EP1456232A2

EP1456232A2 - Genes encoding for membrane synthesis and membrane transport proteins

Info

Publication number: EP1456232A2
Application number: EP02781299A
Authority: EP
Inventors: Oskar Zelder; Markus Pompejus; Hartwig Schröder; Burkhard Kröger; Corinna Klopprogge; Gregor Haberhauer
Original assignee: BASF SE
Current assignee: Paik Kwang Industrial Co Ltd
Priority date: 2001-11-05
Filing date: 2002-10-31
Publication date: 2004-09-15
Also published as: DE10154179A1; WO2003040292A2; AU2002349017A1; WO2003040292A3; KR20040058265A

Abstract

The invention relates to novel nucleic acid molecules, to the use thereof in the construction of genetically improved microorganisms and to a method for producing fine chemical products, in particular amino acids, by means of said genetically improved microorganisms.

Description

Genes that code for membrane synthesis and membrane transport proteins

Background of the Invention

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

Summary of the invention

This invention provides novel nucleic acid molecules that can be used to identify or classify CoryneJbacterium glutamicum or related types of bacteria. C. glutamicum is a gram-positive, aerobic bacterium (eg. In the overflow of crude oil) in the industry for the large scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons and for the oxidation of terpenoids overall ^■ meinhin used becomes. The nucleic acid molecules can therefore be used to identify microorganisms that can be used for the production of fine chemicals, for example by fermentation processes. C. glutamicum itself is not pathogenic, but it is related to other Corynebacterium species, such as Corynebacterium diphtheriae (the causative agent of diphtheria), which are important pathogens in humans. The ability to identify the presence of Corynebacterium species can therefore also be of significant clinical importance, for example in diagnostic applications. These nucleic acid molecules can also serve as reference points for mapping the C. glutamicum genome or organisms related to genomes. These "new nucleic acid molecules encode proteins which are referred to here as membrane construction and membrane transport (MCT) proteins. These MCT proteins can, for example, perform a function which is involved in the metabolism (eg biosynthesis or degradation) of 5 compounds which are necessary for membrane biosynthesis, or support the membrane transport of one or more compounds into or out of the cell due to the availability of cloning vectors for use in Cor _. y22ejbacfce.rium glutamicum, as disclosed, for example, in Sinskey et

10 al., U.S. Patent No. 4,649,119, and techniques for genetically manipulating C. glutamicum and the related Brevibacterium species (e.g., actofermentum) Yoshiha a et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al. J. Gen. Microbiol. 130 (1984)

15 2237-2246), the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to make it better and more efficient as a producer of one or more fine chemicals. The improved production or efficiency of the production of a fine chemical can be attributed to a direct

20 based on the manipulation of a gene according to the invention or on an indirect effect of this manipulation.

There are a number of mechanisms by which the modification of an MCT protein according to the invention reduces the yield, production and /

25 or directly affect the efficiency of producing a fine chemical from a C. glutamicum strain containing such an altered protein. These MCT proteins, which are involved in the export of the fine chemical molecules from the cell, can have a greater number or higher activity, so that larger

30 larger amounts of these compounds are secreted into the extracellular medium, from which they are more easily obtained. These MCT proteins, which are involved in the import of the nutrients necessary for the biosynthesis of one or more fine chemicals (e.g. phosphate, sulfate, nitrogen compounds, etc.),

35 can have a greater number or higher activity, so that these precursors, cofactors or intermediates are present in the cell in a higher concentration. In addition, fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins according to the invention which are involved in the biosynthesis of these compounds, or by impairing the activity of one or more MCT proteins which are involved in the degradation of these compounds, the yield, production and / or efficiency of the production of fatty acids and lipid molecules from C. glutamicum can be increased. The mutagenesis of one or more MCT genes according to the invention can also produce MCT proteins with modified activities which indirectly impair the production of one or more desired fine chemicals from C. glutamicum. For example. MCT proteins according to the invention, which are involved in the export of waste products, have a greater number or higher activity, so that the normal metabolic waste of the cell (possibly in higher quantity due to the overproduction of the desired fine chemical) is efficiently exported before it contains the nucleotides and Can damage proteins in the cell (which would decrease cell viability) or interact with the fine chemical biosynthetic pathways (which would decrease the yield, production or efficiency of production of a desired fine chemical). The relatively large intracellular amounts of the desired fine chemical can even be toxic to the cell. For example, increasing the number of transporters that can export these compounds from the cell can increase the viability of the cell in culture, which in turn causes that a larger number of cells in the culture produce the desired fine chemical. The MCT proteins according to the invention can also be manipulated in such a way that the relative amounts of the different lipid and fatty acid molecules are produced. This can have a significant impact on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change the membrane fluidity. Changes in the membrane fluidity can influence the transport of the molecules across the membrane as well as the integrity of the cell, each of which has a considerable effect on the production of fine chemicals from C. glutamicum in large-scale fermenter cultures.

The invention provides new nucleic acid molecules which encode proteins which are referred to here as MCT proteins and are involved, for example, in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of molecules across these membranes , In a preferred embodiment, the MCT protein is involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of molecules across these membranes. Examples of these proteins include those encoded by the genes shown in Table 1.

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

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

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

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

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

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

Another aspect of the invention relates to an isolated MCT protein or a section, for example a biologically active section thereof. In a preferred embodiment, the isolated MCT protein or its section can be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across the membranes. In a further preferred embodiment, the isolated MCT protein or a section thereof is sufficiently homologous to an amino acid sequence from Appendix B so that the protein or its section continues to participate in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or can be involved in the transport of molecules across the membranes.

The invention also relates to an isolated MCT protein preparation. In preferred embodiments, the MCT protein comprises an amino acid sequence from Appendix B. In a further preferred embodiment, the invention relates to an isolated full-length protein which essentially forms a complete amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is homologous.

The MCT polypeptide or a biologically active portion thereof can be operably linked to a non-MCT polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has a different activity than the MCT protein alone and, in other preferred embodiments, results in increased yields, increased production and / or Efficiency of producing a desired fine chemical from C. glutamicum. The integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.

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

Another aspect of the invention relates to methods for modulating the production of a molecule from a microorganism. These methods involve contacting the cell with a substance that modulates MCT protein activity or MCT nucleic acid expression so that a cell-associated activity changes compared to the same activity in the absence of the substance. In a preferred embodiment, the cell is modulated with respect to one or more C. glutamicum metabolic pathways for cell membrane components or with regard to the transport of compounds across the membranes, so that the yields or the production rate of a desired fine chemical are improved by this microorganism. The substance that modulates the MCT protein activity stimulates, for example, the MCT protein activity or the MCT nucleic acid expression. Examples of substances that stimulate MCT protein activity or MCT nucleic acid expression include small molecules, active MCT proteins, and nucleic acids that encode MCT proteins and have been introduced into the cell. Examples of substances that inhibit MCT activity or expression include small molecules and antisense MCT nucleic acid molecules.

