochloride) are derivatives of a common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R) - (+) - N- (2, 4-dihydroxy-3,3-dimethyl-1-oxobutyl) - (β-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of ATP-regulated condensation of β-alanine and pantoic acid.The enzymes responsible for the biosynthesis stages for conversion to pantoic acid, β-alanine and for condensation to pantothenic acid are known in the art. Coenzyme is a metabolically active form of pantothenate. A, whose biosynthesis proceeds in 5 enzyme stages, pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone, (R) -panthenol (provitamin B ₅ ), panthein (and its derivatives) and coenzyme A.

The biosynthesis of biotin from the pimeloyl-CoA precursor molecule in microorganisms has been thoroughly studied and some genes responsible for this synthesis have been identified. It was found that many of the corresponding proteins are also involved in the synthesis of the Fe cluster and are members of nifS class proteins. Lipoic acid is formed from octanoic acid and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and α-ketoglutarate-dehydrogenase complex. Folates are a group of substances that are derivatives of folic acid, which, in turn, is produced from L-glutamic acid, p-aminobenzoic acid and 6-methylopterin. The biosynthesis of folic acid and its derivatives, starting with the metabolism of intermediate compounds of guanosine 5'-triphosphate (GTP), L-glutamic acid and p-aminobenzoic acid, has been thoroughly studied in some microorganisms.

Corrinoids (such as cobalamins and especially vitamin B ₁₂ ) and porphyrins belong to the group of chemical compounds characterized by the presence of a tetrapyrrole ring system. The biosynthesis of vitamin B ₁₂ is quite complex and not yet well studied, however, many enzymes and substrates involved in this biosynthesis are currently known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin."

Niacin is a precursor of the important coenzymes NAD (nicotinamide-adenine dinucleotide) and NADP (nicotinamide-adenine dinucleotide phosphate) and their reduced forms.

The large-scale production of these compounds is based mainly on cell-free chemical synthesis, although some of these chemical compounds, such as riboflavin, vitamin B ₆ , pantothenate and biotin, have also been produced by large-scale cultivation of microorganisms. Only vitamin B _{12 is} produced only by fermentation, due to the complexity of its synthesis. In vitro methods require significant material costs and a lot of time, and therefore are often expensive.

C. Metabolism of purines, pyrimidines, nucleosides and nucleotides and their use.

Purine and pyrimidine metabolism genes and their corresponding proteins are the main targets for the treatment of tumor diseases and viral infections. The term “purine” or “pyrimidine” means nitrogenous bases that are components of nucleic acids, coenzymes, and nucleotides. The term "nucleotide" includes the basic structural units of nucleic acid molecules, which consist of a nitrogenous base, pentose sugar (in the case of RNA, such sugar is ribose; and in the case of DNA, such sugar is D-deoxyribose) and phosphoric acid. The term “nucleoside” includes molecules that serve as nucleotide precursors but which do not contain the phosphoric acid molecule found in nucleotides. By inhibiting the biosynthesis of these molecules or their mobilization with the formation of nucleic acid molecules, one can inhibit the synthesis of RNA and DNA; and by inhibiting this activity in targeted delivery to cancer cells, the ability of tumor cells to divide and replicate can be inhibited. In addition, there are nucleotides that do not form nucleic acid molecules, but instead serve as a backup energy source (i.e., AMP) or as coenzymes (i.e., FAD and NAD).

Some publications describe the medical use of these chemical compounds because of their effect on the metabolism of purines and / or pyrimidines (eg, Christopherson, RI & Lyons, SD (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents." Med Res. Reviews 10: 505-548). Studies of enzymes involved in the metabolism of purines and pyrimidines have focused on the development of new drugs that can be used, for example, as immunosuppressants or antiproliferative agents (Smith JL, (1995) Enzymes in nucleotide synthesis. Curr.Opin. Struct Biol. 5: 752-757 (1995) Biochem. Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other uses, namely, as intermediate compounds in the biosynthesis of certain fine chemicals (e.g., thiamine, S-adenosylmethionine, folates or riboflavin), as energy carriers for a given cell (e.g. , ATP or GTP), and to obtain the chemical compounds themselves, usually used as flavoring agents (e.g. IMP or GMP) or for some medical purposes (see, for example, Kuninaka A. (1996) Nucleotides and Related Compounds in Biotechnology , vol. 6, Rehm et al., eds. VCH: Weinheim, p. 561-612). In addition, enzymes involved in the metabolism of purines, pyrimidines, nucleosides or nucleotides increasingly serve as targets against which, to protect crops, chemical compounds are being developed, including fungicides, herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized (for a review see, for example, Zaikin H. & Dixon JE (1992) "de novo purine nucleotide biosynthesis", Progress in Nucleic Acid Research and Molecular Biology, vol. 42. Academic Press: p. 259- 287; and Michal G. (1999) Nucieotides and Nucleosides, Chapter 8, Biochemical pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism is an object of intensive research and is important for the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe illness, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate in a series of steps through the inosine-5'-phosphate intermediate compound (IMP), resulting in the production of guanosine-5'-monophosphate (GMP) or adenosine-5'-monophosphate (AMP), from which are easily formed triphosphate forms used as nucleotides. These compounds are also used as energy storage substances, since their degradation provides energy to many different biochemical processes in the cell. The biosynthesis of pyrimidines proceeds with the formation of uridine-5'-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-triphosphate (CTP). The deoxy forms of all these nucleotides are formed in a one-step reduction reaction from the diphosphatribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. After phosphorylation, these molecules are able to participate in DNA synthesis.

D. Metabolism of trehalose and its use

Trehalose consists of two glucose molecules linked to each other by an α, α-1,1-bond. It is widely used in the food industry as a sweetener, an additive for drying or freezing foods, and in beverages. However, it is also used in pharmaceuticals, cosmetics, and biotechnology (see, for example, Nishimoto et al., (1998) US Pat. No. 5,759,610; Singer MA & Lindquist S. (1998) Trends Biotech. 16: 460-467; Paiva CLA & Panek AD (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes in many microorganisms and is usually released into the environment from which it can be isolated by known methods.

II. Resistance to destruction by chemical compounds, to environmental influences and to antibiotics. The production of fine chemicals is usually carried out by large-scale cultivation of bacteria designed for the production and secretion of large quantities of these molecules. However, this type of large-scale fermentation exposes microorganisms to stress loads of various types. Such stresses are environmental exposure and chemical stresses.

A. Environmental Resistance

Examples of environmental influences commonly encountered in large-scale enzymatic cultivation are mechanical stress, heat shock, stress due to oxygen deficiency, stress due to the presence of oxygen radicals, pH stress and osmotic stress. The mixing mechanism used in most large-scale fermentation reactors for aeration of the culture is accompanied by heat generation, which leads to an increase in the temperature of the culture. An increase in temperature induces a well-characterized response in the form of heat shock, in which a number of proteins are expressed that not only contribute to the survival of bacteria at high temperatures, but also contribute to their survival in response to a number of other environmental influences (see Neidhardt, FC, et al., eds. (1996) E. coli and Salmonella. ASM Press: Washington, DC, p. 1382-1399; Wosten MM (1998) FEMS Microbiology Reviews 22 (3): 127-50; Bahl H. et al. (1995) FEMS Microbiology Reviews 17 (3): 341-348; Zimmerman JL, Cohill, PR (1991) New Biologist 3 (7): 641-650; Samali A. & Orrenius, S. (1998) Cell. Stress Chaperones 3 (4): 228-236, and references in each of the cited Abot for each of these cases). The regulation of the heat shock reaction in bacteria is facilitated by specific sigma factors and other cellular regulators of gene expression (Hecker, M. Volker, U. (1998) Molecular Microbiology 29 (5): 1129-1136). One of the main problems that cells encounter when exposed to high temperatures is that protein folding is impaired; growing proteins have a sufficient amount of kinetic energy at high temperatures, as a result of which it is difficult for a growing polypeptide chain to maintain a stable conformation long enough to form correctly. Thus, the proteins expressed during the heat shock reaction are composed of two main types of proteins: chaperones (proteins that promote folding or non-folding of other proteins - see, for example, Fink, AL (1999) Physiol. Rev. 79 (2 ): 425-449) -, and proteases that can destroy any improperly laid proteins. Examples of chaperones expressed during a heat shock reaction are GroEL and DNAK; and the proteases that are known to be expressed during the heat shock reaction are Lon, FtsH, and ClpB.

In addition to heat, stress can cause other environmental influences. Although the mixing process in the fermenter is intended to introduce oxygen into a given culture, oxygen may remain in limited quantities, especially when the culture continues to grow, and the oxygen demand thereby increases; insufficient oxygen delivery causes another stress in microorganisms. Cells in fermenter cultures are also subject to a number of osmotic stresses, especially when nutrients are added to the culture, resulting in high extracellular and low intracellular concentrations of these molecules. In addition, large quantities of the desired molecules produced by these microorganisms during their cultivation can lead to osmotic stress in bacteria. Finally, aerobic metabolism, such as the metabolism that occurs in C. glutamicum, leads to the formation of carbon dioxide as a by-product, and the secretion of this molecule can acidify the culture medium, which is caused by the conversion of this molecule to carboxylic acid. Thus, when cultured, bacteria are also often stressed to achieve an acidic pH. Such a conversion may also be beneficial if high levels of major by-product molecules such as ammonia are present in the culture medium, in which case bacteria in the culture can be stressed to achieve a basic pH.

To combat such environmental influences, bacteria have subtle gene systems that are expressed by one or more stresses, such as the aforementioned heat shock system. Genes expressed in response to osmotic shock, for example, encode proteins that can transport or synthesize the corresponding soluble substances, so that the osmotic absorption or export of a particular molecule slows down to controlled levels. Other examples of stress-induced bacterial proteins are those involved in trehalose biosynthesis; enzymes involved in ppGpp metabolism; proteins involved in signal transduction, and in particular, two-component systems that are sensitive to osmotic pressure; and transcription factors that are responsible for a number of stress factors (for example, RssB analogues and / or sigma factors). Many other genes and their protein products are known to those skilled in the art.

B. Resistance to chemical stress

In addition to environmental stresses, cells can also undergo a number of chemical stresses. These stresses can be divided into two categories. The first category includes natural by-products of metabolism and other cellular processes secreted by this cell into the environment. The second category includes chemicals that are present in the extracellular environment and are not products of this cell. Basically, if cells release toxic by-products from concentrated intracellular cytoplasm into a relatively much more diluted extracellular medium, then these products are distributed so that the extracellular levels of a possible toxic compound are quite low. However, large-scale enzymatic cultivation of bacteria is not the case, since many bacteria grow in a relatively small space and at such a high level of metabolism that by-products can accumulate in this environment to almost toxic levels. Examples of such by-products are carbon dioxide, metallic ions, and reactive oxygen species such as hydrogen peroxide. These compounds can affect the activity or structure of cell surface molecules, or they can again enter the cell, where they can seriously damage proteins and nucleic acids. Some other chemicals harmful to the normal functioning of cells can also be detected in the environment. For example, metal ions, such as mercury, cadmium, nickel or copper, are often found in water sources and can take the form of close complexes with cellular enzymes that interfere with the normal functioning of these proteins.

C. Antibiotic Resistance

Bactericidal proteins or antibiotics can also be found in the extracellular environment, which are present either as a result of their introduction by researchers, or as a natural product from another organism used to increase competitive advantage. Microorganisms have several known mechanisms for their protection against antimicrobial chemicals. Widely used methods for the removal and detoxification of antibiotics are the degradation, modification and export of compounds toxic to a given cell. It is known that some prokaryotes have cytoplasmic "evacuation pumps" and show similarities to the so-called "multidrug resistance" proteins found in higher eukaryotes (Neyfakh, AA et al. (1991) Proc. Natl. Acad. Sci. USA 88 : 4781-4785). Examples of such proteins are emrAB from E. coli (Lomovskaya, O. & K. Lewis (1992) Proc. Natl. Acad. Sci. USA 89: 8938-8942), ImrB from B. subtilis (Kumano M. et al (1997 ) Microbiology 143: 2775-2782), smr from S.aureus (Grinius, LG et al (1992) Piasmid 27: 119-129) or cmr from C. glutamicum (Kaidoh K. et al. (1997) Micro. Drug. Resist. 3: 345-350). The microorganism C. glutamicum itself is non-pathogenic in contrast to some other members of the genus Corynebacterium, such as C. diphtheriae or C. pseudotuberculosis. It is known that some pathogenic corynebacteria have multiple resistance to several antibiotics, such as C. jeikeium and C. urealyticum (Soriano, F. et al., (1995) Antimicrob. Agents Chemother. 39: 208-214).

Lincosamides are known as effective antibiotics against Corynebacterium species (Soriano, F. et al., (1995) Antimicrob. Agents Chemother. 39: 208-214). An unexpected result of the present invention was the identification of a gene encoding a linkozamide resistance protein (in particular, lincomycin resistance protein). The LMRB protein originating from C. glutamicum shows 40% homology with the B. subtilis ImrB gene product (see Genbank accession no. AL009126) calculated using CLUSTALW version 1.7 (Thompson, JD, Higgins, DG, Gibson, TJ (1994) Nucl. Acids Res. 22: 4673-4680) using standard pairwise matching parameters (PAIRWISE ALIGNMENT PARAMETERS: slow / precise comparisons: “space-gap penalty” = 10.0 and “space-extension penalty” = 0.10, protein weight matrix = BLOSUM 30, DNA weight matrix = IUB, quick / approximate comparisons: space penalty = 3, size of K-set (words) = 1, number of upper d agonals = 5, window size = 5, pairwise comparisons with switching “slow / fast” = slow. Multiple matching parameters: “penalty for space-pass” = 10/0 and “fine for space-elongation” = 0.05, sequences , diverging with delay = 40%, weight of DNA transitions = 0.50, weight matrix of protein = BLOSUM series, weight matrix of DNA = IUB, negative usage matrix = OFF).

Environmental exposure, chemical stress, and stress under the influence of antibiotics or other antimicrobial substances can affect the behavior of microorganisms during their cultivation and the production of the desired compound from these microorganisms. For example, the osmotic stress of a microorganism can cause unacceptable or unacceptable rapid absorption of one or more compounds, which ultimately leads to damage or death of cells under the influence of osmotic shock. Similarly, chemical compounds present in a given culture that were either exogenously added (e.g. antimicrobial compounds designed to remove unwanted microbes) or were generated by the bacteria themselves (e.g. by-products such as heavy metals or oxygen radicals, or even antimicrobial compounds) can lead to inhibition of the production of fine chemicals or even to the death of a microorganism. The genes of the present invention encode C. glutamicum proteins that prevent cell damage or death by specifically counteracting the source or stress caused by the environment or a chemical.

