DE10154175A1 - Genes coding for homeostasis and adaptation proteins - Google Patents

Abstract

The invention relates to novel nucleic acid molecules, to their use for constructing genetically improved microorganisms and to methods for producing fine chemicals, in particular, amino acids by using these genetically modified microorganisms.

Description

Genes coding for homeostasis and adaptation proteins Background of the Invention

Certain products and by-products from naturally occurring Metabolic processes in cells are common in many industries used, including food, feed, Cosmetic and pharmaceutical industries. These molecules that collectively referred to as "fine chemicals" organic acids, both proteinogenic and non-proteinogenic Amino acids, nucleotides and nucleosides, lipids and fatty acids, Diols, carbohydrates, aromatic compounds, vitamins and Cofactors as well as enzymes. Their production is best done using Growing bacteria on a large scale that have been developed to to produce large quantities of the desired molecule and secrete. An organism particularly suitable for this purpose is Corynebacterium glutamicum, a gram-positive, non-pathogenic bacterium. About stem selection is a number of Mutant strains have been developed that make an assortment more desirable Produce connections. The selection of tribes regarding production of a particular molecule is improved however, a time consuming and difficult process.

Summary of the invention

This invention provides novel nucleic acid molecules that to identify or classify Corynebacterium Have glutamicum or related types of bacteria used. C. glutamicum is a gram-positive, aerobic bacterium that is found in the Large-scale production of a number of industries Fine chemicals, and also for the degradation of hydrocarbons (e.g. when overflowing crude oil) and for the oxidation of terpenoids is commonly used. The nucleic acid molecules can therefore Identify microorganisms that are used for the production of fine chemicals, for example by Fermentation process. C. glutamicum itself is true non-pathogenic, however, it is with other Cotynebacterium species, such as Related to Corynebacterium diphtheriae (the causative agent of diphtheria), which are major pathogens in humans. The ability to do that Can identify presence of Corynebacterium species therefore also have a significant clinical significance, e.g. B. at diagnostic applications. These nucleic acid molecules can also as reference points for mapping the C. glutamicum genome or served by genomes of related organisms.

These new nucleic acid molecules encode proteins that are here called Homeostasis and adaptation (HA) proteins. This For example, HA proteins can perform a function that Maintaining homeostasis in C. glutamicum or at the Ability of this microorganism to adapt to different Adapting environmental conditions is involved. Due to 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) Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gene. Microbiol. 130 (1984) 2237-2246), the Nucleic acid molecules according to the invention for genetic manipulation use this organism to make him a producer of one or more fine chemicals to make them better and more efficient. The improved production or efficiency of producing one Fine chemical can affect the direct effect of manipulation of a gene according to the invention or on an indirect effect based on such manipulation.

There are a number of mechanisms through which change takes place of an HA protein according to the invention the yield, production, and / or efficiency of producing a fine chemical from a C. glutamicum strain containing an altered protein directly can influence. Through genetic manipulation of enzymes that modify aromatic or aliphatic compounds so that these enzymes have higher or lower activity or number can have the production of one or more Fine chemicals that are the modification or degradation products of this Inventions are, modulate. Accordingly, enzymes that are on Metabolism of inorganic compounds are involved, Key molecules (e.g. phosphorus, sulfur and nitrogen molecules) for the Biosynthesis of such fine chemicals as amino acids, vitamins and nucleic acids ready. By changing the activity or the Number of these enzymes in C. glutamicum can be converted increase of these inorganic compounds (or alternative Use Connections), resulting in improved installation rates inorganic atoms in these fine chemicals are made possible. The genetic manipulation of C. glutamicum enzymes involved in general cellular processes are involved. Can also do that Fine chemicals production directly improve as many of these Modify enzymes fine chemicals directly (e.g. amino acids) or the enzymes involved in fine chemical synthesis or secretion involved. The modulation of the activity or the number Cellular proteases can also have a direct effect on the Fine chemicals have a lot of proteases Fine chemicals or enzymes involved in fine chemicals production or their Degradation are involved, can degrade.

The above enzymes involved in the modification and / or degradation of aromatic or aliphatic compounds, general biocatalysis, metabolism or proteolysis of inorganic Compounds involved are themselves fine chemicals that are responsible for their activity in various in vitro industrial applications are desirable. By changing the copy number of the gene for one or more of these enzymes can be found in C. glutamicum Number of these proteins that are produced by the cell increase, reducing the potential yield or efficiency of the Production of these proteins from large-scale C. glutamicum or related bacterial cultures is increased.

The modification of an HA protein according to the invention can also Yield, production and / or efficiency of producing one Fine chemical from a C. glutamicum strain that is an altered Contains protein, affect indirectly. By modulating the Activity and / or the number of such proteins involved in building or are involved in the rearrangement of the cell wall, for example Modify the structure of the cell wall itself so that the cell mechanical and other stress caused by large fermenter cultures exists, withstands better. The growth of C. glutamicum in Large scale also requires a significant one Cell wall production. The modulation of the activity or the number of cell wall Biosynthesis or degradation enzymes can be faster Cell wall biosynthesis rates allow, which in turn, stronger growth rates this microorganism allows in culture and thereby the number of Cells that produce the desired fine chemical increased.

It is also possible to modify the HA enzymes according to the invention indirectly the yield, production or efficiency of production of one or more fine chemicals from C. glutamicum influence. For example. have many common enzymes in C. glutamicum a significant impact on global cell processes (e.g. regulatory processes), which in turn is significant Has an impact on the fine chemical metabolism.

Accordingly, proteases, enzymes, may increase modify toxic aromatic or aliphatic compounds or degrade the viability of C. glutamicum. The proteases support the selective removal of misfolded or incorrectly regulated proteins, such as those, for example, among the relative stressful environmental conditions in a large fermenter culture occur. By changing these proteins, you can still change them Increase activity and the viability of C. glutamicum in Improve culture. The aromatic / aliphatic modification or degradation proteins are not only used to detoxify them Waste compounds (which in the culture medium as impurities or occur as waste products from the cells themselves), but also allow the cell to have alternative carbon sources uses if the optimal carbon source in the culture is limited. By increasing the number and / or the activity can the survival of C. glutamicum cells in culture is enhanced become. The inorganic metabolic proteins according to the invention supply the cell with the inorganic molecules required for all protein and nucleotide syntheses (among others) necessary and thus for the overall viability of the cell are crucial. An increase in the number of viable cells that produce one or more fine chemicals in large culture, should have a simultaneous increase in yield, production and or efficiency of fine chemical production in culture cause.

This invention provides new nucleic acid molecules that the encode proteins referred to herein as (HA) proteins which For example, perform a function that helps maintain homeostasis is involved in C. glutamicum, or in the ability of this Microorganism is involved in various Adapt environmental conditions. Nucleic acid molecules that are an HA protein encode, are referred to here as HA nucleic acid molecules. at a preferred embodiment is an HA protein at the C. glutamicum cell wall biosynthesis or rearrangements, metabolism inorganic compounds, modification or degradation of aromatic or aliphatic compounds involved, or has a C. glutamicum enzymatic or proteolytic activity. Examples of such proteins are those of those in Genes listed in Table 1 can be encoded.

One aspect of the invention therefore relates to isolated Nucleic acid molecules (e.g. cDNAs), comprising a nucleotide sequence which encodes an HA protein or biologically active portions thereof, as well as nucleic acid fragments, which act as primers or Hybridization probes for the detection or amplification of HA-coding nucleic acid (for example, DNA or mRNA) are suitable. With especially preferred embodiments include the isolated Nucleic acid molecule one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof from one of these nucleotide sequences. In other preferred embodiments the isolated nucleic acid molecule encodes one of those in Appendix B amino acid sequences listed. The preferred Ben HA proteins according to the invention likewise preferably have at least one of the HA activities described here.

In the following, the nucleic acid sequences of the Sequence listing along with those described in Table 1 Sequence changes defined at the respective position.

The polypeptide sequences of the Sequence listing along with those described in Table 1 Sequence changes defined at the respective position.

In another embodiment, this is isolated Nucleic acid molecule at least 15 nucleotides long and hybridizes under stringent conditions on a nucleic acid molecule that a Nucleotide sequence from Appendix A comprises. The isolated Nucleic acid molecule preferably corresponds to a naturally occurring one Nucleic acid molecule. The isolated nucleic acid encodes more strongly preferably a naturally occurring C. glutamicum HA protein or a biologically active section of it.

Another aspect of the invention relates to vectors, for example. recombinant expression vectors that the invention Contain nucleic acid molecules, and host cells, into which these Vectors have been introduced. In one embodiment Production of an HA protein uses a host cell that is in grown in a suitable medium. The HA 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 HA gene is introduced or has been changed. The genome of the microorganism is one Embodiment by introducing at least one nucleic acid molecule according to the invention has been changed that the mutated HA Sequence encoded as a transgene. In another embodiment is an endogenous HA gene in the genome of the microorganism homologous recombination with an altered HA gene changed, e.g. B. functionally disrupted. The microorganism is one of them a preferred embodiment of the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum particularly is preferred. The microorganism is preferred Embodiment also for producing a desired connection, such as an amino acid, particularly preferably lysine.

Another preferred embodiment are host cells that more than one of the nucleic acid molecules described in Appendix A. have. Such host cells can be divided into several Establish ways known to those skilled in the art. For example, they can by vectors that are several of the invention Carrying nucleic acid molecules, being transfected. But it is also possible with a vector each has a nucleic acid molecule according to the invention in to introduce the host cell and therefore multiple vectors either to be used simultaneously or at different times. So it can Host cells can be constructed that are numerous, up to several Carry one hundred of the nucleic acid sequences according to the invention. Such an accumulation often makes it possible to add additives Effects on the host cell in terms of Achieve fine chemical productivity.

Another aspect of the invention relates to an isolated HA protein or a section, for example a biologically active one Section of it. The isolated HA protein or its section can in a preferred embodiment of maintaining the Homeostasis in C. glutamicum may or may not be involved Exercise function in adapting this microorganism different environmental conditions are involved. At a another preferred embodiment is the isolated HA protein or a section thereof sufficiently homologous to one Amino acid sequence from Appendix B so that the protein or its section continue to maintain homeostasis in C. glutamicum may be involved or have a role in the Adaptation of this microorganism to different environmental conditions is involved.

The invention also relates to an isolated HA protein preparation. The HA protein comprises one in preferred embodiments Amino acid sequence from Appendix B. Another preferred The invention relates to an isolated embodiment Full-length protein that forms a complete amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) in is essentially homologous.

The HA polypeptide or a biologically active portion thereof can be operably linked to a non-HA polypeptide to create a fusion protein. This fusion protein has in preferred embodiments, an activity other than that HA protein alone and yields other preferred ones Embodiments increased yields, increased production and / or Efficiency of producing a desired fine chemical from C. glutamicum. The integration of this fusion protein into one In particularly preferred embodiments, the host cell modulates the Production of a desired connection from the cell.

Another aspect of the invention relates to a method for Manufacture of a fine chemical. The process sees the cultivation a cell that contains a vector that expresses of an HA nucleic acid molecule according to the invention, so that a fine chemical is produced. This procedure includes a preferred embodiment also the step of extraction a cell containing such a vector, the cell is transfected with a vector that expresses HA- Causes nucleic acid. This method involves another preferred embodiment also the step in which the Fine chemical is obtained from the culture. The cell belongs a preferred embodiment of the genus Corynebacterium or Brevibacterium.