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

Detailed description of the invention

The present invention provides MCT nucleic acid and protein molecules which can be involved in the metabolism of compounds necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across the membranes. The molecules of the invention can be used in the modulation of the production of fine chemicals from microorganisms, such as C. glutamicum, either directly (e.g. where the overexpression or optimization of a fatty acid biosynthetic protein has a direct effect on the yield, production and / or efficiency of production of the Fatty acid of modified C. glutamicum) or by an indirect effect which nevertheless causes an increase in the yield, production and / or efficiency in the production of the desired compound (for example where the modulation of the metabolism of the cell membrane components changes in the yield, production and / or efficiency of production or composition of the cell membrane, which in turn affects the production of one or more fine chemicals). The aspects of the invention are further explained below.

I. Fine chemicals

The term "fine chemical" is known in the art and includes molecules that are produced by an organism and have applications in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. These compounds include organic acids, such as tartaric acid, itaconic acid and diamino-pimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim and the citations contained therein), lipids, saturated and unsaturated fatty acids (e.g. arachidonic acid), diols (e.g. propanediol and butanediol ), Carbohydrates (e.g. hyaluronic acid and trehalose), aromatic compounds (e.g. aromatic amines, vanillin and indigo), vitamins and cofactors (such as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and the citations contained therein; and Ong, AS, Niki, E. and Packer, L.

(1995) "Nutrition, Lipids, Health and Disease" Proceedings of the UNESCO / Confederation of Scientific and Technological 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 other chemicals described by Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and the references cited therein. The metabolism and uses of certain fine chemicals are further discussed below.

A. Amino acid metabolism and uses

The amino acids comprise the basic structural units of all proteins and are therefore essential for normal cell functions. The term "amino acid" is known in the art. The proteinogenic amino acids, of which there are 20 types, serve as structural units for proteins in which they are linked to one another via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) are usually not found in proteins (see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids can be in the D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)). The "essential" amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), so named, because they have to be taken up in the diet due to the complexity of their biosynthesis, are identified by simple biosynthetic pathways converted into the remaining 11 "non-essential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine). Higher animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested in order for normal protein synthesis to take place.

Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in themselves, and it has been discovered that many are used in various applications in the food, feed, chemical, cosmetic, agricultural, pharmaceutical and pharmaceutical industries. 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, as well as aspartate, phenylalanine, glycine and cysteine. Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are widespread feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol 6, Chapter 14a, VCH: Weinheim). It has been discovered that these amino acids can also be used as precursors for the synthesis of synthetic amino acids and proteins such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others, in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985 are suitable substances.

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

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

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

B. vitamins, cofactors and nutraceutical metabolism and uses

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

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

Thiamine (vitamin Bi) is formed by chemical coupling of pyrimidine and thiazole units. Riboflavin (vitamin B) is synthesized from guanosine 5 'triphosphate (GTP) and ribose 5' phosphate. Riboflavin in turn is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds commonly referred to as "Vitamin B6" (e.g. pyridoxine, pyridoxamine, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride) are all derivatives of the common structural unit 5-hydroxy-6-methylpyride. Panthothenate (antothenic acid, R- (+) -N- (2,4-dihydroxy-3,3,3-dimethyl-l-oxobutyl) -ß-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of ß-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion into pantoic acid, into β-alanine and for the condensation into pantothenic acid are known. The metabolically active form of pantothenate is coenzyme A, whose biosynthesis takes place over 5 enzymatic steps. Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone, (R) - Panthenol (provitamin B ₅ ), Pantethein (and its derivatives) and coenzyme A.

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

Corrinoids (such as the cobalamins and especially vitamin Bχ) and the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system. The biosynthesis of vitamin Bχ is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known. Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also known as "niaσin". 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" encompasses nitrogenous bases which are part of the nucleic acids, coenzymes and nucleotides. The term “nucleotide” includes the basic structural units of the nucleic acid molecules, which comprise a nitrogenous base, a pentose sugar (for RNA the sugar is ribose, for DNA the sugar is D-deoxyribose) and phosphoric acid. The term “nucleoside” encompasses molecules which serve as precursors of nucleotides but which, in contrast to the nucleotides, do not contain a phosphoric acid unit exhibit. By inhibiting the biosynthesis of these molecules or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; if this activity is specifically inhibited in carcinogenic cells, the ability of tumor cells to divide and replicate can be inhibited. There are also ^'ukleotide that no nucleic acid molecules form, but serve as energy stores (ie AMP) or as coenzymes (ie FAD and NAD).

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

The metabolism of these compounds in bacteria has been characterized (for overviews see, for example, Zalkin, H. and Dixon, JE (1992) "De novo purin nucleotide biosynthesis" in Progress in Nucleic Acids Research and Molecular biology, Vol. 42, Academic Press, Pp. 259-287; and Michal, G. (1999) "Nucleotides and Nucleosides"; Chap. 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley, New York). The purine metabolism, the object of intensive research, is essential for the normal functioning of the cell. A disturbed purine metabolism in higher animals can cause serious illnesses, e.g.

Gout. The purine nucleotides are removed via a series of steps via the intermediate compound inosine 5 'phosphate (IMP) from Ri- synthesized bose-5-phosphate, which leads to the production of guanosine 5 'monophosphate (GMP) or adenosine 5' monophosphate (AMP), from which the triphosphate forms used as nucleotides can be easily prepared. These compounds are also used as energy stores so that their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis takes place via the formation of uridine 5 'monophosphate (UMP) from ribose 5-phosphate. UMP in turn is converted into cytidine 5 'triphosphate (CTP). The deoxy forms of all nucleotides are produced in a one-step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. After phosphorylation, these molecules can participate in DNA synthesis.

D. Trehalose metabolism and uses

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

II. Membrane biosynthesis and transmembrane transport

The cell membranes serve a number of functions in a cell. First of all, a membrane differentiates the cell content from the environment, so that the cell maintains integrity. The membranes also serve as barriers so that dangerous or undesired connections cannot flow in and desired connections cannot flow out. Due to their structure, cell membranes are inherently impermeable to the not facilitated diffusion of hydrophilic compounds such as proteins, water molecules and ions: a double layer of lipid molecules in which the polar head groups protrude outwards (out of the cell or into the cell interior) and the non-polar tails protrude to the middle of the double layer and form a hydrophobic core (for a general overview of the structure and function of the membrane see Gennis, RB (1989) Biomembranes, Molecular Structure and Function, Springer: Heidelberg). This barrier allows that the cells contain a relatively higher concentration of desired compounds and a relatively lower concentration of undesired compounds than the surrounding medium, since the diffusion of these compounds through the membrane is efficiently blocked.

However, the membrane also provides an effective barrier against the import of desired molecules and the export of waste molecules. To overcome this difficulty, the cell membranes contain many types of transporter proteins that can facilitate the transmembrane transport of various types of compounds: pores or channels and transporters. The former are integral membrane proteins, sometimes protein complexes, that form a regulated opening through the membrane. This regulation or "gating" is usually specific to those to be transported through the pore or channel, so that these transmembrane constructs are specific to a specific class of substrates; For example, a potassium channel is constructed in such a way that only ions with a charge and size similar to potassium can pass through. Channel and pore proteins have certain hydrophobic and hydrophilic domains so that the hydrophobic portion of the protein can attach to the interior of the membrane, whereas the hydrophilic portion defines the interior of the channel, providing a protected hydrophilic environment through which the selected hydrophilic Molecule can arrive. Many such pores / channels are known in the art, including those for potassium, calcium, sodium and chloride ions.