III. Elements and methods of the present invention

The present invention is based, at least in part, on the discovery of new molecules, called here nucleic acid molecules and SRT protein, that increase the ability of C. glutamicum to survive under adverse conditions of chemical stress or environmental exposure. In one embodiment of the invention, the SRT molecules function so that they give C. qlutamicum microorganism resistance to one or more stresses caused by exposure to the environment or chemicals. In a preferred embodiment of the invention, the activity of the SRT molecules of the present invention affects the production of the desired fine chemicals by this microorganism. In a particularly preferred embodiment, the SRT molecules of the present invention have modulated activity such that the yield, production and / or production efficiency of one or more fine chemicals from C. glutamlcum are also modulated.

The term “SRT protein” or “SRT polypeptide” includes proteins that are involved in communicating C. glutamicum resistance to one or more stresses caused by the environment or a chemical. Examples of SRT proteins are proteins encoded by the SRT genes shown in Table 1 and oddly numbered SEQ ID Nos. The terms “SRT gene” or “SRT nucleic acid sequence” means nucleotide sequences encoding an SRT protein and consisting of a coding region as well as corresponding untranslated 5 ′ and 3 ′ regions of a sequence. Examples of SRT genes are the genes shown in Table 1. The terms "production" or "productivity" are known to specialists and mean the concentration of the fermentation product (for example, the desired chemical product of fine organic synthesis), obtained at this time and in a given volume of fermentation and (for example, kg of product per hour per liter). The term "production efficiency" means the time required to achieve a specific level of production (for example, the time taken by these cells to achieve a specific output rate of chemical products of fine organic synthesis). The term "yield" or a product / carbon yield is well known to those skilled in the art and includes the efficiency of converting a carbon source into a product (that is, a fine organic chemical product). The yield is usually expressed, for example, in kg of product per kg of carbon source. With an increase in the yield or production of the specified compound, the number of isolated molecules or used isolated products of this compound in a given amount of culture increases with respect to a given amount of time. The terms “biosynthesis” or “biosynthesis pathway” are well known to those skilled in the art and mean cell synthesis of a compound, preferably an organic compound from intermediates, which can be a multi-step and highly controlled process. The terms “degradation” or “degradation pathway” are well known to those skilled in the art and mean the destruction of a compound, preferably an organic compound, in a cell to produce degradation products (generally speaking, smaller or less complex molecules), which can be a multi-stage and highly controlled process . The term "metabolism" is well known to specialists and means all biochemical reactions that occur in the body. In addition, the metabolism of a particular compound (for example, the metabolism of an amino acid such as glycine) includes all the pathways of biosynthesis, modification and degradation in a given cell related to that compound. The terms "resistance" and "tolerance" are known to specialists and mean the ability of cells to not be exposed to chemicals or environmental conditions, which should somehow lead to a disruption in the normal functioning of these microorganisms. The terms “stress” or “danger” include factors that adversely affect the normal functioning of cells, such as C. glutamicum. Examples of stress are “chemical stress”, in which a cell is exposed to one or more chemical compounds that adversely affect this cell, and “stress under the influence of the environment,” in which a cell is exposed to environmental conditions that differ from those to which this cell is adapted to. Chemical stresses can be caused either by natural metabolic by-products, such as, but not limited to, reactive oxygen species or carbon dioxide, or by chemical compounds that are somehow present in the environment, including, but not limited to, heavy metal ions or bactericidal proteins such as antibiotics. Environmental stresses can be, but are not limited to, for example, temperatures that go beyond normal temperatures, the presence of suboptimal amounts of oxygen, osmotic pressure, or extreme pH values.

In another embodiment, SRT molecules are capable of modulating the production of the desired molecule, such as a fine chemicals in a microorganism, such as C. glutamicum. In accordance with recombinant DNA methods, one or more SRT proteins of the present invention can be modified so that their function is modulated. A change in the activity of response genes, resistance, or stress tolerance, which consists in increasing the tolerance of a given cell to one or more stresses, can improve the ability of a given cell to grow and reproduce under relatively stressful conditions under large-scale cultivation in a fermenter. For example, by overexpressing or constructing a chaperone molecule induced by heat shock so that it has optimized activity, it is possible to increase the ability of a given bacterium to produce properly laid proteins under temperature conditions that are not optimal. With a decrease in the number of proteins with improper folding (and possibly deregulated or non-functional), the ability of this cell to function normally in such a culture increases, which, in turn, leads to an increase in its viability. This overall increase in the number of cells with greater viability and activity in the culture should also lead to an increase in the yield, production and / or efficiency of the production of one or more desired chemicals of fine organic synthesis, which is due to at least a relatively higher number of cells, producing these chemical compounds in a given culture.

Isolated nucleic acid sequences of the present invention are contained in the genome of the Corynebacterium glutamicum strain available in the American Type Culture Collection under accession number ATCC 13032. The nucleotide sequence of the isolated C. glutamicum SRT DNA and the predicted amino acid sequences of C. glutamicum SRT proteins are shown in the Sequence Listing of the oddly numbered SEQ IDs NO: and evenly numbered SEQ ID NO, respectively.

Computer analyzes were performed that allowed us to classify and / or identify these nucleotide sequences as sequences encoding proteins of chemical stress and environmental stress, as well as proteins of resistance and tolerance.

The present invention also relates to proteins having an amino acid sequence that is substantially homologous to the amino acid sequence of the present invention (for example, SEQ ID NO sequences having an even number in the Sequence List). As used herein, a protein having an amino acid sequence that is substantially homologous to a selected amino acid sequence is a protein whose amino acid sequence is at least 50% homologous to the selected amino acid sequence, i.e. the entire selected amino acid sequence. A protein that has an amino acid sequence substantially homologous to the selected amino acid sequence may also be a protein whose amino acid sequence is at least about 50-60%, and preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more, is homologous selected amino acid sequence. Values of ranges and identities that are intermediate between the above values (for example, 75-80% identity, 85-87% identity, 91-92% identity) are also included in the scope of the present invention. The present invention, for example, also includes values of ranges of identities that are a combination of any of the above values, indicated as an upper and / or lower limit.

SRT proteins or their biologically active parts or fragments of the present invention may exhibit resistance or tolerance to one or more chemical or environmental stresses, or they may have one or more of the activities shown in Table 1.

Various aspects of the present invention will be described in more detail in the following subsections:

A. Isolated nucleic acid molecules

In one of its aspects, the present invention relates to isolated nucleic acid molecules that encode SRT polypeptides or its biologically active parts, as well as nucleic acid fragments sufficient for use as hybridization probes or primers to identify or amplify an SRT-coding nucleic acid acids (e.g. SRT DNA). As used herein, the term “nucleic acid molecule” includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and DNA or RNA analogs generated using nucleotide analogs. This term also covers the untranslated sequence located at the 3 ′ and 5 ′ ends of the coding region of the gene: at least about 100 nucleotides higher from the 5 ′ end of the coding region and at least about 20 nucleotides lower from the 3'-end of the coding region of the gene. The nucleic acid molecule may be single-stranded or double-stranded, but preferably double-stranded DNA. An “isolated” nucleic acid molecule is a molecule that is separated from other nucleic acid molecules present in a natural source of nucleic acid. A preferred “isolated” nucleic acid is a molecule that does not contain sequences that typically flank the nucleic acid (that is, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. So, for example, in various embodiments of the invention, an isolated SRT nucleic acid molecule may contain nucleotide sequences of a size of less than about 5 kb, 4 kb, 3 kb, , 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which usually flank a nucleic acid molecule in genomic DNA the cell from which the nucleic acid originates (for example, C. glutamicum cell}. Moreover, an “isolated” nucleic acid molecule, such as a DNA molecule, may basically not contain another cellular mother la or culture medium if it has been produced by recombinant techniques, or chemical precursors or other chemicals, if it has been produced by chemical synthesis.

The nucleic acid molecule of the present invention, for example, a nucleic acid molecule having the nucleotide sequence of SEQ ID NO, having an odd number in the Sequence List, or part thereof, can be isolated using standard molecular biology methods and using the information given in this description. For example, C. glutamicum SRT DNA can be isolated from a C. glutamicum library using all or one of the oddly numbered sequences of SEQ ID NO: in the List of Sequences as a probe for hybridization and standard hybridization techniques (e.g., as described by Sambrook, J., Fritsh, EF & Maniatis, T., Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule encompassing all or part of one of the nucleic acid nucleic acid sequences of the present invention (e.g., the oddly numbered SEQ ID Nos) can be isolated by polymerase chain reaction using oligonucleotide primers designed based on this sequence (e.g. , a nucleic acid molecule encompassing all or part of one of the nucleic acid sequences of the present invention (e.g., odd numbered (SEQ ID Nos: in the Sequence List), can be isolated by polymerase chain reaction using oligonucleotide primers designed on the basis of the same sequence). For example, mRNA can be isolated from normal endothelial cells (e.g., by guanidinium-thioisocyanate extraction method Chirgwin et al. (1979) Biochemistry 18: 5294-5299, and DNA can be obtained using reverse transcriptase (e.g., mouse transcriptase reverse transcriptase Moloney Leukemia (MLV), supplied by Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, supplied by Seikagaku America, Inc., St. Petersburg, FL) Synthetic oligonucleotide primers for the polymerase chain reaction can be designed based on one of the nucleotide sequences the nucleic acid of the present invention can be amplified using cDNA, or alternatively, genomic DNA as a template and corresponding oligonucleotide primers in accordance with standard PCR amplification methods. Thus amplified nucleic acid can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to the nucleotide sequence of SRT can be obtained by standard methods of synthesis, for example, using an automatic DNA synthesizer.

In a preferred embodiment of the invention, an isolated nucleic acid molecule of the present invention contains one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the present invention presented in the Sequence List correspond to the Corynebacterium glutamicum SRT DNA of the present invention. This DNA includes sequences encoding SRT proteins (that is, the "coding region" shown in each oddly numbered sequence of SEQ ID NO: in the Sequence List), as well as 5'-untranslated sequences and 3'-untranslated sequences, also indicated in each oddly numbered SEQ ID NO: in the Sequence List). Alternatively, the nucleic acid molecule may contain only the coding region of any of the indicated nucleic acid sequences in the Sequence Listing.

It should be noted that for the purposes of the present invention, each of the nucleic acid and amino acid sequences represented in the Sequence List has identification numbers RXA, RXN or RXS, designated "RXA", "RXN" or "RXS" followed by 5 digits (i.e., RXA01524, RXN00493 or RXS01027).

Each of these nucleic acid sequences contains up to 3 parts: the region located above the 5'-end, the coding region, and the downstream region. To avoid confusion, each of these three areas is identified by the same designation as RXA, RXN, or RXS. In addition, the expression “one of the oddly numbered sequences in the Sequence Listing” refers to any nucleic acid sequences in the Sequence Listing that can also be identified by their different designations RXA, RXN or RXS. The coding region of each of these sequences is translated into the corresponding amino acid sequence, which is also presented in the Sequence Listing as evenly numbered SEQ ID NO: and which immediately follows the corresponding nucleic acid sequence in this list. So, for example, the coding region of RXA01524 is represented in SEQ ID NO: 1, and the amino acid sequence that it encodes is represented as SEQ ID NO: 2. The nucleic acid molecule sequences of the present invention are identified by the same designation RXA, RXN or RXS as the amino acid molecules that they encode, so that they can be easily correlated. So, for example, the amino acid sequence designated RXA01524 is a sequence translated from the coding region of the nucleotide sequence of a nucleic acid molecule RXA01524; the amino acid sequence designated RXN00034 is a sequence translated from the coding region of the nucleotide sequence of a nucleic acid molecule RXN00034; and the amino acid sequence designated RXS00568 is a sequence translated from the coding region of the nucleotide sequence of the nucleic acid molecule RXS00568. The correspondence between the nucleotide and amino acid sequences of RXA, RXN and RXS of the present invention and their SEQ ID No. are presented in table 1.

Some genes of the present invention are “F-designated genes”. F-designated genes are genes shown in Table 1 that have the letter “F” preceding the designations RXA, RXN, or RXS. So, for example, SEQ ID NO: 7, designated, as shown in Table 1, “F RXA00498”, is an F-designated gene, like SEQ ID NO: 25, 33 and 37 (indicated in Table 1 as “F RXA01345” , “F RXA02543” and “F RXA02282, respectively).

In one embodiment, the nucleic acid molecules of the present invention do not include the sequences in Table 2. In the case of the dapD gene, the sequence for this gene is published by A. Wehrmann, et al., (1998) J. Bacteriol. 180 (12): 3159-3165. However, the sequence obtained by the authors of the present invention is generally longer than the published version. Obviously, the basis of the published version of the sequence is the wrong start codon, and therefore this sequence is only a fragment of the real coding sequence.

In another embodiment, an isolated nucleic acid molecule of the present invention includes a nucleic acid molecule that is complementary to one of these nucleotide sequences of the present invention (e.g., the oddly numbered sequence SEQ ID NO: in the Sequence List) or part thereof. A nucleic acid molecule that is complementary to one of the nucleotide sequences of the present invention is a molecule that is sufficiently complementary to one of the nucleotide sequences shown in the Sequence List (for example, the oddly numbered sequence SEQ ID NO :), such that it can hybridize with one of the nucleotide sequences of the present invention, and thereby form a stable duplex.

In another preferred embodiment of the invention, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89% or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous and the nucleotide sequence of the invention (e.g., odd numbered sequences of SEQ ID NO: in the Sequence Listing), or a portion thereof. The scope of the present invention also includes values of intervals and identities that are intermediate for the above ranges (for example, 70-90% identity or 80-95% identity). Thus, for example, the scope of the present invention also includes ranges of identity values using a combination of any of the above values, indicated as an upper and / or lower limit. In yet another preferred embodiment of the invention, an isolated nucleic acid molecule of the present invention includes a nucleotide sequence that hybridizes under stringent conditions with one or part of one of the nucleotide sequences of the present invention.

In addition, the nucleic acid molecule of the present invention may include only a portion of the coding region of a given sequence of one of the oddly numbered sequences of SEQ ID NO: in the List of Sequences, for example, a fragment that can be used as a probe or primer, or a fragment encoding a biologically active part SRT protein. The nucleotide sequences determined by cloning SRT genes from C. glutamicum allow the generation of probes and primers designed to identify and / or clone SRT homologs in cells of other types and organisms, as well as SRT homologs from other Corynebacteria or related species. The probe / primer usually contains a substantially purified oligonucleotide. This oligonucleotide typically includes a region of a nucleotide sequence that hybridizes under stringent conditions with at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of the sense strand of one of the nucleotide sequences of the present invention ( for example, one of the oddly numbered sequences of SEQ ID NO: in the List of sequences), the antisense sequence of one of these sequences, or with their natural mutant E. Primers based on the nucleic acid sequence of the present invention can be used in PCR reactions for cloning SRT homologs. Probes based on SRT nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments of the invention, said probe further includes an attached tag group, for example, such a tag group may be a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor. These probes can be used as part of a diagnostic test kit to identify cells that do not sufficiently express the SRT protein, for example, by measuring the level of SRT-coding nucleic acid in a cell sample, for example, by detecting SRT-mRNA levels or determining whether the SRT gene was mutated or deleted.

