WO2003040293A2

WO2003040293A2 - Genes coding for stress resistance and tolerance proteins

Info

Publication number: WO2003040293A2
Application number: PCT/EP2002/012137
Authority: WO
Inventors: Oskar Zelder; Markus Pompejus; Hartwig Schröder; Burkhard Kröger; Corinna Klopprogge; Gregor Haberhauer
Original assignee: Basf Aktiengesellschaft
Priority date: 2001-11-05
Filing date: 2002-10-31
Publication date: 2003-05-15
Also published as: AU2002346804A1; DE10154181A1; EP1453964A2; WO2003040293A3; KR20040053279A

Abstract

The invention relates to nucleic acid molecules, the use thereof in the construction of bio-engineered improved microorganisms and to methods for the production of fine chemicals, especially amino acids, with the aid of said bio-engineered improved microorganisms.

Description

Genes that code for stress resistance and tolerance proteins

Background of the Invention

Certain products and by-products of naturally occurring metabolic processes in cells are used in many industries, including the food, feed, cosmetic and pharmaceutical industries. These molecules, collectively referred to as "fine chemicals", include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, ipide and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. They are best produced using large-scale bacteria that have been developed to produce and secrete large quantities of the desired molecule. A particularly suitable organism for this purpose is Corynebacterium glutamicum, a gram-positive, non-pathogenic bacterium. Through strain selection, a number of mutant strains have been developed that produce a range of desirable compounds. However, selecting strains that are improved in the production of a particular molecule is a time consuming and difficult process.

Summary of the invention

This invention provides novel nucleic acid molecules that can be used to identify or classify Corynebacterium glutamicum or related types of bacteria. C. glutamicum is a gram-positive, aerobic bacterium that is commonly used in industry for the large-scale production of a number of fine chemicals, as well as for the degradation of hydrocarbons (e.g. when crude oil overflows) and for the oxidation of terpenoids. The nucleic acid molecules can therefore be used to identify microorganisms that can be used for the production of fine chemicals, for example by fermentation processes. C. glutamicum itself is not pathogenic, but it is related to other Corynebacterium species, such as Corynebacterium diphtheriae (the causative agent of diphtheria), which are important pathogens in humans. The ability to identify the presence of Corynebacterium species can therefore also be of significant clinical importance, for example in diagnostic applications. These nucleic acid molecules can also serve as reference points for mapping the C. glutamicum geome or genomes of related organisms. These new nucleic acid molecules encode proteins, which are referred to here as stress, resistance and tolerance (SRT) proteins. These SRT proteins can, for example, enable C. glutamicum to survive under conditions that are chemically or environmentally dangerous for this microorganism. Due to the availability of cloning vectors for use in Corynebacterium glutamicum, such as disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetically manipulating C. glutamicum and the related BreviJbacterium species (e.g. lactofermentum) Yoshi - hama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gen. Microbiol. 130 (1984) 2237-2246), the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to make it better and more efficient as a producer of one or more fine chemicals, by the ability of these proteins, the growth and reproduction of C. to enable glutamicum (and also the continuous production of one or more fine chemicals) under conditions which usually hinder the growth of the organism, for example those conditions which are used in fermentative cultivation in

Large-scale encountered frequently. For example, by overexpressing or genetically manipulating a heat shock-induced protease molecule so that it has optimal activity, Mna can improve the bacteria's ability to degrade misfolded proteins when the bacterium is exposed to high temperatures. If fewer misfolded (and possibly incorrectly regulated or non-functional) proteins interact with the normal reaction mechanisms in the cell, the ability of the cell to function normally in such a culture is increased, which in turn offers increased survivability. This overall increase in the number of cells with greater viability and activity in the culture should also increase in the yield, production, and / or efficiency of production of one or more desired fine chemicals due to the relatively larger number of cells that produce these chemicals in the culture cause.

This invention provides new SRT nucleic acid molecules that encode SRT proteins that, for example, can enable C. glutamicum to survive under conditions that are chemically or environmentally dangerous for this microorganism. Nucleic acid molecules that encode an SRT protein are referred to here as SRT nucleic acid molecules. In a preferred embodiment, the SRT protein participates in a metabolic pathway that enables C. glutamicum to survive under conditions that are either chemically or ecologically hazardous to this microorganism. are borrowed. Examples of these proteins are encoded by the genes listed in Table 1.

One aspect of the invention consequently relates to isolated nucleic acid molecules (for example cDNAs) comprising a nucleotide sequence which encodes an SRT protein or biologically active sections thereof, and also nucleic acid fragments which act as primers or hybridization probes for detecting or amplifying SRT-coding nucleic acid (for example DNA or mRNA) are suitable. In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof from one of these nucleotide sequences. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B. The preferred SRT proteins according to the invention also preferably have at least one of the SRT activities described here.

In the following, the nucleic acid sequences of the sequence listing together with the sequence changes at the respective position described in Table 1 are defined as Appendix A.

In the following, the polypeptide sequences of the sequence listing together with the sequence changes at the respective position described in Table 1 are defined as Appendix B.

In a further embodiment, the isolated nucleic acid molecule is at least 15 nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence from Appendix A. The isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule. The isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum SRT protein or a biologically active portion thereof.

Another aspect of the invention relates to vectors, for example recombinant expression vectors which contain the nucleic acid molecules according to the invention, and host cells into which these vectors have been introduced. In one embodiment, a host cell that is grown in a suitable medium is used to produce an SRT protein. The SRT protein can then be isolated from the medium or the host cell.

Another aspect of the invention relates to a genetically modified microorganism in which an SRT gene has been introduced or modified. In one embodiment, the genome of the microorganism can be obtained by introducing at least one ß nucleic acid molecule has been changed, which encodes the mutated SRT sequence as a transgene. In another embodiment, an endogenous SRT gene in the genome of the microorganism has been changed, for example functionally disrupted, by homologous recombination with an altered SRT gene. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also used to produce a desired compound, such as an amino acid, particularly preferably lysine.

Another preferred embodiment is host cells that have more than one of the nucleic acid molecules described in Appendix A. Such host cells can be produced in various ways known to those skilled in the art. For example, they can be transfected by vectors which carry several of the nucleic acid molecules according to the invention. However, it is also possible to introduce one nucleic acid molecule according to the invention into the host cell with one vector and therefore to use several vectors either simultaneously or in a staggered manner. Host cells can thus be constructed which carry numerous up to several hundred of the nucleic acid sequences according to the invention. Such an accumulation often leads to superadditive effects on the host cell with regard to fine chemical productivity.

Another aspect of the invention relates to an isolated SRT protein or a section, for example a biologically active section thereof. In a preferred embodiment, the isolated SRT protein or its section has the ability to

To increase survival of C. glutamicum under conditions that are chemically or ecologically dangerous for these microorganisms. In a further preferred embodiment, the isolated SRT protein or a portion thereof is sufficiently homologous to an amino acid sequence of Appendix B that the protein or its portion still retains the ability to increase C. glutamicum survival under conditions appropriate for it Microorganisms are chemically or ecologically dangerous.

The invention also relates to an isolated SRT protein preparation. In preferred embodiments, the SRT protein comprises an amino acid sequence from Appendix B. In a further preferred embodiment, the invention relates to an isolated full-length protein which essentially forms a complete airtino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is homologous. The SRT polypeptide or a biologically active portion thereof can be operably linked to a non-SRT polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has a different activity than the SRT protein alone and, in other preferred embodiments, results in increased yields, increased production and / or efficiency in the production of a desired fine chemical from C. glutamicum. The integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.

Another aspect of the invention relates to a method for producing a fine chemical. The method provides for the cultivation of a cell which contains a vector which brings about the expression of an SRT nucleic acid molecule according to the invention, so that a fine chemical is produced. In a preferred embodiment, this method also comprises the step of obtaining a cell which contains such a vector, the cell being transfected with a vector which brings about the expression of an SRT nucleic acid. In a further preferred embodiment, this method also comprises the step in which the fine chemical is obtained from the culture. In a preferred embodiment, the cell belongs to the genus Corynebacterium or Brevibacterium.