Another aspect of the invention relates to methods for Modulation of the production of a molecule from a microorganism. These methods involve bringing the cell into contact with one Substance that inhibits HA protein activity or HA nucleic acid Expression modulates so that cell-associated activity compared to the same activity in the absence of the substance is changed. The cell is in a preferred embodiment for one or more C. glutamicum processes involved in the Cell wall biosynthesis or rearrangement, metabolism inorganic compounds, modification or degradation aromatic or aliphatic compounds or on enzymatic or proteolytic activities are modulated. The For example, substance that modulates HA protein activity stimulates. HA protein activity or HA nucleic acid expression. Examples of substances that inhibit HA protein activity or Stimulate nucleic acid expression include small molecules, active HA proteins and nucleic acids encoding HA proteins and have been introduced into the cell. Examples of substances which inhibit HA activity or expression include small ones Molecules and antisense HA nucleic acid molecules.

Another aspect of the invention relates to methods for Modulation of the yields of a desired compound from a cell, comprising inserting a wild-type or mutant HA gene into a cell that 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 done so that the native gene through the integrated copy is replaced what the production of the desired compound from the cell to be modulated. These yields are one preferred embodiment increased. Another preferred embodiment, the chemical is a fine chemical which in a particularly preferred embodiment, an amino acid is. This amino acid is particularly preferred Embodiment L-lysine.

Detailed description of the invention

The present invention provides HA nucleic acid and - Protein molecules ready to participate in cell wall biosynthesis or Rearrangements, on the metabolism of inorganic compounds, on the Modification or degradation of aromatic or aliphatic compounds involved in C. glutamicum, or the one C. have glutamicum-enzymatic or proteolytic activity. The Molecules of the invention can be used to modulate production of fine chemicals from microorganisms, such as C. glutamicum, either directly (e.g. where the overexpression or the optimization of the Activity of a protein involved in the production of a Fine chemical (e.g. an enzyme) has a direct impact on the Yield, production and / or efficiency of producing one Fine chemical from the modified C. glutamicum), use or have an indirect impact that is nevertheless an increase in Yield, production and / or efficiency of production of the desired connection (e.g. where the modulation of the Activity or number of copies of an aromatic or aliphatic modification or degradation protein from C. glutamicum Increase in viability of C. glutamicum cells does what again increased production in a large scale culture allows. The aspects of the invention are as follows further explained.

1. Fine chemicals

The term "fine chemical" is known in the art and contains molecules that are produced by an organism and find applications in various industries, such as but not limited to the pharmaceutical industry that Agriculture, and cosmetics industries. These connections include organic acids such as tartaric acid, itaconic acid and Diaminopimelic acid, both proteinogenic and non-proteinogenic Amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., Ed. VCH: Weinheim and the quotes it contains), 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 (such as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and den quotes contained therein; and Ong, A. S., Niki, E. and Packer, L. (1995) "Nutrition, Lipids, Health and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia, held on 1-3 Sept. 1994 in Penang, Malysia, AOCS Press (1995)), Enzyme and all others by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the references cited therein Chemicals. The metabolism and uses of certain Fine chemicals are further explained below.

A. Amino acid metabolism and uses

The amino acids comprise the basic structural units all proteins and are therefore for the normal Cell functions essential. The term "amino acid" is in the art known. The proteinogenic amino acids, of which there are 20 types, serve as structural units for proteins in which they over Peptide bonds are linked together, whereas the non- proteinogenic amino acids (hundreds of which are known) usually not found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids can be in the D or L configuration although L-amino acids are usually the only type which is found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids are in both prokaryotic and eukaryotic cells well characterized (see e.g. Stryer, L. Biochemistry, 3. Edition, pp. 578-590 (1988)). The "essential" amino acids (Histidine, isoleucine, leucine, lysine, methionine, phenylalanine, Threonine, tryptophan and valine), so called because of the Complexity of their biosynthesis with the diet added must be through simple biosynthetic pathways in the rest 11 "non-essential" amino acids (alanine, arginine, asparagine, Aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and Tyrosine). Higher animals have the ability to do some to synthesize these amino acids, however, the essential amino acids are ingested with food so normal protein synthesis takes place.

Aside from their function in protein biosynthesis these amino acids have interesting chemicals in themselves and you have discovered that many in different applications in the Food, feed, chemical, cosmetic, agricultural and pharmaceutical industry. Lysine is not an important amino acid only for human nutrition, but also for monogastric animals such as poultry and. Pigs.

Glutamate is most commonly used as a flavor additive (Monosodium glutamate, MSG) and widely used in the food industry used, as well as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are all found in the pharmaceutical industry used. Glutamine, valine, leucine, isoleucine, Histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are common Feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rebin et al., (ed.) Biotechnology Vol. 6, Chapter 14a, VCH: Weinheim). It has been discovered that these amino acids also act as precursors for 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, p. 57-97, VCH, Weinheim, 1985 are suitable substances.

The biosynthesis of these natural amino acids in organisms that they can produce, for example bacteria, is well characterized (for an overview of the bacterial Amino acid biosynthesis and its regulation, see Umbarger, H.E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate is obtained through reductive amination of α-ketoglutarate, an intermediate in Citric acid cycle, synthesized. 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 after oxidation, transamination and hydrolysis steps Amino acid. Cysteine and glycine are both made from serine produces, the former by condensation of homocysteine with serine, and the latter by transferring the Side chain β-carbon atom on tetrahydrofolate, in a through Serine transhydroxymethylase catalyzed reaction. Phenylalanine and Tyrosine are derived from the precursors of glycolysis and Pentose phosphate pathway, erythrose 4-phosphate and phosphoenol pyruvate in one 9-step biosynthetic pathway, which is only in the last two steps after the synthesis of prephenate different. Tryptophan is also made from these two Starting molecules produced, but its synthesis takes place in one 11-step pathway. Tyrosine can be passed through in one Phenylalanine hydroxylase catalyzed reaction also from phenylalanine produce. Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis. Aspartate is made Oxaloacetate, an intermediate of the citrate cycle. Asparagine, methionine, threonine and lysine are each made by Conversion of aspartate produced. Isoleucine is made from threonine educated. In a complex 9-step process, the formation of Histidine from 5-phosphoribosyl-1-pyrophosphate, an activated one Sugar.

Amino acids, the amount of which the cell's protein biosynthesis requirements exceeds, cannot and will not be saved degraded instead so that intermediates for the Main cell metabolic pathways are provided (see for an overview Stryer, L., Biochemistry, 3rd ed. Chap. 21 "amino acid Degradation and the Urea Cycle "; S 495-516 (1988)). The cell is though able to convert unwanted amino acids into useful ones Convert metabolic intermediates, however, that is Amino acid production in terms of energy, precursor molecules and of the enzymes required for their synthesis. It is surprising therefore not that amino acid biosynthesis through feedback inhibition is regulated, the presence of a certain amino acid their own production slowed down or stopped completely (for one Overview of the feedback mechanism at Amino acid biosynthetic pathways, see Stryer, L., Biochemistry, 3rd ed., Chap. 24 "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988)). The Ejection of a certain amino acid is therefore determined by the amount this amino acid is restricted in the cell.

B. vitamins, cofactors and nutraceutical metabolism as well uses

Vitamins, cofactors and nutraceuticals include another Group of molecules. Higher animals have lost the ability to synthesize them and thus have to absorb them, though it is easily synthesized by other organisms such as bacteria become. These molecules are either biologically active molecules in itself or precursors of biologically active substances, which as Electron carriers or intermediates in a number of Serve metabolic pathways. These connections have next to hers Nutritional value also has a significant industrial value as colorants, Antioxidants and catalysts or others Processing auxiliaries. (For an overview of the structure, activity and the For industrial applications of these connections see, for example. Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, Pp. 443-613, VCH: Weinheim, 1996). The term "vitamin" is in the Known in the art and includes nutrients derived from a Organism are required for normal function, but not of this organism itself can be synthesized. The group The vitamins can contain cofactors and nutraceutical compounds include. The term "cofactor" includes non-proteinaceous Compounds necessary for the occurrence of normal enzyme activity are. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferred organic. The term "nutraceutical" includes food additives, in plants and animals, especially humans, are good for your health. Examples of such molecules are vitamins, Antioxidants and also certain lipids (e.g. multiple unsaturated fatty acids).

The biosynthesis of these molecules in organisms that contribute to them Production capable, like bacteria, is extensive have been characterized (Ullmann's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996, Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley &Sons; Ong, A. S., Niki, E. and Packer, L. (1995) "Nutrition, Lipids, Health and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for free Radical Research - Asia, held on 1-3 Sept. 1994 in Penang, Malaysia, AOCS Press, Champaign, IL X, 374 S).

Thiamine (vitamin B ₁ ) is formed by chemical coupling of pyrimidine and thiazole units. Riboflavin (vitamin B ₂ ) is synthesized from guanosine 5'-triphosphate (GTP) and ribose 5'-phosphate. Riboflavin in turn is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of connections that collectively 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-1-oxobutyl) -β-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of β-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 studied extensively, and one has identified several of the genes involved. It has found that many of the corresponding proteins on the Fe cluster Synthesis are involved and the class of nifS proteins belong. The lipoic acid is derived from the octanoic acid and serves as a coenzyme in energy metabolism, where it is a component of the pyruvate dehydrogenase complex and α-Ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derived from folic acid, which in turn is derived from L- Glutamic acid, p-aminobenzoic acid and 6-methyl pterine are derived is. The biosynthesis of folic acid and its derivatives, starting out of metabolic metabolic intermediates Guanosine 5'-triphosphate (GTP), L-glutamic acid and p-aminobenzoic acid has been extensively studied in certain microorganisms.

Corrinoids (such as the cobalamins and especially vitamin B12) and the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system. The biosynthesis of vitamin B ₁₂ is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called "niacin". Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.

The production of these compounds on a large scale is largely based on cell-free chemical syntheses, although some of these chemicals have also been produced by large-scale cultivation of microorganisms, such as riboflavin, vitamin B ₆ , pantothenate and biotin. Only vitamin B ₁₂ 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" includes nitrogenous bases that are part of the Are nucleic acids, coenzymes and nucleotides. The term "Nucleotide" contains the basic structural units of the Nucleic acid molecules that contain a nitrogenous base, a pentose Sugar (for RNA, the sugar is ribose, for DNA, the sugar is D- Deoxyribose) and phosphoric acid. The term "nucleoside" includes molecules that serve as precursors to nucleotides that but in contrast to the nucleotides no phosphoric acid unit exhibit. By inhibiting the biosynthesis of these molecules or it is their mobilization to form nucleic acid molecules possible to inhibit RNA and DNA synthesis; will this Targeted activity inhibited in carcinogenic cells, the Inhibit the ability of tumor cells to divide and replicate. It also gives nucleotides that do not form nucleic acid molecules, however, as an energy store (i.e. AMP) or as a coenzyme (i.e. FAD and NAD) serve.

Several publications have used this Chemicals for these medical indications are described, the Purine and / or pyrimidine metabolism is influenced (e.g. Christopherson, R.I. and Lyons, S.D. (1990) "Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents ", Med. Res. Reviews 10: 505-548) Enzymes involved in purine and pyrimidine metabolism have focused on developing new drugs that for example as an immunosuppressant or antiproliferant can be used (Smith, J.L. "Enzymes in Nucleotide Synthesis" Curr. Opin. Struct. Biol. 5 (1995) 752-757; Biochem. Soc. Transact. 23 (1995) 877-902). The purine and pyrimidine bases, However, nucleosides and nucleotides also have others Possible uses: as intermediates in the biosynthesis of various Fine chemicals (e.g. thiamine, S-adenosyl methionine, folate or Riboflavin), as an energy source for the cell (e.g. ATP or GTP) and for chemicals themselves, are commonly called Flavor enhancers used (e.g. IMP or GMP) or for many medical 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 that are based on purine, pyrimidine, Nucleoside or nucleotide metabolism are involved also always stronger than targets against the chemicals for the Plant protection, including fungicides, herbicides and Insecticides are being developed.

