EP1444343A2

EP1444343A2 - Genes coding for metabolic pathway proteins

Info

Publication number: EP1444343A2
Application number: EP02783046A
Authority: EP
Inventors: Oskar Zelder; Markus Pompejus; Hartwig Schröder; Burkhard Kröger; Corinna Klopprogge; Gregor Haberhauer
Original assignee: BASF SE
Current assignee: Paik Kwang Industrial Co Ltd
Priority date: 2001-11-05
Filing date: 2002-10-31
Publication date: 2004-08-11
Also published as: BR0213774A; ZA200404426B; US7141663B2; US7355032B2; KR100868693B1; CN1578834A; WO2003040681A3; AU2002346807A1; US20070072274A1; DE10154292A1; KR20050042245A; WO2003040681A2; US20050019877A1

Abstract

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

Description

Genes that code for metabolic pathway 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 , They are best produced by growing large-scale bacteria that have been developed to produce and secrete large quantities of one or more desired molecules. A particularly suitable organism for this purpose is Corynebacterium glutamicυm, a gram-positive, non-pathogenic bacterium. Through strain selection, a number of mutant strains have been developed that produce a range of desirable compounds. However, selecting strains that are improved in the production of a particular molecule is a time consuming and difficult process.

Summary of the invention

This invention provides novel nucleic acid molecules that can be used to identify or classify Corynebacterium glutamicυm or related types of bacteria. C. glutamicυm 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 (for example, when overflowing crude oil) 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 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 which serve as reference points for mapping the C. glutamicum genome or genomes of related organisms.

These new nucleic acid molecules encode proteins that are referred to here as metabolic pathway (MP) proteins. These MP proteins can, for example, perform a function which is involved in the transcription, translation or post-translational regulation of proteins which are important for the normal metabolic functioning of cells. Due to the availability of cloning vectors for use in Corynebacterium glutamicum, such as disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetically manipulating C. glutamicum and the related Brevibacterium species (e.g. lactofermentum) Yoshi - hama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gen. Microbiol. 130 (1984) 2237-2246), the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to make it better and more efficient as a producer of one or more fine chemicals.

The improved yield, production and / or efficiency of the production of a fine chemical can be based on a direct or indirect effect of the manipulation of a gene according to the invention. Specifically, changes in C. glutamicum MP proteins that usually regulate the yield, production, and / or efficiency of production of a fine chemical of a metabolic fine chemical pathway can have a direct impact on the overall production or production rate of one or more of these desired compounds have this organism. Changes in the proteins involved in these metabolic pathways can also have an indirect effect on the yield, production and / or efficiency of production of a desired fine chemical. The regulation of metabolism is necessarily complex, and the regulatory mechanisms which accomplish the different pathways can overlap in many places, so that more than one metabolic pathway can be set quickly according to a particular cell event. This enables the modification of a regulatory protein for one pathway to have an effect on many other pathways, some of which may be involved in the biosynthesis or degradation of a desired fine chemical. In this indirect manner, modulating the effect of an MP protein can have an impact on the production of a fine chemical that is produced via a metabolic pathway that is different from that which is directly regulated by this MP protein. The nucleic acid and protein molecules according to the invention can be used to directly improve the yield, production and / or efficiency of the production of one or more desired fine chemicals from Corynebacterium glutamicum. By means of genre combination techniques known in the art, one or more regulatory proteins according to the invention can be manipulated in such a way that their function is modulated. For example, the mutation of an MP protein involved in the repression of the transcription of a gene encoding an enzyme necessary for the biosynthesis of an amino acid so that it is no longer capable of repression of the transcription Cause an increase in the production of this amino acid. Accordingly, changing the activity of an MP protein that causes increased translation or activating the post-translational modification of a C. glutamicum protein that is involved in the biosynthesis of a desired fine chemical can in turn increase the production of this chemical. The opposite situation can also be useful: by increasing the repression of transcription or translation or by post-translational negative modification of a C. glutamicum protein, which is involved in the regulation of the degradation pathway for a compound, the production of this chemical can be increased. In any case, the overall yield or the speed of production of the desired fine chemical can be increased.

It is also possible that these changes in the protein and nucleotide molecules according to the invention can improve the yield, production and / or efficiency of the production of fine chemicals by indirect mechanisms. The metabolism of a particular compound is necessarily entangled with other pathways of biosynthesis and degradation within the cell, and necessary cofactors, intermediates, or substrates in one pathway are likely to be provided or restricted by another pathway. The efficiency of the activity of other fine chemical biosynthesis or degradation pathways can therefore be influenced by modulating one or more regulatory proteins according to the invention. The manipulation of one or more regulatory proteins can also increase the overall ability of the cell to grow and to grow in culture, especially in large fermentative cultures, where the growth conditions can be less than optimal. For example. By mutating an MP protein according to the invention, which usually suppresses the biosynthesis of nucleosides in response to a suboptimal extracellular nutrient supply (which prevents cell division), so that it has a lower repressive activity, one can do the biosynthesis of nucleosides and perhaps that Increase cell division. Changes in the MP-Pro Tones that cause increased cell growth and division in culture, at least due to the increased number of cells that produce the chemical in culture, can increase the yield, production and / or efficiency of production of one or more desired fine chemicals evoke from culture.

The invention provides new nucleic acid molecules that encode proteins, which are referred to here as metabolic regulatory (MP) proteins and can, for example, carry out an enzymatic step that is involved in the transcriptional, translational or post-translational regulation of metabolic pathways in C. glutamicum. Nucleic acid molecules that encode an MP protein are referred to here as MP nucleic acid molecules. In a preferred embodiment, the MP protein is involved in the transcription, translation or post-translational regulation of one or more metabolic pathways. 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 codes for an MP protein or biologically active sections thereof, and also nucleic acid fragments which act as primers or hybridization probes for detecting or amplifying MP code - Render nucleic acid (e.g. DNA or mRNA) are suitable. In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof of one of these nucleotide sequences. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B. The preferred MP proteins according to the invention likewise preferably have at least one of the MP 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 olecule 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 mo lekül preferably corresponds to a naturally occurring nucleic acid molecule. The isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum MP protein or a biologically active portion thereof.

Another aspect of the invention relates to vectors, for example recombinant expression vectors, which contain the nucleic acid molecules according to the invention and host cells into which these vectors have been introduced. In one embodiment, a host cell that is grown in a suitable medium is used to produce an MP protein. The MP 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 MP 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 codes the mutated MP sequence as a transgene. In another embodiment, an endogenous MP gene in the microorganism genome is modified by homologous recombination with an altered MP gene, e.g. functionally disrupted. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, Corynebacterium glutamicυm being particularly preferred. In a preferred embodiment, the microorganism is also used to produce a desired compound, such as an amino acid, particularly preferably lysine.