This pore and channel mediated system is limited to very small molecules, such as ions, since pores or channels that are large enough to allow complete proteins to pass through by facilitating diffusion would not be able to pass through smaller molecules prevent. The transport of molecules through this process is sometimes referred to as "facilitated diffusion" because the driving force of a concentration gradient is required for the transport to take place. Permeases also facilitate the easier diffusion of larger molecules, such as glucose or other sugars, into the cell if the concentration of these molecules is greater on one side of the membrane than on the other (also referred to as "uniport"). In contrast to pores or channels, these integral proteins (which often have 6 to 14 membrane-spanning α-helices) do not form open channels through the membrane, but they do bind to the target molecule on the membrane surface and then undergo a conformational change, so that the target molecule is released on the opposite side of the membrane.

However, cells often need to import or export molecules against the existing concentration gradient ("active transport"), a situation in which facilitated diffusion cannot take place. There are two general mechanisms that the cell uses for such membrane transport; Symport or Antiport, and energy-coupled transport, like the one conveyed by ABC transporters. Symport and antiport systems couple the movement of two different molecules across the membrane (via permeases with two separate binding sites for two different molecules); With Symport, both molecules are transported in the same direction, whereas with Antiport, one molecule is imported and the other molecule is exported. This is energetically possible because one of these two molecules moves along a concentration gradient, and this energetically favorable event is only made possible by the simultaneous movement of a desired compound against the prevailing concentration gradient. Individual molecules can be transported against the concentration gradient across the membrane in an energy-driven process, such as with the ABC transporters. In this system, the transport protein located in the membrane has an ATP-binding cassette, when the target molecule is bound, ATP is converted to ADP + Pi, and the resulting released energy is used to drive the movement of the target molecule to the opposite side of the membrane, which is indicated by the Transporter is facilitated. For a more detailed description of all of these transport systems, see Bamberg, E. et al. , (1993) "Charge transport of ion pumps on lipid bilayer membranes", Q. Rev. Biophys. 26: 1-25; Findlay, J.B.C.

(1991) "Structure and function in membrane transport systems", Curr. Opin. Struct. Biol. 1: 804-810; Higgins, C.F. (1992) "ABC transporters from microorganisms to man", Ann. Rev. Cell. Biol. 8: 67-113; Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomebranes, Molecular Structure and Function, Springer: Heidelberg, pp. 270-322; and Nikaido, H. and Saier, H.

(1992) "Transport proteins in bacteria: common themes in their design", Science 258: 936-942, and the references contained in each of these citations.

The synthesis of membranes is a well characterized process involving many components, the most important of which are the lipid molecules. Lipid synthesis can be divided into two parts: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate and the addition or modification of a polar head group. Usual lipids found in bacterial membranes used include phospholipids, glycolipids, sphingolipids and phosphoglycerides. Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA-carboxylase or in acetyl-ACP by acetyl transacylase. After a condensation reaction, these two product molecules together form acetoacetyl-ACP, which is converted by a series of condensation, reduction and dehydration reactions, so that a saturated fatty acid molecule with the desired chain length is obtained. The production of the unsaturated fatty acids from these molecules is catalyzed by specific desaturases, either aerobically using molecular oxygen or anaerobically (for descriptions of fatty acid synthesis see FC Neidhardt et al. (1996) E. coli and Sal onella. ASM Press: Washington, DCS 612-636 and the references cited therein: Lengeier et al. (Ed.) (1999) Biology of Procaryotes, Thieme: Stuttgart, New York and the references cited therein, and Magnuson, K. et al. (1993) Microbiological Reviews 57: 522-542 and the references cited therein). The cyclopropane fatty acids (CFA) are synthesized by a specific CFA synthase with SAM as a cosubstrate. Known fatty acid chains are synthesized from knocked-out deaminated amino acid chains, so that knocked out 2-0xoacids are obtained (see Lengeier et al. (Ed.) (1999) Biology of Procaryotes. Thieme: Stuttgart, New York and the literature references therein). Another essential step in lipid synthesis is that

Transfer of fatty acids to the polar head groups, for example by glycerol phosphate acyl transferases. The combination of different precursor molecules and biosynthetic enzymes causes the production of different fatty acid molecules, which has a significant impact on the membrane composition.

III. Elements and methods according to the invention

The present invention is based, at least in part, on the discovery of new molecules, which are referred to here as MCT nucleic acid and protein molecules and control the production of cell membranes in C. glutamicum and effect the movement of molecules across these membranes. In one embodiment, the MCT molecules are involved in the metabolism of compounds which are involved in the construction of the cell membranes in C. glutamicum or in the transport of the molecules across these membranes. In a preferred embodiment, the activity of the MCT molecules according to the invention for regulating the production of membrane components has an effect on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the activity of the modulated MCT molecules is modulated such that the C. glutamicum Metabolic pathways that are regulated by the MCT proteins according to the invention, are modulated with regard to yield, production and / or production efficiency and are modified with regard to the efficiency of the transport of the compounds through the membranes, which increases the yield, production and / or efficiency of the Production of a desired fine chemical modulated either directly or indirectly by C. glutamicum.

The term "MCT protein" or "MCT polypeptide" encompasses proteins that are involved in the metabolism of compounds that are essential for the construction of

Cell membranes in C. glutamicum are necessary, or are involved in the transport of molecules across these membranes. Examples of MCT proteins include those encoded by the MCT genes listed in Table 1 and Appendix A. The terms "MCT gene" or "MCT nucleic acid sequence" encompass nucleic acid sequences which encode an MCT protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of MCT genes are listed in Table 1. The terms "production" or "productivity" are known in the art and include the concentration of the fermentation product (for example the desired fine chemical which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1) The term "production efficiency" encompasses the time it takes to achieve a certain production quantity (for example how long it takes the cell to set up a certain throughput rate of a fine chemical). The term "yield" or "product / carbon yield". is known in the art and encompasses the efficiency of converting the carbon source to the product (ie, the fine chemical), for example, usually expressed as kg product per kg carbon source. Increasing the yield or production of the compound will increase the amount of molecules or molecules recovered suitable obtained molecules of this compound in a certain Culture volume increased over a fixed period. The terms "biosynthesis" or "biosynthetic pathway" are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step or highly regulated process. The terms "degradation" or "degradation path" are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), e.g. in a multi-step or highly regulated Process. The term "metabolism" is known in the art and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a particular compound (e.g. the metabolism of an amino acid, such as Gly- a) then includes all biosynthesis, modification and degradation pathways of this compound in the cell.