The metabolism of these compounds in bacteria is have been characterized (for overviews see, for example, Zalkin, H. and Dixon, J.E. (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 "; Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley, New York). The Purine metabolism, the object of intensive research, is for the normal Functioning of the cell essential. A disturbed purine metabolism in higher animals can cause serious illnesses, e.g. Gout. The purine nucleotides are made through a series of steps via the intermediate compound inosine 5'-phosphate (IMP) Ribose-5-phosphate synthesized, leading to the production of Guanosine 5'-monophosphate (GMP) or adenosine 5'-monophosphate (AMP) leads from which are used as nucleotides Have triphosphate molds made easily. These connections will also used as an energy store so that their degradation is energy for many delivers different biochemical processes in the cell. The Pyrimidine biosynthesis occurs through the formation of Uridine 5'-monophosphate (UMP) from ribose 5-phosphate. UMP is in turn Cytidine 5'-triphosphate (CTP) converted. The deoxy forms of all Nucleotides are generated in a one-step reduction reaction Diphosphate ribose form of the nucleotide for Diphosphate deoxyribose form of the nucleotide produced. After phosphorylation these molecules can participate in DNA synthesis.

D. Trehalose metabolism and uses

Trehalose consists of two glucose molecules that are about α, α-1,1 bond are linked together. It becomes ordinary in the food industry as a sweetener, as an additive for dried or frozen foods and in beverages used. However, it is also used in the pharmaceutical industry, applied in the cosmetics and biotechnology industry (see e.g. Nishimoto et al., (1998) U.S. Patent No. 5,759,610; Singer, M. A. and Lindquist, S. Trends Biotech. 16 (1998) 460-467; Paiva, C.L.A. and Panek, A.D. Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, M.J. Japan 172 (1997) 97-102). Trehalose gets through Enzymes produced by many microorganisms and based on natural ones Were released into the surrounding medium, from which they are im Methods known in the art can be obtained.

II. Maintaining homeostasis in C. alutamicum and environmental Adaptation

The metabolic and other biochemical processes through which Cells work, are like environmental conditions Temperature, pressure, concentrations of solutes and Oxygen availability sensitive. Become one or more of these Environmental conditions disturbed or changed in a way that with incompatible with the normal functioning of these cell processes the cell must maintain an intracellular environment, which enables her, despite the hostile extracellular Survive environment. Gram-positive bacterial cells, such as C. glutamicum cells have a number of mechanisms by which the internal homeostasis despite unfavorable extracellular conditions can be maintained. These include a cell wall Proteins, the possible toxic aromatic and aliphatic Connections can degrade, proteolysis mechanisms go wrong folded or incorrectly regulated proteins are quickly destroyed can and catalysts that intracellular reactions can enable that under the conditions necessary for bacterial growth are optimal, would not normally take place.

In addition to surviving in a hostile environment, you can bacterial cells (e.g. C. glutamicum cells) adapt frequently, so that they can take advantage of such conditions. Cells in an environment that has the desired carbon sources are missing, for example, can grow on one less adjust suitable carbon source. Cells can also do less use desirable inorganic compounds if the commonly used are not available. C. glutamicum cells possess a number of genes that enable them to express themselves adapt so that they use inorganic molecules to which they usually under optimal growing conditions as nutrients and Precursors for metabolism would not encounter. below Aspects of cellular processes are described in detail are involved in homeostasis and adaptation.

A. Modification and degradation of aromatic and aliphatic links

Bacterial cells are regularly a variety of aromatic and aliphatic compounds exposed in nature. Aromatic compounds are organic molecules with a cyclic one Ring structure, whereas aliphatic compounds are organic Are molecules with open chain structures instead of ring structures. These compounds can be industrial by-products Processes occur (e.g. benzene or toluene), but can also by certain microorganisms (e.g. alcohols) are produced. Many of these compounds are toxic to the cells, especially the aromatic compounds that are due to the high-energy ring structure are highly reactive. Hence certain Bacteria develop mechanisms by which they make these connections can modify or degrade so that they are not for the cell are dangerous longer. Cells can have enzymes that hydroxylate aromatic or aliphatic compounds, Can isomerize or methylate, making them either less toxic be made, or so that the modified form by standard Cell waste and degradation routes can be processed. Cells can also possess enzymes that potentially have one or more of these can specifically degrade toxic substances, causing the cell is protected. Principles and examples of these types of Modification and breakdown processes in bacteria are in several Publications described, for example. Sahm, H .. (1999) "Procaryotes in Industrial Production "in Lengler, J.W. et al., ed. Biology of the Procaryotes, Thieme Published by Stuttgart, and Schlegel, H. G. (1992) General Microbiology, Thieme, Stuttgart).

In addition to simply inactivating harmful aromatic or Aliphatic compounds have many bacteria like this adapted to use these compounds as carbon sources for can use a continuous metabolism if the preferred carbon sources of the cells are not available. For example. Pseudomonas strains are known, the toluene, benzene and Can use 1,10-dichlorodecane as carbon sources (Chang, B.V. et al. (1998) Chemosphere 35 (12): 2807-2815; Wischnak, C. et al. (1988) Appl. Environ. Microbiol. 64 (9): 3507-3511; Churchill, S.A. et al. (1999) Appl. Environ. Microbiol. 65 (2): 549-552). It are similar examples of many other types of bacteria found in Are known in the art.

One has started the ability of certain bacteria to be aromatic and modify or degrade aliphatic compounds, exploit. Crude oil is a complex mixture of chemicals that contains aliphatic molecules and aromatic compounds. By Application of bacteria that these toxic compounds from Leaking or modifying leaking oil can be done in many ways Curb environmental damage with high efficiency and low costs (See, for example, Smith, M.R. (1990) "The biodegradation of aromatic hydrocarbons by bacteria "Biodegradation 1 (2-3): 191-206; and Suyama, T. et al. (1998> "Bacterials isolates degrading aliphatic polycarbonates "FEMS Microbiol. Lett. 161 (2): 255-261).

B. Metabolism of inorganic compounds

Cells (e.g. bacterial cells) contain large amounts different molecules, such as water, inorganic ions and organic substances (e.g. proteins, sugar and other Macromolecules). The main mass of an ordinary cell consists of 4. Atom types: carbon, oxygen, hydrogen and nitrogen. Inorganic substances only make a smaller one Percentage of cell content, however, they are correct Functioning of the cell is equally important. These molecules include phosphorus, sulfur, calcium, magnesium, iron, zinc, Manganese, copper, molybdenum, tungsten and cobalt. Many of these Connections are essential for building molecules, such as nucleotides (Phosphorus) and amino acids (nitrogen and sulfur) from Importance. Others of these inorganic ions serve as cofactors for Enzyme reactions or contribute to osmotic pressure. All these Molecules must be absorbed by the bacterium from the environment become.

For each of these organic compounds, it is desirable that the bacterium takes the form that the Standard cell metabolism machinery is the easiest to use can. However, the bacterium can encounter environments in which these preferred forms are not readily available. Around to survive in such conditions, it is important that the bacteria have additional biochemical mechanisms, the less metabolically active but readily available forms can convert these inorganic compounds into forms that can be used by cell metabolism. Possess bacteria often a set of genes that make enzymes for this purpose encode that are only expressed when the desired ones inorganic species are not available. These genes for the Metabolism of various inorganic compounds serve as another tool, the bacteria to adapt to suboptimal Can use environmental conditions.

After carbon, nitrogen is the most important element. An ordinary bacterial cell contains 12 to 15% nitrogen. It is part of amino acids and nucleotides as well as many other molecules in the cell. Nitrogen can also serve as a replacement for oxygen as a terminal electron acceptor in energy metabolism. Good sources of nitrogen include many organic and inorganic compounds such as ammonia gas or ammonium salts (e.g. NH ₄ Cl, (NH ₄ ) ₂ SO ₄ or NH ₄ OH), nitrates, urea, amino acids or complex nitrogen sources such as masiquell water, soy flour , Soy protein, yeast extract, meat extract etc. Ammonium nitrogen is fixed by the action of certain enzymes: glutamate dehydrogenase, glutamine synthase and glutamine-2-oxoglutarate aminotransferase. Amino nitrogen is transferred from one organic molecule to another by aminotransferases, a class of enzymes that transfer an amino group from an alpha amino acid to an alpha keto acid. Nitrate can be reduced by nitrate reductase, nitrite reductase and other redox enzymes until it is converted to molecular nitrogen or ammonia, which are easily used by the cell in standard metabolic pathways.

Phosphorus becomes intracellular in organic and inorganic Forms found and can be taken up by the cell in both forms although most microorganisms are preferred absorb inorganic phosphate. The conversion of organic phosphate in a form that the cell can use requires the effect of phosphatases (e.g. phytases, the phytate-producing phosphate and hydrolyze inositol derivatives). Phosphate is a Key component of the synthesis of nucleic acids and thus plays a role significant role in cellular energy metabolism (e.g. in the synthesis of ATP, ADP and AMP).

Sulfur is for the synthesis of amino acids (e.g. methionine and Cysteine), vitamins (e.g. thiamine, biotin and lipoic acid) and Iron-sulfur proteins necessary. Bacteria get sulfur predominantly from inorganic sulfate, although thiosulfate, sulfite, and Sulfide is also commonly used. Under conditions, under to whom these compounds are not readily available many bacteria genes that allow them Use sulfonate compounds such as 2-aminosulfonate (taurine) (Kertesz, M. A. (1993) "Proteins induced by sulfate limitation in Escherichia coli. Pseudomonas putida or Staphyloccocus aureus "J. Bacteriol. 175: 1187-1190).

Other inorganic atoms, for example metal or calcium ions, are also crucial for the viability of cells. iron plays a key role in redox reactions and is a Cofactor of iron-sulfur proteins, heme proteins, and Cytochromes. The absorption of iron in the bacterial cell can be caused by the effect of siderophores, chelating agents, the extracellular Bind iron ions and carry them inside the cell, respectively. For the metabolism of iron and other inorganic Compounds, see: Lengler et al., (1999) Biology of Prokaryotes, Thieme Published by Stuttfart; Neidhardt, F.C., et al., Ed. Escherichia coli and Salmonella. ASM Press: Washington, DC; Sonenshein, A.L. et al., Ed. (1996) Bacillus subtilis and other Gram-Positive Bacteria, ASM-Press: Washington, D. C :; Voet, D. and Voet, J.G. (1992) Biochemistry, VCH: Weinheim; Brock, T. D. and Madigan, M. T. (1991) Biology of Microorganisms, 6th Edition Prentice Hall: Eaglewood Cliffs, pp. 267-269; Rhodes, P.M. and Stanbury, P.F. Applied Microbial Physiology - A Practical Approach, Oxford Univ. Press: Oxford.

C. Enzymes and Proteolysis

The intracellular conditions, for the bacteria, like C. glutamicum, are often not conditions under which usually many biochemical reactions would take place. So that these reactions take place under physiological conditions, the cells use enzymes. Enzymes are biological Protein catalysts that spatially orientate the reacting molecules or create a specialized environment so that the energy hurdle for a biochemical reaction. different Enzymes catalyze different reactions, and every enzyme can be a transcription, translation or subject to post-translational regulation, so that the reaction only under the suitable conditions and at certain times. enzymes can be used for degradation (e.g. proteases), synthesis (e.g. synthases), or modification (e.g. transferases or isomerases) of Connections contribute to all the production necessary Enable connections in the cell. This in turn contributes to Maintaining cellular homeostasis.