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

Another aspect of the invention relates to an isolated MP protein or a section, for example a biologically active section thereof. In a preferred embodiment, the isolated MP protein or its portion regulates one or more Metabolic pathways in C. glutamicυm transcriptional, translational or post-translational. In a further preferred embodiment, the isolated MP protein or a portion thereof is sufficiently homologous to an amino acid sequence of Appendix B that the protein or its portion further has the ability to transcriptionally, translationally or post-translationally one or more metabolic pathways in C. glutamicum regulate.

The invention also relates to an isolated MP protein preparation. In preferred embodiments, the MP 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 MP polypeptide or a biologically active portion thereof can be operably linked to a non-MP polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has a different activity than the MP protein alone and, in other preferred embodiments, regulates one or more metabolic pathways in C. glutamicum transcriptionally, translationally or post-translationally. The integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.

Another aspect of the invention relates to a method for producing a fine chemical. The method provides for the cultivation of a cell which contains a vector which brings about the expression of an MP 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 MP nucleic acid. In a further preferred embodiment, this method also comprises the step in which the fine chemical is obtained from the culture. In a preferred embodiment, the cell belongs to the genus Cory-πeJbacteriu 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 MP protein activity or MP nucleic acid expression so that cell-associated activity changes compared to the same activity in the absence of the substance. is changed. In a preferred embodiment, the cell is modulated in C. glutamicum with regard to one or more regulatory systems for metabolic pathways, so that the yields or the speed at which a desired fine chemical is produced by this microorganism is improved. The substance that modulates the MP protein activity stimulates, for example, the MP protein activity or the MP nucleic acid expression. Examples of substances that stimulate MP protein activity or MP nucleic acid expression include small molecules, active MP proteins and nucleic acids that encode MP proteins and have been introduced into the cell. Examples of substances that inhibit MP activity or expression include small molecules and antisense MP nucleic acid molecules.

Another aspect of the invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing into a cell an MP wild-type or mutant gene which either remains on a separate plasmid or is integrated into the genome of the host cell. The integration into the genome can be random or by homologous recombination, so that the native gene is replaced by the integrated copy, which causes the production of the desired compound from the cell to be modulated. In a preferred embodiment, these yields are increased. In a further preferred embodiment, the chemical is a fine chemical, which in an especially preferred embodiment is an amino acid. In a particularly preferred embodiment, this amino acid is L-lysine.

Detailed description of the invention

The present invention provides MP nucleic acid and protein molecules which are involved in the regulation of the Coryne.foacte.rium glutamicum metabolism, including the regulation of the fine chemical metabolism. When modulating the production of fine chemicals from microorganisms, such as C. glutamicυm, the molecules according to the invention can either be used directly (for example where the modulation of the activity of a regulatory protein of the lysine pathway has a direct effect on the yield, production and / or efficiency of production of lysine from this organism) or indirectly, which still has an increase in the yield, production and / or efficiency of production of the desired compound (e.g. where the modulation of the regulation of a nucleotide biosynthetic protein has an effect on the production of an organic acid or an Fatty acid from the bacterium, possibly due to the associated regulatory changes in biosynthesis or construction for these chemical laymen in response to the changed regulation of nucleotide biosynthesis). The aspects of the invention are further explained below.

I. Fine chemicals

The term "fine chemical" is known in the art and includes molecules that are produced by an organism and have applications in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. These compounds include organic acids, such as tartaric acid, itaconic acid and diamino-pimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim and the 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 Ulimann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", p 443-613 (1996) VCH: Weinheim and the citations 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 Associ ations in Malaysia and the Society for Free Radical Research - Asia, held on 1-3. Sept. 1994 in Penang, Malysia, AOCS Press (1995)), Enzymes and all other chemicals described by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the references cited 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 normal cell functions. The term "amino acid" is known in the art. The proteinogenic amino acids, of which there are 20 types, serve as structural units for proteins in which they are linked to one another via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) are usually not found in proteins (see Ullmann's Encyclope - dia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids can be in the D or L configuration, although L-amino acids are usually the only type which is found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)). The "essential" amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), so called because they have to be included in the diet due to the complexity of their biosynthesis, are identified by simple biosynthetic pathways in the rest

10 11 "non-essential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine) converted. Higher animals have the ability to synthesize some of these amino acids, however, the essential amino acids must be ingested with food

15 normal protein synthesis takes place.

Apart from their function in protein biosynthesis, these amino acids are interesting chemicals in themselves, and it has been discovered that many have been used in various applications in the near

20 food, feed, chemical, cosmetic, agricultural and pharmaceutical industries are used. Lysine is not only an important amino acid for human nutrition, but also for monogastric animals such as poultry and pigs. Glutamate is most often used as a taste additive (monosodium

25 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 pharmaceutical

30 eutical and cosmetic industries used. 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

35 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 Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, S. 57-97, VCH, Weinheim, 1985 suitable substances.

40

The biosynthesis of these natural amino acids in organisms that can produce them, e.g. bacteria, has been well characterized (for an overview of bacterial amino acid biosynthesis and its regulation, see Umbarger, H.E. (1978) Ann. Rev.

45 Bioehem. 47: 533-606). Glutamate is synthesized by reductive amination of ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline and arginine are used successively generated from glutamate. The biosynthesis of serine takes place in a three-step process and begins with 3-phosphoglycerate (an intermediate in glycolysis), and gives this amino acid after oxidation, transamination and hydrolysis steps. Cysteine and glycine are each produced from serine, the former by condensation of homocysteine with serine, and the latter by transferring the side chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentosephosphate pathway, erythrose-4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differs only in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in one

11-step pathway. 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 formed from oxa acetate, 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 cell's protein biosynthesis requirements, cannot be stored and are instead broken down, so that intermediates are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd ed. 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 certain amino acid slowing down or completely stopping its own production (for an overview of the feedback mechanism in amino acid biosynthesis pathways, see Stryer, L ., Biochemistry, 3rd ed., Chapter 24, "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988)). The output of a certain amino acid is therefore restricted by the amount of this amino acid in the cell. B. vitamins, cofactors and nutraceutical metabolism and uses

Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and must therefore absorb them, although they are easily synthesized by other organisms such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a number of metabolic pathways. In addition to their nutritional value, these compounds also have a significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure, activity and the industrial applications of these compounds, see, for example, Ullmann'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 (Ulimann'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 pyridine 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 mononu Kleotide (FMN) and flavin adenine dinucleotide (FAD) used. The family of compounds which are collectively referred to as "vitamin B6" (for example pyridoxine, pyridoxamine, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride) are all derivatives of the common structural unit 5-hydroxy-6-methylpyridine. Panthothenate (pantothenic acid, R - (+) - N- (2, 4-dihydroxy-3, 3-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. Lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the CC-ketoglutarate dehydrogenase complex. 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) and the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system. The biosynthesis of vitamin B ₂ is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin". Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms. The production of these compounds on a large scale is largely based on cell-free chemical syntheses, although some of these chemicals have also been produced by large-scale cultivation of microorganisms, such as riboflavin, vitamin Bξ, pantothenate and biotin. Only vitamin Bι ₂ is only produced by fermentation due to the complexity of its synthesis. In-vitro processes require a considerable amount of materials and time and often high costs.