In one embodiment of the invention, the nucleic acid molecule of the present invention encodes a protein or part thereof, which includes an amino acid sequence that is sufficiently homologous to the amino acid sequence of the present invention (for example, the evenly numbered sequence SEQ ID NO: in the Sequence List), so that the protein or part thereof retains the ability to report C. glutamicum resistance or tolerance to one or more stresses caused by chemical compounds niyami or environmental exposure. As used herein, the term “sufficiently homologous” refers to proteins or parts thereof having amino acid sequences that include a minimum number of amino acid residues (eg, an amino acid residue that has a side chain similar to that of the amino acid residue in the evenly numbered sequence of SEQ ID NO: in the List sequences) identical or equivalent to the amino acid sequence of the present invention, so that the protein or part thereof is capable of reporting C. glutamicum res tentnost to one or more stresses caused by chemical or environmental exposure. Proteins that are members of these metabolic pathways and described in this application increase the resistance or tolerance of C. glutamicum to one or more harmful factors or stresses caused by chemicals or environmental influences. Examples of such activity are also described in this application. Thus, the "function of the SRT protein" is to impart C. glutamicum general resistance to its surrounding elements, which may adversely affect its normal growth or function, and / or contribute, directly or indirectly, to increase the yield, production and / or production efficiency one or more fine chemicals. Examples of SRT protein activities are presented in table 1.

In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80- 90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the entire amino acid sequence of the present invention (for example, the evenly numbered sequence SEQ ID NO: in the Sequence List ) The scope of the present invention also includes values of intervals and identities that are intermediate for the above ranges (for example, 75-80% identity or 85-87% identity or 91-92% identity). Thus, for example, the scope of the present invention also includes ranges of identity values using a combination of any of the above values, indicated as an upper and / or lower limit.

Parts of the proteins encoded by the SRT nucleic acid molecules of the present invention are preferably biologically active parts of one of the SRT proteins. As used herein, the “biologically active portion of the SRT protein” means a portion, for example, a domain / motif of an SRT protein that is capable of imparting resistance or tolerance to one or more harmful factors or stresses caused by chemical compounds or environmental exposure, or has the activity described in table 1. In order to determine whether the SRT protein or its biologically active part is able to increase the resistance or tolerance of C. glutamicum to one or more harmful factors or stresses caused by chemical Skim compounds or exposure to the environment can be analyzed for enzyme activity. Methods for carrying out such analyzes are well known to every person skilled in the art and are described in detail in Example 8 of the description of the invention.

Additional nucleic acid fragments encoding the biologically active portions of the SRT protein can be obtained by isolating a portion of one of the amino acid sequences of the present invention (e.g., the evenly numbered sequence SEQ ID NO: in the Sequence List), expressing the encoded portion of the protein or SRT peptide (e.g., by recombinant expression in vitro) and evaluating the activity of the encoded portion of an SRT protein or peptide.

The present invention also relates to nucleic acid molecules that differ from one of the nucleotide sequences of the present invention (for example, the oddly numbered sequence SEQ ID NO: in the Sequence List) (or part thereof), due to the degeneracy of the genetic code, and therefore encode the same protein SRT, which is encoded by the nucleotide sequences of the present invention. In another embodiment, an isolated nucleic acid molecule of the present invention has a nucleotide sequence encoding a protein having the amino acid sequence indicated in the Sequence List (e.g., the evenly numbered sequence SEQ ID NO :). In yet another embodiment, the nucleic acid molecule of the present invention encodes a full-length C. glutamicum protein that is substantially homologous to the amino acid sequence of the present invention (encoded by the open reading frame indicated in the oddly numbered sequence of SEQ ID NO: in the Sequence List).

It will be apparent to one skilled in the art that in one embodiment of the invention, the sequences of the present invention should not include known sequences, such as those found in Genbank, shown in Tables 2 or 4, which were known prior to the invention. In one embodiment, the present invention relates to nucleotide and amino acid sequences that are longer than a known sequence (for example, the sequence available in Genbank) (or the protein encoded by such a sequence) shown in Table 2 or 4). So, for example, the present invention relates to a nucleotide sequence that is larger and / or at least 39% identical to the nucleotide sequence indicated by RXA00084 (SEQ ID NO: 189), a nucleotide sequence that is larger and / or at least , 56% identical to the nucleotide sequence designated RXA00605 (SEQ ID NO: 11), and a nucleotide sequence that is larger and / or at least 50% identical to the nucleotide sequence designated RXA00886 (SEQ ID NO: 39). Any person skilled in the art can calculate the lower limit of percent identity for any given sequence of the present invention by evaluating the GAP-calculated values on the identity scale presented in Table 4 for each of the three best matches for this sequence, and by subtracting the largest GAP-calculated value of percent identity from 100 percent. It is also apparent to each person skilled in the art that nucleotide and amino acid sequences having a percentage of identity greater than the lower threshold thus calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%, preferably at least 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89% or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98% , 99% or more identical) are also included in the scope of this of the invention.

In addition to the nucleotide sequences of C. glutamicum SRTs presented in the Sequence Listing as oddly numbered SEQ ID NO #, it is obvious for every person that polymorphism of DNA sequences can occur in this population (for example, in C. glutamicum population), which leads to changes in the amino acid sequences of SRT proteins. Such genetic polymorphism in the SRT gene may be present in individuals of a given population due to natural modifications. As used herein, the terms “gene” and “recombinant gene” mean nucleic acid molecules containing an open reading frame encoding an SRT protein, preferably C. glutamicum SRT protein. Such natural variations may be mainly the result of a 1-5% variation in the nucleotide sequence of the SRT gene. Any of such variations in the nucleotide sequence leading to polymorphism in SRT, which is a consequence of natural variations, and therefore does not lead to a change in the functional activity of SRT proteins, are included in the scope of the present invention.

Nucleic acid molecules corresponding to naturally occurring variants and not derived from C. glutamicum homologues of the C. glutamicum SRT DNA of the present invention can be isolated based on their homology with the C. qlutamicum SRT nucleic acid described herein using C. glutamicum or parts thereof, as a probe for hybridization in accordance with standard hybridization methods under stringent hybridization conditions. Accordingly, in another embodiment, the isolated nucleic acid molecule of the present invention has a length of at least 15 nucleotides and hybridizes under stringent conditions to a nucleic acid molecule containing the nucleotide sequence represented by the oddly numbered sequence SEQ ID NO: in List of sequences. In other embodiments, the nucleic acid is at least 30, 50, 100, 250, or more nucleotides in length. As used herein, the term “hybridizes under stringent conditions” refers to hybridization and washing under the described conditions under which nucleotide sequences having at least 60% homology typically continue to hybridize to each other. These conditions should preferably be such that these sequences having a homology of at least about 65%, more preferably at least about 70%, and even more preferably at least about 75 % or more, mainly continue to hybridize with each other. Such stringent conditions are known to those skilled in the art and can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1.-6.3.6. A preferred non-limiting example of hybridization under stringent conditions is hybridization in 6x sodium chloride / sodium citrate (SSC) at about 45 ° C followed by one or more washes in 0.2 × SSC, 0.1% SDS at 55-65 ° C. In this case, it is preferable that the isolated nucleic acid molecule of the present invention, which hybridizes under stringent conditions with the nucleotide sequence of the present invention, corresponds to a natural nucleic acid molecule. As used herein, the term “natural” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence found in nature (eg, encoding a natural protein). In one embodiment, the nucleic acid encodes a native C. glutamicum SRT protein.

In addition to the naturally occurring variants of the SRT sequence that may be present in a given population, it is also apparent to one skilled in the art that changes may be made to the nucleotide sequence of the present invention by mutation, which thereby leads to changes in the amino acid sequence of the encoded SRT protein without affecting, at the same time, on the functional ability of this SRT protein. Thus, for example, nucleotide substitutions can be made in the nucleotide sequence of the present invention, resulting in substitutions in “non-essential” amino acid residues. A “non-essential” amino acid residue is an amino acid residue that can be replaced in the wild-type sequence of one of the SRT proteins (for example, in the evenly numbered sequence of SEQ ID NO: in the Sequence List) without changing the activity of said SRT protein, while the “essential” amino acid the residue is the amino acid residue that is necessary for the activity of the SRT protein. However, there are other amino acid residues (for example, residues that are not conserved or are only semi-conserved in a domain with SRT activity) that may not play a major role in protein activity, and therefore, they can probably be replaced without change SRT activity.

Accordingly, in another aspect of its implementation, the present invention relates to nucleic acid molecules encoding SRT proteins that contain changes in amino acid residues that are not important for SRT activity. The amino acid sequence of such an SRT protein is different from the amino acid sequence of the indicated evenly numbered sequence of SEQ ID NO: in the Sequence List, but still retains at least one of the SRT activities described herein. In one embodiment, an isolated nucleic acid molecule has a nucleotide sequence encoding a protein, where the protein comprises an amino acid sequence that is at least about 50% homologous to the amino acid sequence of the present invention, capable of increasing resistance or tolerance to one or more harmful factors or stresses caused by environmental exposure or chemicals, or has one or more activities, represents listed in table 1. Preferably, the protein encoded by the specified nucleic acid molecule. was at least about 50-60% homologous to the amino acid sequence of one of the oddly numbered sequences in the List of sequences, more preferably at least about 60-70%, homologous to one of these sequences, more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of these sequences, and most preferably at least about 96%, 97%, 98% or 99% homologous to one of the amino acid sequences of the present inventions.

To determine the percent homology of two amino acid sequences (for example, one of the amino acid sequences of the present invention and its mutant) or two nucleic acids, the sequences are arranged for their optimal comparison (for example, spaces can be introduced into a given sequence of a single protein or nucleic acid to optimally match it primary sequence with the sequence of another protein or nucleic acid). Then compare amino acid residues or nucleotides at the corresponding positions of amino acids or nucleotides. If a position in one sequence (e.g., one of the amino acid sequences of the present invention) is occupied by the same amino acid residue or nucleotide present in the corresponding position of another sequence (e.g., a mutant form of the amino acid sequence), then these molecules are homologous in this position (i.e. , as used herein, the term “homology” of amino acids or nucleic acids is equivalent to the term “identity” of amino acids or nucleic acids). The percentage of homology between the two sequences depends on the number of identical positions common to these sequences (i.e.,% homology = number (#) of identical positions / number of all (#) positions × 100).

An isolated nucleic acid molecule encoding an SRT protein homologous to the protein sequence of the present invention (e.g., the evenly numbered sequence SEQ ID NO: in the List of Sequences) can be created by introducing one or more nucleotide substitutions, additions or deletions to the nucleotide sequence of the present invention, so so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the present invention by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferably carried out in one or more of the alleged “non-essential” amino acid residues. “Conservative amino acid substitution” means replacing a present amino acid residue with an amino acid residue having a similar side chain. A family of amino acid residues having a similar side chain is known to those skilled in the art. Such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), with acidic side chains (e.g., aspartic acid, glutamic acid), with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), with beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyros in, phenylalanine, tryptophan, histidine). Thus, the putative non-essential amino acid residue in the SRT protein is preferably replaced by another amino acid residue from the family with the same side chain. Alternatively, in another embodiment of the invention, mutations can be arbitrarily introduced into the entire SRT coding sequence or part thereof, for example, by saturating mutagenesis, and the resulting mutants can be screened for the SRT activity described in this application to identify mutants that retain SRT activity. After mutagenesis of the nucleotide sequence, which is one of the oddly numbered sequences of SEQ ID NOs in the Sequence List), the encoded protein can be recombinantly expressed and the activity of this protein can be determined using the assays described here (see Example 8 in the “Illustrative Examples” section).

In addition to the nucleic acid molecules encoding the SRT proteins described above, in another aspect, the present invention relates to isolated nucleic acid molecules that are antisense. An “antisense” nucleic acid includes a nucleotide sequence complementary to a “sense” nucleic acid encoding a protein, for example, a complementary coding strand to a double-stranded DNA molecule or a complementary mRNA sequence. Accordingly, the antisense nucleic acid may be linked to the sense nucleic acid by a hydrogen bond. The antisense nucleic acid may be complementary to the entire SRT coding chain, or only part of it. In one embodiment, an antisense nucleic acid molecule is a molecule that is antisense with respect to the “coding region” of a given coding strand of a nucleotide sequence encoding an SRT protein. The term "coding region" means a region of a nucleotide sequence containing codons that translate to form amino acid residues (for example, the complete coding region of SEQ ID NO: 120 (RXA00600) includes nucleotides 1-1098). In another embodiment, an antisense nucleic acid molecule is a molecule that is antisense with respect to the “non-coding region” of a given coding strand of a nucleotide sequence encoding an SRT. The term "non-coding region" means 5'- and 3'-sequences flanking the coding region, which is not translated into amino acids (that is, also called 5'- and 3'-non-translated regions).

Given that the SRT coding sequences of the coding chain described herein (e.g., sequences represented by the oddly numbered SEQ ID NOs sequences in the Sequence List), the antisense nucleic acids of the present invention can be constructed in accordance with Watson and Crick base pairing rules. The antisense nucleic acid molecule may be complementary to the entire coding region of the SRT mRNA, but it is more preferable that the molecule is an oligonucleotide that is antisense only with respect to the part encoding or non-coding for the SRT mRNA. So, for example, an antisense oligonucleotide can be complementary to the region surrounding the SRT mRNA translation initiation site. An antisense oligonucleotide may, for example, have a length of approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides. The antisense nucleic acid of the present invention can be constructed by chemical synthesis or enzymatic ligation reactions in accordance with known procedures. So, for example, an antisense nucleic acid (for example, an antisense oligonucleotide) can be chemically synthesized using natural nucleotides or variously modified nucleotides designed to increase the biological stability of these molecules or to increase the physical stability of a duplex formed between antisense and sense nucleic acids, for example derivatives of phosphorothioate and nucleotides substituted with acridine can be used. Examples of modified nucleotides that can be used to generate this antisense nucleic acid are 5-fluorouracil, 5-bromouracil, 5-chlororacil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracilethyl, 5-carb -2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylquinosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylguanine, 3 -methylcytosine, N6-adenine, 7-methylguanine, 5-methyl aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylquinosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), pseudo-acetic acid, vibine, pseudo-acetic acid, vibration -thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-hydroxyacetic acid methyl ester, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w and 2,6-diaminopurine. Alternatively, said antisense nucleic acid may be biologically produced using an expression vector in which the nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will have an antisense orientation with respect to the target nucleic acid of interest further described in the following subsection).

The antisense nucleic acid molecules of the present invention are typically introduced into a cell or generated in situ so that they hybridize or bind to cellular mRNA and / or genomic DNA encoding an SRT protein in order to inhibit expression of the protein, for example, by inhibiting transcription and / or translation . Such hybridization can occur due to the corresponding complementarity of nucleotides with the formation of a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the main groove of the double helix. This antisense molecule can be modified so that it specifically binds to a receptor or antigen expressed on the surface of a selected cell, for example, by binding an antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. Said antisense nucleic acid molecule can also be delivered to cells using the vectors described in this application. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is controlled by a strong prokaryotic, viral, or eukaryotic promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of the present invention is an α-anomeric nucleic acid molecule, an α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNAs, in which, unlike ordinary β-units, the chains are parallel to each other (Gaultier et al. (1987) Nucl. Acids Res. 15: 6625-6641). This antisense nucleic acid molecule may also include a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic. Acids Res. 15: 6131-6148) or a chimeric RNA DNA analogue (Inoue et al. (1987) FEBS Lett 215: 327-330).