The fact that enzymes are among the most active physiological conditions are optimized, under which the The most viable bacterium, however, means that the Enzyme activity in the event of a disturbance in the environmental conditions is most likely disturbed. For example. can change temperature produce aberrantly folded proteins, and the same applies to pH Value changes - protein folding is highly dependent on electrostatic and hydrophobic interactions of the amino acids within the polypeptide chain so that any change in charges individual amino acids (as, for example, by changing the cellular pH value) has a significant effect the ability of the protein to fold correctly. Changes in temperature efficiently change the amount kinetic energy that the polypeptide molecule possesses, which the Ability of the polypeptide to affect itself correctly folded, energetically stable configuration. Not correct folded proteins can serve the cell for two reasons be dangerous. First, the aberrantly folded protein can be a similarly aberrant activity or have no activity. Secondly, incorrectly folded proteins cannot Conformational areas that are necessary for the correct regulation by others cellular systems are necessary and can continue to do so be active, but in an uncontrolled manner.

The cell has a mechanism by which misfolded Enzymes and regulatory proteins can be destroyed quickly, before the cell is damaged: proteolysis. Proteins like those of the la / lon family and those of the Clp family and specifically break down incorrectly folded proteins (see e.g. Sherman, M.Y., Goldberg, A.L. (1999) EXS 77: 57-78 and those therein references, as well as Porankiewicz, J. (1999) Molec. Microbiol. 32 (3). 449-58 and contained therein References; Neidhardt, F.C. et al. (1996) E. coli and Salmonella, ASM Press: Washington, D.C. and references therein; and Pritchard, G.G. and Coolbear, T. (1993) FEMS Microbiol. Rev. 12 (1-3): 179-206 and references cited therein). This Enzymes bind to incorrectly folded or unfolded proteins and break them down in an ATP-dependent manner. Proteolysis serves thus as an important mechanism used by the cell is used to restore normal cell functions to environmental changes Preserve damage, and also enables the cell to underneath Conditions and survive in environments that are due to a improperly regulated and / or aberrant enzyme or regulatory activity would otherwise be toxic.

Proteolysis also has important functions in the cells under optimal environmental conditions. Within normal Metabolic processes, proteases support the hydrolysis of Peptide bonds in the catabolism of complex molecules to the necessary Provide breakdown products, and in protein modification. Secreted proteases play in the catabolism of external nutrients even before these compounds enter the cell important role. The proteolytic activity itself can also perform regulatory functions; the sporulation in B. subtilis and the course of the cell cycle in Caulobactet.spp. Become as is well known through proteolytic key events in everyone of these species regulated (Gottesman, S. (1.999) Curr. Opin. Microbiol. 2 (2): 142-147). So proteolytic processes are key for cellular survival under suboptimal and optimal Environmental conditions, and contribute to overall homeostasis conservation Cells at.

D. Cell wall production and rearrangements

The cell's biochemical machinery can adhere easily adjust other or possibly adverse environments, however the cells still need a general mechanism through which they are protected from the environment. Offers for many bacteria the cell wall has such protection, which also plays a role in Adhesion, in cell growth and division and in Transport of the desired solutes and waste materials plays.

The cells need - in order to be functional - intracellular Concentrations of metabolites and other molecules in the Are substantially above those of the surrounding media. This one Metabolites are largely prevented from being due to the cell leaving the hydrophobic membrane there is a tendency that the system water molecules from the external medium into the cell leaves, so that the inner concentrations of the solutes the correspond to external concentrations. Water molecules can easily pass the cell membrane, and this membrane can resulting sources and the pressure do not withstand what osmotic lysis of the cell. The strength of the Cell greatly improves the cell's ability to withstand these pressures to withstand, and offers another barrier for the unwanted diffusion of these metabolites and the desired ones dissolved substances from the cell. The cell wall also prevents unwanted material gets into the cell.

The cell wall also takes part in a number of other cellular Processes such as adhesion, cell growth and division. Due to the fact that the cell wall completely covers the cell surrounds any interaction of the cell with its surroundings are mediated through the cell wall. The cell wall must therefore be on each attachment of the cells to other cells and to desired ones Surfaces to be involved. The cell also cannot grow or divide without simultaneous changes in the cell wall. Because the protection that the wall offers, also with the growth, with the Morphogenesis and propagation must be present is one of the Key steps in cell division in cell wall synthesis Cell so that a new cell splits from the old one. The Cell wall synthesis is thus often associated with cell growth and Cell division tandem regulated (see e.g. Sonenshein, A. L. ed. (1993) Bacillus subtilis and Other Gram-Positive Bacteria, ASM: Washington, D.C.).

The structure of the cell wall varies between gram-positive and gram-negative bacteria. The basic remains in both types Structure similar to the wall: an overlapping grid of two Polysaccharides, N-acetyl-glucosmain (NAG) and N-acetylmuramic acid (NAM), which contains amino acids (mostly L-alanine, D-glutamate, Diaminopimelinsäure and D-alanine) are cross-linked, which as "peptidoglycan" referred to as. The processes involved in cell wall synthesis are known (see, for example, Michal, G. Ed. (1999) Biochmeical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York).

In the case of gram-negative bacteria, the inner cell membrane has one single-layer peptidoglycan layer coated (about 10 nm thick), which is called Murein sacculus. The Peptidoglycan structure is very strong and its structure determines the shape of the organism. The outer surface of the Murein sacculus is with an outer membrane that covers porins and other membrane proteins, Contains phospholipids and lipospolysaccharides. to Maintaining a firm bond with the outer membrane has the gram- negative cell wall, lipid molecules scattered in it, which they in the Anchor the surrounding membrane.

Gram-positive bacteria such as Corynebacterium glutamicum the cytoplasmic membrane with a multilayer, approximately 20 to 80 nm thick peptidoglycan covered (see, for example, Lengeler et al. (1999) Biology of Prokaryotes Theime-Verlag: Stuttgart, pp. 913-918, p. 875-899 and pp. 88-109 and those listed therein References). The gram-positive cell wall also contains tonic acid, a Polymer made from glycerin or ribitol that has a phosphate group are linked together. Pond acid can also be combined with amino acid associate and form covalent bonds with muramic acid. In the cell wall also needs lipoteichoic acids and Ponduronic acids be present. If present, they become cellular Surface structures, such as flagella or capsules, also in this Layer of the wall anchored.

III. Elements and methods according to the invention

The present invention is based at least in part on the Discovery of new molecules, here called HA nucleic acid and Protein molecules are referred to and maintaining the homeostasis involved in C. glutamicum, or a Exercise function in adapting this microorganism different environmental conditions is involved. At a Embodiment take the HA molecules at the C. glutamicum cell wall synthesis or rearrangements, the metabolism of inorganic Compounds, modification or degradation of aromatic or Aliphatic compounds, or have an enzymatic or proteolytic activity. In a particularly preferred Embodiment has the activity of the invention HA molecules with regard to cell wall biosynthesis or rearrangements, the metabolism of inorganic compounds, the modification or the degradation of aromatic or aliphatic compounds or an impact on enzymatic or proteolytic activity on the production of a desired fine chemical by this Organism. In a particularly preferred embodiment the HA molecules according to the invention are modulated in this way Activity that the cellular processes in C. glutamicum to which the HA molecules are involved (e.g. C. glutamicum cell wall biosynthesis or rearrangement, metabolism of inorganic compounds, Modification or degradation of aromatic or aliphatic Compounds or enzymatic or proteolytic activity), also have altered activity, which is either direct or indirectly a modulation of the yield, production and / or Efficiency of producing a desired fine chemical by C. causes glutamicum.

The term "HA protein" or "HA polypeptide" includes proteins involved in a number of cellular processes that with C. glutamicum homeostasis or the ability of C. glutamicum cells for adaptation to unfavorable environmental conditions related. For example. can an HA protein on the C. glutamicum cell wall biosynthesis or rearrangements, metabolism inorganic compounds in C. glutamicum, on the modification or on Degradation of aromatic or aliphatic compounds in C. glutamicum, or an enzymatic or proteolytic C. have glutamicum activity. Examples of HA proteins include those of those listed in Table 1 and Appendix A. HA genes can be encoded. The expression "HA-Gen" or "HA nucleic acid sequence" include nucleic acid sequences that contain an HA protein encode that from a coding area and corresponding untranslated 5 'and 3' sequence regions. Examples for HA genes are listed in Table 1. The terms "Production" or "productivity" are known in the art and include the concentration of the fermentation product (e.g. the desired fine chemical within a set Time period and a fixed fermentation volume formed (e.g. kg product per hour per 1). The term "efficiency of production "comprises the time it takes to achieve a certain production volume is necessary (e.g. how long the cell is to Establishing a certain throughput rate of a fine chemical required). The term "yield" or " Product / Carbon Yield "is known in the art and encompasses the efficiency of the Converting the carbon source into the product (i.e. the Fine chemical). For example, this is usually expressed as kg of product per kg of carbon source. By increasing the yield or Production of the compound is the amount of molecules obtained or the appropriate recovered molecules of this compound in a certain amount of culture over a fixed period of time elevated. The terms "biosynthesis" or "biosynthetic pathway" are used in Known in the art and include the synthesis of a compound preferably an organic compound, through a cell Interconnections, for example in a multi-step or strong regulated process. The terms "degradation" or "degradation route" are in Known in the art and include splitting a connection, preferably an organic compound, through a cell in Breakdown products (more generally, smaller or less complex Molecules), for example in a multi-step or highly regulated Process. The term "metabolism" is known in the art and encompasses all that takes place in an organism biochemical reactions. The metabolism of a certain one Compound (e.g. the metabolism of an amino acid such as glycine) then all the biosynthetic, modification and degradation pathways Connection in the cell. The term "homeostasis" is in the Known in the art and includes all mechanisms by the cell used to maintain a constant intracellular environment despite to maintain the prevailing extracellular conditions. A non-limiting example of these processes is that Use of the cell wall to prevent osmotic lysis due to the high intracellular concentrations of dissolved Substances. The term "adjustment" or "adjustment to a." Environmental Condition "is known in the art and includes mechanisms by the cell cannot be used to make the cell survive enabling preferred environmental conditions (generally speaking, those environmental conditions in which one or more favorable Nutrients are missing or in which an environmental condition such as Temperature, pH, osmolarity, percentage oxygen and the like outside the optimal survival range of the cell lie). Many cells, including C. glutamicum cells, possess genes that encode proteins that are among them Environmental conditions are expressed, and those in these suboptimal Conditions allow for continuous growth.

In a further embodiment, the HA- Molecules to modulate a desired molecule, such as one Fine chemical, in a microorganism such as C. glutamicum, capable. There are a number of mechanisms through which the Change of a HA protein according to the invention the yield, Production and / or efficiency of the production of a fine chemical a C. glutamicum strain that contains such an altered protein contains, are directly influenced. By modifying enzymes, the aromatic or aliphatic compounds like this modify that these enzymes have a higher or lower activity or Number can show the production of one or more Fine chemicals that are the modification or degradation products of this Connections are, modulate. Accordingly, enzymes that are involved in the metabolism of inorganic compounds, Key molecules (e.g. phosphorus, sulfur and Nitrogen molecules) for the biosynthesis of such fine chemicals, such as Amino acids, vitamins and nucleic acids, ready. By changing the Activity or the number of these enzymes in C. glutamicum can be seen increase the conversion of these inorganic compounds (or use alternative inorganic compounds), so improved rates of incorporation of inorganic atoms into them To enable fine chemicals. The genetic manipulation of C. glutamicum enzymes involved in general cellular processes can also directly improve fine chemical production, because many of these enzymes contain fine chemicals (e.g. amino acids) or the enzymes involved in fine chemical synthesis or -secretion involved, modify directly. The modulation of the Activity or number of cellular proteases can also be a have a direct effect on fine chemical production, as many Proteases fine chemicals or enzymes involved in the Involved in the production or mining of fine chemicals. The The above-mentioned enzymes that are involved in the modification or degradation aromatic or aliphatic compounds, on the general Biocatalysis, on the metabolism of inorganic compounds or on the proteolysis involved are each self Fine chemicals because of their activity in various industrial in vitro applications are desirable. By changing the Copy number of the gene for one or more enzymes in C. glutamicum you can see the number of these proteins produced by the cell will increase, increasing the potential yield or efficiency the production of these proteins from large-scale cultures of C; glutamicum or related bacteria is increased.