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

Genes for the purine and pyrimidine metabolism and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The term "purine" or "pyrimidine" encompasses nitrogen-containing bases which are part of the nucleic acids, coenzymes and nucleotides. The term “nucleotide” includes the basic structural units of the nucleic acid molecules, which comprise a nitrogenous base, a pentose sugar (in the case of RNA the sugar is ribose, in the case of 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 carcinogenic 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 that serve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).

Several publications have described the use of these chemicals for these medical indications, the purine and / or pyrimidine metabolism being influenced (for example 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 "Enzymes in Nucleotide Synthesis" Curr. Opin. Struct. Biol. 5 (1995) 752-757; Biochem. Soc. Transact. 23 (1995) 877-902). However, the purine and pyridine bases, nucleosides and nucleotides also have other possible uses: as intermediates in the biosynthesis of various Fine chemicals (e.g. thiamine, S-adenosyl methionine, folate or riboflavin), as energy carriers for the cell (e.g. ATP or GTP) and for chemicals themselves, are usually used as flavor enhancers (e.g. IMP or GMP) or for many medical see applications (see, for example, Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim, pp. 561-612). Enzymes which are based on purine, Pyrimidine, nucleoside or nucleotide metabolism are also increasingly becoming targets against which crop protection chemicals, including fungicides, herbicides and insecticides, are being developed.

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

D. Trehalose metabolism and uses

Trehalose consists of two glucose molecules that are linked together via cc, α-l, 1 binding. 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) U.S. Patent No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech. 16 (1998) 460-467; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium from which it can be obtained by methods known in the art.

II. Mechanisms of metabolic regulation

All living cells have complex catabolic and anabolic abilities with many interconnected metabolic pathways. In order to maintain a balance between the different parts of this extremely complex metabolic network, the cell uses a fine-tuned regulatory network. By regulating enzyme synthesis and enzyme activity, either independently or simultaneously, the cell can regulate the activity of completely different metabolic pathways, so that the changing needs of the cell are satisfied.

The induction or repression of the enzyme synthesis can take place either at the transcription or translation level or at both (for an overview, see Lewin, B. (1990) Genes IV, Part 3: "Controlling prokaryotic genes by transcription", Oxford University Press, Oxford, pp. 213-301, and the references cited therein, and Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons). All of these known regulatory processes are mediated by additional genes, which themselves react to various external influences (e.g. temperature, nutrient supply, or light). Exemplary protein factors that are involved in this type of regulation include the transcription factors. These are proteins that bind to the DNA, which either increases the expression of a gene (positive regulation, as in the case of the ara operon from E. coli) or decreases (negative regulation, as in the case of the lac operon from E) coli). These expression-modulating transcription factors can themselves be subject to regulation. Their activity can be regulated, for example, by binding small-molecule compounds to the DNA-binding protein, which stimulates the binding of these proteins to the appropriate binding site on the DNA (as in the case of arabinose for the ara operon) or inhibits it ( as in the case of lactose for the lac operon) (see, for example, Helmann, JD and Chamberlin, MJ (1988) "Structure and function of bacterial sigma factors" Ann. Rev. Biochem. 57: 839-872; Adhya, S (1995) "The lac and gal operons today" and Boos, W. et al., "The maitose system", both in Regulation of Gene Expression in Escherichia coli (Lin, ECC and Lynch, AS ed.) Chapman & Hall : New York, pp. 181-200 and 201-229; and Moran, CP (1993) "RNA polymerase and transcription factors" in: Bacillus subtilis and other gram-positive bacteria, Sonenshein, AL Hrs. ASM: Washington, DCS 653-667).

Protein synthesis is regulated not only at the level of transcription, but often also at the level of translation. This regulation can occur through many mechanisms, including altering the ability of the ribosome to bind to one or more mRNAs, binding the ribosome to mRNA, maintaining or removing the secondary mRNA structure, using common or less common codons for a particular gene, the degree of abundance of one or more tRNAs and special regulatory mechanisms, such as attenuation (see Vellanoweth, RI (1993) Translation and its regulation in Bacillus subtilis and other gram-positive bacteria, Sonenshein, AL et al. Ed. ASM: Washington, DC, Pp. 699-711 and references cited therein.

The transcription and translation regulation can be directed to a single protein (sequential regulation) or simultaneously to several proteins in different metabolic pathways (coordinated regulation). Genes whose expression is regulated in a coordinated manner are often close together in an operon or regulon in the genome. This up- or down-regulation of gene transcription and translation is controlled by the cellular or extracellular amounts of various factors, such as substrates (precursors and intermediate molecules that are used in one or more metabolic pathways), catabolites (molecules that are caused by biochemical metabolic pathways) which are related to the production of energy from the breakdown of complex organic molecules such as sugar) and end products (the molecules obtained at the end of a metabolic pathway). The expression of genes that encode enzymes that are necessary for the activity of a particular metabolic pathway is induced by high amounts of substrate molecules for this metabolic pathway. Accordingly, this gene expression is repressed when there are high intracellular amounts of the end product of the route (Snyder, L. and Champness, W. (1977) The Molecular Biology of Bacteria ASM: Washington). Gene expression can also be regulated by other external and internal factors, such as environmental conditions (e.g. heat, oxidative stress, or hunger). These global environmental changes cause changes in the expression of specialized modulating genes that trigger gene expression directly or indirectly (via additional genes or proteins) by binding to DNA and thereby induce or repress transcription (see, for example, Lin, ECC and Lynch, AS Ed. (1995) Regulation of Gene Expression in Escherichia coli, Chapman & Hall: New York).

Another mechanism by which cellular metabolism can be regulated is at the level of the protein. This regulation takes place either via the activities of other enzymes or by binding low-molecular components that prevent or enable the normal function of the protein. Examples of protein regulation by binding small molecules include binding GTP or NAD. The binding of low molecular weight chemicals is usually reversible as with the GTP-binding proteins. These proteins occur in two states (with bound GTP or GDP), one state being the active form of the protein and the other the inactive form.

Regulation of protein activity by the action of other enzymes is usually done by covalent modification of the protein (i.e., phosphorylation of amino acid residues such as histidine or aspartate, or methylation). This covalent modification is usually reversible, which is accomplished by an enzyme with the opposite activity. An example of this is the opposite activity of kinases and phosphorylases in protein phosphorylation: protein kinases phosphorylate specific residues on a target protein (e.g. serine or threonine), whereas protein phosphorylases remove the phosphate groups from these proteins. Enzymes that modulate the activity of other proteins are usually modulated by external stimuli themselves. These stimuli are mediated by proteins that act as sensors. A well-known mechanism by which these sensor proteins mediate these external signals is by dimerization, but others are also known (see, for example, Msadek, T. et al. (1993) "Two-component Regulatory Systems" in: Bacillus subtilis and Other Gram-Positive Bacteria, Sonenshein, AL et al., ed., ASM: Washington, pp. 729-745 and references cited therein).