In another embodiment, the MCT molecules according to the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum. There are a number of mechanisms by which the modification of an MCT protein according to the invention can directly influence the yield, production and / or efficiency of the production of a fine chemical from a C. glutamicum staiαπx which contains such a modified protein. These MCT proteins, which are involved in the export of the fine chemical molecules from the cell, can be present in large numbers or have increased activity, so that larger amounts of these compounds are secreted into the extracellular medium from which they can be obtained more easily. MCT proteins that are involved in the import of the nutrients that are necessary for the biosynthesis of one or more fine chemicals (for example phosphate, sulfate, nitrogen compounds, etc.) can accordingly be present in greater numbers or with higher activity, so that these precursor, cofactor or intermediate compounds are present in the cell in higher concentrations. In addition, fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins according to the invention, which are involved in the biosynthesis of these compounds, or by influencing the activity of one or more MCT proteins, which are involved in the degradation of these compounds to increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from C. glutamicum.

The mutagenesis of one or more genes according to the invention can also produce MCT proteins with modified activities which influence the production of one or more fine chemicals from C. glutamicum. MCT proteins according to the invention, which are involved in the export of waste products, can be present in greater number or higher activity, so that the normal metabolic waste of the cell (possibly in higher quantity due to overproduction of the desired fine chemical) can be exported efficiently before being nucleotides and Damage proteins within the cell (which reduces cell viability) or interact with other fine chemical pathways (which reduces the yield, production or efficiency of production of the desired fine chemical). The relatively large intracellular amounts of the desired fine chemical per se can be toxic to the cell. By increasing the number of transporters used to export this connection the cell is capable, the viability of the cell in culture can thus be increased, which in turn brings with it a larger number of cells in culture which produce the desired fine chemical. The MCT proteins according to the invention can be manipulated in such a way that the relative amounts of different lipid and fatty acid molecules are produced. This can have a significant impact on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change the membrane fluidity. Changes in membrane fluidity can affect the transport of molecules across the membrane as well as cell integrity, each of which has a significant impact on the production of fine chemicals from C. glutamicum in large-scale fermenter cultures.

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

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

The MCT protein according to the invention or a biologically active section or fragments thereof can be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across these membranes, or one or more of the molecules in Activities listed in Table 1.

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

A. Isolated nucleic acid molecules

One aspect of the invention relates to isolated nucleic acid molecules which encode MCT polypeptides or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for identifying or amplifying MCT-coding nucleic acids (for example MCT-DNA). The term “nucleic acid molecule” is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg mRNA) as well as DNA or RNA analogs that are generated by means of nucleotide analogs. This term also includes the untranslated sequence located at the 3 'and 5' end of the coding gene region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3' end of the coding gene region. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA. An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. An "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example, sequences that are located on the 5 'or

3 'end of the nucleic acid are located). In various embodiments, for example, the isolated MCT nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of the nucleotide sequences that naturally comprise the nucleic acid molecule in the genomic DNA of the Flank the cell from which the nucleic acid originates (for example a C. glutamicum cell). An "isolated" nucleic acid molecule, such as a cDNA molecule, can also be substantially free of any other cellular material or culture medium if it is produced by recombinant techniques, or of chemical precursors or other chemicals if it is chemically synthesized.

A nucleic acid molecule according to the invention, for example a nucleic acid molecule with a nucleotide sequence from Appendix A or a section thereof, can be produced by means of standard molecular biological techniques and the sequence information provided here. For example. a C. glutamicurπ-MCT cDNA can be isolated from a C. glufcamicum bank by using a complete sequence from Appendix A or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch , EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd to 1st Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Furthermore, a nucleic acid molecule comprising a complete sequence from Annex A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been prepared on the basis of this sequence (for example a nucleic acid molecule comprising a complete sequence can be used Appendix A, or a portion thereof, can be isolated by polymerase chain reaction using oligonucleotide primers made from this same sequence from Appendix A). For example. mRNA can be isolated from normal endothelial cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and the cDNA can be converted using reverse transcriptase (for example Moloney-MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for amplification via polymerase chain reaction can be created on the basis of one of the nucleotide sequences shown in Appendix A. A nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis. Oligonucleotides which correspond to an MCT nucleotide sequence can be produced by standard synthesis methods, for example using an automatic DNA synthesizer.

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

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

In one embodiment, the nucleic acid molecule according to the invention encodes a protein or a portion thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion thereof further participates in the metabolism of compounds which are essential for the construction of the cell membranes in C. glutamicum are necessary, or may be involved in the transport of the molecules across these membranes. As used herein, the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (e.g., an amino acid residue with a side chain similar to an amino acid residue in one of the sequences of Appendix B) to an ami - Show nos acid sequence from Appendix B, so that the protein or a portion thereof can also be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of the molecules across these membranes. Protein components of these pathways for membrane components or membrane transport systems, as described here, can play a role in the production and secretion of one or more fine chemicals. Examples of these Activities are also described here. Thus, the "function of an MCT protein" relates either directly or indirectly to the yield, production and / or efficiency of the production of one or more fine chemicals. Table 1 shows examples of MCT protein activities.

Sections of proteins which are encoded by the MCT nucleic acid according to the invention are preferably biologically active sections of one of the MCT proteins. The term “biologically active section of an MCT protein”, as used here, is intended to include a section, for example a domain or a motif, of an MCT protein which is involved in the metabolism of compounds which are essential for the structure of the cell membranes in C. glutamicum are necessary, or may be involved in the transport of molecules across these membranes, or has an activity given in Table 1. To determine whether an MCT protein or a biologically active portion thereof can be involved in the metabolism of compounds necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across these membranes, a test of the enzymatic activity can be carried out be performed. These test methods, as described in detail in Example 8 of the example part, are familiar to the person skilled in the art.

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

An isolated nucleic acid molecule that encodes an MCT protein that is homologous to a protein sequence from Appendix B can be prepared by introducing one or more nucleotide substitutions,

additions or deletions are generated in a nucleotide sequence from Appendix A, so that one or more amino acid substances ttions, additions or deletions are introduced into the encoded protein. The mutations can be introduced into one of the sequences from Appendix A by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are preferably introduced on one or more of the predicted non-essential amino acid residues. In a "conservative amino acid substitution", the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families do not include amino acids with basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). A predicted non-essential amino acid residue in an MCT protein is thus preferably replaced by another amino acid residue of the same side chain family. In a further embodiment, the mutations can alternatively be introduced randomly over all or part of the MCT coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the MCT activity described here in order to identify mutants, that maintain MCT activity. After mutagenesis of one of the sequences from Appendix A, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here (see Example 8 of the example part).