The modification of an HA protein according to the invention can Yield, production and / or efficiency of producing one Fine chemical from a C. glutamicum strain containing one contains modified protein, also affect indirectly. By Modulating the activity and / or the number of these proteins, which on Building or rearrangement of the cell wall are involved, one can For example, modify the structure of the cell wall itself so that the Cell the mechanical or other stress that occurs in large-scale Prevails, can withstand better. moreover requires the growth of C. glutamicum on a large scale substantial cell wall production. The modulation of the activity or the number of cell wall biosynthesis or degradation enzymes enable faster cell wall biosynthesis rates, which in turn stronger growth rates of this microorganism in culture enables and thereby increases the number of cells that the produce the desired fine chemical.

By modifying the HA enzymes according to the invention, the Yield, production or efficiency of the production of one or indirectly influence several fine chemicals from C. glutamicum. For example. many of the common enzymes in C. glutamicum have one significant impact on global cellular processes (e.g. regulatory processes), which in turn is significant Has an impact on the fine chemical metabolism.

Accordingly, proteases, enzymes, may increase modify toxic aromatic or aliphatic compounds or degrade the viability of C. glutamicum. The proteases support the selective removal of misfolded or incorrectly regulated proteins, such as those, for example, among the relative stressful environmental conditions in a large fermenter culture occur. By changing these proteins, you can still change them Increase activity and the viability of C. glutamicum in Improve culture. The aromatic / aliphatic modification or degradation proteins are not only used to detoxify them Waste compounds (which in the culture medium as impurities or occur as waste products from the cells themselves), but also allow the cell to have alternative carbon sources uses if the optimal carbon source in the culture is limited. By increasing the number and / or the activity can the survival of C. glutamicum cells in culture is enhanced become. The inorganic metabolic proteins according to the invention supply the cell with the inorganic molecules required for all protein and nucleotide syntheses (among others) necessary and thus for the overall viability of the cell are crucial. An increase in the number of viable cells that produce one or more fine chemicals in large culture, should a simultaneous increase in yield, production and / or efficiency of fine chemical production in culture cause.

As a starting point for the production of the invention The genome of a Corynebacterium is suitable for nucleic acid sequences glutamicum strain, owned by the American Type Culture Collection the designation ATCC 13032 is available.

These nucleic acid sequences can be identified by the in Table 1 designates the changes according to the invention Prepare nucleic acid sequences using conventional methods.

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

A. Isolated nucleic acid molecules

One aspect of the invention relates to isolated nucleic acid molecules, the HA polypeptides or biologically active portions thereof encode, as well as nucleic acid fragments for use as Hybridization probes or primers for identification or Amplification of HA-coding nucleic acids (e.g. HA-DNA) are sufficient. The term "nucleic acid molecule" is intended to mean DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g. mRNA) as well as DNA or RNA analogs generated using nucleotide analogs include. This term also includes those at the 3 'and 5' ends of the coding gene region located untranslated sequence: 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. On "Isolated" nucleic acid molecule is made from others Nucleic acid molecules separated in the natural source of the nucleic acid are present. An "isolated" nucleic acid preferably has no sequences that the nucleic acid in the genomic DNA of the Organism from which the nucleic acid originates, naturally flank (e.g. sequences that are at the 5 'or 3' end of the Nucleic acid). In various embodiments for example the isolated HA nucleic acid molecule is less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of Nucleotide sequences that naturally contain the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid originates (e.g. a Flank C. glutamicum cell). An "isolated" Nucleic acid molecule, such as a cDNA molecule, can also be essentially free from another cellular material or culture medium, if made by recombinant techniques, or free of chemical precursors or other chemicals if there is is chemically synthesized.

A nucleic acid molecule according to the invention, for example one Nucleic acid molecule with a nucleotide sequence from Appendix A or Section of it can be done using molecular biological Standard techniques and the sequence information provided here isolated become. For example. can a C. glutamicum HA cDNA from a C. Glutamicum bank can be isolated by a complete sequence Appendix A or a section thereof as a hybridization probe and Standard hybridization techniques (such as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory manual. 2nd edition Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) be used. In addition, a nucleic acid molecule comprising a complete sequence from Appendix A or a section isolate thereof by polymerase chain reaction, the Oligonucleotide primer created based on this sequence have been used (e.g. a nucleic acid molecule, comprising a complete sequence from Appendix A or a section of which are isolated by the polymerase chain reaction by Oligonucleotide primers can be used based on this same sequence from Appendix A). For example. leaves isolate mRNA from normal endothelial cells (e.g. through the Guanidinium thiocyanate extraction method by Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and the cDNA can be determined using reverse transcriptase (e.g. Moloney MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Petersburg, FL) can be produced. Synthetic oligonucleotide primers for the Amplification via polymerase chain reaction can be carried out on the Basis of one of the nucleotide sequences shown in Appendix A. create. A nucleic acid according to the invention can be by means of cDNA or alternatively, genomic DNA as a template and suitable Oligonucleotide primers according to standard PCR amplification techniques be amplified. The nucleic acid amplified in this way can be converted into a appropriate vector can be cloned and by DNA sequence analysis be characterized. Oligonucleotides, one HA nucleotide sequence can be matched by standard synthetic methods, For example, with an automatic DNA synthesizer become.

In a preferred embodiment, a Isolated nucleic acid molecule according to the invention one of the in Appendix A nucleotide sequences listed. The sequences of Appendix A correspond to the Corynebacterium HA cDNAs according to the invention glutamicum. These cDNAs include sequences that contain HA proteins (i.e., the "coding area" as specified in each sequence in Appendix A. ) and the 5 'and 3' untranslated sequences that are also given in Appendix A. The nucleic acid molecule can alternatively only the coding region of one of the sequences in Include Appendix A.

In a further preferred embodiment, a isolated nucleic acid molecule according to the invention one of the in The nucleotide sequences shown in Appendix A are complementary Nucleic acid molecule or a portion thereof, which is a Nucleic acid molecule that is one of those shown in Appendix A. Nucleotide sequences is sufficiently complementary that it is with a of the sequences given in Appendix A can hybridize, whereby a stable duplex is created.

In a further preferred embodiment, a Isolated nucleic acid molecule according to the invention Nucleotide sequence that is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90% or 90-95% and more preferably at least about 95%, 96%, 97%, 98%, 99% or even more homologous to one given in Appendix A. Nucleotide sequence or a portion thereof. Another preferred embodiment comprises an inventive isolated nucleic acid molecule a nucleotide sequence which, for example, under stringent conditions, with one of those shown in Appendix A. Nucleotide sequences or a portion thereof hybridized.

The nucleic acid molecule according to the invention can moreover only have one Section of the coding region from one of the sequences in Appendix A includes, for example, a fragment that acts as a probe or primer or fragment can be used, which a biological encoded active portion of an HA protein. The one from the cloning of the HA genes from C. glutamicum determined nucleotide sequences enable the generation of probes and primers which are used for Identification and / or cloning of HA homologs in other cell types and organisms and HA homologs from other Corynebacteria or related species are designed. The probe or the primer usually include essentially purified oligonucleotide. The Oligonucleotide usually includes a nucleotide sequence region under stringent conditions on at least about 12, preferably about 25, more preferably about 40, 50 or 75 successive nucleotides of a sense strand from one of those in Appendix A sequences indicated, an antisense strand from one of the sequences given in Appendix A or naturally occurring Mutants hybridized therefrom. Primer based on a Nucleotide sequence from Appendix A can be used in PCR reactions for cloning from HA homologs. Probes based on the HA nucleotide sequences can be used to detect transcripts or genomic sequences that are the same or homologous proteins encode, are used. Included in preferred embodiments the probe also includes a label group attached thereto, e.g. Radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. These probes can be part of a diagnostic test kits used to identify cells that misexpress an HA protein, such as by measuring an amount an HA-encoding nucleic acid in a cell sample, e.g. Measure the HA mRNA level or by determining whether a genomic HA gene is mutated or deleted.

In one embodiment, the invention encodes Nucleic acid molecule a protein or a portion thereof, the one Amino acid sequence that is sufficiently homologous to one Amino acid sequence from Appendix B is that the protein or a section of which continue to maintain homeostasis in C. gIutamicum may be involved, or may perform a function who adapt this microorganism to different ones Environmental conditions is involved. As used here, the Term "sufficiently homologous" proteins or sections thereof, whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (e.g. an amino acid test with a similar side chain as an amino acid residue in one of the Sequences from Appendix B) to an amino acid sequence from Appendix B have so that the protein or a portion thereof continues to the Maintaining homeostasis in C. glutamicum may be involved can, or can perform a function related to the adjustment this microorganism involved in various environmental conditions is. Proteins involved in C. glutamicum cell wall biosynthesis or to rearrangements, to the metabolism of inorganic compounds modification or degradation of aromatic or aliphatic Compounds are involved or an enzymatic or have proteolytic C. glutamicum activity, as described here, can play a role in the production and secretion of one or play several fine chemicals. Examples of these activities are also described here. Thus, the "function of a HA protein "either directly or indirectly the yield, Production and / or efficiency of the production of one or more Fine chemicals. In Table 1 are examples of the HA protein activities indicated.

In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90%, 90-95% and most preferably at least about 96%, 97%, 98%, 99% or even more homologous a complete amino acid sequence in Appendix B.

Sections of proteins by those of the invention HA nucleic acid molecules are encoded are preferably biological active sections of one of the HA proteins. The term "Biologically active section of an HA protein" as used here a section, e.g. a domain or a motif, of an HA protein involved in the maintenance of the Homeostasis in C. glutamicum can be involved, or one Can perform function on the adaptation of this microorganism different environmental conditions is involved, or one in Activity shown in Table 1. To determine whether an HA Protein or a biologically active portion thereof at the C. glutamicum cell wall biosynthesis or rearrangements, on Metabolism of inorganic compounds, on modification or on Degradation of aromatic or aliphatic compounds is involved or an enzymatic or proteolytic C. Having glutanicum activity can be a test of enzymatic activity be performed. This test procedure, as described in detail in Example 8 of the example part are familiar to the person skilled in the art.

Additional nucleic acid fragments that are biologically active Coding sections of an HA protein can be isolated a portion of one of the sequences in Appendix B, Express the encoded portion of the HA protein or peptide (e.g. by recombinant expression in vitro) and determining the Activity of the coded portion of the HA protein or peptide produce.

The invention also encompasses nucleic acid molecules that differ from one of the nucleotide sequences (and sections of this) due to the degenerate genetic code and thus encode the same HA protein as that of the nucleotide sequences shown in Appendix A is encoded. In another embodiment has an insulated according to the invention Nucleic acid molecule is a nucleotide sequence that contains a protein an amino acid sequence shown in Appendix B. In a another embodiment encodes the invention Nucleic acid molecule. C. glutamicum full-length protein that leads to a Amino acid sequence from Appendix B (encoded by one in Appendix A shown open reading frame) is essentially homologous.

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

B. Recombinant Expression Vectors and Host Cells

Another aspect of the invention relates to vectors, preferably Expression vectors that contain a nucleic acid that contains an HA Encode protein (or a portion thereof). As used here the term "vector" refers to a nucleic acid molecule that contains a can transport other nucleic acid to which it is bound is. A vector type is a "plasmid", what a circular double-stranded DNA loop is in the additional DNA segments can be ligated. Another type of vector is viral Vector, with additional DNA segments in the viral genome can be ligated. Certain vectors can be in one Autonomously replicate the host cell into which they have been introduced (e.g. bacterial vectors, with bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g. non-episomal mammalian vectors) are inserted into the genome of a host cell integrated into the host cell when it is introduced and thus together replicated with the host genome. In addition, certain vectors the expression of genes with which they are operably linked are, control. These vectors are called "expression vectors" designated. Usually the expression vectors used in Recombinant DNA techniques are used in the form of Plasmids. In the present description, "plasmid" and "Vector" can be used interchangeably because the plasmid is the most most common vector form is. The invention aims at this other expression vector forms, such as viral vectors (e.g. replication-deficient retroviruses, adenoviruses and adeno-related Viruses) that perform similar functions.