Another aspect of the invention relates to methods for modulating the production of a molecule from a microorganism. These methods involve contacting the cell with a substance that modulates SRT protein activity or SRT nucleic acid expression so that a cell-associated activity is changed compared to the same activity in the absence of the substance. In a preferred embodiment, the cell is modulated in terms of resistance to one or more chemicals or in terms of resistance to one or more ecological stress factors in such a way that the yields or the production rate of a desired fine chemical are improved by this microorganism. For example, the substance that modulates SRT protein activity stimulates SRT protein activity or SRT nucleic acid expression. Examples of substances that stimulate SRT protein activity or SRT nucleic acid expression include small molecules, active SRT proteins and nucleic acids that encode SRT proteins and have been introduced into the cell. Examples of substances that inhibit SRT activity or expression include small molecules and antisense SRT nucleic acid molecules. Another aspect of the invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing into a cell an SRT wild-type or mutant gene which either remains on a separate plasmid or is integrated into the genome of the host cell. The integration into the genome can be random or by homologous recombination, so that the native gene is replaced by the integrated copy, which causes the production of the desired compound from the cell to be modulated. In a preferred embodiment, these yields are increased. In a further preferred embodiment, the chemical is a fine chemical, which in an especially preferred embodiment is an amino acid. In a particularly preferred embodiment, this amino acid is L-lysine.

Detailed description of the invention

The present invention provides SRT nucleic acid and protein molecules which are involved in the survival of C. glutamicum when this microorganism is exposed to chemical or ecological pollutants. The molecules according to the invention can be used in the modulation of the production of fine chemicals from microorganisms, since these SRT proteins are a measure for a continuous growth and multiplication of C. glutamicum in the presence of toxic chemicals or dangerous environmental conditions, such as, for example, during fermentative cultivation in Large scale occur. By increasing the rate of growth or at least by maintaining normal growth under poor if not toxic conditions, one can increase the yield, production and / or efficiency of production of one or more fine chemicals from this culture, at least due to the relatively large number of cells that produce the fine chemical in culture, increase. The aspects of the invention are further explained below.

I. fine chemicals

The term "fine chemical" is known in the art and includes molecules that are produced by an organism and have applications in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. These compounds include organic acids, such as tartaric acid, itaconic acid and diamino-pimelic acid, both 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, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., Ed. VCH: Weinheim and the quotes contained therein, 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 (e.g. aro Aatic amines, vanillin and indigo), vitamins and cofactors (as described in Ulimann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and the quotes contained therein; and Ong, AS, Niki, E. and 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 on Sept. 1-3, 1994 in Penang, Malysia, AOCS Press (1995)), Enzyme and all other chemicals described by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the references therein , The metabolism and uses of certain fine chemicals are further discussed below.

Amino acid metabolism and uses

The amino acids comprise the basic structural units of all proteins and are therefore essential for normal cell functions. The term "amino acid" is known in the art. The proteinogenic amino acids, of which there are 20 types, serve as structural units for proteins in which they are linked to one another via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) are usually not found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids can be in the D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)). The "essential" amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), so named, because they have to be taken up in the diet due to the complexity of their biosynthesis, are identified by simple biosynthetic pathways converted into the remaining 11 "non-essential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine). Higher animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested in order for normal protein synthesis to take place. Aside from their function in protein biosynthesis, these amino acids are interesting chemicals per se and it has been discovered that many are used in various applications in the food, feed, chemical, cosmetic, agricultural and pharmaceutical industries. Lysine is not only an important amino acid for human nutrition, but also for monogastric animals such as poultry and pigs. Glutamate is most commonly used as a flavor additive (monosodium glutamate, MSG) and widely used in the food industry, as well as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are widespread feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol 6, Chapter 14a, VCH: Weinheim). It has been discovered that these amino acids are also 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, in Ulimann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985 are suitable substances.

The biosynthesis of these natural amino acids in organisms that can produce them, e.g. bacteria, has been well characterized (for an overview of bacterial amino acid biosynthesis and its regulation, see Umbarger, HE (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate is synthesized by reductive amination of α-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline and arginine are each made up of glutamate one after the other. The biosynthesis of serine takes place in a three-step process and begins with 3-phosphoglycerate (an intermediate in glycolysis), and gives this amino acid after oxidation, transamination and hydrolysis steps. Cysteine and glycine are each produced from serine, the former by condensation of homocysteine with serine, and the latter by transferring the side chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxyethylase. Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentosephosphate pathway, erythrose-4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differs only in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in an 11-step process. Tyrosine can be obtained by phenylalanine droxylase catalyzed reaction also from phenylalanine. Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis. Aspartate is formed from oxa acetate, an intermediate of the citrate cycle. Asparagine, methionine, threonine and lysine are each produced by converting aspartate. Isoleucine is formed from threonine. In a complex 9-step process, histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.

Amino acids, the amount of which exceeds the cell's protein biosynthesis requirement, cannot be stored and are instead broken down, so that intermediate products are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd ed. Chap. 21 "amino acid

Degradation and the Urea Cycle "; S 495-516 (1988)). Although the cell is able to convert undesired amino acids into useful metabolic intermediates, the amino acid production is in terms of energy, precursor molecules and the enzymes necessary for their synthesis It is therefore not surprising that amino acid biosynthesis is regulated by feedback inhibition, the presence of a particular amino acid slowing down or completely stopping its own production (for an overview of the feedback mechanism in amino acid biosynthesis pathways, see Stryer , L., Biochemistry, 3rd ed., Chapter 24, "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988). The output of a certain amino acid is therefore restricted by the amount of this amino acid in the cell ,

B. vitamins, cofactors and nutraceutical metabolism and uses

Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and must therefore absorb them, although they are easily synthesized by other organisms such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a number of metabolic pathways. In addition to their nutritional value, these compounds also have a significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure, activity and the industrial applications of these compounds, see, for example, Ulian's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996). The term "vitamin" is known in the art and includes nutrients derived from an organ must be required for normal function, but cannot be synthesized by this organism itself. The group of vitamins can include cofactors and nutraceutical compounds. The term "cofactor" encompasses non-proteinaceous compounds which are necessary for the occurrence of normal enzyme activity. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferably organic. The term “nutraceutical” encompasses food additives which are harmful to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (eg polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been extensively characterized (Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996, Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, AS, Niki, E. and 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 on September 1-3, 1994 in Penang, Malaysia, AOCS Press, Champaign, IL X, 374 S).

Thiamine (vitamin Bi) is formed by chemical coupling of pyrimidine and thiazole units. Riboflavin (vitamin B) 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). The family of compounds which are collectively referred to as "vitamin B6" (for example pyridoxine, pyridoxamine, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride) are all derivatives of the common structural unit 5-hydroxy-6-methylpyridine. Panthothenate (pantothenic acid, R- (+) -N- (2,4-dihydroxy-3,3,3-dimethyl-1-oxobutyl) -ß-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of ß-alanine and pantoic acid. The enzymes responsible for the biosynthetic steps for the conversion into pantoic acid, into β-alanine and for the condensation into pantothenic acid are known. The metabolically active form of pantothenate is coenzyme A, whose biosynthesis takes place over 5 enzymatic steps. Pantothenate, pyridoxal-5 '-phospha, cysteine and ATP are the 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 ), pantethein (and its derivatives) and coenzyme A.

The biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively investigated and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in the Fe cluster synthesis and belong to the class of the nifS proteins. Lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex. The folates are a group of substances that are all derived from folic acid, which in turn is derived from L-glutanic acid, p-aminobenzoic acid and 6-methylptin. The biosynthesis of folic acid and its derivatives, based on the metabolic intermediates guanosine 5 'triphosphate (GTP), L-glutamic acid and p-aminobenzoic acid, has been extensively investigated in certain microorganisms.