B. Recombinant Expression Vectors and Host Cells

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

The recombinant expression vector according to the invention comprises a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors one or more regulatory sequences, selected on the basis of the host cells to be used for expression, the is operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is bound to the regulatory sequence (s) in such a way that expression of the nucleotide sequence is possible (for example in an in vitro transcription / Translation system - or in a host cell if the vector is introduced into the host cell). The term "regulatory sequence" is intended to encompass promoters, enhancers and other expression control elements (for example polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the direct expression of the nucleotide sequence only in certain host cells. The person skilled in the art is aware that the design of an expression vector can depend on factors such as the choice of the host cell to be transformed, the extent of expression of the desired protein, etc. The expression vectors according to the invention can be introduced into the host cells, so that proteins or peptides are thereby obtained , including fusion proteins or peptides that are encoded by the nucleic acids as described herein (e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins, etc.). The recombinant expression vectors according to the invention can be designed for the expression of MCT proteins in prokaryotic or eukaryotic cells. For example. MCT genes can be found in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel, CAMJJ et al. (1991) "Heterologous gene expression in filamentous fungi" in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure, ed., Pp. 396-428: Academic Press: San Diego; and van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi. In: Applied Molecular Genetics of Fungi, Peberdy, JF et al., Ed., Pp. 1- 28, Cambridge University Press: Cambridge), algal and multicellular plant cells (see Schmidt, R. and Willmitzer, L. (1988) "High efficiency Agrohacterium tumefaciens-meάiateά transformation of Arabidopsis thaliana leaf and cotyledon explants" Plant Cell Rep.: 583 -586) or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

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

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

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

One strategy to maximize the expression of the recombinant protein is to express the protein in a bacterium whose ability to proteolytically cleave the recombinant protein is impaired (Gottesman, S. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to change the nucleic acid sequence of the nucleic acid to be inserted into an expression vector, so that the individual codons for each amino acid are those which are preferably used in a bacterium selected for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). This change in the nucleic acid sequences according to the invention is carried out by standard DNA synthesis techniques.

In a further embodiment, the MCT protein expression vector is a yeast expression vector. Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943 ), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods of constructing vectors suitable for use in other fungi, such as filamentous fungi, suitable include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, JF Peberdy et al., ed. , Pp. 1-28, Cambridge University Press: Cambridge.

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

In a further embodiment, the MCT proteins according to the invention can be expressed in single-cell plant cells (such as algae) or in plant cells of higher plants (for example spermatophytes such as crops). Examples of plant expression vectors include those which are described in detail in: Bekker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium Vectors for Plant Transformation", Nucl. Acids Res. 12: 8711-8721.

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

In a further embodiment, the recombinant mammalian expression vector can preferably bring about the expression of the nucleic acid in a specific cell type (for example, tissue-specific regulatory elements are used for the expression of the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors

(Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulin (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 10 (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. 3:15 537-546).

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

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

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

Control 30 of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a highly effective regulatory region whose activity is determined by the cell type, the vector is introduced into the ^•. 5 For a discussion of the regulation of gene expression using antisense genes, see Weintraub, H. et al. , Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1 (1) 1986.

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

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

Using conventional transformation or transfection methods, vector DNA can be introduced into prokaryotic or eukaryotic cells. The terms "transformation" and "transfection", "conjugation" and "transduction" as used herein are intended to encompass 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-dextran-mediated transfection, lipofection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.

For the stable transfection of mammalian cells it is known that, depending on the expression vector and transfection technique used, only a small part of the cells integrate the foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selectable marker (eg resistance to antibiotics) is usually introduced into the host cells together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. A nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an MCT protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified by drug selection (eg cells that have integrated the selectable marker survive, whereas the other cells die). To generate a homologously recombined microorganism, a vector is produced which contains at least a section of an MCT gene into which a deletion, addition or substitution has been introduced in order to change the MCT gene, for example to functionally disrupt it. This MCT gene is preferably a Corynebacterium glutamicum MCT gene, however a homologue from a related bacterium or even from a mammalian, yeast or insect source can be used. In a preferred embodiment, the vector is designed in such a way that the endogenous MCT gene is functionally disrupted when homologous recombination occurs (ie no longer encodes a functional protein, also referred to as a "knockout" vector). The vector can alternatively be designed in such a way that the endogenous MCT gene is mutated or otherwise altered in the case of homologous recombination, but still codes the functional protein (for example the upstream regulatory region can be altered in such a way that the expression of the endogenous MCT protein is thereby effected is changed.). The modified portion of the MCT gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the MCT gene, which is a homologous recombination between the exogenous MCT gene carried by the vector and one endogenous MCT gene in a microorganism. The additional flanking MCT nucleic acid is long enough for successful homologous recombination with the endogenous gene. The vector usually contains several kilobase flanking DNA (both at the 5 'and 3' ends) (see, for example, Thomas, KR and Capecchi, MR (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (eg, by electroporation), and cells in which the introduced MCT gene is homologously recombined with the endogenous MCT gene are selected using methods known in the art.

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

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

C. Uses and methods according to the invention

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

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

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

The manipulation of the MCT nucleic acid molecules according to the invention can bring about the production of MCT proteins with functional differences from the wild-type MCT proteins. These proteins can be improved in their efficiency or activity, can be present in the cell in larger numbers than usual, or can be weakened in their efficiency or activity.

There are many mechanisms by which the modification of an MCT molecule according to the invention directly influences the yield, production and / or efficiency of the production of one or more fine chemicals from a C. glutamicum strain which contains such a modified protein. The production of fine chemical compounds from large-scale C. glufcamicum cultures is significantly improved if C. glutamicum secretes the desired compounds, since these compounds can be easily purified from the culture medium (in contrast to extraction from the bulk of C. glutamicu cells ). By increasing the number or activity of transporter molecules that the fine chemicals from the Cell export, it may be possible to increase the amount of fine chemical produced that is present in the extracellular medium, thereby facilitating harvesting and cleaning. In contrast, the efficient overproduction of one or more fine chemicals requires increased amounts of cofactors, precursor molecules and intermediates for the appropriate biosynthetic pathways. By increasing the number and / or activity of transporter proteins involved in the import of nutrients such as carbon sources (ie sugars), nitrogen sources (ie amino acids, ammonium salts), phosphates and sulfur, the production of a fine chemical can be reduced improve any nutritional constraints in the biosynthesis process. In addition, fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or by increasing the number of one or more MCT proteins according to the invention which are involved in the biosynthesis of these compounds, or by influencing the activity of one or more MCT proteins which are involved in the degradation of these compounds, can increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from C. glutamicum.

The genetic manipulation of one or more MCT genes according to the invention can likewise produce MCT proteins with modified activities which indirectly influence the production of one or more desired fine chemicals from C. glutamicum. The normal biochemical metabolic processes, for example, produce a large number of waste products (e.g. hydrogen peroxide and other reactive oxygen species) that can actively interact with the same metabolic processes (e.g., peroxynitrite nitrates, as is known, tyrosine side chains, whereby some enzymes are inactivated with tyrosine in the active center (Groves, JT (1999) Curr. Opin. Chem. Biol. 3 (2); 226-235). Although these waste products are usually excreted, the C. glutamicum strains used for large-scale fermentative production are used for the overproduction of one or However, several fine chemicals have been optimized and can thus produce more waste products than is usual for a C. glutamicum wild type. By optimizing the activity of one or more MCT proteins according to the invention, which are involved in the export of waste molecules, the cell's viability can be improved improve and be an efficient metabolic activity The presence of high intracellular amounts of the desired fine chemical can be toxic to the cell, so increasing the cell's ability to secrete these compounds can improve cell viability. The MCT proteins according to the invention can be manipulated so that the relative amounts of different lipid and fatty acid molecules are changed. This can have a significant impact on the lipid composition of the cell membrane. Because each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change membrane fluidity. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained above, can modify the export of waste products or the fine chemicals produced or the import of necessary nutrients. These membrane fluidity changes can also significantly affect cell integrity; Cells with relatively weaker membranes are more susceptible to mechanical stress in a large fermenter environment, which can damage or kill the cells. By manipulating MCT proteins, which are involved in the production of fatty acids and lipids for membrane construction, so that the membrane composition of the resulting membrane should be more sensitive to the environmental conditions in the cultures used to produce fine chemicals a larger proportion of C. glutamicum cells survive and multiply. Larger amounts of C. glutamicum cells in a culture should result in greater yields, production or efficiency in the production of the fine chemical from the culture.

