EP1446421A2 - Mutated gene from corynebacterium glutamicum - Google Patents

Abstract

The invention relates to novel nucleic acid molecules, the use thereof for the construction of genetically improved micro-organisms and methods for the production of fine chemicals, in particular amino acids by means of said genetically improved micro-organisms.

Description

Genes that code for new 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, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors and enzymes. Their production is best accomplished by growing 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 Cornebac e iu-T! 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 Corynebactθrium glutamicum or related types of bacteria. C. glutamicu-n is a gram-positive, aerobic bacterium that is commonly used in industry for the large-scale production of a number of fine chemicals and also 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. glutamicu-n 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. gl u tamicum genome or organisms related to genomes. These new nucleic acid molecules encode proteins that are referred to herein as marker and fine chemical production (MCP) proteins. These MCP-Pro one can, for example, be directly or indirectly involved in the production of one or more fine chemicals in C. glutamicum. The MCP proteins according to the invention can also be involved in the degradation of hydrocarbons or in the oxidation of terpenoids. These proteins can be used to identify Coryneiac e iu-ri glutamicum or organisms related to C. glutamicum; the presence of an MCP protein specific for C. glutamicum and related species in a protein mixture can indicate the presence of one of these bacteria in the sample. Furthermore, these MCP proteins can have homologs in plants or animals which are involved in a disease state or a condition; so these proteins can serve as useful pharmaceutical targets for drug screening and therapeutic compound development.

Due to the availability of cloning vectors useful 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 brevibacterium species (e.g. lactofermentum) (Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Katsu ata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gen. Microbiol. 130: 2237- 2246 (1984)), the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to modulate the production of one or more fine chemicals. This modulation can take place due to a direct effect of the manipulation of a gene according to the invention or due to an indirect effect of such a manipulation. For example. by modifying the activity of a protein involved in the biosynthesis or degradation of a fine chemical (ie, by mutagenesis of the corresponding gene), the cell's ability to synthesize or degrade this compound can be directly modulated, thereby reducing the yield and / or efficiency of production of the fine chemical is modulated. Likewise, by modulating the activity of a protein that regulates a fine chemical pathway, one can directly influence whether the production of the desired compound is up or down regulated, both of which modulate the yield or efficiency of the production of the fine chemical from the cell.

Indirect modulation of fine chemical production can also be done by modifying the activity of a protein of the invention (ie, mutagenizing the corresponding gene) so that the overall ability of the cell to grow and divide, or to remain viable and productive, is increased. The Production of fine chemicals from C. glutamicum is usually achieved by large-scale fermentation culture of these microorganisms, conditions that are often suboptimal for growth and cell division. By modifying a protein of the invention (e.g., a stress reaction protein, a cell wall protein, or a protein involved in the metabolism of compounds necessary for the occurrence of cell division and growth, such as nucleotides and amino acids), so that survival, growth and Increasing in these conditions is possible, it may be possible to increase the number and productivity of these altered C. glufca icum cells in large-scale cultures, which in turn leads to increased yields and / or increased efficiency in the production of one or more desired fine chemicals should lead. Furthermore, the metabolic pathways of a cell are necessarily interdependent and co-regulated. By changing the activity of any metabolic pathway in C. glutamicum (ie by changing the activity of one of the proteins according to the invention which is involved in such a pathway) it is possible to simultaneously change the activity or regulation of another metabolic pathway in this microorganism which may be directly involved in the synthesis or degradation of a fine chemical.

This invention provides new nucleic acid molecules which encode proteins, which are referred to here as MCP proteins and, for example, modulate the production or efficiency of the production of one or more fine chemicals in C. glutamicum or as identification markers for C. glutamicum or related organisms can serve. Nucleic acid molecules that encode an MCP protein are referred to here as MCP nucleic acid molecules. In a preferred embodiment, the MCP protein can modulate the production or efficiency of the production of one or more fine chemicals in C. glutamicum or serve as identification markers for C. glutamicu-n or related organisms. Examples of such proteins are those encoded by the genes shown 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 MCP protein or biologically active sections thereof, and also nucleic acid fragments which can be used as primers or hybridization probes for the detection or application of MCP- 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 of one of these nucleotide sequences or a complement thereof. In other preferred embodiments, co- the isolated nucleic acid molecule detects one of the amino acid sequences listed in Appendix B. The preferred MCP proteins according to the invention likewise preferably have at least one of the MCP 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. gluta-nicu-n MCP protein or a biologically active section 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, this host cell is used to produce an MCP protein by growing the host cell in a suitable medium. The MCP 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 MCP gene has been introduced or modified. In one embodiment, the genome of the microorganism has been changed by introducing at least one nucleic acid molecule according to the invention which encodes the mutated MCP sequence as a transgene. In another embodiment, an endogenous MCP gene in the genome of the microorganism has been changed, for example functionally disrupted, by homologous recombination with an altered MCP gene. In a preferred embodiment, the microorganism belongs to the genus Cory ebacterium 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, lysine being particularly preferred. 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 the person 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 can often achieve superadditive effects on the host cell with regard to fine chemical productivity.

Another aspect of the invention relates to an isolated MCP protein or a section, for example a biologically active section thereof. In a preferred embodiment, the isolated MCP protein or its section can modulate the production or efficiency of the production of one or more fine chemicals in C. glutamicum or serve as identification markers for C. glutamicum or related organisms. In a further preferred embodiment, the isolated MCP protein or a portion thereof is sufficiently homologous to an amino acid sequence from Appendix B that the protein or its portion retains the ability, for example the production or efficiency of the production of one or more fine chemicals in C. to modulate glutamicum or to serve as an identification marker for C. glutamicum or related organisms.

The invention also relates to an isolated MCP protein preparation. In preferred embodiments, the MCP 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 amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is homologous.

The MCP polypeptide or a biologically active portion thereof can be operably linked to a non-MCP polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has a different activity than the MCP protein alone. In other preferred embodiments, this fusion protein can modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum or serve as an identification marker for C. glutamicum or related organisms. Integrating this Fusion protein into a host cell, in particularly preferred embodiments, modulates the production of a desired compound from the cell.

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 MCP 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 MCP 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 particularly 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 MCP protein activity or MCP 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 with respect to one or more C. glutamicum MCP protein activities, so that the yield, production and / or efficiency of the production of a desired fine chemical is improved by this microorganism. The substance that modulates MCP protein activity can be a substance that stimulates MCP protein activity or MCP nucleic acid expression. Examples of substances that stimulate MCP protein activity or MCP nucleic acid expression include small molecules, active MCP proteins and nucleic acids that encode MCP proteins and have been introduced into the cell. Examples of substances that inhibit MCP activity or expression include small molecules and MCP antisense nucleic acid molecules.