The recombinant expression vector according to the invention comprises a Nucleic acid according to the invention in a form which is for 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 Expression to use host cells with the to expressing nucleic acid sequence is operatively linked. In a recombinant expression vector means "functional linked "that the nucleotide sequence of interest to the regulatory sequence (s) is linked to the expression the nucleotide sequence is possible (e.g. in a 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 be promoters, enhancers and others Expression control elements (for example. Polyadenylation signals) include. This regulatory sequences are described, for example, in Goeddel: genes Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which is the constitutive expression of a nucleotide sequence in many Control host cell types, and those that direct expression control the nucleotide sequence only in certain host cells. The Those skilled in the art are aware that the design of a Expression vector can depend on factors such as the choice of transforming host cell, the level of expression of the desired protein, etc. The expression vectors according to the invention can be introduced into the host cells, so that Proteins or peptides, including fusion proteins or -peptides encoded by the nucleic acids as described here are produced (e.g. HA proteins, mutated forms of HA proteins, fusion proteins, etc.).

The recombinant expression vectors according to the invention can for the expression of HA proteins in prokaryotic or eukaryotic cells. For example. can HA genes in bacterial cells, such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A. et al. (1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488; by den Hondel, C.A.M.J. J. et al. (1991) "Heterologous gene expression in filamentous fungi" in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, Ed., Pp. 396-428: Academic Press: San Diego; and from the gondola, C.A.M. J.J. & Punt, P.J. (1991). "Gene transfer systems and vector development for filamentous fungi. in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds. pp. 1-28, Cambridge University Press: Cambridge), algae and multicellular Plant cells (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrobacterium tumefaciens-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). The recombinant expression vector can alternatively, for example with T7 promoter regulatory sequences and T7 polymerase, be transcribed and translated in vitro.

The expression of proteins in prokaryotes is usually done with Vectors that are constitutive or inducible promoters contain the expression of fusion or non-fusion proteins Taxes. Fusion vectors target a number of amino acids a protein encoded therein, usually at the amino terminus of the recombinant protein, at. Have these fusion vectors usually three tasks: 1) enhancing the expression of recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) support the cleaning of the recombinant protein by acting as a ligand in the Affinity purification. Fusion expression vectors often have one proteolytic cleavage site at the junction of the fusion unit and of the recombinant protein, so that the separation of the recombinant protein from the fusion unit after purification of the fusion protein is possible. These enzymes and theirs corresponding recognition sequences include factor Xa, thrombin and Enterokinase.

Common fusion expression vectors include pGEX (Pharmacia Biotech Inc. Smith, D.B. and Johnson, K.S. (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 to the recombinant Target protein is fused. In one embodiment, the coding sequence of the HA protein into a pGEX expression vector cloned so that a vector is generated which is a fusion protein encoded, comprising from the N-terminus to the C-terminus, GST - thrombin- Cleavage site-X protein. The fusion protein can by Affinity chromatography purified using glutathione-agarose resin become. The recombinant HA protein that does not fuse with GST is, by cleaving the fusion protein with thrombin be won.

Examples of suitable inducible ones E. coli non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). The 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 pET11d vector is based on the transcription of one T7-gn10-lac fusion promoter co-expressed by a viral RNA polymerase (T7 gn1) is mediated. This viral Polymerase is derived from the host strains BL 21 (DE3) or HMS174 (DE3) supplied by a resident λ prophage using a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter harbors.

A strategy to maximize the expression of the recombinant Protein is the expression of the protein in a host bacterium, its ability to proteolytically cleave the recombinant Protein is disturbed (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 in an expression vector insert nucleic acid so that the individual codons for each amino acid are those that are preferably in one for expression selected bacteria, such as C. glutamicum, can be used (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). This change The nucleic acid sequences according to the invention are carried out by Standard DNA synthesis techniques.

In a further embodiment, the HA protein expression vector is a yeast expression vector. Examples of vectors for Expression in the yeast S. cerevisiae include pYepSec1 (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 method of constructing vectors that differ for use in other mushrooms, such as filamentous mushrooms, suitable include those detailed in: from den Hondel, C.A.M.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, the HA proteins according to the invention can be used in Insect cells using baculovirus expression vectors be expressed. Baculovirus vectors used for. Expression of Proteins in cultured insect cells (e.g. Sf9 cells) are available include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

In a further embodiment, the inventive HA proteins in single-cell plant cells (such as algae) or in Plant cells of higher plants (e.g. spermatophytes, such as Field crops) are expressed. examples for Plant expression vectors include those 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, an inventive Nucleic acid in mammalian cells with a Mammalian expression vector expressed. 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 using in Mammalian cells become the control functions of the expression vector often provided by viral regulatory elements. Commonly used promoters come, for example, from Polyoma, adenovirus2, Cytomegalovirus and Simian Virus 40. For other suitable ones Expression systems for prokaryotic and eukaryotic cells, see chapters 16 and 17 from Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

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

The invention also provides a recombinant expression vector ready comprising a DNA molecule according to the invention, which in Antisense direction is cloned into the expression vector. This means that the DNA molecule has such a regulatory sequence is functionally connected that the expression (by Transcription of the DNA molecule) of an RNA molecule that leads to HA mRNA is antisense is possible. There can be regulatory sequences be selected that are functional to one in Antisense direction cloned nucleic acid and which are bound continuous expression of the antisense RNA molecule in a variety of Control cell types, for example. Viral promoters and / or Enhancers or regulatory sequences that are selected constitutive, tissue-specific or cell type-specific expression control of antisense RNA. The antisense expression vector can in the form of a recombinant plasmid, phagemid or attenuated Virus are present in the antisense nucleic acids under the Control of a highly effective regulatory area whose activity is determined by the cell type in which the vector is introduced. For a discussion of regulation 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, in which is a recombinant expression vector according to the invention has been introduced. The terms "host cell" and "recombinant Host cell "are used interchangeably here. It it goes without saying that these terms are not just one certain target cell, but also the offspring or potential Descendants of this cell affect. Because in successive Generations determined due to mutation or environmental influences Modifications can occur, these offspring are not absolutely identical to the parental cell, but are in scope of the term as used herein.

A host cell can be a prokaryotic or eukaryotic cell his. For example. can an HA protein in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as ovarian cells Chinese hamster (CHO) or COS cells) become. Other suitable host cells are known to the person skilled in the art. Microorganisms related to Corynebacterium glutamicum and are suitable as host cells for the invention Nucleic acid and protein molecules can be used are in the table 3 listed.

Through conventional transformation or transfection processes vector DNA can be expressed in prokaryotic or eukaryotic cells contribute. The terms "transformation", "transfection", "Conjugation" and "transduction" as used here are intended to be a variety of methods known in the art for introducing foreign nucleic acid (e.g. DNA) into a Host cell, including calcium phosphate or Calcium chloride coprecipitation, DEAE-Dektran-mediated transfection, Lipofection or electroporation. Suitable procedures for Transformation or transfection of 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 others Laboratory manuals.

For the stable transfection of mammalian cells it is known that depending on the expression vector used and Transfection technology only a small part of the cells have the foreign DNA in it Integrated genome. To identify and select them Integrants usually become a gene that has a selectable marker (e.g. resistance to antibiotics) encoded together with the gene of interest introduced into the host cells. preferred selectable markers include those that are resistant to Grant drugs such as G418, hygromycin and methotrexate. A Nucleic acid encoding a selectable marker can be converted into a host cell can be introduced on the same vector as the one that encodes an HA protein, or can be on a separate Vector. Cells brought in with the Nucleic acid have been stably transfected by Drug selection can be identified (e.g. cells that make up the integrated selectable markers survive, whereas the other cells die).

To generate a homologously recombined microorganism made a vector that has at least a portion of an HA Contains gene in which a deletion, addition or substitution has been introduced to change the HA gene, e.g. functionally disrupt. This HA gene is preferably a Corynebacterium glutamicum-HA gene, however, can be a homologue of a related bacterium or even from a mammalian, yeast or source of insects. In a preferred one Embodiment, the vector is designed such that the endogenous HA gene is functionally disrupted in homologous recombination (i.e. no longer encodes a functional protein, either referred to as the "knockout" vector). The vector can alternatively be designed such that the endogenous HA gene in homologous Recombination is mutated or otherwise changed, however encodes the functional protein (e.g., upstream located regulatory area be changed so that the expression of the endogenous HA protein is changed.). The altered section of the HA gene is in the homologue Recombination vector at its 5 'and 3' ends of additional nucleic acid of the HA gene flanked by a homologous recombination between the exogenous HA gene carried by the vector and one endogenous HA gene in a microorganism. The Additional flanking HA nucleic acid is essential for successful homologous recombination with the endogenous gene is sufficiently long. 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 in one Microorganism (e.g. by electroporation) and cells, in which the introduced HA gene is homologous with the endogenous HA gene recombined are known in the art using Process selected.

In another embodiment, recombinant Microorganisms are produced that contain selected systems that enable regulated expression of the introduced gene.

The inclusion of an HA gene in a vector under control des Lac-Operons enables z. B. the expression of the HA gene only in Presence of IPTG. These regulatory systems are in the Known in the field.

A host cell according to the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used for production (i.e. Expression) of an HA protein can be used. The invention also prescribes processes for the production of HA proteins Ready to use the host cells of the invention. At a The method comprises growing the embodiment host cell according to the invention (in which a recombinant expression vector, the encoded, inserted, or in an HA protein Genome has been introduced that is a wild-type or modified HA protein) in a suitable medium until the HA protein has been produced. The process comprises one another embodiment isolating the HA 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 can used in one or more of the following procedures: Identification of C. glutamicum and related organisms, Mapping genomes of organisms with C. glutamicum are related, identification and localization of C. glutamicum sequences of interest, evolution studies, determination of HA protein areas necessary for function, modulation the activity of an HA protein; Modulation of the metabolism of one or more organic compounds; Modulation of the Modification or degradation of one or more aromatic or aliphatic compounds; Modulation of cell wall synthesis or rearrangements; Modulation of enzyme activity or proteolysis; and modulation of cellular production Desired compound, such as a fine chemical: The invention HA nucleic acid molecules have a variety of uses. she can first be used to identify an organism as Corynebacterium glutamicum or close relatives thereof are used become. You can also use it to identify C. glutamicum or of a relative of them in a mixed population of Microorganisms can be used. The invention represents the Nucleic acid sequences of a number of C. glutamicum genes. By Probe the extracted genomic DNA from a culture of one uniform or mixed population of microorganisms among stringent conditions with a probe covering an area of a C. glutamicum gene that is unique to this organism one can determine whether this organism is present. Corynebacterium glutamicum itself is not pathogenic, but it is it with pathogenic species like Corynebacterium diptheriae, related. The detection of such an organism is from significant clinical importance.

The nucleic acid and protein molecules according to the invention can serve as markers for specific areas of the genome. This is not only when mapping the genome, but also for functional studies of C. glutamicum proteins useful. to Identification of the genome area to which a specific C. glutamicum DNA-binding protein binds, the C. glutamicum genome, for example. to be cleaved and the fragments containing the DNA binding protein be incubated. Those who bind the protein can additionally with the nucleic acid molecules according to the invention, preferably probed with easily detectable markings; the binding of such a nucleic acid molecule to the Genome fragment allows localization of the fragment on the genomic map of C. glutamicum, and if this occurs several times different enzymes, it makes it easier rapid determination of the nucleic acid sequence to which the protein is attached binds. The nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum, can serve.