A thorough understanding of the regulatory networks that control cell metabolism in microorganisms is crucial for the production of chemicals in high yield through fermentation. Control systems for downward regulation of the metabolic pathways can be removed or reduced to improve the synthesis of desired chemicals, and accordingly those for the upward regulation of the metabolic pathway for a desired product can be constitutively activated or optimized with regard to activity (as shown in Hirose, Y. and Okada, H. (1979) "Microbial Production of Amino Acids", in: Peppler, HJ and Perlman, D. (ed.) Microbial Technology 2nd ed., vol. 1, chap. 7, Academic Press, New York).

III. Elements and methods according to the invention

The present invention is based at least in part on the discovery of new molecules, which are referred to here as MP nucleic acid and protein molecules and which regulate one or more metabolic pathways in C. glutamicum by means of transcriptional, translational or post-translational measures. In one embodiment, the MP molecules regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally. In a preferred embodiment, the activity of the MP molecules according to the invention for regulating one or more metabolic pathways in C. glutamicum has an effect on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the MP molecules according to the invention have modulated activity, so that the metabolic pathways of C. glutamicum, which regulate the MP proteins according to the invention, are modulated in terms of their efficiency or their throughput, which is either directly or indirectly Yield, production and / or efficiency of production of a desired fine chemical modulated by C. glutamicum.

The term "MP protein" or "MP polypeptide" encompasses proteins that regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally. Examples of MP proteins include those encoded by the MP genes listed in Table 1 and Appendix A. The terms "MP gene" or "MP nucleic acid sequence" encompass nucleic acid sequences that encode an MP protein that consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of MP genes are listed in Table 1. The terms “production” or “productivity” are known in the art and include the concentration of the fermentation product (for example the desired fine chemical which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1) The term "production efficiency" encompasses the time it takes to achieve a certain amount of production (for example how long it takes the cell to set up a certain throughput rate of a fine chemical). The term "yield" or "product / carbon Yield "is known in the art and includes the efficiency of converting the carbon source to the product (ie, the fine chemical). For example, this is usually expressed as kg product per kg carbon source. Increasing the yield or production of the compound increases the amount of compound obtained. NEN molecules or the appropriate obtained molecules of this compound in a certain amount of culture over a specified period of time. The terms “biosynthesis” or “biosynthetic pathway” are known in the art and include 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 certain compound (eg the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways of this compound in the cell. The term "regulation" is known in the art and encompasses the activity of one protein to control the activity of another protein. The term "transcription regulation" is known in the art and includes the activity of a protein to inhibit or activate the conversion of a DNA encoding a target protein into mRNA. The term "translation regulation" is known in the art and includes the activity of a protein to inhibit or activate the conversion of an mRNA encoding a target protein into a protein molecule. The term "post-translational regulation" is known in the art "and includes the activity of a protein to inhibit or improve the activity of a target protein by covalently modifying the target protein (for example by methylation, glycosylation or phosphorylation).

In another embodiment, the MP molecules according to the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum. Genre combination techniques can be used to manipulate one or more regulatory proteins according to the invention for metabolic pathways in such a way that their function is modulated. For example. For example, a biosynthesis enzyme can be improved in efficiency or its allosteric control region can be destroyed so that the inhibition of the production of the compound is prevented. Correspondingly, a degradation enzyme can be deleted or modified by substitution, deletion or addition in such a way that its degradation activity for the desired compound is reduced without the viability of the cell being impaired. In any case, the total yield or the production rate of one of these desired fine chemicals can be increased.

It is also possible that these changes in the protein and nucleotide molecules according to the invention can improve the production of fine chemicals in an indirect manner. The regulatory mechanisms of the metabolic pathways in the cell are necessarily linked, and the activation of one metabolic pathway can cause the repression or activation of another in an accompanying manner. By modulating the activity of one or more proteins of the invention, the production or efficiency of the activity of other fine chemical biosynthesis or degradation pathways can be affected. By lowering the ability of an MP protein to repress the transcription of a gene encoding a particular amino acid biosynthetic protein, other amino acid biosynthetic pathways can be simultaneously depressed because these pathways are linked. By modifying the MP proteins according to the invention, the growth and division of cells from their extracellular surroundings can be decoupled to a certain degree; by influencing an MP protein that usually represents the biosynthesis of a nucleotide when the extracellular conditions for growth and cell division are suboptimal so that it now lacks this function, growth can be allowed to occur even if the extracellular Conditions are bad. This is particularly important in large-scale fermentation farming, where the conditions in the culture are often suboptimal in terms of temperature, nutrient supply or aeration, but which still promote growth and cell division when the cellular regulatory systems for these factors are eliminated.

The genome of a Corynebacterium glutamicum strain, which is available from the American Type Culture Collection under the name ATCC 13032, is suitable as a starting point for producing the nucleic acid sequences according to the invention.

From these nucleic acid sequences, the nucleic acid sequences according to the invention can be produced by conventional methods using the changes described in Table 1.

The MP protein according to the invention or a biologically active section or fragments thereof can regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally, or can have one or more of the activities listed in Table 1. Various aspects of the invention are described in more detail in the subsections below:

A. Isolated nucleic acid molecules

One aspect of the invention relates to isolated nucleic acid molecules which encode MP polypeptides or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for the identification or amplification of MP-coding nucleic acids (e.g. MP-DNA). The term “nucleic acid molecule” as used here is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg 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 gene region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. An "isolated" nucleic acid preferably has no sequences 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 MP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotide sequences that naturally contain the nucleic acid molecule in the genomic Flank the DNA of the cell from which the nucleic acid originates (for example a C. glutamicum cell). An "isolated" nucleic acid molecule, such as a cDNA molecule, can also be essentially free of another cellular material or culture medium if it is produced by recombinant techniques, or free of chemical precursors or other chemicals if it is chemically synthesized becomes.

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 produced using standard molecular biological techniques and the sequence information provided here. For example. a C. glutamiσum MP cDNA can be isolated from a C. glutamicum library by using a complete sequence from Appendix A or a portion thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Furthermore, a nucleic acid molecule comprising a complete sequence from Annex A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been prepared 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 OH gonucleotide 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 , available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for the 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 MP nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule according to the invention comprises one of the nucleotide sequences listed in Appendix A.

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

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

Amino acid sequence that is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion of which still has the ability to regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally. As used herein, the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (e.g. an amino acid residue with a side chain similar to an amino acid residue in one of the sequences of Appendix B) to an amino acid sequence from Appendix B so that the protein or a portion thereof can regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally. Protein components of these pathways, as described here, can regulate the biosynthesis or degradation of one or more fine chemicals. Examples of these activities are also described here. Thus, the "function of an MP protein" relates to the overall regulation of one or more metabolic fine chemical pathways. Table 1 shows examples of MP protein activities.