Corrinoids (such as the cobalamins and especially vitamin Bχ) and the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system. The biosynthesis of vitamin Bχ is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin". Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The production of these compounds on a large scale is largely based on cell-free chemical syntheses, although some of these chemicals have also been produced by large-scale cultivation of microorganisms, such as riboflavin, vitamin Bg, pantothenate and biotin. Only vitamin Bχ is only produced by fermentation due to the complexity of its synthesis. In-vitro processes require a considerable amount of materials and time and often high costs.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Genes for the purine and pyrimidine metabolism and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The term "purine" or "pyrimidine" encompasses nitrogen-containing bases which are part of the nucleic acids, coenzymes and nucleotides. The term "nucleo- tid "contains the basic structural units of the nucleic acid molecules, which comprise a nitrogenous base, a pentose sugar (for RNA the sugar is ribose, for DNA the sugar is D-deoxyribose) and phosphoric acid. The term" nucleoside "includes molecules that act as precursors by inhibiting the biosynthesis of these molecules or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis if this activity is targeted at carcinogens Inhibited cells inhibit the ability of tumor cells to divide and replicate, and there are nucleotides that do not form nucleic acid molecules, but that serve as energy sources (ie AMP) or as coenzymes (ie FAD and NAD).

Several publications have described the use of these chemicals for these medical indications, the purine and / or pyrimidine metabolism being influenced (e.g. Christopherson, RI and Lyons, SD (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents ", Med. Res. Reviews 10: 505-548). Studies on enzymes that are involved in the purine and pyrimidine metabolism have focused on the development of new drugs that can be used, for example, as immunosuppressants or antiproliferants (Smith, JL "Enzymes in Nucleotide Synthesis" Curr. Opin. Struct. Biol. 5 (1995) 752-757; Biochem. Soc. Transact. 23 (1995) 877-902). However, the purine and pyridine bases, nucleosides and nucleotides also have other possible uses: as intermediates in the biosynthesis of various fine chemicals (e.g. thiamine, S-adenosyl methionine, folate or riboflavin), as energy sources for the cell (e.g. ATP or GTP) and for chemicals themselves, are commonly used as flavor enhancers (e.g. IMP or GMP) or for many medical applications (see e.g. Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., VCH: Weinheim, pp. 561-612) Enzymes that are involved in the purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly used as targets against the chemicals for crop protection, including fungicides, herbicides and insecticides are developed.

The metabolism of these compounds in bacteria has been characterized (for overviews see, for example, Zalkin, H. and Dixon, JE (1992) "De novo purin nucleotide biosynthesis" in Progress in Nucleic Acids Research and Molecular biology, Vol. 42, Academic Press, Pp. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides"; Chap. 8 in: Biochemical Pathways: An Atlas of Bioche is- try and Molecular Biology, Wiley, New York). The purine metabolism, the object of intensive research, is essential for the normal functioning of the cell. A disturbed purine metabolism in higher animals can cause serious illnesses, e.g. gout. The purine nucleotides are synthesized from ribose 5-phosphate via a series of steps via the intermediate compound inosine 5 'phosphate (IMP), which leads to the production of guanosine 5'-monophosphate (GMP) or adenosine 5' -monophosphate (AMP), from which the triphosphate forms used as nucleotides can be easily produced. These compounds are also used as energy stores so that their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis takes place via the formation of uridine 5 'monophosphate (UMP) from ribose 5-phosphate. UMP in turn is converted into cytidine 5 'triphosphate (CTP). The deoxy forms of all nucleotides are produced in a one-step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. After phosphorylation, these molecules can participate in DNA synthesis.

D. Trehalose metabolism and uses

Trehalose consists of two glucose molecules which are linked to one another via an α, ot-1, 1 bond. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen foods, and in beverages. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, e.g., Nishi oto et al., (1998) US Patent No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech 16 (1998) 460-467; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium from which it can be obtained by methods known in the art.

II. Resistance to chemical damage and environmental stress

Fine chemicals are usually produced by large-scale culture of bacteria that have been developed for the production and secretion of large quantities of these molecules. However, this type of large-scale fermentation means that the microorganisms are subjected to various types of stress. These stress factors include environmental and chemical stress. Examples of environmental stress commonly encountered in large fermentation cultures include mechanical stress, heat stress, stress due to lack of oxygen, stress due to oxygen radicals, pH stress and osmotic stress. The stirring mechanism used to aerate the culture in most large fermenters generates heat, which increases the temperature of the culture. Temperature increases induce the well-described heat shock response, in which a set of proteins are expressed that are responsible for the survival of the bacterium in the face of high temperatures

10 supports, but also increases survival in response to a number of other environmental stress factors (see Neidhardt, FC et al., Ed. (1996) E. coli and Salmonella. ASM Press: Washington DC: pp. 1382-1399; Wosten , MM (1998) FEMS Microbiology Reviews 22 (3): 127-50; Bahl, H. et al. (1995) FEMS Microbiology Reviews 17 (3):

15 341-348; Zimmerman, J.L., Cohill, P.R. (1991) New Biologist 3 (7): 641-650; Samali, A. and Orrenius, S. (1998) Cell. Stress Chaperones 3 (4): 228-236, and the references cited in each of the citations). The regulation of the heat shock response in bacteria is determined by specific sigma factors and other cellular ones

20 regulators of gene expression are facilitated (Hecker, M., Volker, U. (1998). Molecular Microbiology 29 (5): 1129-1136). One of the biggest problems the cell experiences when exposed to high temperatures is deteriorated protein folding; nascent proteins have one under high temperature conditions

Sufficient kinetic energy that the growing polypeptide chain does not remain in a stable conformation long enough to fold correctly. Two of the key protein types that are expressed in the heat shock reaction are therefore made up of chaperones (proteins that cause the folding or unfolding of others

30 proteins support - s. e.g. Fink, A.L. (1999) Physiol. Rev. 79 (2): 425-449) and proteases that destroy all misfolded proteins. Examples of chaperones that are expressed in the heat shock reaction are GroEL and DNAK; Proteases, which are known to express during the response to heat shock

35 will include Lon, FtsH and ClpB.

In addition to heat, other environmental stress factors can also provoke a stress reaction. Although the fermenter stirring process is said to introduce oxygen into the culture, the oxygen supply can be limited, especially if the culture has reached an advanced stage of growth and its oxygen requirement is thus increased; an insufficient supply of oxygen is another stress for the microorganism. The cells in fermenter cultures are also subjected to a number of osmotic stress factors, especially when the nutrients are added to the culture, causing a high extracellular and a low intracellular concentration of these molecules. The large amounts of the desired molecules that are produced in culture by these organisms can contribute to the osmotic stress of bacteria. Finally, aerobic metabolism, such as that used in C. glutamicum, produces carbon dioxide as a waste product; the secretion of this molecule can acidify the culture medium due to the conversion of this molecule into carboxylic acid. Thus, bacteria in culture are also often subject to an acidic pH stress. The opposite can also be true - if large amounts of basic waste materials are present in the culture medium, the bacteria in the culture can also be subjected to a basic pH stress.

In addition to the environmental stress factors, the cells can also be subject to a number of chemical stress factors. These can fall into two categories. The first are natural waste products of metabolism and other processes that are secreted by the cell into the surrounding medium. The second are chemicals in the extracellular medium that do not come from the cell. When the cells secrete toxic waste products from the concentrated intracellular cytoplasm into the relatively much more dilute extracellular medium, these products spread so that the extracellular amounts of the potentially toxic compound are quite low. However, this cannot be the case with large-scale fermenter cultures of the bacterium: in a relatively small environment, so many bacteria grow with such a high metabolic rate that the waste products accumulate in the medium in almost toxic quantities. Examples of such waste products are carbon dioxide, metal ions and reactive oxygen species such as hydrogen peroxide. These compounds can disrupt the activity or structure of the cell surface molecules, or can re-enter the cell, where they can severely damage proteins and also nucleic acids. Certain other chemicals that are dangerous to the normal functioning of the cells can of course be found in the extracellular medium. For example. Metal ions such as mercury, cadmium, nickel or copper are often found in water sources that form solid complexes with cellular enzymes that prevent the normal functioning of these proteins. Bacteriocidal proteins can also be present in the extracellular milieu, either through the intervention of the researcher or as a natural product from other organisms that are used to gain a competitive advantage. The latter bacteriocidal compounds are probably not a stress that occurs in fermentative growth (unless the researcher applies selective pressure to the culture during growth), whereas metal ions are common, which is caused by the Purity of water and other compounds that are added to the fermenter system depends.