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

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

Examples

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

A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30 ° C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml of buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml of culture volume). The composition of buffer I: 140.34 g / 1 sucrose, 2.46 g / 1 MgS0 ₄ • 7 H ₂ 0, 10 ml / 1 KH ₂ P0 ₄ solution (100 g / l, adjusted to pH with KOH 6.7), 50 ml / 1 Ml2 concentrate (10 g / 1 (NH ₄ ) ₂ S0, 1 g / 1 NaCl, 2 g / 1 MgS0 • 7 H ₂ 0, 0.2 g / 1 CaCl ₂ , 0.5 g / 1 yeast extract (Difco), 10 ml / 1 trace element mixture (200 mg / 1 FeS0 ₄ • H0, 10 mg / 1 ZnS0 ₄ • 7 H ₂ 0, 3 mg / 1 MnCl ₂ • 4 H ₂ 0, 30 mg / 1 H ₃ B0 ₃ , 20 mg / 1

CoCl ₂ • 6 H ₂ 0, 1 mg / 1 NiCl • 6 H ₂ 0, 3 mg / 1 Na ₂ Mo0 ₄ • 2 H ₂ 0, 500 mg / 1 complexing agent (EDTA or citric acid), 100 ml / 1 vitamin mixture ( 0.2 ml / 1 biotin, 0.2 mg / 1 folic acid, 20 mg / 1 p-aminobenzoic acid, 20 mg / 1 riboflavin, 40 mg / 1 Ca panthothenate, 140 mg / 1 nicotinic acid, 40 mg / 1 Pyridoxol hydrochloride, 200 mg / 1 myoinositol). Lysozyme was added to the suspension in a final concentration of 2.5 mg / ml. After about 4 hours of incubation at 37 ° C, the cell wall was broken down and the protoplasts obtained were harvested by centrifugation. The pellet was washed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added. After adding proteinase K at a final concentration of 200 μg / l, 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 precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by incubation for 30 min at -20 ° C and 30 min centrifugation at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) , The DNA was dissolved in 1 ml of TE buffer containing 20 μg / ml RNase A and dialyzed against 1000 ml of TE buffer at 4 ° C. for at least 3 hours. During this time the buffer was exchanged 3 times. 0.4 ml of 2 M LiCl and 0.8 ml of ethanol were added to 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, prepared as described in Example 1, "Molecular Cloning: A Laboratory Manual" was used according to known and well-established methods (see, for example, Sambrook, J. et al. (1989). Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", John Wille & Sons) Cosmid and plasmid banks.

Any plasmid or cosmid could be used. Plasmids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741) found particular use; pACYC177 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or

Cosmids such as SuperCosl (Stratagene, LaJolla, USA) or Loristδ (Gibson, T.J. Rosenthal, A., and Waterson, R.H. (1987) Gene 53: 283-286.

Example 3: DNA sequencing and computer function analysis

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

Example 4: In Vivo Mutagenesis

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

Example 5: DNA transfer between Escherichia coli and Corynebacterium glutamicum

Several Corynebacterium and Breviipaσfcerium species contain endogenous plasmids (such as pHMl519 or pBLl) that replicate autonomously (for an overview see, for example, Martin, J.F. et al. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel , FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons), to which an origin of replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevijbacfcertium species. Particular uses as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn-903 transposon) or for chloramphenicol (Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim) There are numerous examples in the literature for the production of a wide variety of shuttle vectors which are replicated in E. coli and C. glutamicum and which can be used for various purposes, including gene overexpression ( see, e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, JF et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ et al. (1992) Gene 102: 93-98).

Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to introduce such hybrid vectors into Corynebacterium glutamicum strains. The transformation of C. glutamicum can be carried out by protoplast transformation (Kastsumata, R. et al., (1984) J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et al., (1989) FEMS Microbiol. Letters , 53: 399-303) and, in cases where special vectors are used, can also be achieved by conjugation (as described, for example, in Schaefer, A., et (1990) J. Bacteriol. 172: 1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods known in the art) and transforming it into E. coli. This transformation step can be carried out using standard methods, but an Mcr deficit is advantageously the E. coli strain was used, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166: 1-19).

Example 6: Determination of expression of the mutant protein

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

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

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

Genetically modified Corynebacteria are grown in synthetic or natural growth media. A number of different growth media for Corynebakterian are known and easy available (Lieb et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) "The Genus Corynebacterium ", in: The Procaryotes, Vol. II, Balows, A., et al., Ed. Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH ₄ CI or (NH ₄ ) 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 that may be included in the media include the chloride, phosphorus, or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating agents can be added to the medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols such as catechol or protocatechuate or organic acids such as citric acid. The media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine. Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like. The exact composition of the media connections depends heavily on the respective experiment and is decided individually for each case. Information about media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and the like. All media components are sterilized, either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration. The components can be sterilized either together or, if necessary, separately. All media components can be present at the beginning of the cultivation or can be added continuously or in batches.

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

The incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth. The disclosed growth experiments can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes. To screen a large number of clones, the microorganisms should be grown in microtiter plates, glass tubes or shake flasks with or without baffles. Preferably 100 ml shake flasks are used, which are filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, a mathematical correction should be carried out for the evaporation losses.

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

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

Determining the activities and kinetic parameters of enzymes is well known in the art. Experiments to determine the activity of a particular altered enzyme must be adapted to the specific activity of the wild-type enzyme, which is within the ability of the person skilled in the art. Overviews of enzymes in general as well as specific details relating to the structure, kinetics, principles, processes, applications and examples for determining many enzyme activities can be found, for example, in the following literature references: Dixon, M., and Webb, EC: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction Mechanisms. Freeman, San Francisco; Price, N.C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P.D-. Ed. (1983) The Enzymes, 3rd 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 measured by many well-established methods, such as DNA band shift assays (also referred to as gel retardation assays). The effect of these proteins on the expression of other molecules can be measured using reporter gene assays (as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904 and the references cited therein). Reporter gene test systems are well known and established for use in pro- and eukaryotic cells using enzymes such as beta-galactosidase, green fluorescent protein and several others.