Sections of proteins which are encoded by the MP nucleic acid molecules according to the invention are preferably biologically active sections of one of the MP proteins. The term "biologically active section of an MP protein", as used here, is intended to include a section, for example a domain or a motif, of an MP protein that transcriptionally, translationally or post-translationally a metabolic pathway in C. glutamicum can regulate, or has an activity given in Table 1. To determine whether an MP protein or a biologically active portion thereof can regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally, a test of the enzymatic activity can be carried out. These test methods, as described in detail in Example 8 of the example part, are familiar to the person skilled in the art.

In addition to naturally occurring variants of the MP 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 MP protein without affecting the functionality of the MP 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 can be changed in a wild-type sequence from one of the MP proteins (Appendix B) without changing the activity of the MP protein, whereas an "essential" amino acid residue is required for the MP protein activity. However, other amino acid residues (e.g. non-preserved or single Semiconserved amino acid residues in the domain with MP activity) cannot be essential for the activity and can therefore probably be changed without changing the MP activity.

An isolated nucleic acid molecule that encodes an MP protein that is homologous to a protein sequence from Appendix B can be generated by incorporating one or more nucleotide substitutions, additions or deletions into a nucleotide sequence from Appendix A such that one or more amino acid substitutions , additions or deletions are introduced into the encoded protein. The mutations can be introduced into one of the sequences from Appendix A by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferably introduced on one or more of the predicted non-essential amino acid residues. 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 do not include amino acids with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) 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 MP 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 MP coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the MP activity described here in order to identify mutants, that maintain MP activity. After mutagenesis of one of the sequences from Appendix A, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here (see Example 8 of the example section).

B. Recombinant Expression Vectors and Host Cells

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

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

The recombinant expression vectors according to the invention can be designed for the expression of MP proteins in prokaryotic or eukaryotic cells. For example. MP genes can be found in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, CAMJJ et al. (1991) "Heterologous gene expression in filamentous fungi" in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, ed., Pp. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi. In: Applied Molecular Genetics of Fungi, Peberdy, JF et al., Ed., Pp. 1- 28, Cambridge University Press: Cambridge), algal and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefacieizs-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell

Rep.: 583-586) or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Proteins are usually expressed in prokaryotes using vectors that contain constitutive or inducible promoters that 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 terminal 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 Detection 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 MP protein is cloned into a pGEX expression vector, so that a vector is generated which encodes a fusion protein, comprising from the N-terminus to the C-terminus, GST - thrombin cleavage site - X- Protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. The recombinant MP protein that is not fused to GST can be obtained by cleaving the fusion protein with thrombin.

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

One strategy to maximize the expression of the recombinant protein is to express the protein in a host bacterium whose ability to proteolytically cleave the recombinant protein is impaired (Gottesman, S. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 119-128). Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those which are preferably used in a bacterium selected for expression, such as C. glutamicum (Wada et al. (1992 ) Nucleic Acids Res. 20: 2111-2118). This change in the nucleic acid sequences according to the invention is carried out by standard DNA synthesis techniques. In a further embodiment, the MP protein expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 ( Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., ed., pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, the MP 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 MP proteins according to the invention can be expressed in single-cell plant cells (such as algae) or in plant cells of higher plants (for example spermatophytes such as crops). Examples of plant expression vectors include those which are described in detail in: Bekker, D., 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, Adenovirus2, Cytomegalievirus and Si ian Virus 40. For further suitable expression systems for prokaryotic and eukaryotic cells, see chapters 16 and 17 from Sambrook, J., Fritsch, EF 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

10 gnete tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275) , in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulin

15 linen (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g. neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473 -5477), pancreatic-specific promoters (Edlund et al., (1985) Science 230: 912-916) and mammary-specific promoters

20 (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:

25 537-546).

The invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction. This means

30 tet that the DNA molecule is operably linked to a regulatory sequence such that expression (by transcription of the DNA-.Molecule) of an RNA molecule that is antisense to the MP-mRNA is possible. Regulatory sequences can be selected that are functional to an antisense rich

35 cloned nucleic acid and which control the continuous expression of the antisense RNA molecule in a variety of cell types, for example. Viral promoters and / or enhancers or regulatory sequences can be selected which are constitutive, tissue-specific or cell-type-specific expression

Control 40 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 in which

45 the vector is introduced. For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.

Another aspect of the invention relates to the host cells into which a recombinant expression vector according to the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably here. It goes without saying that these terms refer not only to a specific target cell, but also to the descendants or potential descendants of this cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, these offspring are not necessarily identical to the parental cell, but are still included in the scope of the term as used here.

A host cell can be a prokaryotic or eukaryotic cell. For example. an MP protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to the person skilled in the art. Microorganisms which are related to Corynebacterium glutamicum and which can be suitably used as host cells for the nucleic acid and protein molecules according to the invention are listed in Table 3.

Using conventional transformation or transfection methods, vector DNA can be introduced into prokaryotic or eukaryotic cells. The terms "transformation" and "transfection" as used here are intended to encompass a large number of methods known in the prior art for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

For the stable transfection of mammalian cells it is known that, depending on the expression vector and transfection technique used, only a small part of the cells integrate the foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selectable marker (eg resistance to antibiotics) is usually introduced into the host cells together with the gene of interest. Preferred selectable markers include those that are resistant to medicinal products. elements 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 MP protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection (eg 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 MP gene into which a deletion, addition or substitution has been introduced in order to change the MP gene, for example to functionally disrupt it. This MP gene is preferably a Corynebacterium glutamicum MP 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 such that the endogenous MP gene is functionally disrupted when homologous recombination occurs (i.e., 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 MP gene is mutated or otherwise altered in the case of homologous recombination, but still encodes the functional protein (for example the upstream regulatory region can be altered in such a way that the expression of the endogenous MP protein is thereby effected is changed.). The modified portion of the MP gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the MP gene, which is a homologous recombination between the exogenous MP gene carried by the vector. and an endogenous MP gene in a microorganism. The additional flanking MP nucleic acid is long enough for successful homologous recombination with the endogenous gene. The vector usually contains several kilobases of flanking DNA (at both the 5 'and 3' ends) (see e.g. Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced MP gene is homologously recombined with the endogenous MP gene are selected using methods known in the art.

In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene. The inclusion of an MP gene in a vector under control of the Lac operon, for example, allows expression of the MP 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 to produce (i.e., express) an MP protein. The invention also provides methods of producing MP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell according to the invention (into which a recombinant expression vector which encodes an MP protein has been introduced, or into whose genome a gene has been introduced which is a wild-type or modified MP protein encoded) in a suitable medium until the MP protein has been produced. In a further embodiment, the method comprises isolating the MP proteins from the medium or the host cell.

C. Uses and methods according to the invention

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

The nucleic acid and protein molecules according to the invention can serve as markers for specific regions of the genome. This is useful not only when mapping the genome, but also for functional studies of C. glutamicum proteins. To identify the genome region to which a specific C. glutamicum DNA-binding protein binds, the C. gluta icum 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 nucleic acid molecules according to the invention can moreover be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.