Another aspect of the invention relates to methods for modulating the yields, the production and / or the efficiency of the production of a desired compound from a cell, comprising introducing a wild-type or mutant MCP gene into a cell, which is either on a separate Plasmid remains or is integrated into the genome of the host cell. The integration into the genome can be done randomly or by homologous recombination, so that the native gene is replaced by the integrated copy, which reduces the production of the desired compound modulating cell. 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 MCP nucleic acid and protein molecules that can be used to identify Corynebacterium glutamicum or related organisms, to map the C. glutamicum genome (or the genome of a closely related organism), or to identify microorganisms - Those who are responsible for the production of fine chemicals, e.g. by fermentation processes can be used. The proteins encoded by these nucleic acids can be used for direct or indirect modulation of the production or efficiency of the production of one or more fine chemicals in C. glutamicum, as an identification marker for C. glutamicum or related organisms, for the oxidation of terpenoids or for the degradation of hydrocarbons or as Targets can be used to develop therapeutic pharmaceutical compounds. 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 are used 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 diaminopimelic 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 citations 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. aromatic amines, vanillin and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins" , Pp. 443-613 (1996) VCH: Weinheim and the quotations 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, held on 1-3. Sept. 1994 in Penang, Malaysia, AOCS Press (1995)), Enzymes and all others by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the literature references therein. The metabolism and uses of certain fine chemicals are further discussed below.

A. Amino acid metabolism and uses

The amino acids comprise the basic structural units of all proteins and are therefore essential for the normal cell functions in all organisms. 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 Ulmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids can be in the optical D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthesis 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 called because they usually have to be included in the diet due to the complexity of their biosynthesis, are identified by simple biosynthetic routes in the remaining 11 "non-essential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine) were converted. 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 cosmetics 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 Ulmann'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, for example bacteria, has been well characterized (for an overview of bacterial amino acid biosynthesis and its regulation see Umbarger, HE (1978) Ann. Rev. Bio- former 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, begins with 3-phosphoglycerate (an intermediate product of glycolysis) and, after oxidation, transamination and hydrolysis steps, gives this amino acid. 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 transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentose phosphate pathways, erythrosis-4-phosphate and phosphoenolpyruvate, in a 9-step biosynthetic pathway that only differs 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 also be produced from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis. Aspartate is made from oxaloacetate, an intermediate of the citrate cycle. Asparagine, methionine, threonine and lysine are each produced by converting aspartate. Isoleucine is made 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 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 edition, chapter 21 "Amino Acid Degradation and the Urea Cycle"; S 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, the production of amino acids is expensive in terms of energy, precursor molecules and the enzymes required 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 feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L., Biochemistry , 3rd edition, 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. Metabolism and uses of vitamins, cofactors and nutraceuticals

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 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, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996). The term "vitamin" is known in the art and encompasses nutrients which are required by an organism for normal function, but which cannot be synthesized by this organism itself. The group of vitamins can include cofactors and nutraceutical compounds. The term "cofactor" includes non-proteinaceous compounds that are necessary for normal enzyme activity to occur. 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 beneficial to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been extensively characterized (Ullman'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 B" (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-l-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 '-phosphate, 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. The lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it is a component of the pyruvate dehydrogenase complex and the α-ketoglutarate dehydro- complex of genes. The folates are a group of substances that are all derived from folic acid, which in turn is derived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterine. The biosynthesis of folic acid and its derivatives, starting from 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 ₂ ) and the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system. The biosynthesis of vitamin B ₁₂ 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 synthesis, although some of these chemicals, such as riboflavin, vitamin B ₆ , pantothenate and biotin, have also been produced by large-scale cultivation of microorganisms. Only vitamin Bι is only produced by fermentation due to the complexity of its synthesis. In vitro methods require a considerable amount of materials and time and often high costs.

C. Puri-i, 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 nitrogenous bases which are components of the nucleic acids, coenzymes and nucleotides. The term “nucleotide” includes 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" encompasses molecules which serve as precursors of nucleotides, but which, in contrast to the nucleotides, have no phosphoric acid unit. 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 specifically inhibited in cancer cells, the ability of tumor cells to divide and replicate can be inhibited. There are also nucleotides that do not form nucleic acid molecules, but serve as energy stores (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 involved in purine and pyrimidine metabolism have focused on the development of new drugs that can be used, for example, as immunosuppressants or antiproliferants (Smith, JL (1995) "Enzymes in Nucleotide Synthesis" Curr. Opin Struct. Biol. 5: 752-757; (1995) Biochem. Soc. Transact. 23: 877-902). However, the purine and pyrimidine bases, nucleosides and nucleotides also have other possible uses: as intermediates in the biosynthesis of various fine chemicals (eg thiamine, S-adenosyl methionine, folate or riboflavin), as an energy source for the cell (e.g. ATP or GTP) ) and for chemicals themselves, which are usually used as flavor enhancers (for example IMP or GMP) or for many medical applications (see for example Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Reh et al VCH: Weinheim, pp. 561-612) Enzymes that are involved in the purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly serving as targets against the chemicals for crop protection, including fungicides , Herbicides and insecticides.

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 Biochemistry and Molecular Biology, Wiley, New York). 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, for example 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 breakdown energy for many different which provides 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 to 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 that are linked by an α, α-l, 1 bond. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen food and in beverages. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, e.g., Nishimoto et al., (1998) US Patent No. 5,759,610; Singer, MA and Lindquist, S. (1998) Trends Biotech. 16: 460-467; Paiva, CLA and Panek, AD (1996) Biotech Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 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. Elements and methods of the invention

The present invention is based, at least in part, on the discovery of new molecules which are referred to here as MCP nucleic acid molecules. These MCP nucleic acid molecules are not only suitable for the identification of C. glutamicum or related types of bacteria, but also as markers for mapping the C. glutamicum genome and for the identification of bacteria which are suitable for the production of fine chemicals by, for example, fermentative methods. The present invention is based at least in part on the MCP protein molecules which are encoded by these MCP nucleic acid molecules. These MCP molecules can modulate the yield, production and / or efficiency of the production of one or more fine chemicals in C. glutamicum, serve as identification markers for C. glutamicum or related organisms, degrade hydrocarbons and serve as targets for the development of therapeutic pharmaceutical compounds , In one embodiment, the MCP molecules according to the invention are directly or indirectly involved in the metabolic pathway of one or more fine chemicals in C. glutamicum. In a preferred embodiment, the activity of the MCP molecules according to the invention to participate indirectly or directly in such metabolic pathways has an impact on the production of a desired fine chemical by this microorganism. In a particularly preferred embodiment, the activity of the MCP molecules according to the invention is modulated so that the C. glutamicum metabolic pathways, in which the MCP proteins according to the invention are involved, are modulated in terms of efficiency or output, which directly or indirectly affects the Production or efficiency of production of a desired fine chemical modulated by C. glutamicum.

The term "MCP protein" or "MCP polypeptide" includes proteins that modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, as a target protein for drug screening or design or can serve as identification markers for C. glutamicum or related organisms. Examples of MCP proteins include those encoded by the MCP genes listed in Table 1 and Appendix A. The terms "MCP gene" or "MCP nucleic acid sequence" include nucleic acid sequences which encode an MCP protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of MCP genes are those 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 chemicals) which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1 ). The term "production efficiency" encompasses the time it takes to achieve a certain amount of production (for example, how long it takes the cell to establish a certain rate of ejection of a fine chemical). The term "yield" or "product / carbon yield" is known in the art and encompasses the efficiency of converting the carbon source into the product (ie, the fine chemical). For example, this is usually expressed as kg of product per kg of carbon source. Increasing the yield or production of the compound increases the amount of molecules or suitable molecules of this compound obtained in a given amount of culture over a predetermined period of time. 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 term "Metabolism" is known in the art and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a particular compound (for example the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways in the cell which concern this compound.