Thus, each of these stress factors can influence the behavior of the microorganism during fermenter culture and can disrupt the production of the desired compound from these organisms. For example. For example, osmotic stress from a microorganism can cause inappropriate or unsuitable rapid ingestion of one or more compounds, eventually leading to cellular damage or death from osmotic shock. To combat these environmental stress factors, bacteria have elegant gene systems that are expressed under the influence of one or more stress factors, such as the heat shock system mentioned above. Genes that are expressed in response to osmotic stress encode, for example, proteins that can transport or synthesize compatible solutes, so that the osmotic import or export of a specific molecule is reduced to manageable amounts. Other examples of genes for stress-induced bacterial proteins are those involved in trehalose biosynthesis, those encoding enzymes involved in the ppGpp mechanism, those involved in signal transduction, particularly those encoding two-component systems that are sensitive to osmotic pressure are sensitive, and those that code transcription factors that react to a variety of stress factors (for example, RssB analogs and / or sigma factors). Many other genes and their protein products are known.

III. Elements and methods according to the invention

The present invention is based, at least in part, on the discovery of new molecules, referred to herein as SRT nucleic acid and protein molecules, which enhance C. glutamicum's ability to survive in chemically or ecologically hazardous environments. In one embodiment, the SRT molecules confer C. glutamicum resistance to one or more ecological or chemical stress factors. In a preferred embodiment, the activity of the SRT molecules according to the invention has an effect on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the SRT molecules according to the invention have a modulated activity such that the yield, production and / or efficiency of production of one or more fine chemicals from C. glutamicum is also modulated.

The term "SRT protein" or "SRT polypeptide" encompasses proteins that are involved in the resistance of C. glutamicum to one or more ecological or chemical stress factors. Examples of SRT proteins include those encoded by the SRT genes listed in Table 1 and Appendix A. The terms "SRT gene" or "SRT nucleic acid sequence" encompass nucleic acid sequences which encode an SRT protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of SRT genes are listed in Table 1. The terms "production" or "productivity" are known in the art and include the concentration of the fermentation product (for example the desired fine chemical which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1) The term "efficiency of production" encompasses the time it takes to achieve a certain amount of production (e.g. how long it takes the cell to set up a certain throughput rate of a fine chemical). The term "yield" or "product / carbon yield" is in the Known in the art and includes the efficiency of converting the carbon source to the product (ie, the fine chemical), for example, usually expressed as kg product per kg carbon source, increasing the yield or production of the compound will increase the amount of molecules or the appropriate ones recovered Molecules of this compound in a particular cul quantity increased over a specified period. The terms "biosynthesis" or "biosynthetic pathway" are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step or highly regulated process. The terms "degradation" or "degradation path" are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), e.g. in a multi-step or highly regulated Process. The terms "degradation" or "degradation path" are known in the art and include the degradation of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules) in a multi-step or highly regulated process, for example. The term "metabolism" is known in the art and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a certain compound (eg the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways of this compound in the cell. The terms "resistance" and "tolerance" are well known in the art and include the ability of a cell to withstand exposure to a chemical or environment that would otherwise be detrimental to the normal functioning of this organism. The terms "stress" or "pollutant" include factors that are normal for the Function of cells such as C. glutamicum are harmful. Examples of stress factors include "chemical stress" in which the cell is exposed to one or more chemicals that are harmful to the cell, and "environmental stress" in which the cell is exposed to an environmental condition to which it is not adapted. Chemical stressors can be either natural metabolic waste products, such as, but not limited to, reactive oxygen species or carbon dioxide, or chemicals that are otherwise present in the environment, including but not limited to heavy metal ions or bacteriocidal proteins such as antibiotics. Environmental stress factors can be, but are not limited to, temperatures outside the normal range, suboptimal oxygen availability, osmotic pressures, or, for example, pH extremes.

In another embodiment, the SRT molecules according to the invention can modulate the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum. By means of recombinant genetic engineering, one or more SRT proteins according to the invention can be manipulated in such a way that their function is modulated. Changing the activity of stress response, resistance, or tolerance genes to increase the tolerance of the cell to one or more stress factors can improve the cell's ability to grow and multiply under the relatively stressful conditions of a large fermenter culture , Overexpression or manipulation of a heat shock-induced chaperone molecule so that it obtains optimal activity can, for example, increase the ability of a bacterium to fold proteins correctly under non-optimal temperature conditions. With fewer misfolded (and possibly improperly regulated or non-functional) proteins, the ability of the cell to function normally in such a culture is increased, which in turn provides increased viability. This overall increase in the number of cells with greater viability and activity in culture should also increase in the yield, production, and / or efficiency of production of one or more desired fine chemicals, at least due to the relatively larger number of cells that these chemicals produce in culture - Ren, effect.

The genome of a Corynebacterium glutamicum strain, which is available from the American Type Culture Collection under the name ATCC 13032, is suitable as a starting point for producing the nucleic acid sequences according to the invention. The nucleic acid sequences according to the invention can be produced from these nucleic acid sequences by conventional methods using the changes described in Table 1.

The SRT protein according to the invention or a biologically active section or fragments thereof can confer resistance and / or tolerance to one or more chemical or ecological stress factors, or can have one or more of the activities listed in Table 1.

Various aspects of the invention are described in more detail in the subsections below:

A. Isolated nucleic acid molecules

One aspect of the invention relates to isolated nucleic acid molecules which encode SRT polypeptides or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for identifying or amplifying SRT-encoding nucleic acids (for example SRT-DNA). The term “nucleic acid molecule” is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg RNA) as well as DNA or RNA analogs which are generated by means of nucleotide analogs. This term also includes the untranslated sequence located at the 3 'and 5' ends of the coding region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. An "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the geno i- see DNA of the organism from which the nucleic acid originates (for example, sequences that are located at the 5 'or 3' end of the nucleic acid are located) . In various embodiments, for example, the isolated SRT nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotide sequences that naturally contain the nucleic acid molecule in the genomic Flank the DNA of the cell from which the nucleic acid originates (for example a C. glutamicum cell). An "isolated" nucleic acid molecule, such as a cDNA molecule, can moreover be essentially free of another cellular material or culture medium if it is produced by recombinant techniques, or be free of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule according to the invention, for example a nucleic acid molecule with a nucleotide sequence from Appendix A or a section thereof, can be isolated using standard molecular biological techniques and the sequence information provided here. For example. a C. glutamicum SRT cDNA can be isolated from a C. glutamicum bank by using a complete sequence from Appendix A or a portion thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J. , Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Furthermore, a nucleic acid molecule comprising a complete sequence from Appendix A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been created on the basis of this sequence (for example a nucleic acid molecule can comprise a complete sequence) Appendix A, or a portion thereof, can be isolated by polymerase chain reaction using oligonucleotide primers made from this same sequence from Appendix A). For example. mRNA can be isolated from normal endothelial cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and the cDNA can be obtained by means of reverse transcriptase (for example Moloney-MLV reverse transcriptase) Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification via polymerase chain reaction can be created on the basis of one of the nucleotide sequences shown in Appendix A. A nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so modified can be cloned into a suitable vector and characterized by DNA sequence analysis. Oligonucleotides which correspond to an SRT nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule according to the invention comprises one of the nucleotide sequences listed in Appendix A. In a further preferred embodiment, an isolated nucleic acid molecule according to the invention comprises a nucleic acid molecule which is complementary to one of the nucleotide sequences shown in Appendix A or a section thereof, which is a nucleic acid molecule which is sufficiently complementary to one of the nucleotide sequences shown in Appendix A, that it can hybridize to one of the sequences given in Appendix A, creating a stable duplex.

In a further preferred embodiment, an isolated nucleic acid molecule according to the invention comprises a nucleotide sequence which is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90% or 90-95% and even more preferably at least about 95%, 96%, 97%,

15 is 98%, 99% or even more homologous to a nucleotide sequence specified in Appendix A or a section thereof. In a further preferred embodiment, an isolated nucleic acid molecule according to the invention comprises a nucleotide sequence which, for example under stringent conditions, with one of the nucleic acids shown in Appendix A

20 kleotide sequences or a portion thereof hybridized.