In another embodiment, the antisense nucleic acid molecule is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as mRNA, and with which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described by Haselhoff & Gerlach (1988) Nature 334: 585-591)) can be used to catalyze the cleavage of SRT mRNA transcripts to inhibit the translation of SRT mRNA. A ribozyme specific for the SRT coding nucleic acid can be constructed based on the nucleotide sequence of the SRT cDNA described herein (i.e., SEQ ID NO: 119 (RXA00600)). Thus, for example, an IVS Tetrahymena L-19 RNA derivative can be constructed in which the nucleotide sequence of the active center is complementary to the nucleotide sequence cleaved in the SRT-coding mRNA. See, for example, Cech et al., US Pat. No. 4,987,071 and Cech et al., US Pat. No. 5,116,742. Alternatively, SRT mRNA can be used to select catalytic RNA having specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel, D. & Szostak, J.W. (1993) Science 261: 1411-1418.

Alternatively, the expression of the SRT gene can be inhibited by targeted delivery of nucleotide sequences complementary to the regulatory region of the SRT nucleotide sequence (e.g., promoter and / or enhancer for the SRT gene) with the formation of three-strand helical structures that inhibit transcription of the SRT gene in target cells. In general terms, see Helene, C (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad.Sci. 660: 27-36; and Maher, L.J. (1992) Bioassays 14 (12): 807-15.

B. Recombinant expression vectors and host cells.

In another aspect, the present invention relates to vectors, preferably, expression vectors containing nucleic acid encoding an SRT protein (or part thereof). As used herein, the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid that has been attached to it. One type of vector is a “plasmid," which is a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, where additional DNA segments can be inserted into a given viral genome. Some vectors are capable of autonomous replication in the host cell into which they were introduced (for example, bacterial vectors having a bacterial replication origin and episomal mammalian vectors). Other vectors (for example, non-episomal mammalian vectors), when introduced into the host cell, are integrated into the genome of this host cell and are thereby replicated along with the host genome. In addition, some vectors are able to regulate the expression of genes that have been functionally integrated into them. Such vectors are referred to herein as “expression vectors”. In general, expression vectors used in recombinant DNA technology are often present in the form of plasmids. As used herein, the terms “plasmid” and “vector” are used interchangeably because the plasmid is the most commonly used form of the vector. However, the present invention also includes other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that have equivalent functions.

The recombinant expression vectors of the present invention contain the nucleic acid of the present invention in a form suitable for expression of a nucleic acid in a host cell, which means that these recombinant expression vectors include one or more regulatory sequences that have been selected based on the host cells used for expression , and which were functionally attached to the expressed nucleic acid sequence. In said recombinant expression vector, a “functionally linked” sequence means that the desired nucleotide sequence is attached to the regulatory sequence (s) such that the nucleotide sequence is expressed (for example, in an in vitro transcription / translation system or in a cell- after introduction of this vector into a given host cell). The term "regulatory sequence" includes promoters, enhancers and other elements that control expression (for example, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gen Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences are those that are responsible for the constitutive expression of nucleotide sequences in many types of host cells and which are responsible for the expression of the nucleotide sequence only in certain host cells. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, Ipp-, lac-, lpp-lac-, lacI ^q -, T7-, T5-, T3 -, gal-, trc-, ara-, SP6-, arny, SPO2, λ-p _R - or λ-P _L , which are preferably used in bacteria. Additional regulatory sequences are, for example, promoters derived from yeast and fungi, such as ADC1, MFα, AC, P60, CYC1, GAPDH, TEF, rp28, ADH, plant derived promoters, such as CAMV / 35S, SSU, OCS , lib4, usp, STLS1, B33, nos, or ubiquitin or phaseolin promoters. Artificial promoters may also be used. It is obvious to each person skilled in the art that the construction of an expression vector may depend on factors such as the choice of a host cell that is transformed and the level of expression of the desired protein, etc. The expression vectors of the present invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides encoded by the nucleic acids described herein (e.g., SRT proteins, mutant forms of SRT proteins, fusion proteins, etc.) .

Recombinant expression vectors of the present invention can be designed to express SRT proteins in prokaryotic or eukaryotic cells. For example, SRT genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanes, MA et al. (1992) “Foreign gene expression in yeast: a review. ”Yeast 8: 423-488; van den Hondel, CAMJJ et al. (1991)“ Heterologous gene expression in filamentous fungi ”: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, eds. , p. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer systems and vector development for filamentous fungi: Applied Molecular Genetics of Fungi", Peberdy, JF et al. , eds., p.1-28, Cambridge University Press: Cambridge), algal cells and multicellular plants (see Schmidt R. & Willmitzer L. (1988) High efficiency Agrobacterium tumefaciens - mediated transformation of Arabidopsis thaliana leaf and cotyledon explants "Plant Cell Rep: 583-586) or mammalian cells. Suitable host cells are discussed in more detail in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the indicated recombinant expression vector can be transcribed and translated in vitro, for example, using the T7 promoter regulatory sequences and T7 polymerase.

The expression of proteins in prokaryotes, in most cases, is carried out using vectors containing constitutive or inducible promoters responsible for the expression of hybrid or non-hybrid proteins. Hybrid vectors add a number of amino acids to the protein encoded in this vector, usually to the amino terminus of a given recombinant protein. Such hybrid vectors are typically used for three purposes: 1) to increase the expression of a recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to facilitate the purification of the recombinant protein due to its action as a ligand in affinity purification. Often, the proteolytic cleavage site is introduced into the hybrid expression vectors at the junction between the fusion moiety and the recombinant protein, which allows the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. Such enzymes and their cognate recognition sequences are factor Xa, thrombin and enterokinase.

Typical vectors for expressing hybrid molecules are pGEX (Pharmacia Biotech Inc .; Smith, DB & Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia Piscataway, NJ) which attach glutathione S-transferase (GST), binding to maltose E protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the SRT protein coding sequence is cloned into a pGEX expression vector to create a vector encoding a fusion protein, comprising, from the N-terminus to the C-terminus, a “GST thrombin-protein X cleavage site”. This fusion protein can be purified by glutathione agarose affinity chromatography. A recombinant SRT protein that is not attached to GST can be isolated by cleaving the fusion protein with thrombin.

Examples of suitable inducible non-hybrid E. coli expression vectors are pTrc (Amann et al., (1988) Gene 69: 301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, Px30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pMBL24, , pUR290, pIN-III113-B1, λgt11, pBdCl and Pet11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San. Diego, California (1990) 60-89; and Pouwels et al. , eds. (1985) Cloning Vectors, Elsevier: New York IBSN 0444904018). The expression of the target gene from the pTrc vector is based on transcription using host RNA polymerase from the trp-lac hybrid promoter. The expression of the target gene from the pET 11d vector is based on transcription from the T7 gn10-lac hybrid promoter mediated by coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from the present prophage λ containing the T7 gnl gene under the transcriptional control of the lacUV5 promoter. To transform other types of bacteria, appropriate vectors can be selected. For example, it is known that plasmids pIJ101, pIJ364, pIJ702 and pIJ361 can be used to transform streptomycetes, and plasmids pUB110, pC194 or pBD214 are suitable for transformation of Bacillus species. Some plasmids used to transfer genetic information to Corynebacterium are pHM1519, pBLl, pSA77 or pAJ667 (Pouwels et al., Eds. (1985) Cloning Vectors, Elsevier: New York IBSN 0444904018).

One strategy for maximizing the expression of a recombinant protein is to express the protein in a host bacterium with impaired proteolytic cleavage of the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119 -128). Another strategy is to change the nucleic acid sequence inserted into the expression vector so that the individual codons for each amino acid are codons, preferably used in the bacterium selected for expression, such as C. glutamicum (Wada et al., (1992) Nucleic. Acids Res. 20: 2111-2118). Such a change in the nucleic acid sequences of the present invention can be carried out by standard DNA synthesis methods.

In another embodiment, the SRT protein expressing vector is a yeast expression vector. Examples of vectors for expression in S. cerevisiae yeast are pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), 2μ, pAGI, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan & Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods for constructing vectors suitable for use in other fungi, such as hyphomycetes, are vectors and methods described in detail by van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, Applied Molecular Genetics of Fungi", J.F. Peberdy, et al., Eds., P. 1-28, Cambridge University Press: Cambridge and Pouwels et al., Eds. (1985) Cloning Vectors. Elsevier: New York (IBSN 0444904018).

Alternatively, the SRT proteins of the present invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors suitable for expression of proteins in cultured insect cells (e.g., Sf9 cells) are pAC series vectors (Smith et al. (1983) Mol. Cell. Biol. 3: 2156-2165) and pVL series vectors (Lucklow & Summers (1989) Virology 170: 31-39).

In another embodiment of the invention, the SRT proteins of the present invention can be expressed in cells of unicellular plants (such as algae) or in cells of higher plants (for example, spermatophytes, such as cultivated plants). Examples of plant expression vectors are vectors described in detail by Becker, D. Kemper, E., Schell, J. & Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border". Plant Mol. Biol. 20: 1195-1197; and Bevan M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acid Res. 12: 8711-8721 and include pLGV23, pGHlac +, pBIN19, pAK2004 and pDH51 (Pouwels et al., Eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0444904018).

In yet another embodiment, the nucleic acid of the present invention is expressed in mammalian cells using an expression vector derived from a mammal. Examples of mammalian expression vectors are pCDM8 (Seed, B. (1987) Nature 329-840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, functions regulated by the expression vector are in most cases provided by viral regulatory elements. So, for example, commonly used promoters come from poliomavirus, adenovirus 2, cytomegalovirus and monkey virus SV40. Other suitable expression systems for prokaryotic and eukaryotic cells are described in chapters 16 and 17 of Sambrook, J., Fritsh, E.F. & Maniatis T., Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Laboratory Press, Cold Spring Harbor, NY, 1989.

In another embodiment of the invention, the recombinant mammalian expression vector has the ability to direct nucleic acid expression preferably in cells of this particular type (for example, tissue-specific regulatory elements are used to express the nucleic acid). Such tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters are the albumin promoter (liver-specific; Pinkert et al., (1987) Genes Dev. 1: 268-277), the lymphoid-specific promoters (Calame & Eaton (1988) Adv. Immunol, 43: 235 -275), and in particular, promoters of T-cell receptors (Winoto & Baltimore (1989) EMBO J. 8: 728-733) and immunoglobulins (Banerji et al., (1983) Cell 33: 729-740; Queen & Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g. Wugpe & Ruddle neurofilament promoter (1989) PNAS 86: 5473-5477), pancreatic specific promoters (Edlund et al., (1985) Science 230: 912-916), and promoters, specific mammalian mammary glands (for example, a whey promoter; US patent No. 4873316 and publication of European application No. 264166). Also included within the scope of the present invention are stage-specific regulated promoters, for example, mouse hox promoters (Kessel & Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes & Tilghman (1989) Genes Dev. 3: 537-546) .

The present invention also relates to a recombinant expression vector containing the DNA molecule of the present invention, cloned into the expression vector in antisense orientation. That is, the DNA molecule is functionally attached to the regulatory sequence so that the expression (by transcription of this DNA molecule) of the RNA molecule is provided that is antisense to SRT mRNA. Regulatory sequences functionally linked to an antisense cloned nucleic acid can be selected that regulate the continuous expression of antisense RNA molecules in various cell types, for example, viral promoters and / or enhancers or regulatory sequences that regulate constitutive tissue-specific or cell-specific expression of antisense RNA. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a highly efficient regulatory region, the activity of which can be determined by the type of cells into which the vector is introduced. A discussion of the regulation of gene expression using antisense genes can be found in Weintraub, H. et al., Antisence RNA as a molecular tool for genetic analysis, Rewiews - Trends in Genetics., Vol. 1 (1) 1986.

In another aspect, the present invention relates to host cells into which a recombinant expression vector of the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be noted that these terms refer not only to the particular cell in question, but also to their offspring or their potential offspring. Since some modifications may be present in subsequent generations due to either a mutation or environmental influences, such progeny may not actually be identical to the progeny of the parent cells, but, nevertheless, it is also included in the scope of the term used here.

A host cell can be either a prokaryotic or eukaryotic cell. For example, SRT protein can be expressed in bacterial cells, such as C. glutamicum, in insect cells, in yeast or mammalian cells (such as Chinese hamster ovary (CHO) cells or COS cells). Other suitable host cells are known in the art. Microorganisms related to Corynebacterium glutamicum, which can be successfully used as host cells for expression of the nucleic acid molecules and the protein of the present invention, are presented in table 3.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by standard methods of transformation or transfection. As used herein, the terms “transformation” and “transfection” refer to a number of well-known methods for introducing foreign nucleic acid (eg, linear DNA or RNA (eg, a linearized vector or gene construct without a vector) or nucleic acid in the form of a vector (eg, plasmid, phage , phasemids, phagemids, transposon or other DNA)) into the host cell, including co-precipitation with calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran, lipofection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., (1989). Molecular Cloning: A Laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and other laboratory manuals.

It is known that for stable transfection of mammalian cells, depending on the expression vector used and the transfection method, only a small part of the cells is able to integrate foreign DNA into their genome. To identify and select cells with integrated DNA, a gene encoding a selectable marker (e.g., an antibiotic resistance gene) is usually introduced into the host cells along with the desired gene. Preferred selective markers are markers that report resistance to drugs such as G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker can be introduced into the host cell using the same vector that encodes the SRT protein, or it can be introduced using a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by selection in the presence of the drug (for example, cells into which the selective marker gene has been introduced will survive and other cells will die).

To create a homologous recombinant microorganism, a vector is obtained containing at least a portion of the SRT gene in which a deletion, addition or substitution has been made in order to modify it, for example, functional destruction of the SRT gene. Such an SRT gene is preferably the Corynebacterium glutamicum SRT gene, but it may be a homologous gene derived from a related bacterium or even from a mammal, yeast or insect. In a preferred embodiment of the invention, the vector is designed so that after homologous recombination, the endogenous SRT gene is functionally destroyed (that is, so that it no longer encodes a functional protein, and such a vector is also called a knockout vector). Alternatively, this vector can be designed so that after homologous recombination, the endogenous SRT gene is mutated or otherwise modified, but encodes a functional protein (for example, the upstream regulatory region can be modified with a change in the expression of the endogenous SRT protein). In a vector for homologous recombination, a modified portion of the SRT gene is flanked at its 5 ′ and 3 ′ ends by the additional nucleic acid of the SRT gene, resulting in homologous recombination between the exogenous SRT gene contained in this vector and the endogenous SRT gene in the microorganism. The additional flanking nucleic acid SRT is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several thousand base pairs of flanking DNA (from the 5 ′ and 3 ′ ends) are included in the vector (for a description of the vectors for homologous recombination, see, for example, Thomas K.R. & Capecchi M.R. (1987) Cell 51: 503). This vector is introduced into the microorganism (for example, by electroporation), and cells in which the introduced SRT gene undergoes homologous recombination with the endogenous SRT gene are selected by known methods.