In another embodiment, the MCP molecules according to the invention are capable of directly or indirectly modulating the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum. Using gene recombination techniques, one or more MCP proteins of the invention can be manipulated so that their function is modulated. This modulation of the function can lead to modulation of the yield, production and / or efficiency of the production of one or more fine chemicals from C. glutamicum.

For example, by modifying the activity of a protein involved in biosynthesis or degradation of a fine chemical (i.e., mutagenesis of the corresponding

Gens) directly modulate the ability of the cell to synthesize or degrade this compound, thereby modulating the yield and / or efficiency of fine chemical production. Likewise, by modulating the activity of a protein that regulates a fine chemical pathway, one can directly influence whether the production of the desired compound is up or down, both of which modulate the yield or efficiency of the production of the fine chemical from the cell.

Indirect modulation of fine chemical production can also be done by modifying the activity of a protein of the invention (ie, mutagenizing the corresponding gene) so that the overall ability of the cell to grow and divide, or to remain viable and productive, is increased. The production of fine chemicals from C. glutamicum is usually achieved by large-scale fermentation culture of these microorganisms, conditions that are often suboptimal for growth and cell division. By changing a protein according to the invention (for example a stress reaction protein, a cell wall protein or proteins which are involved in the metabolism of compounds which are necessary for the occurrence of cell growth and division, such as nucleotides and amino acids), so that a better one Surviving, growing and multiplying in these conditions is possible, it may be possible to increase the number and productivity of these altered C. glutamicum cells in large-scale cultures, which in turn leads to increased yields and / or increased efficiency in the production of one or several desired fine chemicals should lead. Furthermore, the metabolic pathways of a cell are necessarily interdependent and co-regulated. By changing the activity of any metabolic pathway in C. glutamicum (ie by changing the activity of one of the proteins according to the invention which is involved in such a pathway) it is possible to simultaneously change the activity or regulation of another metabolic pathway in this microorganism which is directly involved may be involved in the synthesis or degradation of a fine chemical.

The isolated nucleic acid sequences according to the invention are located in the genome of a Corynebacterium glutamicum staimxes, which is available from the American Type Culture Collection under the name ATCC 13032. The nucleotide sequence of the isolated C. glutamicum MCP nucleic acid molecules and the predicted amino acid sequences of the C. glutamicum MCP proteins are shown in Appendix A and B, respectively. Computer analyzes were carried out which classified and / or identified many of these nucleotide sequences as sequences with homology to E. coli or Bacillus subtilis genes.

The present invention also relates to proteins whose amino acid sequence is essentially homologous to an amino acid sequence in Appendix B. As used herein, a protein whose amino acid sequence is essentially homologous to a selected amino acid sequence is at least about 50% homologous to the selected amino acid sequence, for example the entire selected amino acid sequence. A protein whose amino acid sequence is essentially homologous to a selected amino acid sequence can also be at least about 50-60%, preferably at least about

60-70%, more preferably at least about 70-80%, 80-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or even more homologous to the selected amino acid sequence.

An MCP protein according to the invention or a biologically active section or fragment thereof can modulate the yield, production and / or efficiency of the production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or as an identification marker serve for C. glutamicum or related organisms.

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 MCP molecules or biologically active sections thereof, and to nucleic acid fragments which are used as hybridization probes or primers for the identification or amplification of MCP-coding nucleic acids (for example MCP-DNA ) are sufficient. These nucleic acid molecules can be used to identify C. glutamicum or related organisms, to map the genome of C. glutamicum or related organisms or to identify microorganisms that are used to produce fine chemicals, e.g. by fermentation processes are suitable. The term "nucleic acid molecule" as used herein is intended to encompass DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g. mRNA) 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 region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably 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 which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example, sequences which are located at the 5 'or 3' end of the nucleic acid ). In various embodiments, for example, the isolated MCP 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 comprise 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 also be substantially free of other cellular material or culture medium if it is produced by recombinant techniques, or of chemical precursors or other chemicals if it is 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 MCP cDNA can be isolated from a C. glutamicum bank by using a complete sequence from Appendix A or a section 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) can be used. Furthermore, a nucleic acid molecule comprising a complete sequence from Annex A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been produced on the basis of this sequence (for example a nucleic acid molecule comprising a complete sequence can be used 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) at Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL) and by means of random polynucleotide primers or oligonucleotide primers based on one of the nucleotide sequences shown in Appendix A. getting produced. 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 amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis. Oligonucleotides which correspond to an MCP nucleotide sequence can also 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. The sequences of Appendix A correspond to the MCP cDNAs according to the invention from Corynebacterium glutamicum. These cDNAs include sequences, the MCP proteins (ie the "coding region" shown in each sequence in Appendix A), and the 5 'and 3' untranslated sequences, which are also listed in Appendix A. The nucleic acid molecule can alternatively comprise only the coding region of one of the sequences in Appendix A. The nucleic acid molecule according to the invention can moreover only comprise a portion of the coding region of one of the sequences in Appendix A, for example a fragment which can be used as a probe or primer or fragment which encodes a biologically active portion of an MCP protein. The nucleotide sequences determined from the cloning of the MCP genes from C. glutamicum enable the generation of probes and primers which are used to identify and / or clone MCP homologs in other cell types and organisms and MCP homologs from other Corynebacteria or related species are designed. The probe or primer usually comprises essentially purified oligonucleotide. The oligonucleotide usually comprises a nucleotide sequence region which, under stringent conditions, comprises at least about 12, preferably about 25, more preferably about 40, 50 or 75 successive nucleotides of a sense strand from one of the sequences given in Appendix A, an antisense Stranges hybridized from one of the sequences listed in Appendix A or naturally occurring mutants thereof. Primers based on a nucleotide sequence from Appendix A can be used in PCR reactions for cloning MCP homologs. Probes based on the MCP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe also comprises a label group attached to it, for example a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. These probes can be used as part of a diagnostic test kit to identify cells that misexpress an MCP protein, e.g., by measuring an amount of an MCP-encoding nucleic acid in a cell sample, e.g., by detecting MCP mRNA levels or by determining whether a genomic MCP gene is mutated or deleted.

In one embodiment, the nucleic acid molecule according to the invention encodes a protein or a section thereof, the one

Amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion thereof retains the ability to modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum, hydrocarbon degradation, Terpe - Oxidize noide, serve as a target for drug development, or serve as an identification marker for C. glutamicum or related organisms. As used herein, the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences are a minimal number of identical or equivalent (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 amino acid residues to an amino acid sequence from Appendix B, so that the protein or a portion thereof modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or as Identification markers for C. glutamicum or related organisms can serve. Examples of these activities are also described here. The "function of an MCP protein" thus contributes to the overall regulation of the metabolic pathway of one or more fine chemicals or to the degradation of a hydrocarbon or to the oxidation of a terpenoid.

Sections of proteins which are encoded by the MCP nucleic acid according to the invention are preferably biologically active sections of one of the MCP proteins. The term "biologically active section of an MCP protein", as used here, is intended to include a section, for example a domain / motif of an MCP protein, which determines the yield, production and / or efficiency of the production of a or several fine chemicals modulated in C. glutamicum, degrades hydrocarbons, oxidizes terpenoids, serves as a target for drug development or as an identification marker for C. glutamicum or related organisms. To determine whether an MCP protein or a biologically active portion thereof can modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons or oxidize terpenoids, 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.