In one embodiment, the nucleic acid molecule according to the invention encodes a protein or a portion thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence from Appendix B that the protein or a portion thereof maintains the ability to be resistant or tolerant to one or to impart several chemical or environmental stress factors to C. glutamicum. As used herein, the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences refer to a minimal number of identical or equivalent amino acid residues (e.g. an amino acid residue with a side chain similar to an amino acid residue in one of the sequences in Appendix B) have an amino acid sequence from Appendix B so that the protein or a portion thereof can participate in the resistance of C. glutamicum to one or more chemical or environmental stress factors. Protein components of these metabolic pathways increase the resistance or tolerance of C. glutamicum to one or more environmental or chemical pollutants or stress factors. Examples of these activities are also described here. Thus, the "function of an SRT protein" refers to the overall resistance of C. glutamicum to components in its environment that interfere with its normal growth or function. Table 1 shows examples of SRT protein activities. 5 Sections of proteins which are encoded by the SRT nucleic acid molecules according to the invention are preferably biologically active sections of one of the SRT proteins. The term “biologically active section of an SRT protein”, as used here, is intended to include a section, for example a domain or a motif, of an SRT protein which is used to confer resistance or tolerance to one or more environments - or chemical stress factors, or has an activity as shown in Table 1. To determine whether an SRT protein or a biologically active portion thereof can increase the resistance or tolerance of C. glutamicum to one or more chemical or environmental stressors or pollutants, a test of the enzymatic activity can be carried out. These test methods, as described in detail in Example 8 of the example part, are familiar to the person skilled in the art.

In addition to naturally occurring variants of the SRT sequence that may exist in the population, those skilled in the art are also aware that mutation changes can be introduced into a nucleotide sequence of Appendix A, which leads to a change in the amino acid sequence of the encoded SRT protein without affecting the functionality of the SRT protein. For example. nucleotide substitutions which lead to amino acid substitutions at "non-essential" amino acid residues can be produced in a sequence from Appendix A. A "non-essential" amino acid residue can be modified in a wild-type sequence from one of the SRT proteins (Appendix B) without changing the activity of the SRT protein, whereas an "essential" amino acid residue is required for the SRT protein activity. However, other amino acid residues (for example non-conserved or only semi-preserved amino acid residues in the domain with SRT activity) cannot be essential for the activity and can therefore probably be changed without the SRT activity being changed.

An isolated nucleic acid molecule encoding an SRT protein that is homologous to a protein sequence from Appendix B can be generated by incorporating one or more nucleotide substitutions, additions or deletions into a nucleotide sequence from Appendix A such that one or more amino acid substitutions , additions or deletions are introduced into the encoded protein. The mutations can be introduced into one of the sequences from Appendix A by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferably introduced on one or more of the predicted non-essential amino acid residues. With a "conservative amino acid substitution" the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non- polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). A predicted non-essential amino acid residue in an SRT protein is thus preferably replaced by another amino acid residue of the same side chain family. In a further embodiment, the mutations can alternatively be introduced randomly over all or part of the SRT coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the SRT activity described here in order to identify mutants. certify that maintain SRT activity. After mutagenesis of one of the sequences from Appendix A, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here (see Example 8 of the example section).

B. Eekom inante expression vectors and host cells

Another aspect of the invention relates to vectors, preferably expression vectors, containing a nucleic acid encoding an SRT protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached. One type of vector is a "plasmid", which stands for a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, whereby additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they have been introduced (e.g. bacterial vectors with bacterial origin of replication and episomal mammalian vectors). Other vectors (eg non-episomal mammalian vectors) are integrated into the genome of a host cell when it is introduced into the host cell and thereby replicated together with the host genome. In addition, certain vectors can control the expression of genes to which they are operably linked. These vectors are called "expression vectors". Usually the expression vectors used in recombinant DNA techniques take the form of pias- miden. In the present description, "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used vector form. The invention is intended to encompass these other expression vector forms, such as viral vectors (for example replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.

The recombinant expression vector according to the invention comprises a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors one or more regulatory sequences, selected on the basis of the host cells to be used for expression, the is operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is bound to the regulatory sequence (s) in such a way that expression of the nucleotide sequence is possible (for example in an in vitro transcription / Translation system or in a host cell if the vector is introduced into the host cell). The term "regulatory sequence" is intended to encompass promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the direct expression of the nucleotide sequence only in certain host cells. The person skilled in the art is aware that the design of an expression vector can depend on factors such as the choice of the host cell to be transformed, the extent of expression of the desired protein, etc. The expression vectors according to the invention can be introduced into the host cells, so that proteins are thereby or peptides, including fusion proteins or peptides, encoded by the nucleic acids as described herein (e.g., SRT proteins, mutated forms of SRT proteins, fusion proteins, etc.).

The recombinant expression vectors according to the invention can be designed for the expression of SRT proteins in prokaryotic or eukaryotic cells. For example. SRT genes can be found in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, 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" in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, Ed., Pp. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi. in: Applied Molecular Genetics of Fungi, Peberdy, JF et al., eds., pp. 1-28, Cambridge University Press : Cambridge), algae and multicellular

Plant cells (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-meδ. ± ate & transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep .: 583-586) or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Proteins are usually expressed in prokaryotes using vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins. Fusion vectors contribute a number of amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein. These fusion vectors usually have three functions: 1) to increase the expression of recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) supporting the purification of the recombinant protein by acting as a ligand in affinity purification. In the case of fusion expression vectors, a proteolytic cleavage site is often introduced at the junction of the fusion unit and the recombinant protein, so that the recombinant protein can be separated from the fusion unit after the fusion protein has been purified. These enzymes and their corresponding recognition sequences include factor Xa, thrombin and enterokinase.

Common fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ), in which glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein. In one embodiment, the coding sequence of the SRT protein is cloned into a pGEX expression vector so that a vector is generated which encodes a fusion protein, comprising from the N-terminus to the C-terminus, GST - thrombin cleavage site - X- Protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. The recombinant SRT protein that does not fuse with GST can be obtained by cleaving the fusion protein with thrombin.

Examples of suitable inducible Nic t-fusion expression vectors from E. coli include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET III (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter. The target gene expression from the pETlld vector is based on the transcription from a T7-gnl0-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by the BL 21 (DE3) or HMS174 (DE3) host strains from a resident λ prophage which harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize the expression of the recombinant protein is to express the protein in a host bacterium whose ability to proteolytically cleave the recombinant protein is impaired (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 of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those which are preferably used in a bacterium selected for expression, such as C. glutamicum (Wada et al. (1992 ) Nucleic Acids Res. 20: 2111-2118). This change in the nucleic acid sequences according to the invention is carried out by standard DNA synthesis techniques.

In a further embodiment, the SRT protein expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and 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 of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., ed., pp. 1-28, Cambridge University Press: Cambridge. Alternatively, the SRT proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g. Sf9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

In a further embodiment, the SRT proteins according to the invention can be expressed in single-cell plant cells (such as algae) or in plant cells of higher plants (for example spermatophytes such as crops). Examples of plant expression vectors include those which are described in detail in: Bekker, D., Ke per, E., Schell, J. and 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. Acids Res. 12: 8711-8721.

In a further embodiment, a nucleic acid according to the invention is expressed in mammalian cells with a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. Commonly used promoters come, for example, from Polyoma, Adenovirus2, Cytomegalievirus and Simian Virus 40. For further suitable expression systems for prokaryotic and eukaryotic cells, see chapters 16 and 17 from Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

In a further embodiment, the recombinant mammalian expression vector can preferably bring about the expression of the nucleic acid in a specific cell type (for example, tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore ( 1983) Cell 33: 741-748), neuron-specific promoters (e.g. neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477), pancreatic-specific promoters (Edlund et al., (1985) Science 230: 912-916) and mammary-specific promoters (e.g. milk serum promoter; US -Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Development-regulated promoters are also included, for example the mouse hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546).

The invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction. This means that the DNA molecule is operably linked to a regulatory sequence in such a way that expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the SRT mRNA is possible. Regulatory sequences can be selected which are operably linked to a nucleic acid cloned in the antisense direction and which control the continuous expression of the antisense RNA molecule in a multiplicity of cell types, for example viral promoters and / or enhan - Cer or regulatory sequences are selected that control the constitutive, tissue-specific or cell type-specific expression of antisense RNA. The antisense expression vector can 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 effective regulatory region, the activity of which is determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al. , Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention relates to the host cells into which a recombinant expression vector according to the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably here. It goes without saying that these terms refer not only to a specific target cell, but also to the descendants or potential descendants of this cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, these offspring are not necessarily identical to the parental cell, but are still included in the scope of the term as used here. A host cell can be a prokaryotic or eukaryotic cell. For example. an SRT protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to the person skilled in the art. Microorganisms which are related to Corynebacterium glutamicum and which can be suitably used as host cells for the nucleic acid and protein molecules according to the invention are listed in Table 3.