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

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

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

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

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

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

The identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

equivalent

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

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

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

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

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

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

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

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

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

A alanine

C cysteine

D aspartate

E glutamate

F phenylalanine

G glycine

H His

I isoleucine

K lysine

L leucine

M methionine

N asparagine

P proline

Q glutamine

R arginine

S serine

T threonine

V valine

W tryptophan

Y tyrosine

Table 1

Genes that code for membrane synthesis and membrane transport proteins

DNA AS identification: AS AS AS function:

ID: ID: Pos: Wild-type Mutai

1 2 RXA00149 75 P S METHYL ALONYL-COA MUTASE (EC 5.4.99.2)

413 G E ETHYLMALONYL-COA MUTASE (EC 5.4.99.2)

417 A V METHYLMALONYL-COA MUTASE (EC 5.4.99.2)

3 4 RXA00186 189 G E ACETYL HYDROLASE (EC 3.1.1.-)

5 6 RXA00203 288 R C RIBOSE TRANSPORT SYSTEM PERMEASE PROTEIN RBSC

7 8 RXA00228 109 S F TRANSPORTER g 10 RXA00298 143 A T TRANSPORTER

11 12 RXA00345 27 D N MANGANESE BINDING PERIPLASMIC PROTEIN PRECURSOR

13 14 RXA00354 67 G E Hydrolase (HAD superfamily)

15 16 RXA00368 523 A T IRON (III) TRANSPORT SYSTEM PERMEASE PROTEIN SFUB

17 18 RXA00378 570 D N Cation transport ATPases

570 D N Cation transport ATPases

19 20 RXA00412 346 G S ABC TRANSPORTER ATP-BINDING PROTEIN

346 G S ABC TRANSPORTER ATP-BINDING PROTEIN

21 22 RXA00413 247 D N ABC TRANSPORTER SUBSTRATE BINDING PROTEIN

23 24 RXA00432 495 SN NA (+) - LINKED D-ALANINE GLYCINE PERMEASE

26 RXA00453 243 A T TRANSPORTER

246 G D TRANSPORTER

28 RXA00456 99 A T ABC TRANSPORTER ATP-BINDING PROTEIN

30 RXA00477 158 S F PHYTOENE DEHYDROGENASE (EC 1.3.-.-)

32 RXA00525 93 P S ABC TRANSPORTER PERMEASE PROTEIN

93 P s ABC TRANSPORTER PERMEASE PROTEIN

208 E K ABC TRANSPORTER PERMEASE PROTEIN

277 A V ABC TRANSPORTER PERMEASE PROTEIN

34 RXA00526 58 D N ABC TRANSPORTER ATP-BINDING PROTEIN

38 RXA00634 452 G D DI / TRIPEPTIDE TRANSPORTER

40 RXA00654 399 S F INNER MEMBRANE PROTEIN

42 RXA00690 220 D N COBALT TRANSPORT PROTEIN CBIQ

44 RXA00732 453 A V ABC TRANSPORTER ATP-BINDING PROTEIN

46 RXA00758 398 D N PERIPLASMIC OLIGOPEPTIDE-BINDING PROTEIN PRECURSOR

418 G C PERIPLASMIC OLIGOPEPTIDE-BINDING PROTEIN PRECURSOR

48 RXA00759 129 D N OLIGOPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN OPPB

301 L F OLIGOPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN OPPB

50 RXA00777 35 S L PHOSPHATE TRANSPORT SYSTEM PERMEASE PROTEIN PSTC

52 RXA00792 52 G D ACYL-COA DEHYDROGENASE (EC 1.3.99.-)

54 RXA00819 170 G D ACYL-COA DEHYDROGENASE (EC 1.3.99.-)

56 RXA00832 53 AV CALCIUM / PROTON ANTIPORTER

53 A V CALCIUM / PROTON ANTIPORTER

58 RXA00880 314 ATL LONG CHAIN FATTY ACID COA LIGASE (EC 6.2.1.3)

60 RXA00931 278 P S ACYL-COA THIOESTERASE II (EC 3.1.2.-)

62 RXA00939 103 A T CADMIUM RESISTANCE PROTEIN

335 A V CADMIUM RESISTANCE PROTEIN

64 RXA00941 27 G D SODIUM-DEPENDENT PHOSPHATE TRANSPORT PROTEIN

134 A V SODIUM-DEPENDENT PHOSPHATE TRANSPORT PROTEIN

66 RXA00980 409 G R CADMIUM RESISTANCE PROTEIN

409 G R CADMIUM RESISTANCE PROTEIN

68 RXA01013 87 G R DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN DPPC

70 RXA01070 369 A V PROTON / SODIUM-GLUTAMATE SYMPORT PROTEIN

72 RXA011 2 310 G E SULFATE PERMEASE

1

74 RXA01220 43 C Y AMIDE-UREA TRANSPORT SYSTEM PERMEASE PROTEIN

76 RXA01285 250 G E FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING PROTEIN FEPC

250 G E FERRIC ENTEROBACTIN TRANSPORT ATP-BINDING PROTEIN FEPC

78 RXA01298 69 G E TRANSPORTER

69 G E TRANSPORTER

80 RXA01323 143 Q H COPPER UPTAKE ATPASE (EC 3.6.1.-)

144 W C COPPER UPTAKE ATPASE (EC 3.6.1.-)

145 A P COPPER UPTAKE ATPASE (EC 3.6.1.-)

717 P S COPPER UPTAKE ATPASE (EC 3.6.1.-)

82 RXA01338 121 TI CATION-TRANSPORTING ATPASE PACS (EC 3.6.1.-) 121 TI CATION-TRANSPORTING ATPASE PACS (EC 3.6.1.-)

83 84 RXA01339 64 L P TYROSINE-SPECIFIC TRANSPORT PROTEIN

85 86 RXA01425 85 E K 60 KDA INNER MEMBRANE PROTEIN YIDC

87 88 RXA01467 48 T 1 ACYLCARRIER PROTEIN

89 90 RXA01629 3 P s PROLINE / ECTOINE TRANSPORTER

91 92 RXA01667 201 M 1 SODIUM / GLUTAMATE SYMPORT CARRIER PROTEIN

417 G D SODIUM / GLUTAMATE SYMPORT CARRIER PROTEIN

93 94 RXA01677 209 G R THIOLEDISULFIDE INTERCHANGE PROTEIN DSBA

95 96 RXA01727 116 G D BRANCHED-CHAIN AMINO ACID TRANSPORT SYSTEM CARRIER PROTEIN

97 98 RXA01746 146 G E LIPOATE PROTEIN LIGASE B (EC 6.-.-.-)

99 100 RXA01749 189 A V INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

212 PS INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

420 AT INTEGRAL MEMBRANE PROTEIN WITH TRKA DOMAINS

101 102 RXA01822 296 G D TRANSPORTER

103 104 RXA01853 119 E K HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6)

192 P L HYDROXYACYLGLUTATHIONE HYDROLASE (EC 3.1.2.6)

105 106 RXA01936 276 L F MACROLIDE-EFFLUX PROTEIN

107 108 RXA02020 52 A V AROMATIC AMINO ACID TRANSPORT PROTEIN AROP

109 110 RXA02029 37 E K CYCLOPROPANE-FATTY-ACYL-PHOSPHOLIPID SYNTHASE (EC 2.1.1.79)