Additional nucleic acid fragments encoding biologically active portions of an MCP protein can be obtained by isolating a portion of one of the sequences in Appendix B, expressing the encoded portion of the MCP protein or peptide (e.g., by recombinant expression in vitro) and determining the activity produce the encoded portion of the MCP protein or peptide.

The invention also encompasses nucleic acid molecules which differ from one of the nucleotide sequences shown in Appendix A (and sections thereof) because of the degenerate genetic code and thus encode the same MCP protein as that encoded by the nucleotide sequences shown in Appendix A. , In another embodiment, an isolated nucleic acid molecule according to the invention has a nucleotide sequence which encodes a protein with an amino acid sequence shown in Appendix B. In a further embodiment, the nucleic acid according to the invention encodes remolecule a full length C. glutamicum protein which is essentially homologous to an amino acid sequence from Appendix B (encoded by an open reading frame shown in Appendix A).

In addition to naturally occurring variants of the MCP 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 MCP protein without affecting the functionality of the MCP protein. For example. nucleotide substitutions which lead to amino acid substitutions at "non-essential" amino acid residues can be prepared in a sequence from Appendix A. A "non-essential" amino acid residue is a residue that can be changed in the wild-type sequence by one of the MCP proteins (Appendix B) without the activity of the MCP protein being changed, whereas an "essential" amino acid residue for the MCP protein activity is required. However, other amino acid residues (for example non-conserved or only semi-preserved amino acid residues in the domain with MCP activity) cannot be essential for the activity and can therefore probably be changed without changing the MCP activity.

Another aspect of the invention consequently relates to nucleic acid molecules which code for MCP proteins which contain modified amino acid residues which are not essential for MCP activity. These MCP proteins differ in amino acid sequence from a sequence in Appendix B, but still retain at least one of the MCP activities described here. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein that comprises an amino acid sequence that has at least about 50% homology to an amino acid sequence from Appendix B and the yield, production and / or efficiency of production of one or more fine chemicals modulate in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or can serve as an identification marker for C. glutamicum or related organisms.

An isolated nucleic acid molecule that encodes an MCP protein that is homologous to a protein sequence from Appendix B can be generated by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence from Appendix A so that one or more A inoic acid substitutions, additions or deletions are introduced into the encoded protein. The mutations can be converted into one of the sequences from Appendix A using standard techniques such as site-directed tagenese and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are introduced on one or more of the predicted non-essential amino acid residues. In 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 (eg lysine, arginine, histidine), acidic side chains (eg 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 MCP 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 MCP-coding sequence, for example by saturation uagenesis, and the resulting mutants can be examined for MCP activity described here in order to identify mutants who maintain MCP 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 part).

B. jRe.ko-7Lbinaz.te expression vectors and host cells

Another aspect of the invention relates to vectors, preferably expression vectors, containing a nucleic acid encoding an MCP 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 is 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 (for example bacterial vectors with a 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 introduced into the host cell and thereby replicated together with the host genome. In addition, agreed vectors control the expression of genes to which they are operably linked. These vectors are referred to here as "expression vectors". The expression vectors that can be used in recombinant DNA techniques are usually in the form of plasmids. In the present description, "plasmid" and "vector" can be used interchangeably because the plasmid is the most commonly used vector form. However, the invention is intended to encompass other expression vector forms, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.

The recombinant expression vectors according to the invention comprise a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, i.e. that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, “functionally linked” means that the nucleotide sequence of interest is linked 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 include promoters, repressor binding sites, activator binding sites, enhancer areas and other expression control elements (for example terminators, other elements of the m-RNA secondary structure or 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 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 desired level of protein expression etc. The expression vectors according to the invention can be introduced into the host cells so that proteins or peptides are thereby produced , including the fusion proteins or peptides encoded by the nucleic acids as described herein (e.g., MCP proteins, mutated forms of MCP proteins, fusion proteins, etc.).

The recombinant expression vectors according to the invention can be designed for the expression of MCP proteins in prokaryotic or eukaryotic cells. For example. can MCP genes in bacterial nial 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), algal cells and cells of multicellular plants (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.: 583-586) or mammalian cells are expressed. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). As an alternative, the recombinant expression vector can be transcribed and translated in vitro, for example using regulatory sequences of the T7 promoter 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 MCP protein is cloned into a pGEX expression vector to produce a vector that is a fusion protein encoded, 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 MCP protein that is not fused to GST can be obtained by cleaving the fusion protein with thrombin.

Examples of suitable inducible non-fusion-- ?. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET lld (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 pETIld 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 can be carried out using standard DNA synthesis techniques.

In a further embodiment, the MCP 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 Molecu- lar Genetics of Fungi, JF Peberdy et al., eds., pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, the MCP proteins according to 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 pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

In a further embodiment, the MCP proteins according to the invention can be expressed in cells of single-cell plants (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., Kemper, 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, Adenovirus 2, Cytomegalievirus and Simian Virus 40. Further suitable expression systems for prokaryotic and eukaryotic cells see in chapters 16 and 17 of 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. Immuno1. 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. the 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; U.S. 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. That 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 MCP mRNA becomes 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 enhancers or regulatory sequences can be 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 can occur in successive generations due to mutation or environmental influences, these offspring are not conditionally 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. For example, an MCP 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", "conjugation" and "transduction" 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 natural competence, chemically mediated transmission, 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.

It is known that for the stable transfection of mammalian cells, depending on the expression vector and the transfection technique used, only a small part of the cells can integrate the foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (eg 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 MCP protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified, for example, by drug selection (for example, 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 MCP gene into which a deletion, addition or substitution has been introduced in order to change the MCP gene, for example to functionally disrupt it. This MCP gene is preferably a Corynebacterium glu amicum MCP gene, however a homologue from a related bacterium or even from a mammalian, yeast or insect source can be used. In a preferred embodiment, the vector is designed in such a way that the endogenous MCP gene is functionally disrupted when homologous recombination occurs (ie no longer encodes a functional protein; also referred to as a "knockout" vector). The vector can alternatively be designed in such a way that the endogenous MCP gene is mutated or otherwise changed during homologous recombination, but still encodes the functional protein (for example the upstream regulatory region can be changed in such a way that the expression of the endogenous MCP protein thereby is changed.). The modified portion of the MCP gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the MCP gene, which is a homologous recombination between the exogenous MCP gene carried by the vector and one endogenous MCP gene in a microorganism. The additional flanking MCP nucleic acid is long enough for successful homologous recombination with the endogenous gene. Usually the vector contains less than a kilobase of flanking DNA (at both 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 (eg, by electroporation), and cells in which the introduced MCP gene is homologously recombined with the endogenous MCP 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. Inclusion of an MCP gene in a vector, thereby placing it under the control of the Lac operon, enables e.g. expression of the MCP 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 MCP protein. The invention also provides methods for producing MCP proteins using the host cells according to the invention. In one embodiment, the method comprises growing the invention host cell (into which a recombinant expression vector encoding an MCP protein has been introduced, or into whose genome a gene encoding a wild-type or modified MCP protein has been introduced) in a suitable medium until the MCP- Protein has been produced. In a further embodiment, the method comprises isolating the MCP proteins from the medium or the host cell.