Using conventional transformation or transfection methods, vector DNA can be introduced into prokaryotic or eukaryotic cells. The terms "transformation" and "transfection" as used here are intended to encompass a large number of methods known in the prior art for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (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.

For the stable transfection of mammalian cells it is known that, depending on the expression vector and transfection technique used, only a small part of the cells integrate the foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selectable marker (e.g. resistance to antibiotics) is usually introduced into the host cells together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SRT protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection (e.g. cells that have integrated the selectable marker survive, whereas the other cells die).

To generate a homologously recombined microorganism, a vector is produced which contains at least a section of an SRT gene into which a deletion, addition or substitution has been introduced in order to change the SRT gene, for example to functionally disrupt it. This SRT gene is preferably a co- ryneJbacterium glutamicum SRT gene, however, a homologue from a related bacterium or even from a source of mammals, yeasts or insects can be used. In a preferred embodiment, the vector is designed such that the endogenous SRT gene is functionally disrupted when homologous recombination occurs (ie, no longer encodes a functional protein, also referred to as a "knockouf 'vector). The vector may alternatively be designed such that the endogenous SRT gene is mutated or otherwise altered during homologous recombination, but still encodes the functional protein (for example, the upstream regulatory region can be altered in such a way that the expression of the endogenous SRT protein is altered thereby) The SRT gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the SRT gene, which is a homologous recombination between the exogenous SRT gene carried by the vector and an endogenous SRT gene in a microorganism. The additional flanking SRT nucleic acid is successful for a oak homologous recombination with the endogenous gene sufficiently long. The vector usually contains several kilobase flanking DNA (both at the 5 'and 3' ends) (see, for example, Thomas, KR and Capecchi, MR (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g., by electroporation), and cells in which the introduced SRT gene is homologously recombined with the endogenous SRT gene are selected using methods known in the art.

In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene. The inclusion of an SRT gene in a vector under the control of the Lac operon enables e.g. expression of the SRT gene only in the presence of IPTG. These regulatory systems are known in the art.

A host cell according to the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used for the production (ie expression) of an SRT protein. The invention also provides methods for producing SRT proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell according to the invention (into which a recombinant expression vector which encodes an SRT protein has been introduced, or into whose genome a gene has been introduced which is a wild-type or modified SRT protein encoded) in a suitable medium until the SRT protein has been produced. The process comprises in a further embodiment isolating the SRT proteins from the medium or the host cell.

C. Uses and methods according to the invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors and host cells described here can be used in one or more of the following methods: identification of C. glutamicum and related organisms, mapping of genomes of organisms that are related to C. glutamicum related, identification and localization of C. glutamicum sequences of interest, evolution studies, determination of SRT protein areas that are necessary for function, modulation of the activity of an SRT protein; Modulating the activity of an SRT path; and modulating the cellular production of a desired compound, such as a fine chemical. The SRT nucleic acid molecules according to the invention have a multitude of uses. They can initially be used to identify an organism as Corynebacterium glutamicum or close relatives thereof. They can also be used to identify C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes. By probing the extracted genomic DNA of a culture of a uniform or mixed population of microorganisms under stringent conditions with a probe comprising a region of a C. glutamicum G & ns that is unique to this organism, one can determine whether this organism is present is. Corynebacterium glutamicum itself is not pathogenic, but it is related to pathogenic species such as Corynebacterium diptheriae. The detection of such an organism is of significant clinical importance.

The nucleic acid and protein molecules according to the invention can serve as markers for specific regions of the genome. This is useful not only when mapping the genome, but also for functional studies of C. glutamicum proteins. The C. glutamicum genome can be used to identify the genome region to which a specific C. glutamicum DNA-binding protein binds. e.g. cleaved, and the fragments are incubated with the DNA-binding protein. Those that bind the protein can additionally be probed with the nucleic acid molecules according to the invention, preferably with easily detectable markings; the binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genomic map of C. glutamicum, and if this is carried out several times with different enzymes, it facilitates one rapid determination of the nucleic acid sequence to which the protein binds. The nucleic acid molecules according to the invention can moreover be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.

The SRT nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies. The resistance processes in which the molecules of the invention are involved are exploited by a number of cells; By comparing the sequences of the nucleic acid molecules according to the invention with those which code similar enzymes from other organisms, the degree of evolutionary kinship of the organisms can be determined. Accordingly, such a comparison enables the determination of which sequence regions are conserved and which are not, which can be helpful in determining those regions of the protein which are essential for the enzyme function. This type of determination is valuable for protein technology studies and can provide an indication of which protein can tolerate mutagenesis without losing function.

The manipulation of the SRT nucleic acid molecules according to the invention can bring about the production of SRT proteins with functional differences from the wild-type SRT proteins. These proteins may be improved in efficiency or activity, may be present in the cell in greater numbers than usual, or may be weakened in efficiency or activity. The aim of these manipulations is to increase the viability and activity of the cell when it is exposed to environmental and / or chemical stress factors and pollutants, which are common in large-scale fermenter cultures. Increasing the activity or copy number of a heat shock regulated protease can increase the cell's ability to ^• destroy misfolded proteins that would otherwise interfere with normal cell functions (e.g. further binding of substrates and cofactors, although the protein has activity) these molecules do not act appropriately). The same applies to the overexpression or optimization of the activity of one or more chaperone molecules induced by heat or cold shock. These proteins serve to correctly fold nasal polypeptide chains, and thus their increased activity or presence increases the percentage of properly folded proteins in the cell, which in turn increases the overall metabolic efficiency and viability of the cells in culture. Overexpression or optimization of the transporter molecules activated by otic shock should be the ability of the part of the cell that maintain intracellular homeostasis, thereby increasing the viability of these cells in culture. The overexpression or increase in activity by mutagenesis of proteins involved in the development of cellular resistance to various stress factors (either by transporting the attacking chemical out of the cell or by modifying the chemical into a less dangerous substance) should affect the organism's performance the environment containing the dangerous substance (e.g. a large-scale fermenter culture), and thus enable relatively more cells to survive in such a culture. The net effect of all mutagenesis strategies is to increase the quantity of fine chemical producing compounds in the culture, which increases the yield, production and / or efficiency of the production of one or more desired fine chemicals from the culture.

This above list of mutagenesis strategies for SRT proteins, which are said to bring about increased yields of a desired compound, is not intended to be limiting; Variations on these mutagenesis strategies are readily apparent to those skilled in the art. By means of these mechanisms, the nucleic acid and protein molecules according to the invention can be used to generate C. glutamicum or related bacterial strains which express mutated SRT nucleic acid and protein molecules, so that the yield, production and / or efficiency of the production of a desired one Connection is improved. The desired compound can be a natural product of C. glutamicum, which comprises the end products of the biosynthetic pathways and intermediates of naturally occurring metabolic routes, as well as molecules which do not occur naturally in the metabolism of C. glutamicum, but which are derived from a C. glutamicum according to the invention. Trunk are produced.