In another embodiment of the invention, recombinant microorganisms can be produced that contain selected systems that allow for controlled expression of the introduced gene. For example, the inclusion of the SRT gene in a vector under the control of the lac operon promotes the expression of the SRT gene only in the presence of IPTG. Such regulatory systems are well known in the art.

In another embodiment, the endogenous SRT gene in the host cell is destroyed (for example, by homologous recombination or other genetic means known to those skilled in the art) so that expression of its protein product does not occur. In another embodiment, the endogenous or introduced SRT gene in the host cell has been modified by one or more point mutations, deletions or inversions, but it still encodes a functional SRT protein. In yet another embodiment, one or more regulatory regions (e.g., a promoter, repressor, or inducer) of an SRT gene in a microorganism has been modified (e.g., by deletion, truncation, inversion, or point mutation) to modulate SRT gene expression. It will be apparent to any person skilled in the art that host cells containing more than one modification of the described gene and SRT protein can be easily produced by the methods of the present invention and are within the scope of the present invention.

A host cell of the present invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an SRT protein. Accordingly, the present invention further relates to methods for producing SRT proteins using host cells of the present invention. In one embodiment, the method comprises culturing a host cell of the present invention (into which a recombinant expression vector encoding an SRT protein has been introduced, or into a genome of which a gene encoding a wild-type SRT protein or a modified SRT protein has been introduced) in a suitable medium until until the SRT protein is produced. In another embodiment, the method further comprises isolating SRT proteins from the medium or from the host cell.

C. Isolation of SRT Proteins

In another aspect, the present invention relates to isolated SRT proteins and their biologically active parts. An “isolated” or “purified” protein or its biologically active part mainly does not contain cellular material when produced by recombinant DNA methods, or chemical precursors or other chemical compounds in chemical synthesis. The expression “substantially free of cellular material” refers to preparations of the SRT protein in which the protein has been separated from the cellular components of the cell in which it was produced naturally or recombinantly. In one embodiment, the expression “substantially free of cellular material” refers to formulations of an SRT protein containing about less than 30% (dry weight) of a non-SRT protein (also called an “impurity protein”), more preferably about less than 20% of a non-SRT protein, even more preferably about less than 10% of a non-SRT protein, and most preferably about less than 5% of a non-SRT protein. If the SRT protein or its biologically active part was produced by the recombinant method, it is preferable that they also do not contain the culture medium, that is, the culture medium is approximately less than 20%, more preferably approximately less than 10%, and most preferably , approximately less than 5% by volume of the protein preparation. The expression "basically does not contain chemical precursors or other compounds" refers to preparations of the SRT protein in which the protein was separated from chemical precursors or other compounds that were involved in the synthesis of this protein. In one embodiment, the expression “substantially free of chemical precursors or other compounds” refers to SRT protein formulations containing about less than 30% (dry weight) of chemical precursors or non-SRT chemical compounds, more preferably about less than 20% of chemical precursors or non-protein chemical compounds of SRT, even more preferably, less than about 10% of chemical precursors or non-protein chemical compounds ohm SRT, and most preferably less than about 5% chemical precursors or chemicals other than the protein SRT. In preferred embodiments of the invention, the isolated proteins or their biologically active parts do not contain impurity proteins from the same organism from which the given SRT protein is derived. Typically, these proteins are produced by recombinant expression of, for example, C. glutamicum SRT protein in a microorganism such as C. glutamicum.

An isolated SRT protein of the present invention or part thereof may impart resistance or tolerance to one or more harmful factors or stresses caused by chemicals or environmental influences, and has one or more activities indicated in Table 1. In preferred embodiments of the invention, the protein or part of it includes an amino acid sequence that is significantly homologous to the amino acid sequence of the present invention (e.g., evenly numbered sequence SEQ ID NO: in the Sequence List), such that the protein or part thereof retains the ability to mediate this resistance or tolerance to one or more harmful factors or stresses caused by chemical compounds or environmental exposure. The specified portion of the protein is, preferably, the biologically active part described in this application. In another preferred embodiment, the SRT protein has an amino acid sequence encoded by a nucleic acid represented by the evenly numbered sequence SEQ ID NO: in the Sequence Listing. In another preferred embodiment, the SRT protein has an amino acid sequence encoded by a nucleic acid that, for example, hybridizes under stringent conditions to one of the nucleotide sequences of the present invention (e.g., the oddly numbered sequence SEQ ID NO: in the Sequence List). In another preferred embodiment, the SRT protein has an amino acid sequence encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57% , 58%, 59% or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89% or 90%, 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98 %, 99% or more is homologous to one and nucleotide sequences of the invention or parts thereof. The scope of the present invention also includes ranges and values of identities that are intermediate between the above ranges (for example, 70-90% identity or 80-95% identity). Thus, for example, the scope of the present invention also includes ranges of identity values using a combination of any of the above values indicated as upper and / or lower limits. In yet another preferred embodiment of the invention, the SRT proteins of the present invention also have at least one of the SRT activities described in this application. For example, a preferred SRT protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence that hybridizes, for example, hybridizes under stringent conditions, with the nucleotide sequence of the present invention, and which increases the resistance or tolerance of C. glutamicum to one or more harmful factors or stresses caused by exposure to the environment or chemicals, or which has one or more activities listed in table 1.

In other embodiments, the SRT protein is substantially homologous to the amino acid sequence of the present invention (for example, the evenly numbered sequence SEQ ID NO: in the Sequence List) and retains the functional activity of the protein in which one of the amino acid sequences of the present invention is already different in amino acid sequences due to natural change or mutagenesis, as described in detail above in subsection I. Accordingly, in another embodiment, o of the invention, an SRT protein is a protein containing an amino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89% or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to the entire amino acid sequence of the present invention, and to Thoraya has at least one of the above activities SRT. The scope of the present invention also includes ranges and values of identities that are intermediate between the above ranges (for example, 70-90% identity or 80-95% identity). Thus, for example, the scope of the present invention also includes ranges of identity values using a combination of any of the above values indicated as upper and / or lower limits. In another embodiment, the present invention relates to a full-sized protein of C. qlutamicum, which is essentially homologous to the entire amino acid sequence of the present invention.

The biologically active portions of the SRT protein include peptides containing amino acid sequences derived from the amino acid sequence of the SRT protein, for example, the amino acid sequence of SEQ ID NO having an even number in the List of sequences or the amino acid sequence of a protein homologous to the SRT protein, which contains fewer amino acids than full length SRT protein, or a full-sized protein that is homologous to the SRT protein and has at least one of the activities of the SRT protein. Typically, biologically active parts (peptides, for example, peptides that are 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100, or more amino acids in length) include a domain or motif having at least one of the activities of the SRT protein. In addition, other biologically active parts in which other regions of this protein have been deleted can be obtained by recombinant DNA methods and evaluated for one or more of the activities described in this application. Preferably, the biologically active parts of the SRT protein include one or more selected domains / motifs or parts thereof having biological activity.

SRT proteins are preferably produced by recombinant DNA methods. So, for example, a nucleic acid molecule encoding a given protein is cloned into an expression vector (as described above), this expression vector is introduced into the host cell (as described above), and the SRT protein is expressed in this host cell. The SRT protein can then be isolated from cells in accordance with a suitable purification scheme using standard protein purification methods. As an alternative to recombinant expression, the SRT protein, polypeptide or peptide can be synthesized by a standard chemical peptide synthesis method. In addition, a native SRT protein can be isolated from cells (eg, endothelial cells), for example, using an anti-SRT antibody, which can be produced by standard methods using the SRT protein of the present invention or a fragment thereof.

The present invention also relates to chimeric or hybrid SRT proteins. As used herein, the term “chimeric protein” or “fusion protein” of SRT means an SRT polypeptide operably linked to a non-SRT polypeptide. The term "SRT polypeptide" means a polypeptide having an amino acid sequence corresponding to SRT, and the term "non-SRT polypeptide" means a polypeptide having an amino acid sequence corresponding to a protein that is generally not homologous to an SRT protein, for example, a protein that different from SRT protein and which comes from the same or different organism. As for the fusion protein, the term “operably linked” means that the SRT polypeptide and non-SRT polypeptide are attached to each other while maintaining a reading frame. Said non-SRT polypeptide may be attached to the N-terminus or to the C-terminus of the SRT polypeptide. For example, in one embodiment, the fusion protein is a GST-SRT fusion protein in which SRT sequences are attached to the C-terminus of the GST sequences. Such fusion proteins may facilitate the purification of recombinant SRT proteins. In another embodiment, the fusion protein is an SRT protein containing a heterologous signal sequence at its N-terminus. In some host cells (e.g., mammalian host cells), the expression and / or secretion of the SRT protein can be increased using a heterologous signal sequence.

The chimeric or hybrid SRT protein of the present invention is preferably produced by standard recombinant DNA methods. So, for example, DNA fragments encoding other polypeptide sequences are ligated while maintaining the reading frame by standard methods, for example, by using blunt or sticky ends for ligation, cleavage with restrictase enzymes to obtain the corresponding ends, building up sticky ends, if necessary, treatment with alkaline phosphatase to avoid undesired attachment, and enzymatic ligation. In another embodiment of the invention, the hybrid gene can be synthesized by standard methods, including the use of automatic DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that form complementary protruding regions between two consecutive gene fragments, which can then be annealed and re-amplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons, 1992). In addition, many expression vectors are commercially available and already encode part of the hybrid (e.g., GST polypeptide).

An SRT-coding nucleic acid may be cloned into said expression vector so that this portion of the hybrid is attached to the SRT protein while maintaining the reading frame.

Homologous SRT proteins can be generated by mutagenesis, for example, by a single point mutation or truncation of the SRT protein. As used herein, the term “homologue” means a variant form of the SRT protein that acts as an agonist or antagonist of the activity of the SRT protein. An SRT protein agonist can basically retain the same activities, or sub-series, of the biological activities of the SRT protein. An SRT protein antagonist can inhibit one or more activities of the naturally occurring form of the SRT protein, for example, by competitively binding to a lower or upstream member of the SRT system that includes the SRT protein. So, for example, C. glutamicum SRT protein and its homologues of the present invention can increase the tolerance or resistance of C. glutamicum to one or more stresses caused by chemicals or environmental influences.

In an alternative embodiment of the invention, SRT protein homologues can be identified by screening combinatorial libraries of mutants, for example, truncated mutants of the SRT protein, for agonistic or antagonistic activity of the SRT protein. In one embodiment of the invention, a library of diverse SRT variants is generated by combinatorial mutagenesis at the nucleic acid level, where these diverse variants are encoded by a library of diverse genes. A library of diverse SRT variants can be created, for example, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences so that a degenerate series of potential SRT sequences is expressed as separate polypeptides, or alternatively, as a series of larger fusion proteins (e.g. for phage display) containing a series of SRT sequences. There are a number of methods that can be used to produce libraries of potential SRT homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be carried out on an automatic DNA synthesizer, and then this synthetic gene is ligated into the corresponding expression vector. Using a degenerate series of genes makes it possible to obtain, in a single mixture, all sequences encoding the desired series of potential SRT sequences. Methods for the synthesis of degenerate oligonucleotides are known in the art (see, for example, Narang SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056 ; Ike et al. (1983) Nucleic. Acid. Res. 11: 477.

In addition, libraries of fragments of the SRT coding protein can be used to generate diverse populations of SRT fragments for screening and subsequent selection of homologues of the SRT protein. In one embodiment of the invention, a library of fragments of the coding sequence can be created by processing a double-stranded PCR fragment of the SRT coding sequence with nuclease under conditions under which nick-break occurs approximately once per molecule, denaturation of double-stranded DNA, renaturation of DNA to form double-stranded DNA , which may include semantic / antisense pairs from various nick-gap products, removing single-stranded parts from newly formed duplexes by processing S1 nuclease and ligation of the resulting fragment library into an expression vector. Using this method, a library of expressed sequences can be obtained that encodes the N-terminal, C-terminal and internal fragments of the SRT protein of various sizes.

Several methods are known to those skilled in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and screening cDNA libraries for the presence of gene products with selected properties. Such methods can be adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of SRT homologs. The most widely used methods, which are suitable for high-throughput analysis used to screen large gene libraries, usually involve cloning the gene library into replicable expression vectors, transforming the corresponding cells with the obtained vector library, and expressing combinatorial genes under conditions in which detection of the desired activity facilitates vector isolation encoding a given gene whose product has been detected. Recursive combined mutagenesis (REM), a new method that increases the frequency of functional mutants in these libraries, can be used in combination with screening assays to identify SRT homologs (Arkin & Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

In another embodiment, cell assays may be used to analyze a variety of SRT libraries using methods well known in the art.

D. Applications and methods of the present invention

Nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors and host cells can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of the genomes of organisms related to C. glutamicum; identification and localization of the desired C. glutamicum sequences; evolutionary research; determining areas of the SRT protein necessary for its activity; modulating SRT protein activity; modulating SRT pathway activity; and modulating cell production of a desired compound, such as a fine chemicals.

The SRT nucleic acid molecules of the present invention have various uses. First, they can be used to identify the microorganism Corynebacterium glutamicum or a closely related microorganism. In addition, they can be used to identify the presence of C. glutamicum or a closely related microorganism in a mixed population of microorganisms. The present invention relates to the nucleotide sequences of a number of C. glutamicum genes; moreover, by probing the extracted genomic DNA culture of an individual or mixed population of microorganisms under stringent conditions with a probe in the region of the C. glutamicum gene, which is unique to this organism, one can detect the presence of this microorganism.

Although Corynebacterium glutamicum itself is non-pathogenic, however, it is akin to a pathogenic species such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute infectious disease with fever, which is both a local and systemic pathology. With this disease, local lesions develop in the upper respiratory tract and cause necrotic lesions in the epithelial cells; these bacilli secrete a toxin that spreads through these lesions to the distal sensitive tissues of the body. Degenerative changes caused by inhibition of protein synthesis in tissues, such as the heart, muscles, tissues of the peripheral nervous system, adrenal glands, kidneys, liver and spleen, turn this disease into a systemic pathology. Cases of diphtheria are very common in many parts of the world, including Africa, Asia, Eastern Europe and the now independent states of the former Soviet Union. Currently, since 1990, the diphtheria epidemic in the last two indicated regions has resulted in the deaths of at least 5,000 people.

In one embodiment, the present invention relates to a method for identifying the presence or activity of Corynebacterium diphtheriae in a given individual. This method involves the detection of one or more nucleotide or amino acid sequences of the present invention (for example, sequences represented as odd or even, respectively, numbered sequences of SEQ ID NO: in the Sequence List) of a given individual, and thereby detecting the presence or activity of Corynebacterium diphtheriae in this individual. C. glutamicum and C. diphtheriae are related bacteria, and many nucleic acid and protein molecules in C. glutamicum are homologous to the nucleic acid and C. diphtheriae protein, and therefore can be used to detect C. diphtheriae in an individual.