Uses and methods according to the invention

The nucleic acid molecules, proteins, protein hologa, 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. glutamic - m-sequences of interest, evolution studies, determination of MCP protein areas that are necessary for the function, modulation of the activity of an MCP protein; Modulating the activity of one or more metabolic pathways and modulating the cellular production of a desired compound, such as a fine chemical. The MCP 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 a close relative of it. They can also be used to identify the presence of 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 spanning a region of a C. glu amicum gene that is unique to this organism, one can determine whether this organism is present. 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.

Techniques known in the art can be used to detect the presence of C. glutamicum in a sample. In particular, the cells in the sample can first be grown in a suitable liquid or on a suitable solid culture medium in order to increase the number of cells in the culture. These cells are lysed and all of the DNA contained is extracted and, if necessary, purified to remove cell debris and protein material that could interfere with subsequent analysis. Polymerase chain reaction or a similar, in the specialist The known technique is carried out (see a general overview of methodologies commonly used for nucleic acid sequence amplification in Mullis et al., US Patent No. 4683195, Mullis et al., US Patent No. 4965188 and In - nis, MA, and Gelfand, DH (1989) PCR-Protocols, A guide to Methods and Applications, Academic Press, pp. 3-12, and (1988) Bio-technology 6: 1197, and International Patent Application No. WO89 / 01050), wherein primers which are specific for an MCP nucleic acid molecule according to the invention are incubated with the nucleic acid sample so that this specific MCP nucleic acid sequence, if present in the sample, is amplified. The particular nucleic acid sequence to be amplified is selected based on its exclusive presence in the genome of C. glutamicum and only a few closely related bacteria. The presence of the desired amplification product indicates the presence of C. glutamicum or an organism closely related to C. glutamicum.

The nucleic acid and protein molecules according to the invention can also serve as markers for specific regions of the genome. Using techniques known in the art, it is possible to demonstrate the physical localization of the MCP nucleic acid molecules according to the invention on the C. glutamicum genome, which in turn can be used for easier localization of other nucleic acid molecules and genes on the map. The nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can also enable the construction of a genomic map in such bacteria (e.g. Brevibacterium lactofermentum).

The nucleic acid and protein molecules according to the invention are not only suitable for mapping the genome, but also for functional studies of C. glutamicum proteins. To identify the genome region to which a specific C. gluta icum DNA-binding protein binds, the C. glutamicum genome can be cleaved, for example, and the fragments 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 labels; 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 done several times with different enzymes, it facilitates a rapid determination of the nucleic acid sequence to which the protein binds. The MCP nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies. The metabolic processes in which the molecules according to the invention are involved are used by a large number of prokaryotic and eukaryotic cells; By comparing the sequences of the nucleic acid molecules according to the invention with those which encode 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 give an indication of how much mutagenesis the protein can tolerate without losing its function.

The MCP proteins according to the invention can be used as markers for classifying an unknown bacterium as C. glutamicum or for identifying C. glutamicum or closely related bacteria in a sample. For example, using techniques known in the art, cells in a sample may optionally be amplified (eg, by growing in a suitable medium) to increase the sample size, and may then be lysed to release the proteins contained therein. If necessary, this sample can be cleaned to remove cell debris and nucleic acid molecules that could interfere with subsequent analysis. Antibodies that are specific for a selected MCP protein according to the invention can be incubated with the protein sample in a typical Western test format (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York) where the antibody binds to its target protein when that protein is present in the sample. An MCP protein is selected for this test type if it is unique or almost unique for C. glutamicum or C. glutamicum and very closely related bacteria. The proteins in the sample are then separated by gel electrophoresis and transferred to a suitable matrix, such as nitrocellulose. A suitable second antibody with a detectable label (eg chemilucescent or colorimetric) is incubated with the matrix, followed by stringent washing. The presence or absence of the label indicates the presence or absence of the target protein in the sample. If the protein is present, this indicates the presence of C. glutamicum. A similar procedure allows an unknown bacterium to be classified as C. glutamicum; if a series of C. glutamicum specific proteins is not detected in the protein samples obtained from the unknown bacterium was prepared, this bacterium is probably not C. glutamicum.

The genetic manipulation of the MCP nucleic acid molecules according to the invention can bring about the production of MCP proteins with functional differences from the wild-type MCP 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.

These changes in activity can directly modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum. For example, by modifying the activity of a protein involved in the biosynthesis or degradation of a fine chemical (ie, by mutagenesis of the corresponding gene), the ability of the cell to synthesize or degrade this compound can be directly modulated, thereby reducing the yield and / or modulate the efficiency of the production of fine chemicals. Likewise, by modulating the activity of a protein that regulates a fine chemical pathway, one can directly influence whether the production of the desired compound is up- or down-regulated, both of which modulate the yield or efficiency of the fine chemical production from the cell.

Indirect modulation of fine chemical production can also be done by modifying the activity of a protein of the invention (ie, mutagenizing the corresponding gene) so that the overall ability of the cell to grow and divide, or to remain viable and productive, is increased. The production of fine chemicals from C. glutamicum is usually achieved by large-scale fermentation culture of these microorganisms, conditions that are often suboptimal for growth and cell division. By changing a protein according to the invention (for example a stress reaction protein, a cell wall protein or proteins which are involved in the metabolism of compounds which are necessary for the occurrence of cell growth and division, such as nucleotides and amino acids), so that a better one If survival, growth and proliferation are possible in these conditions, it may be possible to increase the number and productivity of these altered C. gluta nicum cells in culture on a large scale, which in turn leads to increased yields and / or increased production efficiency one or more desired fine chemicals. Furthermore, the metabolic pathways of a cell are necessarily interdependent and co-regulated. By changing the activity of any metabolism either in C. glutamicum (ie by changing the activity of one of the proteins according to the invention, which is involved in such a pathway), it is possible at the same time to change the activity or regulation of another metabolic pathway in this microorganism which is directly involved in the synthesis or degradation a fine chemical can be involved.

The above-mentioned mutagenesis strategies for MCP proteins, which are said to bring about increased yields of a fine chemical from C. glutamicum, are not intended to be restrictive; Variations on these mutagenesis strategies are readily apparent to those skilled in the art. Using these strategies, and including the mechanisms disclosed herein, the nucleic acid and protein molecules of the invention can be used to generate C. glutamicum or related bacterial strains expressing mutant MCP nucleic acid and protein molecules so that the yield, production and / or improving the efficiency of producing a desired connection. The desired compound can be any product made by C. glutamicum, including the end products of biosynthetic pathways and intermediates of naturally occurring metabolic pathways, as well as molecules that do not occur naturally in the metabolism of C. glutamicum, but which are derived from a C. glu amicum 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). 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, with KOH to pH 6, 7 adjusted), 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 • H ₂ 0, 10 mg / 1 ZnS0 ₄ • 7 H ₂ 0, 3 mg / 1 MhCl 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 myo-inositol) .Lysozyme was added to the suspension at a final concentration of 2.5 mg / ml. After about 4 hours of incubation at 37 ° C., the The cell wall was broken down and the protoplasts obtained were harvested by centrifugation The pellet was washed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 inM Tris-HCl, 1M EDTA, pH 8) The pellet was resuspended in 4 ml of TE buffer and 0.5 ml of SDS solution (10%) and 0.5 ml of NaCl solution (5 M) were added at 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, pheno1-chloroform-isoamyl 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 made by this method could be used for all purposes, including Southern blotting or to construct genomic libraries.