The MP 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 many 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 provide an indication of which protein can tolerate mutagenesis without losing function.

The manipulation of the MP nucleic acid molecules according to the invention can cause the production of MP proteins with functional differences from the wild-type MP proteins. These proteins can be improved in terms of their efficiency or activity, and can be present in the cell in greater numbers than usual. against, or may be weakened in their efficiency or activity.

These changes in activity can be such that the yield, production and / or efficiency of production of one or more fine chemicals of C. glutamicum is improved. By optimizing the activity of an MP protein that activates the transcription or translation of a gene that encodes a biosynthesis protein for a desired fine chemical, or by influencing or abolishing the activity of an MP protein that triggers the transcription or translation of such a protein Repressed gene, the activity or activity rate of this biosynthetic pathway can be increased due to the presence of increased amounts of a restricting enzyme, for example. Correspondingly, one can change the activity of an MP protein so that it constitutively inactivates post-translationally a protein which is involved in the degradation pathway for a desired fine chemical, or by changing the activity of an MP protein so that it constitutes the transcription or Translation of such a gene represses, increasing the yield and / or rate of production of the fine chemical from the cell due to the reduced degradation of the compound.

By modulating the activity of one or more MP proteins, one can indirectly stimulate production or improve the production rate of one or more fine chemicals from the cell by linking different metabolic pathways. For example. By increasing the yield, production and / or efficiency of production by activating the expression of one or more lysine biosynthesis enzymes, one can simultaneously increase the expression of other compounds, such as other amino acids, which the cell usually needs in larger amounts if lysine is needed in larger quantities. The regulation of the metabolism can also be changed by in the whole cell in such a way that the cell under the environmental conditions of a fermentative culture (where the nutrient and oxygen supply can be poor and toxic waste products can possibly be present in large quantities in the environment) and can grow or replicate better. For example. can be repressed (to block cell growth and division in suboptimal growth conditions) by mutagenizing an MP protein that rejects the synthesis of molecules necessary for cell membrane production in response to high levels of waste product in the extracellular medium so that it does not is longer able to repress this synthesis, increase the growth and proliferation of cells in cultures, even if the growth conditions are suboptimal. Such increased growth or viability should also affect the yields and / or the rate of production of one desired fine chemical from a fermentative culture due to the relatively large number of cells that produce this compound in the culture.

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

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

Examples

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

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 ° C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml of culture volume). The composition of buffer I: 140.34 g / 1 sucrose, 2.46 g / 1 MgS0 ₄ • 7 H ₂ 0, 10 ml / 1 KH ₂ P0 ₄ solution (100 g / l, adjusted to pH with KOH 6.7), 50 ml / 1 M12 concentrate (10 g / 1 (NH) ₂ S0, 1 g / 1 NaCl, 2 g / 1 MgS0 ₄ ^• 7 H ₂ 0, 0.2 g / 1 CaCl, 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 • 6 H ₂ 0, 3 mg / 1 Na ₂ Mo0 ₄ • 2 H ₂ 0, 500 mg / 1 complexing agent (EDTA or citric acid), 100 ml / 1 vitamin mixture (0.2 ml / 1 biotin, 0.2 mg / 1 folic acid, 20 mg / 1 p-aminobenzoic acid, 20 mg / 1 riboflavin, 40 mg / 1 Ca panthothenate, 140 mg / 1 nicotinic acid, 40 mg / 1 pyridoxol hydrochloride , 200 mg / 1 myoinositol). Lyszy was added to the suspension at a final concentration of 2.5 mg / ml. After about 4 hours of incubation at 37 ° C., the cell wall was broken down and the protoplasts obtained were harvested by centrifugation. The pellet was washed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 mM Tris-HCl, 1 M EDTA, pH 8). The pellet was resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added. After adding proteinase K 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, phenol-chloroform-isoayl alcohol and chloroform-isoamyl alcohol using the standard method. 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, prepared as described in Example 1, "Molecular Cloning: A Laboratory Manual" was used according to known and well-established methods (see, for example, Sambrook, J. et al. (1989). Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", John Wille & Sons) Cosmid and plasmid banks.

Any plasmid or cosmid could be used. Plasmids pBR322 (Sutcliffe, J.G. (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

Cosmids, 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 (eg Bacillus spp. Or yeasts such as Saccharomyces cerevisiae), which maintain the integrity of their cannot maintain genetic information. Usual 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, pp. 2277-2294 , ASM: Washington). These strains are known to the person skilled in the art. The use of these strains is, for example, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34 illustrates.

Example 5: DNA transfer between Escherichia coli and Corynebacterium glutamicum

Several Corynebacterium and Brevibacterium species contain endogenous plasmids (such as pHMl519 or pBLl) which replicate autonomously (for an overview see, for example, Martin, JF et al. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel , FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley &. Sons), to which an origin of replication for and a suitable marker from Corynebacterium glutamicυm is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibactertium species. Particular uses as transformation markers for these species are genes for kanamycin resistance (such as those derived from 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 large variety of shuttle vectors, 5 which are replicated in E. coli and C. glutamicum and which 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 10 102: 93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to hybridize such vectors in Corynebacterium glutamicum.

Bring 15 tribes. The transformation of C. glutamicυm 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 conju-

Achieve 20 gation (e.g. as described 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 Plas id DNA from C. glutamicum (using standard methods known in the art) and transforming it into E. coli

25 is lubricated. 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).

30 Example 6: Determination of the Expression of the Mutated Protein

The observations of the activity of a mutant protein in a transformed host cell are based on the fact that the mutant protein expresses in a similar manner and in a similar amount.

35 is lubricated like the wild-type protein. A suitable method for determining the amount of transcription of the mutated gene (an indication of the amount of mRNA available for the translation of the gene product) is to carry out a Northern blot (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Bio

40 logy, Wiley: New York), wherein 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

45 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 Mation is evidence of the extent of the transcription of the mutant gene. Total cell RNA can be isolated from Corynebacterium glutamicυm by various methods known in the art, as described 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 via complex compounds such as molasses or other by-products from sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH ₄ C1 or (NH ₄ ) ₂ SO ₄ , NH ₄ OH, nitrates, urea, amino acids or complex nitrogen sources, such as maize spring water, soy flour, soy protein, yeast extracts, meat extracts and others.