The nucleic acid and protein molecules of the present invention can also serve as markers for specific regions of a given genome. This is important not only for genome mapping, but also for functional studies of C. glutamicum proteins. So, for example, to identify the region of a given genome to which C. glutamicum DNA-binding protein binds, the C. glutamicum genome must be hydrolyzed, and fragments incubated with the DNA-binding protein. Those fragments that bind to this protein can be further probed with the nucleic acid molecules of the present invention, preferably with easily detectable labels; at the same time, the binding of such a nucleic acid molecule to a given fragment of the genome makes it possible to determine the localization of this fragment on the map of the C. qlutamicum genome, and when such probing is carried out repeatedly using various enzymes, it facilitates the rapid identification of the nucleotide sequence to which this protein binds. In addition, the nucleic acid molecules of the present invention can be sufficiently homologous to sequences of related species, such that these nucleic acid molecules can serve as markers for constructing a genomic map for related bacteria, such as Brevibacterium lactofermentum.

The SRT nucleic acid molecules of the present invention can also be used to study the evolution and structure of the protein. The resistance processes in which the molecules of the present invention are involved are used by a wide variety of cells; and by comparing the sequences of the nucleic acid molecules of the present invention with sequences encoding similar enzymes from other organisms, the evolutionary similarity of these organisms can be estimated. Similarly, such a comparison makes it possible to determine which regions of a given sequence are conservative and which are not; it will help to determine those regions of a given protein that are essential for the functioning of this enzyme. This method of determination is important for biotechnological studies of proteins and may indicate that this protein may be tolerant of mutagenesis without losing its function.

The genes of the present invention, for example, the gene encoding LMRB (SEQ ID NO: 1), or another gene of the present invention, encoding resistance or tolerance to stress caused by chemical compounds or environmental exposure (for example, resistance to one or more antibiotics), be used as genetic markers for genetic transformation (for example, to transfer additional genes or to destroy pre-existing genes) of a microorganism such as C. glutamicum or another bacterial species. The use of these nucleic acid molecules that were included in this transgenic cluster (for example, in a plasmid, phage, phasmid, phagemid, transposon or other nucleic acid element) allows for the efficient selection of microorganisms on the basis of the trait that allows this microorganism to survive in harmful conditions or a toxic environment for it (for example, in the presence of an antimicrobial compound). By using one or more of the genes of the present invention as genetic markers, it is possible to increase the speed and ease of constructing and isolating those microorganisms that have the desired transformed traits (for example, the production of modulated chemical products of fine organic synthesis). Although the use of the genes of the present invention for the selection of transformed C. glutamicum and related bacteria is preferred, however, as described herein, homologs (e.g., homologs from other microorganisms), allelic variants or gene fragments that retain the desired activity can be used. In addition, to modulate the transcription of a given gene, the 5 ′ and 3 ′ regulatory elements of the genes of the present invention can be modified as described above (for example, by substitution, insertion, deletion or replacement of nucleotides with a more desirable genetic element). So, for example, an LMRB variant can be applied in which the nucleotide sequence in the region from -1 to -200 located at the 5'-end with respect to the start codon has been modified to modulate (preferably, increase the level) transcription and / or LMRB translations, and constructs can also be applied in which the gene of the present invention (e.g., the LMRB gene (SEQ ID NO: 1) is operably linked to one or more regulatory signals (e.g., sequences that bind to an inducer or repressor), and who can b be used to modulate gene expression. Likewise may be used the present invention, multiple copies of the gene (or functional inactivated).

Another use of the genes of the present invention (for example, a gene encoding LMRB (SEQ ID NO: 1) or another drug or antibiotic resistance gene) is the development of new antibiotics that have activity against Corynebacteria and / or other bacteria. For example, the gene of the present invention can be expressed (or overexpressed) in a suitable host to generate a microorganism with increased resistance to one or more drugs or antibiotics (in the case of LMRB, in particular, lincosamides, and especially lincomycin). A resistant host of this type can then be used for screening for chemical compounds with bacteriostatic and / or bactericidal activity, such as new antibiotic compounds. The genes of the present invention (for example, the LMRB gene) can, in particular, be used to identify new antibiotics whose activity is directed against those microorganisms that already have resistance to standard antibiotics.

The present invention relates to methods for screening molecules that modulate the activity of an SRT protein either by interacting with the protein itself or with its substrate or binding partner of the SRT protein, or by modulating the transcription or translation of the SRT nucleic acid molecule of the present invention. In these methods, a microorganism expressing one or more SRT proteins of the present invention is contacted with one or more test compounds, and the effect of each test compound on the activity or expression level of the SRT protein is evaluated.

Manipulation of SRT nucleic acid molecules can lead to the production of SRT proteins functionally different from wild-type SRT proteins. These proteins may have increased efficiency or activity and may be present in the cell in a greater amount than is usually the case, or they may have reduced efficiency or activity. The purpose of such manipulations is to increase the viability and activity of a cell when exposed to chemical stresses and harmful environmental influences, which usually accompany large-scale enzymatic cultivation. Thus, with an increase in the activity or number of copies of a protease regulated by heat shock, the ability of a given cell to destroy improperly laid proteins that may interfere with the normal functioning of the cell in one way or another (for example, as a result of maintaining binding to substrates or cofactors, despite that this protein does not have corresponding activity with respect to these molecules). The same applies to overexpression or optimization of the activity of one or more chaperone molecules induced by heat or cold shock. These proteins contribute to the proper folding of growing polypeptide chains, and therefore their increased activity or their presence should contribute to an increase in the percentage of correctly laid proteins in a given cell, which, in turn, should increase the overall metabolic efficiency and viability of these cells in culture. Overexpression or optimization of carrier molecules activated by osmotic shock should lead to an increased ability of some cells to maintain intracellular homeostasis, and thereby increase their viability in culture. Similarly, overproduction or increased activity through mutagenesis of proteins that impart resistance to various types of chemical stresses to cells (either by removing harmful chemicals from a given cell, or by modifying these chemicals to reduce the level of harmful substances) should increase the adaptability of the given microorganism to conditions environment containing harmful substances (that is, with large-scale enzymatic cultivation), and thus it can contribute s survival of a relatively large number of cells in a culture. A common effect of all these mutagenesis strategies is to increase the number of compounds producing fine chemicals in a given culture, and thereby increase the yield, production and / or efficiency of producing one or more desired fine chemicals from a given culture.

The above list of mutagenesis strategies for SRT proteins in order to increase the yields of the desired compound is not limited to these strategies; and options for these mutagenesis strategies will be apparent to every person skilled in the art. Thanks to the mechanisms of these mutagenesis strategies, the nucleic acid and protein molecules of the present invention can be used to generate C. glutamicum or related bacterial strains expressing mutant nucleic acid and SRT proteins in order to improve the yield, production and / or production efficiency of the desired compound. The desired compound can be any natural product of C. glutamicum, which is the end products of the biosynthesis pathway and intermediate compounds of the natural metabolic pathways, as well as molecules that are not usually involved in the metabolism of C. glutamicum, but which are produced by the C. glutamicum strain of the present invention.

The present invention is further illustrated by the following examples, which should not be construed as limiting it. The contents of all works, patent applications, patents, published patent applications, tables, and the list of sequences cited in this application are hereby incorporated by reference.

ILLUSTRATION BY EXAMPLES

Example 1: Obtaining the full genomic DNA of Corynebacterium glutamicum ATCC 13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 ° C. with vigorous shaking in BHI medium (Difco). These cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer-1 (5% of the original culture volume; all indicated volumes were calculated for 100 ml of culture volume). The composition of buffer-1: 140.34 g / l of sucrose, 2.46 g / l of MgSO ₄ × 7 H ₂ O, 10 ml / l of a solution of KH ₂ PO ₄ (100 g / l, adjusted to pH 6.7 by adding KOH), 50 ml / l of M12 concentrate (10 g / l (NH ₄ ) ₂ SO ₄ , 1 g / l NaCl, 2 g / l MgSO ₄ × 7 Н ₂ О, 0.2 g / l CaCl ₂ , 0 , 5 g / l of yeast extract (Difco), 10 ml / l of a mixture of trace elements (200 mg / l FeSO ₄ × H ₂ O, 10 mg / l ZnSO ₄ × 7 H ₂ O, 3 mg / l MnCl ₂ × 4 H ₂ O, 30 mg / L H ₃ VO ₃ , 20 mg / L CoCl ₂ × 6 H ₂ O, 1 mg / L NiCl ₂ × 6 H ₂ O, 3 mg / L Na ₂ MoO ₄ × 2 Н ₂ О) , 500 mg / l of complexing agent (EDTA or citric acid), 100 ml / l of a mixture of vitamins (0.2 mg / l of biotin, 0.2 mg / l of folic acid, 20 mg / l of p-aminobenzoic acid, 20 mg / l riboflavin, 40 mg / l calcium antotenate, 140 mg / l nicotinic acid, 40 mg / l pyridoxol hydrochloride, 200 mg / l myoinositol) Then lysozyme was added to the suspension to a final concentration of 2.5 mg / ml After incubation for approximately 4 hours at 37 ° C. , cell walls were destroyed and the resulting protoplasts were collected by centrifugation. The precipitate was washed once with 5 ml of buffer-1 and once with 5 ml of TE-buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8). Then, the precipitate was resuspended in 4 ml of TE buffer and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCI solution (5M) were added. After proteinase K was added to a final concentration of 200 μg / ml, the suspension was incubated for approx. for 18 hours at 37 ° C. DNA was purified by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol using standard procedures. Then, DNA was precipitated by adding 1/50 volume of 3M sodium acetate and 2 volumes of ethanol, after which they were incubated for 30 minutes at -20 ° C and centrifuged for 30 minutes at 12,000 rpm in a high speed centrifuge using an SS34 rotor (Sorvall ) Then, the DNA was dissolved in 1 ml of TE buffer containing 20 μg / ml RNase A and dialyzed at 4 ° C. against 1000 ml of TE buffer for at least 3 hours. During this time, the buffer was changed 3 times. Aliquots of 0.4 ml of dialyzed DNA solution were added with 0.4 ml of 2M LiCl and 0.8 ml of ethanol. After incubation for 30 minutes at -20 ° C, DNA was collected by centrifugation (13000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). Precipitated DNA was dissolved in TE buffer. DNA obtained from this procedure can be used for any purpose, including southern blotting or the construction of genomic libraries.

Example 2: Construction of genomic libraries of Corynebacterium glutamicum ATCC 13032 in Esherichia coli

Using DNA obtained as described in Example 1, cosmid and plasmid libraries were constructed using well-known and well-developed methods (see, for example, Sambrook J. et al., (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Ausubel FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons).

Any plasmid or cosmid can be used. In particular, pBR322 plasmids were used (Sutcliffe J.G. (1979) Proc. Natl. Acad. Sci. USA, 75: 3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156), pBS series plasmids (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or cosmids such as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, TJ, Rosenthal A. & Waterson, RH (1987) Gene 53: 283-286. Gene libraries, in particular for use in C. glutamicum, can be constructed using plasmid pSL109 (Lee, HS & AJSinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).

Example 3: DNA sequencing and computer functional analysis

The genomic libraries described in Example 2 were used for DNA sequencing in accordance with standard methods, in particular, chain termination using AB1377 sequencers (see, for example, Fleischman, RD et al. (1995) “Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd. ”, Science, 269: 496-512). Sequencing primers with the following nucleotide sequences of 5'-GGAAACAGTATGACCATG-3 'or 5'-GTAAAACGACGGCCAGT-3' were used.

Example 4: In vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum can be carried out by transferring plasmid DNA (or another vector) from E. coli or other microorganisms (e.g. Bacillus spp. Or yeast, such as Saccharomyces cerevisiae), which have impaired the ability to maintain the integrity of their genetic information. Conventional mutant strains have mutations in the genes of the DNA repair system (e.g. mutHLS, mutD, mutT, etc. see Rupp, WD (1996), DNA repair mechanisms, in: Escherichia coli and Salmonella, p.2277-2294 , ASM: Washington). These strains are well known to every specialist. The use of such strains is illustrated, for example, by Greener, A. & Callahan, M (1994) Strategies 7: 32-34.

Example 5: DNA Transfer from Escherichia coli to Corynebacterium glutamicum

Some species of Corynebacterium and Brevibacterium contain endogenous plasmids (such as pHM1519 or pBLl) that autonomously replicate (see, for example, review by Martin, J.F. et al., (1987) Biotechnology, 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be easily constructed using standard vectors for E. coli (Sambrook J. et al., (1989). Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Ausubel FM et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons) to which a replication initiation site and a suitable marker from Corynebacterium glutamicum have been added. Said replication initiation sites are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. The specific transformation markers used for these species are kanamycin resistance genes (such as genes derived from transposons Tn5 or Tn903) or chloramphenicol (Winnacker, EL (1987) From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim) . The literature provides numerous examples of the construction of a wide variety of shuttle vectors that replicate in both E. coli and C. glutamicum and which can be used for many purposes, including overexpression of genes (see, for example, Yoshihama et al., (1985 ) J. Bacteriol. 162: 591-597; Martin, JF et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ et al. (1991) Gene, 102: 93-98).

Using standard methods, you can clone the desired gene in any of the shuttle vectors described above and introduce such a hybrid vector into Corynebacterium glutamicum strains. Transformation of C. glutamicum can be accomplished by transformation of protoplasts (Kastsumata, R. et al. (1984), J. Bacteriol. 159: 306-311), electroporation (Liebl, E. et al., (1989) FEMS Microbiol. Letters 53: 399-303), and in cases where special vectors are used, this transformation can also be carried out by conjugation (as described, for example, Schafer, A. et al. (1990) J. Bacteriol. 172: 1663-1666 ) Shuttle vectors for C. glutamicum can also be transferred to E. coli by obtaining plasmid DNA from C. glutamicum (standard well-known methods) and transforming them into E. coli. Such a transformation step can be carried out by standard methods, but it is preferable to use a Mcr-deficient strain of E. coli, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19).

Genes can be overexpressed in C. glutamicum strains using plasmids that contain pCGl (US Pat. No. 4,617,267) or fragments thereof, and optionally a kanamycin resistance gene from TN903 (Grindley, ND & Joyce, C.M. (1980) Proc. Natl. Acad. Sci., USA, 77 (12): 7176-7180). In addition, genes can be overexpressed in C. glutamicum strains using the plasmid pSL109 (Lee, H.-S. & A.J.Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).