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

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

Any plasmid or cosmid could be used. Plasmids pBR322 (Sutcliffe, JG (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741) found particular use; pACYC177 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or Cosmide, 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-genome Random Sequencing and Assembly 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 (e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae), which maintain the integrity of their genetic information cannot maintain. Common mutator strains have mutations in the genes for the DNA repair system (eg, mutHLS, mutD, mutT, etc., for comparison see Rupp, WD (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277 -2294, ASM: Washington). These strains are known to the person skilled in the art. The use of these strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.

Example 5: DNA transfer between Escherichia coli and Corynebacterium glutamicum

Several Corynebacterium and Brevibacterium species contain endogenous plasmids (such as, for example, pHM1519 or pBLl) that replicate autonomously (for an overview, see, for example, Martin, JF et al. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coli and Corynebac eri m 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 that originate from Corynebacterium and Brevibactertium species have been isolated. 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 that replicate in E. coli and C. glutamicum and can be used for various purposes, including gene overexpression (see, for example, 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. gluta-nicum 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, also by conjugation (as described, for example, in Schäfer, 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-deficient E. coli strain is advantageously 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 to the wild-type protein. A suitable method for determining the amount of transcription of the mutant 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), whereby a primer which is designed in such a way that it binds to the gene of interest is provided with a detectable (usually radioactive or chemiluminescent) label so that - if the Total RNA of a culture of the organism extracted, separated on a gel, on a stable matrix is transferred and incubated with this probe - the binding and the quantity of binding of the probe indicate the presence and also the amount of mRNA for this gene. This information is an indication of the extent of the transcription of the mutant gene. Total cell RNA can be isolated from Corynebacterium glutamicum by various methods known in the art, such as in Bormann, ER 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 Corynebakterian are known and readily 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 through complex compounds such as molasses or other by-products of 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 contain. Exemplary nitrogen sources include ammonia gas or ammonium salts such as NH ₄ C1 or (NH ₄ ) ₂ S0, NH ₄ OH, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract 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 strongly depends on the respective experiment and is decided individually for each specific case. Information on media optimization is available from the textbook "Applied Microbiological Physiology, A Practical Approach" (ed. P.M. Rhodes, P.F. 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 value 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 bindings 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 either 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 ODgoo ^of 0.5-1.5 using cells that are placed 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 had been incubated at 30 ° C., were grown. 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 mutant 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 skill of the skilled person. 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, Longman, London; Fersht (1985) Enzyme Structure and Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, San Francisco; Price, NC, Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, PD: Ed. (1983) The Enzymes, 3rd Edition, Academic Press, New York; Bisswanger, H. (1994) Enzyme Kinetics, 2nd Edition VCH, Weinheim (ISBN 3527300325); Bergmeyer, HU, Bergmeyer, J., Graßl, M. Ed. (1983-1986) Methods of Enzymatic Analysis, 3rd edition Vol. I-XII, Verlag Chemie: Weinheim; and Ulimann'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 determined with reporter gene assays (as in Kolmar, H. et al., (1995)

EMBO J. 14: 3895-3904 and the references cited therein) can be measured. 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 techniques as described in Gennis, R.B. (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 culturing the modified microorganisms under suitable conditions (such as those described above) and the medium and / or the cellular components is examined for the increased production of the desired product (ie an amino acid). 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; Rehm 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 Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, FJ (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to measuring the final fermentation product, it is also possible to analyze other metabolic pathway components used to produce the desired compound, such as intermediates and by-products, to determine the overall efficiency of the compound's production. The analytical 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 of biosynthetic pathways and measurements of gases that are generated during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. 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 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 treatment. 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 many contaminants in the sample, or the contaminants remaining on the resin, the sample however not. If necessary, these chromatographic steps can be repeated using the same or different chromatographic resins. The person skilled in the art is skilled in the selection of the suitable chromatography resins and their most effective application 5 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.

10 Many cleaning procedures are known in the art and the previous cleaning procedure is not intended to be limiting. These cleaning techniques are described, for example, in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

15

The identity and purity of the isolated compounds can be determined by prior art techniques. These include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS,

20 enzyme test or microbiological test. 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. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27,

25 VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 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.

30 17th

equivalent

Those skilled in the art will recognize or can - using only routine procedures - 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:

40

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.

45 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" lists 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 new proteins

DNA AS identification: AS AS AS function:

ID: ID: Pos: Wild-type Mutai

1 2 RXA00033 158 L F TRANSPORTER

382 G E TRANSPORTER

3 4 RXA00054 613 A V DNA / RNA HELICASE (DEAD / DEAH BOX FAMILY)

5 6 RXA00056 262 G D Hypothetical Exported Protein

7 8 RXA00065 101 S F hypot etical protein

9 10 RXA00068 35 S N hypothetical protein

11 12 RXA00077 374 A T HUNTINGTIN INTERACTING PROTEIN HYPE

13 14 RXA00080 70 G D INTEGRAL MEMBRANE PROTEIN

253 P S INTEGRAL MEMBRANE PROTEIN

15 16 RXA00110 172 S F Hypothetical Membrane Spanning Protein

17 18 RXA00118 53 V I Hypothetical Cytosolic Protein

19 20 RXA00121 164 G D hypothetical protein

21 22 RXA00150 85 G D Hypothetical Membrane Spanning Protein

23 24 RXA00151 110 V 1 hypothetical protein

25 26 RXA00155 231 PS hypothetical protein

28 RXA00185 508 D N Hypothetical cytosolic protein

30 RXA00197 322 AV MEMBRANE METALLOPROTEASE

32 RXA00199 12 P S hypothetical protein

370 L F hypothetical protein

34 RXA00211 44 V I GLUTAMYL-TRNA (GLN) AMIDOTRANSFERASE SUBUNIT B (EC 6.3.5.-)

36 RXA00220 122 G D ACETYL TRANSFERASE (EC 2.3.1.-)

38 RXA00240 8 R K hypothetical protein

40 RXA00354 67 G E Hydrolase (HAD superfamily)

42 RXA00397 236 A V XAA-PRO AMINOPEPTIDASE (EC 3.4.11.9)

44 RXA00409 217 G D TRANSPORTER

46 RXA00416 223 AV Hypothetical Membrane Spanning Protein

48 RXA00424 4 R K hypothetical protein

50 RXA00451 78 S N CYTOSINE DEAMINASE (EC 3.5.4.1)

52 RXA00476 274 G E hypothetical protein

287 G R hypothetical protein

54 RXA00486 80 R H TRANSCRIPTIONAL REGULATORY PROTEIN, LYSR FAMILY

56 RXA00493 363 A T 60 KD CHAPERONIN GROEL

58 RXA00496 340 P L hypothetical protein

60 RXA00503 186 AT putative sugar kinase

62 RXA00507 224 A V LIPASE (EC 3.1.1.3)

64 RXA00510 17 G s hypothetical protein

66 RXA00550 554 S F PENICILLIN BINDING PROTEIN 1A

68 RXA00552 295 AT THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1)

69 70 RXA00576 157 E K hypothetical protein

71 72 RXA00611 291 IT TRANSPORTER

73 74 RXA00614 86 P S PHOSPHOESTERASE

75 76 RXA00637 148 E K TRANSCRIPTIONAL REGULATOR, MERR FAMILY

77 78 RXA00654 399 S F INNER MEMBRANE PROTEIN

79 80 RXA00678 127 AT hypothetical protein

81 82 RXA00691 214 AT Hypothetical cytosolic protein

83 84 RXA00719 306 D N GTP-BINDING PROTEIN

468 AT GTP BINDING PROTEIN

85 86 RXA00731 550 A T TRANSPORTER

776 L F TRANSPORTER

87 88 RXA00740 93 AT hypothetical protein

89 90 RXA00812 106 A V INHIBITION OF MORPHOLOGICAL DIFFERENTIATION

91 92 RXA00831 166 AT Hypothetical cytosolic protein

93 94 RXA00835 54 A V Hypothetical Membrane Spanning Protein

95 96 RXA0091 635 D N hypothetical protein

97 98 RXA00943 55 P S hypothetical protein

123 L F hypothetical protein

99 100 RXA00963 211 PS 2-HYDROXYHEPTA-2,4-DIENE-1, 7-DIOATE ISOMERASE (EC 5.3.3.-) / 5-CARBOXYMETHYL-2-OXO-HEX-3-ENE-1, 7-DIOATE DECARBOXYLASE (EC 4.1.1.-)