Inorganic salt compounds that may be included in the media include the chloride, phosphorus, or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate or organic acids such as citric acid. The media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. The exact composition of the media connections depends heavily on the respective experiment and is decided individually for each case. Information on media optimization is available from the textbook "Applied Microbiol. Physiology, A Practiσal 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 can also be kept constant during the cultivation by adding NaOH or NH ₄ OH. If complex media components such as yeast extract are used, the need for additional buffers is reduced, since many complex compounds have a high buffer capacity. When using a fermenter for the cultivation of microorganisms, the pH value can also be regulated with gaseous ammonia. The incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth. The growth experiments disclosed can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes. To screen a large number of clones, the microorganisms should be grown in microtiter plates, glass tubes or shake flasks with or without baffles. Preferably 100 ml shake flasks are used, which contain 10%

(based on the volume) of the required growth medium are filled. 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 made 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 grown on agar plates, such as CM plates (10 g / 1 glucose, 2.5 g / 1 NaCl, 2 g / 1 Urea, 10 g / 1 polypeptone, 5 g / 1 yeast extract, 5 g / 1 meat extract, 22 g / 1 agar pH 6.8 with 2 M NaOH), which were incubated at 30 ° C. The inoculation of the media is carried out either by introducing a saline solution of C. glutamicum cells from CM plates or by adding a liquid preculture of this bacterium.

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

Determining the activities and kinetic parameters of enzymes is well known in the art. Experiments to determine the activity of a particular altered enzyme must be adapted to the specific activity of the wild-type enzyme, which is within the ability of the person skilled in the art. Overviews of enzymes in general as well as specific details relating to the structure, kinetics, principles, processes, applications and examples for determining many enzyme activities can be found, for example, in the following references: Dixon, M., and Webb, EC: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and Mechanism, Free, New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, San Francisco; Price, N.C., 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 Ullmann's Encyclopedia of Industrial Chemistry (1987) Vol. A9, "Enzymes", VCH, Weinheim, pp. 352-363.

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

The activity of membrane transport proteins can be determined according to the techniques 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 growing the modified microorganisms under suitable conditions (such as those described above) and the medium and / or the cellular components are 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 Ulimann'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 components of the metabolic ^■ pathways that are used to produce the desired compound, such as intermediates and by-products, to determine the overall productivity of the organism, the yield and / or to determine the efficiency of the production of the connection. 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 from biosynthetic pathways and measurements of gases that are generated during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein.

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

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

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

Many cleaning methods are known in the art that are not limited to the previous cleaning method. These are described, for example, in Bailey, J.E. & Ollis, D.F. 10 Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined by standard techniques in the art. These include

15 sen high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzyme test or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya

20 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581- 587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;

25 Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

equivalent

30

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

35

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

In column 1 "DNA-ID" the respective number refers to the SEQ ID NO of the attached sequence listing. A "5" in column 40 "DNA-ID" therefore means a reference to SEQ ID NO: 5.

In column 2 "AS-ID", the respective number refers to the SEQ ID NO of the attached sequence listing. A "6" in the "AS-ID" column therefore means a reference to SEQ ID NO: 6. 45 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 metabolic pathway proteins

DNA AS identification: AS AS AS function: ID: ID: Pos: wild type mutant

1 2 RXA00116 26 L F ASPARTATE AMINOTRANSFERASE (EC 2.6.1.1)

3 4 RXA00147 144 I L CARBAMOYL-PHOSPHATE SYNTHASE SMALL CHAIN (EC 6.3.5.5)

239 G S CARBAMOYL PHOSPHATE SYNTHASE SMALL CHAIN (EC 6.3.5.5)

5 6 RXA00152 96 P S STOMATIN LIKE PROTEIN

237 G s STOMATIN LIKE PROTEIN

7 8 RXA00166 103 T 1 METHYL TRANSFERASE (EC 2.1.1.-)

9 10 RXA00278 144 G D GLUTAMINE BINDING PROTEIN PRECURSOR

11 12 RXA00323 41 A V GLUTAMINE SYNTHETASE (EC 6.3.1.2)

80 G E GLUTAMINE SYNTHETASE (EC 6.3.1.2)

13 14 RXA00330 492 T 1 THREONINE SYNTHASE (EC 4.2.99.2)

15 16 RXA00383 57 A T PROTOPORPHYRINOGEN OXIDASE (EC 1.3.3.4)

17 18 RXA00389 55 A T oxoglutarate semialdehyde dehydrogenase (EC 1.2.1.-)

481 G E oxoglutarate semialdehyde dehydrogenase (EC 1.2.1.-)

19 20 RXA00416 223 A V Hypothetical Membrane Spanning Protein

21 22 RXA00489 338 S F GMP REDUCTASE (EC 1.6.6.8)

23 24 RXA00552 295 AT THIOSULFATE SULFURTRANSFERASE (EC 2.8.1.1)

26 RXA00579 490 P L PARA-AMINOBENZOATE SYNTHASE COMPONENT I (EC 4.1.3.-)

28 RXA00620 141 G D PHOSPHORIBOSYLAMINOIMIDAZOLE-SUCCINOCARBOXAMIDE SYNTHASE (EC 6.3.2.6)

30 RXA00636 146 E K oxidoreductase FAD-binding

32 RXA00708 256 G S 2,5-DIKETO-D-GLUCONIC ACID REDUCTASE (EC 1.1.1.-)

34 RXA00727 78 P S GLUTAMINE BINDING PERIPLASMIC PROTEIN PRECURSOR

149 A V GLUTAMINE BINDING PERIPLASMIC PROTEIN PRECURSOR

280 AV GLUTAMINE BINDING PERIPLASMIC PROTEIN PRECURSOR

36 RXA00867 513 P S POLYRIBONUCLEOTIDE NUCLEOTIDYLTRANSFERASE (EC 2.7.7.8)

38 RXA00955 180 A V INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC 4.1.1.48) / N- (5'-PHOSPHO-RIBOSYL) ANTHRANILATE ISOMERASE (EC 5.3.1.24)

490 INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC 4.1.1.48) / N- (5'-PHOSPHO-RIBOSYL) ANTHRANILATE ISOMERASE (EC 5.3.1.24)

40 RXA00957 220 P L ANTHRANILATE SYNTHASE COMPONENT I (EC 4.1.3.27)

42 RXA00970 133 A V HOMOSERINE KINASE (EC 2.7.1.39)

138 P S HOMOSERINE KINASE (EC 2.7.1.39)

44 RXA00997 117 A V 3-DEMETHYLUBIQUINONE-93-METHYLTRANSFERASE (EC 2.1.1.64)

46 RXA01095 262 P s INDOLE-3-GLYCEROL PHOSPHATE SYNTHASE (EC 4.1.1.48)

48 RXA01105 270 A V HISTIDINOL-PHOSPHATE AMINOTRANSFERASE (EC 2.6.1.9)

50 RXA01115 72 T I 3-OXOADIPATE ENOL-LACTONE HYDROLASE (EC 3.1.1.24) / 4-CARBOXYMUCONOLACTONE DECARBOXYLASE (EC 4.1.1.44)