In addition to using replicative plasmids, overexpression of genes can also be achieved by integrating them into the genome. Genomic integration into C. glutamicum or other species of Corynebacterium or Brevibacterium can be accomplished by well-known methods, such as homologous recombination with genomic region (s), restriction enzyme mediated integration (REMI) (see, for example, DE 19823834) or by using transposons. Modulation of the activity of a desired gene can also be accomplished by modifying regulatory regions (e.g., promoter, repressor and / or enhancer), by modifying, insertion or deletion of sequences using site-directed methods (such as homologous recombination) or random-event-based methods (such as transposon mutagenesis or REMI). Nucleotide sequences that function as transcription terminators can also be inserted at the 3 ′ end with respect to the coding region of one or more genes of the present invention; and such terminators are well known in the art and are described, for example, by Winnacker, E.L. (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim.

Example 6: Assessment of Expression of Mutant Protein

Assessment of the activity of a mutated protein in a transformed host cell is based on the fact that this mutant protein is expressed in the same amount as the wild-type protein. The method used to determine the level of transcription of this mutant gene (an indicator of the amount of mRNA required for translation to form a gene product) involves Northern blot analysis (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley; New York), in which a primer designed to bind the desired gene is labeled with a detectable label (usually radioactive or chemiluminescent), so that when extracting total RNA from the culture of a given microorganism, its gel electrophoresis, transfer to a stable When the matrix was incubated and incubated with this probe, the binding and amount of the binding probe indicated the presence, as well as the amount of mRNA of this gene. This information indicates the degree of transcription of this mutant gene. Total cellular RNA can be obtained from Corynebacterium glutamicum by several methods well known in the art, such as those described by Bormann, E.R. et al., (1992) Mol. Microbiol. 6: 317-326.

Standard methods such as Western blot analysis can be used to determine the presence or relative amount of protein translated from this mRNA (see, e.g., Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley; New York) . In this method, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody that specifically binds to the desired protein. This probe is usually labeled with a chemiluminescent or colorimetric label, which can be easily detected. The presence and amount of the observed label indicates the presence and amount of the desired mutant protein in a given cell.

Example 7: Growth of genetically modified Corynebacterium glutamicum - medium and cultivation conditions

Genetically modified bacteria, Corynebacterium, were cultured in synthetic or natural culture media. There are a number of different well-known and readily available culture media for Corynebacterium (Lieb et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998) Biotechnology Letters 11: 11-16 ; patent DE 4120867; Liebl (1992) "The Genus Corynebacterium: The Procaryotes", Volume II, Balows, A. et al., eds. Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins, and trace elements. Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose are excellent carbon sources. Sugar can also be delivered to these media via complex compounds, such as molasses or other by-products from sugar refining. These compounds may also be preferred for the delivery of mixtures of various carbon sources. Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of nitrogen sources are gaseous ammonia or ammonia salts, such as NH ₄ Cl or (NH ₄ ) ₂ SO ₄ , NH ₄ OH, nitrates, urea, amino acids or complex nitrogen sources, such as corn extract, soy flour, soy protein, yeast extract, meat extract, etc. Compounds of inorganic salts that can be included in these environments are chlorides, phosphates or sulfates of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to this medium to maintain metal ions in solution. Particularly valuable chelating compounds are dihydroxyphenols, such as catechin or protocatechuate, or organic acids, such as citric acid. This medium also typically contains other growth factors, such as vitamins or growth stimulants, examples of which are biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from components of complex media, such as yeast extract, molasses, corn extract and others. The exact composition of the compounds in the media is highly dependent on this particular experiment and is determined separately for each specific case. Information on optimizing environments can be found in the Applied. Microbiol. Physiology ”, A Practical Approach (eds., PMRhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 199635773). Culture media can also be purchased from commercial suppliers, for example, standard medium 1 (Merck) or BHI (kernel extract, DIFCO) or the like.

All media components are sterilized either by heating (20 minutes at 1.5 bar and 121 ° C) or by sterile filtration. These components can be sterilized together, or, if necessary, separately. All media components may be present at the beginning of cultivation, or they may, but not necessarily, be added continuously or periodically.

Cultivation conditions are determined separately for each experiment. The temperature should be between 15 ° C and 45 ° C. This temperature may be constant, or it may vary during the experiment. The pH of this medium should be in the range of 5 to 8.5, and preferably about 7.0, and can be maintained by adding buffers to the medium. An example of a buffer used for these purposes is potassium phosphate buffer. Alternatively or simultaneously, synthetic buffers such as MOPS, HEPES, ACES and others can be used. During the cultivation process, a constant pH of the culture can also be maintained by adding NaOH or NH ₄ OH. If components of a complex medium are used, such as yeast extract, the need for additional buffers can be reduced due to the fact that many complex compounds have a high buffer capacity. If a fermenter is used for the cultivation of microorganisms, the pH can also be adjusted by adding gaseous ammonia.

The incubation time is usually from several hours to several days. This time is chosen so that the maximum amount of product is accumulated in this broth. The culture experiments described herein can be carried out in a number of vessels, such as microtiter plates, glass tubes, glass flasks, or glass or metal fermenters of various sizes. For screening a large number of clones, microorganisms should be cultured in microtiter plates, in glass tubes or in shaker flasks, either with or without septa. Preferably, 100 ml flask-shakers are used, filled with 10% (by volume) of the desired culture medium. These flasks should be shaken on a rotary shaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, mathematical correction of evaporation losses should be carried out.

When testing genetically modified clones, an unmodified control clone or control clone containing the main plasmid without any insertion should also be tested. The medium is inoculated to OD ₆₀₀ = 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g / l glucose, 2.5 g / l NaCl, 2 g / l urea, 10 g / l polypeptone, 5 g / l yeast extract, 5 g / l meat extract, 22 g / l agar, pH 6.8 with 2M NaOH), which are incubated at 30 ° C. Inoculation of this medium is carried out either by introducing a salt suspension of C. glutamicum cells from CM plates, or by adding a liquid pre-culture of this bacterium.

Example 8 - In Vitro Analysis of the Function of Mutant Proteins

Determining the activities and kinetic parameters of enzymes is a well-known procedure. The experiments to determine the activity of any given modified enzyme must be specifically adapted for the specific activity of the wild-type enzyme, which can be easily carried out by any specialist. A general description of enzymes, as well as specific details regarding structures, kinetics, principles, methods, applications and examples of the determination of many enzymatic activities, can be found, for example, in the following works: Dixon, M. & Webb, EC (1979) Enzymes. Longmans: London; Fersht (1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price NC Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, PD ed. (1983) The Enzymes, 3 ^rd ed. Academic Press: New York; Bisswanger H. (1994) Enzymkinetik, 2 ^nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, HU, Bergmeyer, J. Graβl, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3 ^rd ed. vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, Enzymes VCH: Weinheim, p. 352-363.

The activity of DNA-binding proteins can be determined by several well-developed methods, such as DNA strip shift assays (so-called gel motility retardation assays). The effect of such proteins on the expression of other molecules can be measured by assays for the presence of a reporter gene (such as the one described by Kolmar H. et al. (1995) EMBO J. 14: 3895-3904 and the references therein). Testing systems using the reporter gene are well known and designed for their use in both prokaryotic and eukaryotic cells using enzymes such as beta-galactosidase, a fluorescent protein emitting in the green range of light, and some other enzymes.

Determination of the activity of membrane transport proteins can be carried out by known methods described by Gennis, R.B. (1989) Pores, Channels and Transporters, Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234 and 270-322.

Example 9: Analysis of the effect of mutant protein on the production of the desired product

The effect of genetic modification in C. glutamicum on the production of the desired compound (such as an amino acid) can be evaluated by culturing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and / or cellular component for increased production of the desired product (i.e., amino acid ) Such methods of analysis are well known to any person skilled in the art and include spectroscopy, thin layer chromatography, various types of staining methods, enzyme and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullmann, Encyclopedia of Industrial Chemistry (1987) vol. A2, p. 89-90 and p. 433-613, VCH: Weinheim, (1985); Fallon A. et al. (1987) “Applications of HPLC in Biochemistry”: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17 ; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III: “Product recovery and purification”, page 469-714, VCH: Weinheim; Belter, PA et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley a nd Sons; Kennedy JF & Cabral JMS (1992) Recovery processes for biological materials, John Wiley &Sons; Shaiewitz, JA & Henry, JD (1988) Biochemical separations, Ullmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1 -27, VCH: Weinheim & Dechow, FJ (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to evaluating the final fermentation product, you can also analyze other components of the metabolic pathways used to produce the desired compound, such as intermediates and by-products, in order to determine the total yield, level and / or production efficiency of this compound. Methods of analysis are measuring levels of nutrients in a given environment (e.g. sugars, hydrocarbons, nitrogen sources, phosphates and other ions), measuring the composition and growth of biomass, analyzing the production of common metabolites of biosynthetic pathways, and measuring the gases produced during fermentation. Standard methods for these measurements are described in Applied. Microbiol. Physiology, A Practical Approach, P.M. Rhodes & P.F. Stanbury, eds., IRL Press p. 103-129, 131-163 and 165-192 (ISBN 0 199635773) and in the works cited there.

Example 10: Purification of the desired product from a culture of C. glutamicum

Isolation of the desired product from C. glutamicum cells or the supernatant of the culture described above can be carried out by various methods well known in the art. If the desired product is not secreted from the cells, then these cells can be harvested from the culture by centrifugation at low speed, and then they can be lysed by standard methods such as mechanical disruption or sonication. Cell debris is removed by centrifugation, and the supernatant fraction containing soluble proteins is left for subsequent isolation of the desired compound. If this product is secreted from C. glutamicum cells, then these cells are removed from the culture by centrifugation at low speed, and the supernatant fraction is left for subsequent purification.

The supernatant fraction obtained by any purification method is subjected to chromatography using a suitable resin, where either the desired molecule is retained on a chromatographic resin, while many impurities are not retained in the sample, or where impurities are retained on this resin and the sample is not. Such chromatography steps may be repeated, if necessary, using the same or other chromatographic resins. Any specialist can independently select resins for chromatography and their most effective application for the purification of a particular molecule. The purified product can be concentrated by filtration or ultrafiltration and stored at a temperature at which this product is as stable as possible.

There are a wide range of cleaning methods known to those skilled in the art, and these methods are not limited to the above method. Such cleaning methods are described, for example. Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be evaluated by standard methods. Such methods are high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic analysis or microbiological methods. For a description of these assay methods, see Patek et al. (1994), Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al., (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) vol. A27, VCH: Weinheim, p.89-90, p.521-540, p.540-547, p.559-566, p.575-581 and p.581- 587; Michal G. (1999) Biochemical pathway: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Fallon A. et al. (1987) "Applications of HPLC in Biochemistry": Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

Example 11: Cloning of the Corynebacterium qlutamicum gene encoding lincomycin resistance using the reporter gene A. Identification of the gene encoding the LMRB protein

Plasmid pSL 130 was constructed by ligation of the ACeB (racec) promoter region of C. glutamicum (Kirn HJ et al. (1997) J. Microbiol. Biotechnol. 7: 287-292) into the polyninker of the pRS415 hybrid vector with an Iac operon that lacks a promoter (Simon, RW et al. (1987) Gene 53: 85-96). Plasmid pSL145 was constructed by inserting the resulting paceB-lac region into the E. coli pACC184 cloning vector. E. coli DH5αF 'was transformed with pSL145 and the resulting strain was used as a host for screening the C. glutamicum genomic library (in pSL109).

Transformants were screened by cultivation on agar medium containing 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). A white colony containing DNA affecting lacZ expression was selected for subsequent analysis. This clone was found to contain a 4 kbp fragment. from the gene library. Subclones were constructed in pSL109 and a subclone was identified that retained the white bacteria phenotype on an X-Gal plate. This subclone was found to contain a 2.6 kb BamHI-XhoI fragment. (plasmid pSL149-5). This fragment was sequenced and identified as a gene encoding a membrane protein (LMRB gene).

1442 nucleotides of the coding sequence of the LMRB gene encode a polypeptide of 481 amino acid residues with a high percentage of hydrophobic amino acids. A Genbank search found that the LMRB protein is 40% identical to the protein product of the Bacillus subtilis ImrB gene (Genbank Accession AL009126, TREMBL Accession P94422), as determined using the CLUSTAL W assay (using standard parameters).

The LMRB protein contains the sequence motif 158-A-P-A-LGPTLSG-167 (SEQ ID N0: 301), which is similar to the well-known consensus motif of the multidrug resistance protein GXXXGPXXGG (SEQ ID NO: 302) Paulsen I.T., & Skurray RA (1993) Gene 124: 1-11). Therefore, the LMRB protein has been classified as a drug resistance protein.

B. In vivo analysis of ImrB function

The ImrB gene was overexpressed in C. glutamicum ASO19E12 (Kim H.J. et al. (1997) J. Microbiol. Biotechnol. 7: 287-292) using the plasmid pSL149-5 described above.

The destruction of the LMRB gene was carried out using the vector PsL18-lmrB. This vector was constructed as follows: the internal fragment of the LMRB gene was amplified by PCR under standard conditions using primers 5′-CTCCAGGATTGCTCCGAAGG-3 ′ (SEQ ID NO: 303) and 5′-CACAGTGGTTGACCACTGGC-3 ′ (SEQ ID NO: 304). The resulting PCR product was treated with T7 DNA polymerase and T7 polynucleotide kinase and cloned into the SmaI site of pSL18 plasmid (Kim Y.H. & H.- S. Lee. (1996) J. Microbiol. Biotechnol. 6: 315-320). The destruction of the LMRB gene in C. glutamicum ASO19E12 was carried out by conjugation as previously described (Schwarzer & Punler (1991) Bio / Technology 9: 84-87).

C. glutamicum cells transformed with pSL149-5 showed the same as non-transformed cells, resistance to erythromycin, penicillin G, tetracycline, chloramphenicol, spectinomycin, nalidixic acid, gentamicin, streptomycin, USSR ethidium bromidebromide bromide sodium dodecyl sulfate. However, significant differences were observed in the resistance of transformed and non-transformed cells to lincomycin.

C. glutamicum cells overexpressing LMRB were found to grow in the presence of 20 μg / ml lincomycin. In contrast, cells that do not express excess LMRB (or cells that carry the destruction of the LMRB gene) are not able to grow on agar medium containing 5 μg / ml lincomycin. This effect was clearly noticeable in liquid culture. Overexpression of LMRB led to a 9-fold increase in resistance (compared with wild type) to lincomycin, and the destruction of the LMRB gene led to a decrease in resistance (28% of wild type) to this antibiotic.

Example 12: Analysis of the gene sequences of the present invention

Comparison of sequences and determination of percent homology between two sequences are well known methods, and they can be carried out using a mathematical algorithm such as Karlin & Altschul (1990) Proc.Natl. Acad. Sci., USA, 87: 2264-68, modified as described by Karlin & Altschul (1993) Proc.Natl. Acad. Sci., USA, 90: 5873-77. Such an algorithm is introduced into the NBLAST and XBLAST programs (version 2.0) by Altschul et al. (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the SRT nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to the SRT protein molecules of the present invention. To obtain primary sequences with “gaps” in order to compare them, the “Gapped BLAST” program described by Altschul et al. Can be used. (1997) Nucleic. Acids Res. 25 (17): 3389-3402. When using the BLAST and Gapped BLAST programs, every specialist knows how to optimize the parameters of this program (for example, XBLAST and NBLAST) to analyze a specific sequence.