101 102 RXA01011 201 AT Hypothetical cytosolic protein

103 104 RXA01017 37 V I Hypothetical Cytosolic Protein

105 106 RXA01037 28 GD hypothetical protein

107 108 RXA01046 408 A V ATP-DEPENDENT CLP PROTEASE ATP-BINDING SUBUNIT CLPA

109 110 RXA01109 113 AV hypothetical protein

111 112 RXA01122 104 R c hypothetical protein

113 114 RXA01127 60 D N 3-ISOPROPYLMALATE DEHYDROGENASE (EC 1.1.1.85)

115 116 RXA01128 221 T 1 TAURINE-BINDING PERIPLASMIC PROTEIN PRECURSOR

117 118 RXA01129 2 V 1 cyclic nucleotide binding protein / 2 CBS domains

119 120 RXA01173 146 AT Hypothetical Secreted Protein

121 122 RXA01174 174 L F Hypothetical membrane glycoprotein

123 124 RXA01184 363 R K ABC TRANSPORTER INTEGRAL MEMBRANE PROTEIN

430 G D ABC TRANSPORTER INTEGRAL MEMBRANE PROTEIN

125 126 RXA01196 81 G D Hypothetical cytosolic protein

127 128 RXA01263 74 S F O-ANTIGEN ACETYLASE

129 130 RXA01268 150 D N CAPM PROTEIN

131 132 RXA01271 307 S F CAPD PROTEIN

131 132 RXA01271 525 L F CAPD PROTEIN

133 134 RXA01273 106 A V Hypothetical Membrane Associated Protein

135 136 RXA01315 20 A T TRANSCRIPTIONAL REGULATOR, TETR FAMILY

101 G D TRANSCRIPTIONAL REGULATOR, TETR FAMILY

137 138 RXA01318 67 AV putative membrane protein

139 140 RXA01326 103 A V hypothetical protein

141 142 RXA01331 13 T 1 Hypothetical Membrane Spanning Protein

403 P S Hypothetical Membrane Spanning Protein

544 T 1 Hypothetical Membrane Spanning Protein

143 144 RXA01400 549 G R Hypothetical cytosolic protein

145 146 RXA01425 85 EK 60 KDA INNER MEMBRANE PROTEIN YIDC

147 148 RXA01434 957 S F VIRULENCE FACTOR MVIN

1012 P S VIRULENCE FACTOR MVIN

149 150 RXA01439 116 L F ACETYL TRANSFERASE (EC 2.3.1.-)

151 152 RXA01441 104 CR hypothetical protein

153 154 RXA01448 159 G D INTEGRAL MEMBRANE PROTEIN

155 156 RXA01518 148 AT Hypothetical Membrane Spanning Protein

157 158 RXA01536 121 A V hypothetical protein

308 AV hypothetical protein

375 AT hypothetical protein

159 160 RXA01544 78 V 1 hypothetical protein

161 162 RXA01548 153 G D hypothetical protein

163 164 RXA01549 31 S G Hypothetical Membrane Spanning Protein

53 AT Hypothetical Membrane Spanning Protein

159 AT Hypothetical Membrane Spanning Protein

165 166 RXA01554 124 G D GLUCAN ENDO-1, 3-BETA-GLUCOSIDASE A1 PRECURSOR (EC 3.2.1.39)

486 G R GLUCAN ENDO-1, 3-BETA-GLUCOSIDASE A1 PRECURSOR (EC 3.2.1.39)

167 168 RXA01574 123 P S GLYCOSYL TRANSFERASE

169 170 RXA01585 112 V M hypothetical protein Rv2474c

171 172 RXA01590 1432 E K hypothetical protein

173 174 RXA01628 67 D N Hypothetical membrane glycoprotein

224 GD Hypothetical Membrane Glycoprotein

175 176 RXA01642 165 G E EXTRACELLULAR SUBTILISIN-LIKE PROTEASE PRECURSOR (EC 3.4.21.-)

294 AT EXTRACELLULAR SUBTILISIN-LIKE PROTEASE PRECURSOR (EC 3.4.21.-)

177 178 RXA01647 112 D N Hypothetical Membrane Associated Protein

167 GS Hypothetical Membrane Associated Protein

179 180 RXA01672 56 G D Hypothetical Membrane Spanning Protein

68 L F Hypothetical Membrane Spanning Protein

181 182 RXA01677 209 G R THIO DISULFIDE INTERCHANGE PROTEIN DSBA

183 184 RXA01693 102 AT Hypothetical cytosolic protein

185 186 RXA01741 17 D N hypothetical protein

187 188 RXA01 49 189 A V INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

212 PS INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

420 AT INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

189 190 RXA01754 596 D N hypothetical protein

191 192 RXA01761 441 R C hypothetical protein

1376 GS hypothetical protein

193 194 RXA01770 243 D N DNA HELICASE II (EC 3.6.1.-)

195 196 RXA01771 222 P S membrane protease subunits, stomatin / prohibitin homologs

197 198 RXA01776 169 L F Hypothetical Membrane Spanning Protein

199 200 RXA01778 81 S F hypothetical protein

201 202 RXA01 79 229 T A Hypothetical Exported Protein

203 204 RXA01789 104 L F hypothetical protein

205 206 RXA01791 60 A V hypothetical protein

207 208 RXA01809 128 RQ Hypothetical Membrane Associated Protein

209 210 RXA01825 75 G D Hypothetical Membrane Spanning Protein

211 212 RXA01842 246 PS QUINONE OXIDOREDUCTASE (EC 1.6.5.5)

213 214 RXA01899 233 S N Hypothetical cytosolic protein

215 216 RXA01905 103 V I Hypothetical Exported Protein

219 220 RXA01910 220 G S hypothetical protein

221 222 RXA01945 629 E K Hypothetical Exported Protein

1382 G D Hypothetical Exported Protein

223 224 RXA01966 46 A V OLIGORIBONUCLEASE (EC 3.1.-.-)

225 226 RXA01982 341 AT hypothetical protein

227 228 RXA02021 305 S F 2,3,4,5-TETRAHYDROPYRIDINE-2-CARBOXYLATE N-SUCCINYLTRANSFERASE (HOMOLOG)