The HA nucleic acid molecules according to the invention are suitable also for evolution and protein structure studies. The Processes involved in the adaptation and maintenance of the Homeostasis are involved in which the molecules of the invention are used by many species; by Comparison of the sequences of the nucleic acid molecules according to the invention with those that code similar enzymes from other organisms, can determine the degree of evolutionary kinship of the organisms become. Accordingly, such a comparison enables Determining which sequence areas are conserved and which are not, which is helpful in determining such areas of the protein that are essential for the enzyme function. This guy the determination is valuable for protein technology studies and can give an indication of which protein mutagenesis can tolerate without losing function.

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

The modulation of the activity or the number of HA proteins that are involved in cell wall biosynthesis or rearrangements, can reduce production, yield and / or production efficiency of one or more fine chemicals from C. glutamicum cells influence. By changing the cell activity of these proteins the structure or the thickness of the cell wall can be modulated. The cell wall serves as a protective device against osmotic lysis and external sources of injury; by modifying the cell wall can test the ability of C. glutamicum, mechanical stress and the stress caused by shear forces, which this Microorganism is subject to large fermenter cultivation, to withstand, strengthen. Each C. glutamicum cell is also one thick cell wall surround, and so there is a significant Share of biomass that is present in a large culture Cell wall. By increasing the rate at which the cell wall is synthesized, or by activating cell wall synthesis (by genetic manipulation of the HA cell wall proteins according to the invention) you can see the growth rate of the microorganism increase. Accordingly, by lowering the activity or the Number of proteins involved in cell wall degradation, or by lowering the repression of cell wall biosynthesis Achieve an overall increase in cell wall production. An increase in Number of viable C. glutamicum cells (as indicated by a the previously described protein changes take place) should cause an increased number of cells that the produce the desired fine chemical in large fermenter cultures, what the yields or the efficiency of production of this Cultural connections should increase.

The modulation of the activity or number of C. glutamicum-HA- Proteins involved in the modification or breakdown of aromatic or aliphatic compounds may also be involved direct or indirect effects on the production of one or have several fine chemicals from these cells. Certain aromatic or aliphatic modification or degradation products are desirable fine chemicals (e.g. organic acids or modified aromatic or aliphatic compounds); Consequently can be done by modifying the enzymes that make these modifications (e.g. hydroxylation, methylation or isomerization) or Perform degradation reactions, the yields of these desired Increase connections. Accordingly, one can lower the Activity or the number of proteins involved in metabolic pathways are involved, the modified degradation products of the above further reduce the reactions mentioned, the yields of these To improve fine chemicals from C. glutamicum cells in culture.

These aromatic and aliphatic modification and Degradation enzymes are fine chemicals themselves. Can in purified form these enzymes to break down aromatic and aliphatic Compounds (e.g. of toxic chemicals such as crude oil products) for biological restoration of contaminated areas, for technical decomposition of waste or for large-scale or economically feasible production of the desired modified aromatic or aliphatic compounds or their Degradation products, some of which are suitable as carbon or Energy sources for other fine chemical producing compounds in Culture can be used, used (see e.g. Faber, K. (1995) Biotransformations in Organic Chemistry, Springer: Berlin and the quotes it contains; and Roberts, S. M. ed. (1992-1996) Preparative Biotransformations, Wiley: Chichester and the quotes it contains). Through such genetic engineering of these proteins that their regulation by others cellular mechanisms can be reduced or prevented, one can increase the total number or activity of these proteins, thereby not only does the yield of these fine chemicals improve, but also the activity of these harvested proteins.

The modification of these aromatics and aliphatic modifiers and degrading enzymes can also have an indirect effect on the Have production of one or more fine chemicals. Lots aromatic and aliphatic compounds (such as those described in Culture media as contaminants or from the cellular Metabolism as waste products) are toxic to the cells; by modifying and / or dismantling these connections so that they can be easily removed or destroyed if the Have cell viability increased. These enzymes can do this Modify or remove connections so that the resulting products in normal carbon metabolism can get to the cell. This enables the cell to do this Use compounds as alternative carbon or energy sources. For large-scale crops, where limiting quantities of the optimal Carbon sources can be present, these enzymes Provide a process by which the cells continue to grow and can share, being aromatic or aliphatic Compounds are used as nutrients. In any case the resulting increase in the number of C. glutamicum cells in the culture that produces the desired fine chemical, again increased yields or increased efficiency of the Production of the fine chemical (s) effect.

The modifications of the activity or the number of HA proteins, involved in the metabolism of inorganic compounds, can manufacture one or more fine chemicals Cultures of C. glutamicum or related bacteria directly or affect indirectly. For example. can be many desirable Fine chemicals such as nucleic acids, amino acids, cofactors and Vitamins (e.g. thiamine, biotin and lipoic acid) not without inorganic Molecules such as phosphorus, nitrate, sulfate and iron are synthesized become. The inorganic proteins according to the invention Metabolism enables the cell to extract these molecules a variety of inorganic compounds and their Redistribution into various fine chemical biosynthetic pathways. By Increase the activity or the number of enzymes that on Metabolism of these organic compounds are involved, you can do that Offer this possibly limiting inorganic Enlarge molecules, increasing the production or efficiency of the Production of various fine chemicals from C. glutamicum cells, that contain these modified proteins directly increase. The Modification of the activity or the number of inventive Proteins of inorganic metabolism can also cause C. glutamicum a limited supply of inorganic Compounds can use better, or that non-optimal inorganic Compounds for the synthesis of amino acids, vitamins, cofactors or nucleic acids, all for continuous growth and cell replication are necessary. By improving the viability of these cells in Large cultures can also count the number of C. glutamicum cells that have one or produce several fine chemicals in the culture, enlarged what, in turn, the yields or the efficiency of Production of one or more fine chemicals increased.

The C. glutamicum enzymes for general processes are self desirable fine chemicals. The specific characteristics of the Enzymes (i.e., regio and stereospecificity, among others) close them suitable catalysts for chemical reactions in vitro. Either complete C. glutamicum cells with a suitable one Incubate substrate so that the desired product from the Enzymes are produced in the cell, or the desired enzymes can from C. glutamicum cultures (or those of a related Bacteria) are overproduced and cleaned and then in in vitro reactions under industrial conditions (either in solution or immobilized on a suitable immobile Phase) can be used. In these situations, the enzyme either be a natural C. glutamicum protein, or it can be be mutagenized to have a changed activity. This Enzymes are commonly used in the industry as Catalysts in the chemical industry (e.g. organic Synthetic chemistry), as food additives, as feed components, for Fruit processing, for leather production, in detergents, in analysis and medicine and in the textile industry (see e.g. Yamada, H. (1993) "Microbial reactions for the production of useful organic compounds ", Chimica 47: 5-10; Roberts, S.M. (1998) Preparative biotransformation: the employment of enzymes and wholecells in synthetic chemistry ", J. Chem. Soc. Perkin Trans. 1: 157-169; Zaks, A. and Dodds, D.R. (1997) "Application of biocatalysis and biotransformations to the synthesis of pharmaceuticals, "DDT 2: 513-531; Roberts, S.M. and Williamson, N.M. (1997) "The use of enzymes for the preparation of biologically active natural products and analogues in optically active form, "Curr. Organ. Chemistry 1: 1-20; Faber, K. (1995) Biotransformation in Organic Chemistry, Springer, Berlin Roberts, S. M. ed. (1992-1996) Preparative Biotransformations, Wiley: Chichester; Cheetham, P. S.J. (1995) "The applications of enzymes in industry" in Handbook of Enzyme technology, 3rd ed., Wiseman A. Ed. Elis: Horwood, pp. 419-552 and Ullman's Encyclopedia of Industrial Chemistry (1987) Vol. 9, Enzymes, pp. 390-457). By increasing the Activity or the number of these enzymes can also be seen Ability of the cell to increase the substrates provided convert desired products, or these enzymes for increased Overproduce yields in large culture. By mutagenesis of this Proteins can also be used to inhibit feedback or others eliminate repressive cellular regulatory controls so that Larger amounts of these enzymes are produced and produced by the cell are activated, which means greater yields, production or Efficiency of producing these fine chemical proteins Large cultures are preserved. By manipulating these enzymes you can the activity of one or more C. glutamicum metabolic pathways, such as those for biosynthesis or the breakdown of one or several fine chemicals.

The mutagenesis of the proteolytic enzymes according to the invention, see that they changed in terms of activity or number yield, production and / or efficiency the production of one or more fine chemicals from C. Affect glutamicum directly or indirectly. By enlarging the activity or the number of these proteins can be seen Increase the ability of the bacterium to survive in a large culture, This is because the cell can break down proteins more quickly due to high temperatures, non-optimal pH and others Stress factors that can prevail in fermenter cultivation are folded incorrectly. A larger number of cells in these Cultures can have increased yields or increased efficiency of Production of one or more desired fine chemicals because of the relatively larger number of cells that these Produce connections in culture, result. C. glutamicum cells also have multiple Cell surface proteases that break down external nutrients into molecules made by the Cell lighter than carbon / energy sources or nutrients other type can be used. An increase in activity or the number of these enzymes can improve this turnover and increases the amount of available nutrients, which makes Cell growth or production is improved. The modifications of the proteases according to the invention can thus indirectly the C, affect glutamicum fine chemical production.

A more direct impact on fine chemicals production in Response to modification of one or more Proteases according to the invention can take place if the proteases on the Production or degradation of a desired fine chemical are. By lowering the activity of a protease, the one Fine chemical or a protein that breaks down in the synthesis of a Fine chemical is involved, you can see the levels of this Increase fine chemical (due to reduced degradation or enhanced synthesis of the compound). Accordingly, one can go through Increasing the activity of a protease that degrades a compound which results in a fine chemical or a protein, involved in the breakdown of a fine chemical Get similar result: increased amounts of the desired Fine chemical from C. glutamicum cells that contain these modified proteins contain.

The above-mentioned mutagenesis strategies for HA proteins, the increased yields of a fine chemical from C. glutamicum intended to be effective should not be restrictive; variations these strategies for mutagenesis are readily apparent to the person skilled in the art. Through these mechanisms, the inventive Nucleic acid and protein molecules are used to make C. glutamicum or related strains of bacteria, the mutant HA nucleic acid and Express protein molecules, so that the yield, Production and / or production efficiency of a desired one Connection is improved. The desired connection can be a Product of C. glutamicum, which is the end product of Biosynthetic pathways and intermediates occurring naturally metabolic pathways as well as molecules involved in the metabolism of C. glutamicum does not occur naturally, but from one C. glutamicum strain according to the invention are produced.

This invention is further illustrated by the examples below illustrated, which are not to be considered restrictive should. The contents of all in this patent application cited references, patent applications, patents and published patent applications are hereby incorporated by reference added.