Another example of a mathematical algorithm used to compare sequences is the Meyers & Miller (1988) Comput.Appi.Biosci algorithm. 4: 11-17). Such an algorithm is introduced into the ALIGN program (version 2.0), which is part of the software package for matching the GCG sequence. When using the ALIGN program for comparing amino acid sequences, a table of weight residues PAM120, a penalty for a space length = 12, and a penalty for a space = 4 can be used. Other algorithms for sequence analysis are known to those skilled in the art, and these are ADVANCE and ADAM described by Torelli & Robotti (1994) Comput. Appl. Biosci. 10: 3-5, and FASTA described by Pearson & Lipmann (1988) P.N.A.S. 85: 2444-8.

The percent homology between two amino acid sequences can also be determined using the GAP program available in the GCG software package (available at http://www.gcg.com), using either the Blosum 62 matrix or the PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4, and weights of length 2, 3, or 4. The percent homology between two nucleotide sequences can be determined using the GAP program in the GCG software package using standard parameters such as space weight = 50 and length weight = 3.

Comparative analyzes of gene sequences of the present invention with sequences present in Genbank were performed using well-known methods (see, for example, Bexevanis & Ouellette, eds., (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley &Sons; New York). The gene sequences of the present invention were compared with genes available in Genbank in three stages. In a first step, a BLASTN assay (eg, an assay for local comparison) was performed to compare each sequence of the present invention with the nucleotide sequences present in Genbank, and 500 best matches were left for subsequent analysis. For these top 500 matches, a subsequent search was performed using FASTA (for example, a combined analysis for local and global comparison of primary sequences, in which limited regions of sequences were compared). Then, each gene sequence of the present invention was subjected to global comparison with each of the best three matches in FASTA using the GAP program in the GCG software package (using standard parameters). To obtain the correct results, the lengths of the sequences obtained from Genbank were adjusted to the length of the requested sequences by well known methods. The results of these analyzes are presented in Table 4. The data obtained are identical to the data that would have been obtained if the (global) GAP analysis had been carried out separately for each of the genes of the present invention compared to each of the genes available in Genbank, but this would require much less computer time than for (global) analysis with an extensive GAP database. The sequences of the present invention, for each of which a comparison is not made above the cutoff value, can be determined in Table 4 by the lack of information. In addition, it should be noted that the percent homology obtained by comparison using GAP and presented in Table 4 under the heading “% homology (GAP)” are given in the European numbering format, where “,” represents the decimal point comma. So, for example, the value “40.345” in this column means “40.345%”.

Example 13: Construction and operations with microarrays with DNA

The sequences of the present invention can also be used for the construction and use of DNA microarrays (the construction, methodology and use of DNA templates are well known in the art and are described, for example, by Schena M. et al. (1995) Science 270: 467-470; Wodicka L. et al. (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi JL et al. (1997) Science 278: 680-686).

Microarrays with DNA are solid or flexible carriers consisting of nitrocellulose, nylon, glass, silicone or other materials. Nucleic acid molecules can be attached to a given surface in a specific order. After appropriate labeling, other nucleic acids or mixtures of nucleic acids can be hybridized with immobilized nucleic acid molecules, and this label can be used to monitor and measure the intensities of individual signals of hybridized molecules in certain areas. This technique allows you to simultaneously determine the relative or absolute amount of all or separately selected nucleic acids in the used nucleic acid sample or mixture of nucleic acids. Therefore, DNA microarrays allow parallel analysis of the expression of multiple (up to 6800 or more) nucleic acids (see, for example, Schena M. (1996) Bioassays 18 (5): 427-431).

The sequences of the present invention can be used to construct oligonucleotide primers capable of amplifying specific regions of one or more C. glutamicum genes by a nucleic acid amplification reaction, such as a polymerase chain reaction. The selection and construction of 5'- or 3'-oligonucleotide primers or their corresponding linkers allows covalent binding of the obtained PCR products to the surface of the carrier medium described above (as well as described, for example, Schena M. et al. (1995) Science 270 : 467-470).

Nucleic acid microarrays can also be constructed by in situ oligonucleotide synthesis described by Wodicka L. et al. (1997) Nature Biotechnology 15: 1359-1367. When using photolithographic methods, precisely defined areas of this matrix are subjected to light irradiation. Protective groups that were photolobed were activated and attached nucleotides, while the areas that were protected from light exposure did not undergo any modification. Successive cycles of light protection and light activation allow the synthesis of various oligonucleotides in certain positions. Specific small regions of the genes of the present invention can be synthesized on microarrays via solid-phase oligonucleotide synthesis.

The nucleic acid molecules of the present invention, present in the sample or in a mixture of nucleotides, can be hybridized with these microarrays. These nucleic acid molecules can be labeled by standard methods. For this, nucleic acid molecules (e.g., mRNA molecules or DNA molecules) are labeled by introducing labeled isotopes or fluorescently labeled nucleotides, for example, during reverse transcription or DNA synthesis. Hybridization of labeled nucleic acids with microarrays is described, for example, by Schena M. et al. (1995) see above; Wodicka L. et al. (1997) see above; and DeSaizieu A. et al. (1998) (see above). Detection and quantification of hybridized molecules are associated with a specific label introduced. Radioactive labels can be detected, for example, as described by Schena M. et al. (1995) (see above), and fluorescence labels can be detected, for example, as described by Shalon et al. (1996) Genome Research 6: 639-645.

The use of the sequences of the present invention in the technology using DNA microarrays described above allows comparative analyzes of various strains of C. glutamicum or other corynebacteria. For example, studies of differences between strains based on the profiles of individual transcripts and identification of genes that are important in terms of the specific and / or desired properties of the strains, such as pathogenicity, productivity and stress tolerance, can be facilitated by applying a matrix technique nucleic acids. Thanks to the technique using nucleic acid matrices, it is also possible to carry out comparative analyzes of the gene expression profile of the present invention during the fermentation reaction.

Example 14: Analysis of the dynamics of cell populations of proteins (proteomics)

The genes, compositions and methods of the present invention can be used to study the interactions and dynamics of populations of proteins called “proteomics”. The populations of these proteins of interest are, but are not limited to, a general population of C. glutamicum proteins (e.g., compared to populations of proteins of other microorganisms), a population of proteins that are active under specific environmental or metabolic conditions (e.g., during fermentation , at high or low temperature or at high or low pH) or a population of proteins that are active in the process of specific phases of growth and development.

Protein populations can be analyzed by various well-known methods, such as gel electrophoresis. Cellular proteins can be obtained, for example, by lysis or extraction, and can be separated from each other by a number of electrophoretic methods. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) separates proteins mainly by their molecular weight. Isoelectric focusing in a gel (IEF-PAGE) separates proteins according to their isoelectric point (which makes it possible to identify not only this amino acid sequence, but also post-translational modifications of this protein). Another, more preferred method of protein analysis is the sequential combination of both IEF-PAGE and SDS-PAGE, known as two-dimensional gel electrophoresis (described, for example, Hermann et al. (1998) Electrophoresis 19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other methods of separation can also be used to separate proteins, such as capillary gel electrophoresis; and such methods are well known in the art.

Proteins separated using this technique can be visualized using standard methods such as staining or labeling. Suitable dyes are known to those skilled in the art and are Coomassie brilliant blue, silver dye, or fluorescent dyes such as Ruby dye (Sypro Ruby) (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., ³⁵ S-methionine, ³⁵ S-cysteine, ¹⁴ C-labeled amino acids, ¹⁵ N-amino acids, ¹⁵ NO ₃ or ¹⁵ NH ₄ ⁺ or ¹³ C-labeled amino acids) in medium C glutamicum allows the labeling of proteins derived from these cells before separation. Similarly, fluorescent labels can be used. These labeled proteins can be extracted, isolated and separated by previously described methods.

Proteins visualized by these methods can also be analyzed by determining the amount of dye or label used. The amount of a given protein can be quantified, for example, by optical methods, and a comparison can be made with the amount of other proteins in the same gel or in other gels. Comparison of proteins on gels can be carried out, for example, using optical comparison, by spectroscopy, visualizing scanning and analysis of gels, or using photographic films and screens. Such methods are well known in the art.

Direct sequencing or other standard methods can be used to determine the identity of any given protein. For example, sequencing of N- and / or C-terminal amino acids (e.g., Edman degradation) or mass spectrometry (in particular, MALDI or ESI methods (see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). In accordance with these methods, the protein sequences obtained in this application can be used to identify C. glutamicum proteins.

The data obtained by these methods can be used to compare the nature of the presence, activity or modification of proteins in various samples under various biological conditions (for example, in various microorganisms, at a certain period of time of fermentation, in certain environmental conditions or in different biotopes, etc. .). The data obtained as a result of such experiments, separately or in combination with other methods, can be used for various purposes, for example, to compare the behavior of different organisms in a given (for example, metabolic) situation, to increase the productivity of strains that produce thin chemical products organic synthesis, or to increase the efficiency of the production of chemical products of fine organic synthesis.

Equivalents

Many equivalents of the specific embodiments of the invention described in this application are obvious or can be obtained by each specialist by routine experimentation. Such equivalents are included in the scope of the following claims.

Claims (43)

1. An isolated Corynebacterium glutamicum nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary to it, wherein said molecule encodes a polypeptide having lincomycin resistance protein activity. 2. An isolated nucleic acid molecule that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 2 and having the activity of lincomycin resistance protein, or a sequence complementary to it. 3. An isolated nucleic acid molecule that encodes a natural allelic variant of a polypeptide containing the amino acid sequence of SEQ ID NO: 2 and having the activity of lincomycin resistance protein, or a sequence complementary to it. 4. An isolated nucleic acid molecule containing a nucleotide sequence that is at least 50% identical to the full-length nucleotide sequence of SEQ ID NO: 1, or a sequence complementary to it, wherein said nucleic acid molecule encodes a polypeptide having lincomycin resistance protein activity. 5. An isolated nucleic acid molecule containing at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary to it, wherein said nucleic acid molecule encodes a peptide having lincomycin resistance protein activity. 6. The selected nucleic acid molecule, which under stringent conditions, hybridizes with the nucleic acid molecule according to any one of claims 1 to 5. 7. The selected nucleic acid molecule containing the nucleic acid molecule according to any one of claims 1 to 6 and a nucleotide sequence encoding a heterologous polypeptide. 8. A recombinant expression vector comprising a nucleic acid molecule according to any one of claims 1 to 7 and one or more regulatory sequences. 9. The recombinant vector of claim 8, which is a plasmid. 10. A method of obtaining a recombinant host cell, comprising transfecting the host cell with the recombinant vector of claim 8 or 9. 11. The method of claim 10, wherein said cell is a microorganism. 12. The method according to claim 11, where the specified cell belongs to the genus Corynebacterium or Brevibacterium. 13. The method of claim 10, where the expression of the indicated nucleic acid molecule modulates the production of amino acids. 14. The method according to item 13, where the specified amino acid is a proteinogenic or non-proteinogenic amino acid. 15. A method of obtaining a recombinant host cell, comprising transfecting the host cell with a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1, where the nucleic acid molecule has one or more modifications of the nucleic acid compared to the sequence shown in SEQ ID NO: 1 , and where the nucleic acid molecule encodes a polypeptide having the activity of lincomycin resistance protein. 16. A method of producing a recombinant host cell, comprising transfecting the host cell with a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1, where the regulatory region of the nucleic acid molecule is modified relative to the regulatory region of this wild-type molecule and where the nucleic acid molecule encodes a polypeptide having lincomycin resistance protein activity. 17. A method for producing a polypeptide comprising culturing a recombinant host cell transfected with the vector of claim 8 in a suitable culture medium to produce a polypeptide having lincomycin resistance protein activity. 18. The selected polypeptide containing the amino acid sequence of SEQ ID NO: 2, where the specified polypeptide has the activity of protein resistance to lincomycin. 19. The selected polypeptide containing a natural allelic variant of the polypeptide containing the indicated amino acid sequence of SEQ ID NO: 2, where the specified natural allelic variant has the activity of lincomycin resistance protein. 20. An isolated polypeptide that is encoded by a nucleic acid molecule containing a nucleotide sequence that is at least 50% identical to the full-length nucleic acid sequence of SEQ ID NO: 1, wherein said polypeptide has lincomycin resistance protein activity. 21. An isolated polypeptide comprising an amino acid sequence that is at least 50% identical to the entire amino acid sequence of SEQ ID NO: 2, wherein said polypeptide has lincomycin resistance protein activity. 22. The selected polypeptide containing a fragment of the amino acid sequence of SEQ ID NO: 2, where the specified fragment retains the activity of the protein resistance to lincomycin. 23. The selected polypeptide encoded by a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1, where the specified peptide has the activity of protein resistance to lincomycin. 24. An isolated polypeptide according to any one of claims 18-23, further comprising heterologous amino acid sequences. 25. A method for producing an amino acid, comprising culturing a recombinant host cell transfected with the vector of claim 8, wherein the amino acid is produced. 26. The method according A.25, where the specified method further comprises the step of isolating the amino acid from the specified culture. 27. The method according A.25, where the specified cell belongs to the genus Corynebacterium or Brevibacterium. 28. The method of claim 25, wherein said cell is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum and the strains shown in table 3. 29. The method according A.25, where the expression of a nucleic acid molecule from the specified vector leads to modulation of the production of amino acids. 30. The method according A.25, where the specified amino acid is a proteinogenic or non-proteinogenic amino acid. 31. The method according A.25, where the specified amino acid is selected from the group comprising lysine, glutamate, glutamine, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, valine, leucine, isoleucine, arginine, proline, histidine, tyrosine, phenylalanine and tryptophan. 32. A method of producing an amino acid comprising culturing a cell whose genomic DNA has been altered by introducing a nucleic acid according to any one of claims 1 to 7. 33. A method for diagnosing Corynebacterium diphtheriae in a patient, comprising determining at least one nucleic acid molecule according to claims 1-5 or at least one polypeptide molecule according to claims 18-23, that is, diagnosing or determining the activity of Corynebacterium diphtheriae in a patient. 34. An isolated nucleic acid molecule consisting of a fragment of at least 15 nucleotides of the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary to it, wherein said nucleic acid molecule can be used as a primer. 35. An isolated nucleic acid molecule consisting of a fragment of at least 15 nucleotides of the nucleotide sequence of SEQ ID NO: 1, or a sequence complementary to it, wherein said nucleic acid molecule can be used as a probe. 36. An isolated nucleic acid molecule consisting of a fragment of at least 15 nucleotides of the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary to it, wherein said nucleic acid molecule can be used as an antisense molecule. Priority on points: 06/25/1999 according to claims 1-36; 07/01/1999 according to claims 1-36; 07/08/1999 according to claims 1-36; 07/09/1999 according to claims 1-36; 07/14/1999 according to claims 1-36; 08/27/1999 according to claims 1-36; 08/31/1999 according to claims 1-36.

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