229 230 RXA02032 208 G S INTEGRAL MEMBRANE PROTEIN

231 232 RXA02053 66 HY DRGA PROTEIN

233 234 RXA02058 188 S F hypothetical protein

235 236 RXA02059 24 G R Hypothetical Membrane Spanning Protein

237 238 RXA02070 150 A T MRP PROTEIN HOMOLOG

239 240 RXA02084 105 A V hypothetical protein

241 242 RXA02091 170 E K hypothetical protein

243 244 RXA02097 28 A V TRANSCRIPTIONAL REGULATOR

427 G R TRANSCRIPTIONAL REGULATOR

245 246 RXA02104 39 T I GLYCERATE KINASE (EC 2.7.1.31)

247 248 RXA02123 678 L F MAGNESIUM CHELATASE SUBUNIT CHLI

249 250 RXA02138 88 A V HESB PROTEIN

251 252 RXA02151 165 AT Hypothetical Membrane Spanning Protein

253 254 RXA02163 23 P S hypothetical protein

255 256 RXA02185 104 GS RPF PROTEIN PRECURSOR

257 258 RXA02186 39 S F Hypothetical cytosolic protein

259 260 RXA02203 36 D N hypothetical protein

261 262 RXA02206 73 G D OXIDOREDUCTASE (EC 1.1.1.-)

114 A T OXIDOREDUCTASE (EC 1.1.1.-)

314 R C OXIDOREDUCTASE (EC 1.1.1.-)

263 264 RXA02217 65 E K quinate dehydrogenase (pyrroloquinoline-quinone) (EC 1.1.99.25) 92 T I quinate dehydrogenase (pyrroloquinoline-quinone) (EC 1.1.99.25)

265 266 RXA02219 292 V M Hypothetical cytosolic protein

267 268 RXA02221 176 L F hypothetical protein

269 270 RXA02267 65 AT DNA (CYTOSINE-5) METHYL TRANSFERASE (EC 2.1.1.37)

271 272 RXA02271 126 G D Hypothetical Exported Protein

273 274 RXA02280 502 A V HEAT SHOCK PROTEIN HTPG

275 276 RXA02294 57 E K hypothetical protein

277 278 RXA02295 130 G D TRANSPORTER

279 280 RXA02296 61 S N Hypothetical Exported Protein

281 282 RXA02298 447 L F Hypothetical cytosolic protein

283 284 RXA02302 279 G E CHLORAMPHENICOL SENSITIVE PROTEIN RARD

285 286 RXA02303 268 G D hypothetical protein

289 290 RXA02339 38 P S Hypothetical cytosolic protein

291 292 RXA02360 243 V I TYPE I RESTRICTION-MODIFICATION SYSTEM DNA METHYLASE

293 294 RXA02362 525 MI TRANSCRIPTIONAL REGULATOR

295 296 RXA02367 3 I V PIRIN

297 298 RXA02387 10 AT Hypothetical cytosolic protein

299 300 RXA02390 195 G D THREONINE EFFLUX PROTEIN

301 302 RXA02395 533 A V TRANSPORTER

303 304 RXA02425 34 G E Heavy-Metal Cation Transporter

305 306 RXA02433 9 S N hypothetical protein

307 308 RXA02486 141 T I Hypothetical Cytosolic Protein

309 310 RXA02488 27 A T HEME TRANSPORT ASSOCIATED PROTEIN

311 312 RXA02495 81 G s ABC TRANSPORTER INTEGRAL MEMBRANE PROTEIN

313 314 RXA02570 184 R c ABC TRANSPORTER INTEGRAL MEMBRANE PROTEIN

315 316 RXA02591 377 P s PHOSPHOENOLPYRUVATE CARBOXYKINASE [GTP] (EC 4.1.1.32)

317 318 RXA02604 570 G D Hypothetical Membrane Spanning Protein

319 320 RXA02678 3 M 1 Hypothetical cytosolic protein

321 322 RXA02683 81 GS hypothetical protein

323 324 RXA02721 181 GS Hypothetical cytosolic protein

325 326 RXA02725 208 AV hypothetical protein

327 328 RXA02735 27 P S 6-PHOSPHOGLUCONOLACTONASE (EC 3.1.1.31)

329 330 RXA02736 312 SF PUTATIVE OXPPCYCLE PROTEIN OPCA

331 332 RXA02744 231 M I PUTATIVE INTEGRAL MEMBRANE TRANSPORT PROTEIN

333 334 RXA02757 73 P S hypothetical protein

335 336 RXA02777 306 G D Hypothetical cytosolic protein

337 338 RXA02784 4 A V hypothetical protein

339 340 RXA02786 292 E K hypothetical protein

341 342 RXA02798 457 L S SULFITE REDUCTASE (NADPH) FLAVOPROTEIN ALPHA-COMPONENT (EC 1.8.1.2)

343 344 RXA02817 227 G D AEFA PROTEIN

345 346 RXA02825 362 S F HEME TRANSPORT ASSOCIATED PROTEIN

347 348 RXA02951 26 L F Hypothetical cytosolic protein

349 350 RXA03010 106 GS hypothetical protein

351 352 RXA03029 6 K E TRANSCRIPTIONAL REGULATOR

7 P A TRANSCRIPTIONAL REGULATOR

8 C 1 TRANSCRIPTIONAL REGULATOR

353 354 RXA03053 99 M 1 hypothetical protein

355 356 RXA03098 164 S N DNA alkylation repair enzyme

357 358 RXA03113 374 P L INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

359 360 RXA03184 109 s F TRANSPORTER

361 362 RXA03316 6 D N

363 364 RXA03343 171 S F

365 366 RXA03377 55 A V

367 368 RXA03463 53 V 1

369 370 RXA03506 80 P L Hypothetical cytosolic protein

371 372 RXA03523 167 AT

375 376 RXA03577 72 A T

377 378 RXA03584 12 S F

379 380 RXA03591 902 S F

381 382 RXA03613 106 S Y

383 384 RXA03682 18 P s

385 386 RXA03700 139 E K

269 A T

387 388 RXA03803 44 P s

391 392 RXA03886 23 A V

393 394 RXA03940 86 T 1

395 396 RXA03947 81 A T

84 S N

397 398 RXA03955 126 T 1

399 400 RXA03975 55 V 1

401 402 RXA04007 145 s N

403 404 RXA04075 66 G s

173 A V

405 406 RXA04085 55 P S

407 408 RXA04092 18 AV

409 410 RXA04095 30 RK

413 414 RXA04169 21 T 1

415 416 RXA04172 46 P S

417 418 RXA04181 40 G D

419 420 RXA04319 40 R C

421 422 RXA04337 19 P S

423 424 RXA04352 2 G s

425 426 RXA04354 50 T 1

427 428 RXA04370 102 P L

429 430 RXA04546 85 A V Hypothetical cytosolic protein

362 PL Hypothetical cytosolic protein

431 432 RXA04549 214 G R

435 436 RXA04642 112 P L

437 438 RXA06004 148 D N

439 440 RXA06040 58 S P 59 1 M

93 V E

Claims

claims

1. An isolated nucleic acid molecule coding for a polypeptide with that referred to in Table / Column 2

Amino acid sequence in which the nucleic acid molecule in the amino acid position specified in Table / column 4 codes a different proteinogenic amino acid than the respective amino acid specified in Table / column 5 in the same line.

The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule in the amino acid position indicated in Table / Column4 encodes the amino acid indicated in Table / Column6 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.