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

54 RXA0240 23 A T GTP PYROPHOSPHOKINASE (EC 2.7.6.5)

56 RXA01253 112 AT COBYRIC ACID SYNTHASE (EC 3.-.-.-)

58 RXA01321 156 V A FORMYL TETRAHYDROFOLATE DEFORMYLASE (EC 3.5.1.10)

60 RXA01381 180 G D THIF PROTEIN

62 RXA01434 957 S F VIRULENCE FACTOR MVIN

1012 P S VIRULENCE FACTOR MVIN

64 RXA01442 122 R H PHOSPHORIBOSYLGLYCINAMIDE FORMYLTRANSFERASE 2 (EC 2.1.2.-)

66 RXA01483 200 R C DEOXYGUANOSINETRIPHOSPHATE TRIPHOSPHOHYDROLASE (EC 3.1.5.1)

326 P S DEOXYGUANOSINETRIPHOSPHATE TRIPHOSPHOHYDROLASE (EC 3.1.5.1)

68 RXA01515 239 A V DIHYDROPTEROATE SYNTHASE (EC 2.5.1.15)

70 RXA01518 148 AT Hypothetical Membrane Spanning Protein

72 RXA01690 184 G D BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE (EC 2.6.1.42)

74 RXA01746 146 G E LIPOATE PROTEIN LIGASE B (EC 6.-.-.-)

76 RXA01810 263 G S HEMIN-BINDING PERIPLASMIC PROTEIN HMUT PRECURSOR o

78 RXA01850 1 V M L-SERINE DEHYDRATASE (EC 4.2.1.13)

45 S N L-SERINE DEHYDRATASE (EC 4.2.1.13)

80 RXA01940 269 A V INOSINE-URIDINE PREFERRING NUCLEOSIDE HYDROLASE (EC 3.2.2.1)

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

84 RXA02095 409 A V ABC TRANSPORTER ATP-BINDING PROTEIN

914 A V ABC TRANSPORTER ATP-BINDING PROTEIN

86 RXA02156 48 A T ACETYL GLUTAMATE KINASE (EC 2.7.2.8)

88 RXA02162 434 T I ARGININOSUCCINATE LYASE (EC 4.3.2.1)

90 RXA02176 136 P S PHOSPHOSERINE AMINOTRANSFERASE (EC 2.6.1.52)

92 RXA02194 129 L F ATP PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.17)

94 RXA02206 73 GD OXIDOREDUCTASE (EC 1.1.1.-)

114 A T OXIDOREDUCTASE (EC 1.1.1.-)

314 R c OXIDOREDUCTASE (EC 1.1.1.-)

95 96 RXA02237 78 D N GUANYLATE KINASE (EC 2.7.4.8)

97 98 RXA02239 168 P S PHOSPHOPANTOTHENATE - CYSTEINE LIGASE (EC 6.3.2.5) / PHOSPHOPANTOTHENOYLCYSTEINE DECARBOXYLASE (EC 4.1.1.36)

99 100 RXA02250 24 P S RIBX PROTEIN

101 102 RXA02295 130 G D TRANSPORTER

103 104 RXA02299 36 A V ASPARTATE 1 -DECARBOXYLASE (EC 4.1.1.11)

105 106 RXA02382 416 E K GAMMA-GLUTAMYL PHOSPHATE REDUCTASE (GPR) (EC .2.1.41)

107 108 RXA02390 195 G D THREONINE EFFLUX PROTEIN

109 110 RXA02499 65 G S PYRROLINE-5-CARBOXYLATE REDUCTASE (EC 1.5.1.2)

216 AT PYRROLINE-5-CARBOXYLATE REDUCTASE (EC 1.5.1.2)

111 112 RXA02516 158 P s CYSTEINE DESULFHYDRASE (EC 4.4.1.-) / SELENOCYSTEINE LYASE (EC 4.4.1.16)

113 114 RXA02532 284 P s CYSTATHIONINE GAMMA SYNTHASE (EC 4.2.99.9)

115 116 RXA02536 132 P s carbon-nitrogen hydrolase

117 118 RXA02560 138 G E OXYGEN-INSENSITIVE NAD (P) H NITROREDUCTASE (EC 1.-.-.-) / DIHYDROPTERIDINE REDUCTASE (EC 1.6.99.7)

192 OXYGEN-INSENSITIVE NAD (P) H NITROREDUCTASE (EC 1.-.-.-) / DIHYDROPTERIDINE REDUCTASE (EC 1.6.99.7)

119 120 RXA02653 448 G E pyridoxal-dependent decarboxylase

121 122 RXA02754 374 P S NICOTINATE PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.11)

123 124 RXA02758 110 G E PHOSPHOSERINE PHOSPHATASE (EC 3.1.3.3)

125 126 RXA02790 30 LF 4-AMINO-4-DEOXYCHORISMATE LYASE (EC 4.-.-.-)

127 128 RXA02912 62 G D METHYL TRANSFERASE (EC 2.1.1.-)

129 130 RXA02970 233 E K AMINOTRANSFERASE

131 132 RXA03003 209 P L ASPARTATE AMINOTRANSFERASE (EC 2.6.1.1)

133 134 RXA03171 70 P S URACIL PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.9)

144 L F URACIL PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.9)

199 A V URACIL PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.9)

135 136 RXA03282 312 G D CYSTATHIONINE GAMMA SYNTHASE (EC 4.2.99.9)

137 138 RXA03465 224 P S MOLYBDENUM COFACTOR BIOSYNTHESIS PROTEIN A

261 P S MOLYBDENUM COFACTOR BIOSYNTHESIS PROTEIN A

139 140 RXA03677 389 A T TREHALOSE / MALTOSE BINDING PROTEIN

141 142 RXA03695 70 T I METHYL TRANSFERASE (EC 2.1.1.-)

143 144 RXA03754 459 G D 3-ISOPROPYLMALATE DEHYDRATASE LARGE SUBUNIT (EC 4.2.1.33)

145 146 RXA03829 36 A T PROBABLE ACETOLACTATE SYNTHASE FAMILY

147 148 RXA03871 33 R H MOLYBDOPTERIN BIOSYNTHESIS MOEA PROTEIN

360 T I MOLYBDOPTERIN BIOSYNTHESIS MOEA PROTEIN

149 150 RXA04120 348 G D GAMMA-GLUTAMYL TRANSPEPTIDASE (EC 2.3.2.2)

151 152 RXA041 3 41 T I GMP synthase - Glutamine amidotransferase domain

153 154 RXA04174 856 G S ACONITATE HYDRATASE (EC 4.2.1.3)

155 156 RXA04186 385 A T 5-METHYLTETRAHYDROFOLATE - HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.13)

492 V I 5-METHYLTETRAHYDROFOLATE— HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.13)

990 S N 5-METHYLTETRAHYDROFOLATE— HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.13)

157 158 RXA04228 279 AT GLUTAMINASE (EC 3.5.1.2) 159 160 RXA04360 161 P s ANTHRANILATE PHOSPHORIBOSYL TRANSFERASE (EC 2.4.2.18)

161 162 RXA07000 398 P S GLUTAMATE SYNTHASE [NADPH] LARGE CHAIN (EC 1.4.1.13)

163 164 RXA07001 110 A τ MALTOSE-BINDING PROTEIN PRECURSOR

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.

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

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

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

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

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

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

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