This invention is further illustrated by the following examples, which are not to be construed as limiting. The contents of all references, patent applications, patents and published patent applications cited in this patent application are hereby incorporated by reference. Examples

Example 1: Preparation of the entire genomic DNA from Corynebacterium glutamicum ATCC13032

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 ° C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml of culture volume). The composition of buffer I: 140.34 g / 1 sucrose, 2.46 g / 1 MgS0 ₄ • 7 H ₂ 0, 10 ml / 1 KH ₂ P0 ₄ solution (100 g / l, adjusted to pH with KOH 6.7), 50 ml / 1 M12 concentrate (10 g / 1 (NH ₄ ) ₂ S0 ₄ , 1 g / 1 NaCl, 2 g / 1 MgS0 ₄ • 7 H ₂ 0, 0.2 g / 1 CaCl ₂ , 0.5 g / 1 yeast extract

(Difco), 10 ml / 1 trace element mixture (200 mg / 1 FeS0 ₄ • H0, 10 mg / 1 ZnS0 ₄ • 7 H ₂ 0, 3 mg / 1 MnCl ₂ • 4 H ₂ 0, 30 mg / 1 H ₃ B0 ₃ , 20 mg / 1 CoCl ₂ • 6 H ₂ 0, 1 mg / 1 NiCl ₂ • 6 H ₂ 0, 3 mg / 1 Na ₂ Mo0 ₄ • 2 H ₂ 0, 500 mg / 1 complexing agent (EDTA or Citric acid), 100 ml / 1 vitamin mixture (0.2 ml / 1 biotin, 0.2 mg / 1 folic acid, 20 mg / 1 p-aminobenzoic acid, 20 mg / 1 riboflavin, 40 mg / 1 Ca-panthothenate, 140 mg / 1 nicotinic acid, 40 mg / 1 pyridoxol hydrochloride, 200 mg / 1 myoinositol) Lysozyme was added to the suspension in a final concentration of 2.5 mg / ml. After about 4 hours of incubation at 37 ° C., the cell wall was broken down and the protoplasts obtained were harvested by centrifugation and the pellet was washed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 mM Tris-HCl, 1 M EDTA, pH 8) ml of TE buffer was resuspended, and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5 M) were added With a final concentration of 200 μg / ml, the suspension was incubated at 37 ° C. for about 18 hours. The DNA was purified by extraction with phenol, phenol-chloroform-isoayl alcohol and chloroform-isoamyl alcohol using standard procedures. Then the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by incubation for 30 min at -20 ° C and 30 min centrifugation at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) , The DNA was dissolved in 1 ml of TE buffer containing 20 μg / ml RNase A and dialyzed against 1000 ml of TE buffer at 4 ° C. for at least 3 hours. During this time the buffer was exchanged 3 times. 0.4 ml of 2 M LiCl and 0.8 ml of ethanol were added to 0.4 ml aliquots of the dialyzed DNA solution. After 30 min incubation at -20 ° C, the DNA was collected by centrifugation (13000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE buffer. DNA produced by this method could be used for all purposes used, including Southern blotting or to construct genomic libraries.

Example 2: Construction of genomic Corynebacterium glutamicum (ATCC13032) banks in Escherichia coli

Starting from DNA, prepared as described in Example 1, "Molecular Cloning: A Laboratory Manual" was used according to known and well-established methods (see, for example, Sambrook, J. et al. (1989). Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", John Willey & Sons) Cosmid and Plas id banks.

Any plasmid or cosmid could be used. The plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741) were used in particular; pACYCl77 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or Cos ide, such as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, TJ Rosenthal, A., and Waterson, RH (1987) Gene 53: 283-286.

Example 3: DNA sequencing and computer function analysis

Genomic banks, as described in Example 2, were used for DNA sequencing according to standard methods, in particular the chain termination method with ABI377 sequencing machines (see, for example, Fleischman, RD et al. (1995) "Whole-geno e Random Sequencing and Asse bly of Haemophilus Influenzae Rd., Science 269; 496-512) The sequencing primers with the following nucleotide sequences were used: 5 '-GGAAACAGTATGACCATG-3' or 5 '-GTAAAACGACGGCCAGT-3'.

Example 4: In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum can be carried out by passing a plasmid (or other vector) DNA through E. coli or other microorganisms (eg Bacillus spp. Or yeasts such as Saccharomyces cerevisiae) which reduce the integrity of their cannot maintain genetic information. Usual mutator strains have mutations in the genes for the DNA repair system (e.g., utHLS, mutD, mutT, etc., for comparison see Rupp, WD (1996) DNA repair mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM : Washington). These strains are known to the person skilled in the art. The use of these strains is, for example, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34 illustrates.

Example 5: DNA transfer between Escherichia coli and Corynebacterium glutamicum

Several Corynebacterium and Brevibacierium species contain endogenous plasmids (such as pHM1519 or pBLl) that replicate autonomously (for an overview see, for example, Martin, J.F. et al. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel , FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons), to which an origin of replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibactertium species. Particular uses as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn-903 transposon) or for chloramphenicol (Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim) There are numerous examples in the literature for the production of a wide variety of shuttle vectors which are replicated in E. coli and C. glutamicum and which can be used for various purposes, including gene overexpression ( see, e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, JF et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ et al. (1992) Gene 102: 93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to introduce such hybrid vectors into Corynebacterium glutamicum strains. The transformation of C. glutamicum can be carried out by protoplast transformation (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, can also be achieved by conjugation (as described, for example, in Schaefer, A., et (1990) J. Bacteriol. 172: 1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods known in the art) and transforming it into E. coli. This transformation step can be carried out using standard methods, but an Mcr deficit is advantageously the E. coli strain was used, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19).

Example 6: Determination of expression of the mutant protein

The observations of the activity of a mutant protein in a transformed host cell are based on the fact that the mutant protein is expressed in a similar manner and in a similar amount as the wild-type protein. A suitable method for determining the amount of transcription of the mutated gene (an indication of the amount of mRNA available for the translation of the gene product) is to carry out a Northern blot (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York), wherein a primer that is designed to bind to the gene of interest is provided with a detectable (usually radioactive or chemiluminescent) label so that - if the total RNA is one Culture of the organism extracted, separated on a gel, transferred to a stable matrix and incubated with this probe - the binding and the quantity of binding of the probe indicates the presence and also the amount of mRNA for this gene. This information is evidence of the extent of transcription of the mutant gene. Total cell RNA can be isolated from Corynebacterium glutamicum by various methods known in the art, as described in Bormann, E.R. et al., (1992) Mol. Microbiol. 6: 317-326.

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

Example 7: Growth of genetically modified Corynebacterium glutamicum media and growing conditions

Genetically modified Corynebacteria are grown in synthetic or natural growth media. A number of different growth media for Corynebacteri n are known and easy available (Lieb et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) "The Genus Corynebacterium ", in: The Procaryotes, Vol. II, Balows, A., et al., Ed. 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. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH ₄ CI or (NH ₄ ) SO ₄ , NH ₄ OH, nitrates,

Urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extracts, meat extracts and others.

Inorganic salt compounds that may be included in the media include the chloride, phosphorus, or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate or organic acids such as citric acid. The media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. The exact composition of the media connections depends heavily on the respective experiment and is decided individually for each case. Information about media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like. All media components are sterilized, either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration. The components can be sterilized either together or, if necessary, separately. All media components can be present at the beginning of the cultivation or can be added continuously or in batches.

The growing conditions are defined separately for each experiment. The temperature should be between 15 ° C and 45 ° C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by adding buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES; ACES etc. can be used alternatively or simultaneously. The cultivation pH can also be kept constant during the cultivation by adding NaOH or NH ₄ OH. If complex media components, such as yeast extract, are used, the need for additional buffers is reduced, since many complex compounds have a high buffer capacity. When using a fermenter for the cultivation of microorganisms, the pH value can also be regulated with gaseous ammonia.

The incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth. The disclosed growth experiments can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes. To screen a large number of clones, the microorganisms should be grown in microtiter plates, glass tubes or shake flasks with or without baffles. Preferably 100 ml shake flasks are used, which are filled with 10% (by volume) of the required growth medium. The 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, a mathematical correction should be carried out for the evaporation losses.

If genetically modified clones are examined, an unmodified control clone or a control clone which contains the base plasmid without insertion should also be tested. The medium is inoculated to an ODeoo ^of 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g / 1 glucose, 2.5 g / 1 NaCl, 2 g / 1 Urea, 10 g / 1 polypeptone, 5 g / 1 yeast extract, 5 g / 1 meat extract, 22 g / 1 agar pH 6.8 with 2 M NaOH), which have been incubated at 30 ° C. The inoculation of the media is carried out either by introducing a saline solution of C. glutamicum cells from CM plates or by adding a liquid preculture of this bacterium.