111 112 RXA02034 193 T 1 DIPEPTIDE TRANSPORT SYSTEM PERMEASE PROTEIN DPPB

113 114 RXA02035 129 T 1 PERIPLASMIC DIPEPTIDE TRANSPORT PROTEIN PRECURSOR

365 A V PERIPLASMIC DIPEPTIDE TRANSPORT PROTEIN PRECURSOR

115 116 RXA02062 269 T 1 GLYCOSYL TRANSFERASE (EC 2.4.1.-) 269 T 1 GLYCOSYL TRANSFERASE (EC 2.4.1.-)

117 118 RXA02074 341 S N TRANSPORTER

119 120 RXA02095 409 A V ABC TRANSPORTER ATP-BINDING PROTEIN

914 A V ABC TRANSPORTER ATP-BINDING PROTEIN

121 122 RXA02119 209 G E TRANSPORTER

241 G D TRANSPORTER

123 124 RXA02128 10 S N SULFATE PERMEASE

125 126 RXA02173 195 A T ACETYL COENZYME A CARBOXYLASE CARBOXYL TRANSFERASE SUBUNIT BETA (EC 6.4.1.2)

127 128 RXA02220 674 G S CALCIUM-TRANSPORTING ATPASE (EC 3.6.1.38)

129 130 RXA02233 323 A V URACIL PERMEASE

131 132 RXA02309 276 P S OCTAPRENYL-DI PHOSPHATE SYNTHASE (EC 2.5.1.-)

133 134 RXA02335 313 D N acetyl-CoA carboxylase alpha chain (EC 6.4.1.2) / propionyl-CoA carboxylase alpha chain (EC 6.4.1.3)

135 136 RXA02394 127 G E C4-DICARBOXYLATE TRANSPORTER LARGE SUBUNIT

137 138 RXA02395 533 A V TRANSPORTER

139 140 RXA02426 207 A V NA + / H + ANTI PORTER NHAP

320 S L NA + / H + ANTIPORTER NHAP

141 142 RXA02447 185 A V GALACTOSE-PROTON SYMPORTER

185 A V GALACTOSE-PROTON SYMPORTER

143 144 RXA02487 362 G S LONG-CHAIN-FATTY-ACID - COA LIGASE (EC 6.2.1.3)

145 146 RXA02512 23 A V LIPID A BIOSYNTHESIS LAUROYL ACYLTRANSFERASE (EC 2.3.1.-)

147 148 RXA02566 287 S F TETRACYCLINE RESISTANCE PROTEIN, CLASS A

149 150 RXA02570 184 R C ABC TRANSPORTER INTEGRAL MEMBRANE PROTEIN

151 152 RXA02613 210 G R TAURINE-BINDING PERIPLASMIC PROTEIN PRECURSOR

153 154 RXA02627 338 EK DTXR / IRON-REGULATED LIPOPROTEIN PRECURSOR

155 156 RXA02676 387 A V HIGH-AFFINITY GLUCONATE TRANSPORTER

157 158 RXA02677 270 G s GLYCEROPHOSPHORYL DIESTER PHOSPHODIESTERASE (EC 3.1.4.46)

159 160 RXA02684 143 D N MEMBRANE-BOUND PROTEIN LYTR

403 E K MEMBRANE-BOUND PROTEIN LYTR

161 162 RXA02718 355 V M FARNESYL PYROPHOSPHATE SYNTHETASE (EC 2.5.1.1) I GERANYLTRANSTRANSFERASE (EC 2.5.1.10)

163 164 RXA02767 251 E K TRANSPORTER

165 166 RXA02795 240 L F DIPEPTIDE TRANSPORT ATP-BINDING PROTEIN DPPF

167 168 RXA02922 72 A V DIBENZOTHIOPHENE DESULFURIZATION ENZYME C

169 170 RXA02925 429 A V COPPER-SILVER EFFLUX ATPASE (EC 3.6.1.-)

460 P s COPPER-SILVER EFFLUX ATPASE (EC 3.6.1.-)

171 172 RXA02991 138 P s GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP

313 G E GLUTAMINE TRANSPORT SYSTEM PERMEASE PROTEIN GLNP

173 174 RXA03129 209 L F TRANSPORTER

175 176 RXA03157 23 A T PHOSPHOPANTETHEINE ADENYLYL TRANSFERASE (EC 2.7.7.3)

177 178 RXA03285 469 D N TRANSPORTER

179 180 RXA03299 359 G D Hypothetical membrane spanning protein

181 182 RXA03376 1484 P S FATTY ACID SYNTHASE (EC 2.3.1.85) [INCLUDES: EC 2.3.1.38; EC 2.3.1.39; EC 2.3.1.41; EC 1.1.1.100; EC 4.2.1.61; EC 1.3.1.10; EC 3.1.2.14]

183 184 RXA03393 203 A V TRANSPORTER

185 186 RXA03763 47 N S TRANSPORTER

187 188 RXA03864 5 L F Hypothetical Membrane Spanning Protein

189 190 RXA03916 243 S N LIPOPOLYSACCHARIDE BIOSYNTHESIS PROTEIN WZXC

191 192 RXA04102 36 TA PERMEASE

193 194 RXA04164 49 L F TRANSPORTER

195 196 RXA04275 482 L F TRANSPORTER

197 198 RXA04314 69 V I SODIUM-DEPENDENT PHOSPHATE TRANSPORT PROTEIN

199 200 RXA04323 360 S F SODIUM / PROTON ANTIPORTER PROTEIN SHAA I SODIUM / PROTON ANTIPORTER PROTEIN SHAB

575 S F SODIUM / PROTON ANTIPORTER PROTEIN SHAA I SODIUM / PROTON ANTIPORTER PROTEIN SHAB

201 202 RXA04359 171 H Y PUTATIVE TRANSPORT PROTEIN SGAT 311 V I PUTATIVE TRANSPORT PROTEIN SGAT

203 204 RXA04482 310 P S TRANSPORT ATP-BINDING PROTEIN CYDC

312 S F TRANSPORT ATP-BINDING PROTEIN CYDC

205 206 RXA04624 67 A T TRANSPORTER

207 208 RXA07009 345 N F PUTATIVE UNDECAPRENYL-PHOSPHATE ALPHA-N-ACETYLGLUCOSAMINYLTRANSFERASE (EC 2.4.1,

209 210 RXA07010 111 P L PHOSPHONATES TRANSPORT SYSTEM PERMEASE PROTEIN PHNE

211 212 RXA07012 263 A V PEROSAMINE SYNTHETASE PER

213 214 RXA07013 49 R H POTASSIUM CHANNEL BETA SUBUNIT

215 216 RXA07022 127 E K ABC TRANSPORTER ATP-BINDING PROTEIN

455 E K ABC TRANSPORTER ATP-BINDING PROTEIN

Claims

claims

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

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

2. The isolated nucleic acid molecule according to claim 1, wherein the nucleic acid molecule in the amino acid position specified in table / column 4 encodes the amino acid specified in table / column δ in the same line.

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

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

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

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

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

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