Examples example 1 Preparation of all 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 / l sucrose, 2.46 g / l MgSO _4. 7 H ₂ O, 10 ml / l KH ₂ PO ₄ solution (100 g / l, adjusted to pH with KOH Value 6.7), 50 ml / l M12 concentrate (10 g / l (NH ₄ ) ₂ SO ₄ , 1 g / l NaCl, 2 g / l MgSO ₄ .7 H ₂ O, 0.2 g / l CaCl ₂ , 0.5 g / l. Yeast extract (Difco), 10 ml / l trace element mixture (200 mg / l FeSO ₄ .H ₂ O, 10 mg / l ZnSO ₄ .7 H ₂ O, 3 mg / l MnCl ₂ .4 H ₂ O, 30 mg / l H ₃ BO ₃ , 20 mg / l CoCl ₂ .6 H ₂ O, 1 mg / l NiCl ₂ .6 H ₂ O, 3 mg / l Na ₂ MoO ₄ .2 H ₂ O, 500 mg / l complexing agent (EDTA or citric acid), 100 ml / l vitamin mixture (0.2 ml / l biotin, 0.2 mg / l folic acid, 20 mg / l p-aminobenzoic acid, 20 mg / l riboflavin, 40 mg / l Ca-panthothenate, 140 mg / l nicotinic acid, 40 mg / l pyridoxol hydrochloride, 200 mg / l myoinositol) Lysozyme was added to the suspension in a final concentration of 2.5 mg / ml The cell wall was degraded for 1 hour incubation at 37 ° C. and the protoplasts obtained were harvested by centrifugation 5 ml of buffer I and washed once with 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffet 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-isoamyl alcohol and chloroform-isoamyl alcohol using standard procedures. Then the DNA was added 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 1 at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) like. The DNA was dissolved in 1 ml TE buffer containing 20 µg / ml RNase A and dialyzed against 1000 ml 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 aliquots of 0.4 ml 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, produced as described in Example 1, were carried out according to known and well established procedures (see e.g. Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory Manual ". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons) Cosmid and plasmid banks.

Any plasmid or cosmid could be used. Special The plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids from the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or Cosmids, such as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T.J. Rosenthal, A., and Waterson, R.H. (1987) Gene 53: 283-286.

Example 3 DNA sequencing and computer function analysis

Genomic libraries as described in Example 2 were used for DNA Sequencing according to standard procedures, especially the Chain termination process with ABI377 sequencing machines (see e.g. Fleischman, R.D. et al. (1995) "Whole-Genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science 269; 496-512) used. 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 performed by plasmid (or other vector) DNA E.coli or other microorganisms (e.g. Bacillus spp. Or Yeasts, such as Saccharomyces cerevisiae), which the Maintain integrity of their genetic information can. Usual mutator strains show mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc., to For a comparison, see Rupp, W. D. (1996) DNA repair mechanisms in Escherichia coli and Salmonella, S. 2277-2294, ASM: Washington). This 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

Contain several species of Corynebacterium and Brevibacterium endogenous plasmids (such as pHM1519 or pBL1) which are autonomous replicate (for an overview see, for example, Martin, J.F. et al. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum are easy to use Construct standard vectors for E. coli (Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biology ", John Wiley & Sons), which a Origin of replication for and a suitable marker Corynebacterlum glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids which are derived from Corynebacterium and Brevibactertium species have been isolated. Special use as a transformation marker for these species are genes for kanamycin resistance (such as those derived from Tn5 or Tn-903 transposon) or for chloramphenicol (Winnacker, E.L. (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, that are replicated in E. coli and C. glutamicum, and those for various purposes can be used, including genetic Overexpression (see, e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, J.F. et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, B.J. et al. (1992) Genes 102: 93-98).

Using standard procedures it is possible to find a gene of interest into one of the shuttle vectors described above clone and such hybrid vectors in Corynebacterium glutamicum Bring tribes. The transformation of C. glutamicum leaves 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 Achieve conjugation (as described, for example, in Schäfer, A., et (1990) J. Bacteriol. 172: 1663-1666). It is also possible that Transfer shuttle vectors for C. glutamicum to E. coli, by plasmid DNA from C. glutamicum (using in the specialist field known standard method) is prepared and in E. coli is transformed: This transformation step can be done with Standard procedures are used, however, one is advantageously Mcr deficient E. coli strain used, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19).

Example 6 Determination of expression of the mutant protein

The observations of the activity of a mutant protein in one transformed host cell rely on the fact that the mutated protein in a similar manner and in a similar amount is expressed like the wild-type protein. A suitable method for Determination of the amount of transcription of the mutant gene (a Evidence of the amount of mRNA required for translation of the gene product is available is a Northern blot (see. e.g., Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York), being a primer designed like this is that it binds to the gene of interest with a detectable (usually radioactive or chemiluminescent) Label is provided so that - if the total RNA of a culture of the organism extracted, separated on a gel, on a stable matrix is transferred and incubated with this probe the binding and the quantity of binding of the probe and also indicates the amount of mRNA for that gene. This Infomration is evidence of the extent of transcription of the mutated gene. Total cell RNA can be broken down by several Isolate methods from Corynebacterium glutamicum, which in the Are known in the art, as described in Bormann, E.R. et al., (1992) Mol. Microbiol. 6: 317-326.

To determine the presence or the relative amount of Protein that is translated from this mRNA can be standard Techniques such as Western blot are used (see e.g. Ausubel et al. (1988) "Current Protocols in Molecular Biology", Wiley, New York). In this procedure, total cell proteins are produced extracted, separated by gel electrophoresis, onto a matrix, such as Nitrocellulose, transferred and with a probe such as one Antibody incubated that specifically binds to the desired protein. This probe is usually chemiluminescent or Colorimetric marking that are easy to prove leaves. The presence and the observed amount of label shows the presence and the 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 41 20 867; 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 may 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 Cl or (NH ₄ ) ₂ SO ₄ , NH ₄ OH, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extracts, meat extracts and others.

Inorganic salt compounds contained in the media may 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. Especially 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, for example biotin, Riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine belong. Growth factors and salts often come from complex ones Media components such as yeast extract, molasses, corn steep liquor and like. The exact composition of the media connections depends heavily on the particular experiment and will be for each case decided individually. Information about media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach "(Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can be also available from commercial providers, 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 either be together or if necessary be sterilized separately. All media components can Be present at the beginning of the cultivation or optionally continuously or be added in batches.

The growing conditions are defined separately for each experiment. The temperature should be between 15 ° C and 45 ° C and can be kept constant or changed during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by adding buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES; ACES etc. can be used alternatively or simultaneously. The cultivation pH value can also be kept constant during the cultivation by adding NaOH or NH ₄ OH. If complex media components such as yeast extract are used, the need for additional buffers is reduced since many complex 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 from several hours to several days. This time is chosen that the maximum amount of product accumulates in the broth. The disclosed growth experiments can be in a variety of Containers such as microtiter plates, glass tubes, glass flasks or Glass or metal fermenters of different sizes carried out become. To screen a large number of clones, the Microorganisms in microtiter plates, glass tubes or Shake flasks are grown with or without baffles: It is preferable to use 100 ml shake flasks with 10% (by volume) of the required growth medium are filled. The flask should be on a rotary shaker (Amplitude 25 mm) with a speed in the range of 100-300 rpm are shaken. Evaporation losses can be caused by Maintaining a moist. Atmosphere are reduced; alternatively, a mathematical should be used for the evaporation losses Correction can be carried out.

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 OD ₆₀₀ of 0.5-1.5 using cells grown on agar plates, such as cm plates (10 g / l glucose, 2.5 g / l NaCl, 2 g / l urea, 10 g / l polypeptone, 5 g / l yeast extract, 5 g / l meat extract, 22 g / l agar pH 6.8 with 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

Determination of activities and kinetic parameters of Enzymes are well known in the art. Determination experiments the activity of a certain modified enzyme must be related to the specific activity of the wild-type enzyme to be adjusted what is within the skill of the professional. Overviews of Enzymes in general as well as specific details that the Structure, kinetics, principles, processes, applications and Examples for determining many enzyme activities can relate to can be found in the following references, for example: Dixon, M., and Webb, E. C: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D: Ed. (1983) The Enzymes, 3rd ed. Academic Press, New York; Bisswanger, H. (1994) Enzyme Kinetics, 2nd edition VCH, Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graßl, M. Ed. (1983-1986) Methods of Enzymatic Analysis, 3rd edition Vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) Vol. A9, "Enzymes", VCH, Weinheim, pp. 352-363.

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

The determination of the activity of membrane transport proteins can according to the techniques as described in Gennis, R. B. (1989) "Pores, Channels and Transporters"; in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137; 199-234; and 270-322.

Example 9 Analysis of the influence of mutant 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 the modified microorganisms under appropriate conditions (such as those described above are grown) and the medium and / or the cellular Components on the increased production of the desired product (i.e. an amino acid) is examined. Such analysis techniques are well known to those skilled in the art and include spectroscopy, Thin layer chromatography, staining methods of various types, enzymatic and microbiological processes as well as analytical Chromatography, such as high-performance liquid chromatography (see e.g. Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, pp. 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, P.A. et al. (1988) Bioseparations: downstream processing for Biotechnology, John Wiley and Sons; Kennedy, J.F. and Cabral, J.M.S. (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical Separations, in Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, Noyes Publications).

In addition to measuring the final fermentation product, it is also possible to add other components of the metabolic pathways analyze that is used to produce the desired compound as intermediate and by-products to the Total productivity of the organism, the yield and / or the efficiency of To determine production of the connection. The analysis method include measurements of the amounts of nutrients in the medium (e.g. sugar, Hydrocarbons, nitrogen sources, phosphate and others Ions), measurements of biomass composition and growth, Analysis of the production of ordinary metabolites Biosynthetic pathways and measurements of gases generated during fermentation become. Standard procedures for these measurements are in Applied Microbial physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the references cited therein described.

Example 10 Purification of the desired product from C. glutamicum Culture

Obtaining the desired product 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, Remove the cells from the culture by slow centrifugation The cells can be harvested by standard techniques such as mechanical force or ultrasound, can be lysed. The Cell debris is removed by centrifugation, and the The supernatant fraction, which contains the soluble proteins, becomes the receive further purification of the desired compound. Will that Product secreted by the C. glutamicum cells Cells removed from the culture by slow centrifugation, and the The supernatant fraction is kept for further purification.

The supernatant fraction from both cleaning processes becomes one Chromatography with a suitable resin, which desired molecule either on the chromatography resin is retained, but not many impurities in the sample, or wherein the impurities remain on the resin, which However, no rehearsal. These chromatography steps can if necessary, be repeated, the same or different Chromatography resins are used. The expert is in the Selection of suitable chromatography resins and the most effective Application for a specific molecule to be purified. The purified product can by filtration or ultrafiltration concentrated and kept at a temperature at which the stability of the product is maximum.

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

The identity and purity of the isolated compounds can be determined by Standard techniques of the field are determined. This include high performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzyme test or microbiological tests. This Analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya  11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, Pp. 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 517th

equivalent

The skilled person recognizes or can - simply by Routine method used - many equivalents of the invention determine specific embodiments. These equivalents are supposed to be covered by the following claims.

The information in Table 1 is to be understood as follows:
In column 1 "DNA-ID" the respective number refers to the SEQ ID NO of the attached sequence listing. A "5" in the "DNA ID" column therefore means a reference to SEQ ID NO: 5.
In column 2 "AS-ID" the respective number refers to the SEQ ID NO of the attached sequence listing. A "6" in the "AS-ID" column therefore means a reference to SEQ ID NO: 6.
Column 3 "Identification" 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 corresponding 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

Claims (8)

1. Isolated nucleic acid molecule coding for a polypeptide with that referred to in Table 1 / Column 2 Amino acid sequence with the nucleic acid molecule in the in Table 1 / column 4 given another amino acid position proteinogenic amino acid encoded as the respective in Table 1 / column 5 amino acid given in the same row. 2. Isolated nucleic acid molecule according to claim 1, wherein the Nucleic acid molecule in that given in Table 1 / column 4 Amino acid position in Table 1 / Column 6 in the same Line specified amino acid coded. 3.. A vector that has at least one nucleic acid sequence Claim 1 contains. 4. A host cell with at least one vector after Claim 3 is transfected. 5. A host cell according to claim 4, wherein the expression of the said nucleic acid molecule for modulating production a fine chemical from said cell. 6. A process for producing a fine chemical, which the Culturing a cell involves using at least one The vector of claim 3 has been transfected 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.