Example 8: In vitro analysis of the function of imitated proteins

Determining the activities and kinetic parameters of enzymes is well known in the art. Experiments to determine the activity of a particular altered enzyme must be adapted to the specific activity of the wild-type enzyme, which is within the ability of the person skilled in the art. Overviews of enzymes in general as well as specific details relating to the structure, kinetics, principles, processes, applications and examples for determining many enzyme activities can be found, for example, in the following literature references: Dixon, M., and Webb, EC: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P.D: Ed. (1983) The Enzymes, 3rd Edition Academic Press, New York; Bisswanger, H. (1994) Enzyme Kinetics, 2nd Edition VCH, Weinheim (ISBN 3527300325); Bergmeyer, H.U., Bergmeyer, J., Graßl, M. Ed. (1983-1986) Methods of Enzymatic Analysis, 3rd edition Vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) Vol. A9, "Enzymes", VCH, Weinheim, pp. 352-363.

The activity of proteins that bind to DNA can be measured by many well-established methods, such as DNA band shift assays (also referred to as gel retardation assays). The effect of these proteins on the expression of other molecules can be measured using reporter gene assays (as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904 and the references cited therein). Reporter gene test systems are well known and established for use in pro- and eukaryotic cells using enzymes such as beta-galactosidase, green fluorescent protein and several others.

The activity of membrane transport proteins can be determined according to the techniques as described in Gennis, RB (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137; 199-234; and 270-322. Example 9: Analysis of the influence of mutated protein on the production of the desired product

The effect of the genetic modification in C. glutamicum on the production of a desired compound (such as an amino acid) can be determined by growing the modified microorganisms under suitable conditions (such as those described above) and the medium and / or the cellular Components for the increased production of the desired product (ie an amino acid) is examined. Such analysis techniques are well known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89) -90 and pp. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry" in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17; Reh et al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery and purification", pp. 469-714, VCH: Weinheim; Belter, PA et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, JF and Cabral, JMS (1992) Recovery processes for biological Materials, John Wiley and Sons; Shaeiwitz, JA and Henry, JD (1988) Biochemical Separations, in Ulimann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11 , Pp. 1-27, VC H: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to measuring the final fermentation product, it is also possible to analyze other components of the metabolic pathways that are used to produce the desired compound, such as intermediates and by-products, to determine the overall productivity of the organism, the yield and / or the efficiency of the To determine production of the connection. The analysis methods include measurements of the amount of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of common metabolites from biosynthetic pathways and measurements of gases that are produced during fermentation , Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein. Example 10: Purification of the desired product from a C. glutamicum culture

The desired product can be obtained from C. glutamicum cells or from the supernatant of the culture described above by various methods known in the art. If the desired product is not secreted by the cells, the cells can be harvested from the culture by slow centrifugation, the cells can be lysed by standard techniques such as mechanical force or ultrasound. The cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is obtained for further purification of the desired compound. If the product is secreted by the C. glutamicum cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is kept for further purification.

The supernatant fraction from both purification procedures is subjected to chromatography with an appropriate resin, either with the desired molecule retained on the chromatography resin but not with many contaminants in the sample, or with the contaminants remaining on the resin but not the sample. If necessary, these chromatography steps can be repeated using the same or different chromatography resins. The person skilled in the art is skilled in the selection of the suitable chromatography resins and the most effective application for a particular molecule to be purified. The purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.

Many cleaning methods are known in the art that are not limited to the previous cleaning method. These are described, for example, in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: 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. Ul ann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

equivalent

Those skilled in the art will recognize or can - by using only routine methods - determine many equivalents of the specific embodiments of the invention. These equivalents are intended to be encompassed by the claims below.

The information in Table 1 is to be understood as follows:

In column 1 "DNA-ID" the respective number refers to the SEQ ID NO of the attached sequence listing. A "5" in the "DNA-ID" column therefore means a reference to SEQ ID NO: 5.

In column 2 "AS-ID", the respective number refers to the SEQ ID NO of the attached sequence listing. A "6" in the "AS-ID" column therefore means a reference to SEQ ID NO: 6.

Column 3 "Identification" contains a unique internal name for each sequence.

In column 4 "AS-POS" the respective number refers to the amino acid position of the polypeptide sequence "AS-ID" in the same line. A “26” in the “AS-POS” column consequently means the amino acid position 26 of the correspondingly indicated polypeptide sequence. The position of the N-Terminal starts at +1.

In column 5 "AS wild type", the respective letter denotes the amino acid - represented in the one-letter code - at the position indicated in column 4 in the corresponding wild type strain.

In column 6 "AS mutant" the respective letter denotes the amino acid - shown in the one-letter code - at the position indicated in column 4 in the corresponding mutant strain.

Column 7 "Function" lists the physiological function of the corresponding polypeptide sequence. One letter code of proteinogenic amino acids:

A alanine

C cysteine

D aspartate

E glutamate

F phenylalanine

G glycine

H His

I isoleucine

K lysine

L leucine

M methionine

N asparagine

P proline

Q glutamine

R arginine

S serine

T threonine

V valine

W tryptophan

Y tyrosine

Table 1

Genes that code for stress resistance and tolerance proteins

DNA AS identification: AS AS AS function:

ID: ID: Pos: Wild-type Mutai

1 2 RXA00165 130 V I MULTIDRUG RESISTANCE- IKE ATP-BINDING PROTEIN MDL

410 G E MULTIDRUG RESISTANCE-LIKE ATP-BINDING PROTEIN MDL

3 4 RXA00404 188 R H CARBON STARVATION PROTEIN A

5 6 RXA00453 243 A T TRANSPORTER

246 Θ D TRANSPORTER

7 8 RXA00493 363 A T 60 KD CHAPERONIN GROEL

9 10 RXA00803 264 P s METHYLENOMYCIN A RESISTANCE PROTEIN

11 12 RXA00829 408 L F DAUNORUBICIN RESISTANCE DNA-BINDING PROTEIN DRRC

13 14 RXA00886 75 G D CHAPERONE PROTEIN DNAJ

242 G D CHAPERONE PROTEIN DNAJ

15 16 RXA01054 402 T I MERCURIC REDUCTASE (EC 1.16.1.1)

17 18 RXA01345 308 A V DNAK PROTEIN

19 20 RXA01559 400 T A PROTEIN TRANSLOCASE SUBUNIT SECD

21 22 RXA01578 130 R C MULTIDRUG RESISTANCE PROTEIN B

23 24 RXA01936 276 LF MACROLIDE-EFFLUX PROTEIN

26 RXA02119 209 G E TRANSPORTER

241 G D TRANSPORTER

28 RXA02202 72 V I ARSENATE REDUCTASE

30 RXA02280 502 A V HEAT SHOCK PROTEIN HTPG

32 RXA02431 73 A V DNA POLYMERASE IV

34 RXA02541 114 G E CHAPERONE PROTEIN DNAJ

308 A V CHAPERONE PROTEIN DNAJ

36 RXA02736 312 S F PUTATIVE OXPPCYCLE PROTEIN OPCA

38 RXA02964 306 V I QUINOLONE RESISTANCE NORA PROTEIN

40 RXA03359 107 V I Universal stress protein family

42 RXA03824 34 G D MULTIDRUG RESISTANCE PROTEIN B

44 RXA06014 24 AT Rhodanese-related sulfur transferases

36 E K Rhodanese-related sulfurtraπsferases

46 RXA07004 150 G D MULTIDRUG RESISTANCE PROTEIN B

Claims

claims

1. Isolated nucleic acid molecule coding for a polypeptide with the amino acid sequence referred in each case in table / column 2, the nucleic acid molecule in the amino acid position indicated in table 1 / column 4 coding for a different proteinogenic amino acid than the respective one in table 1 / column 5 in the same line given amino acid.

2. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule in the amino acid position given in Table 1 / column 4 encodes the amino acid given in Table 1 / column 6 in the same row.

3. A vector containing at least one nucleic acid sequence according to claim 1.

4. A host cell that is transfected with at least one vector according to claim 3.

5. A host cell according to claim 4, wherein the expression of said nucleic acid molecule leads to modulation of the production of a fine chemical from said cell.

6. A method for producing a fine chemical which comprises culturing a cell which has been transfected with at least one vector according to claim 3, so that the fine chemical is produced.

7. The method according to claim 6, characterized in that the fine chemical is an amino acid.

8. The method according to claim 7, characterized in that the amino acid is lysine.