AU2002368410A1

AU2002368410A1 - Methods for the production of methionine

Info

Publication number: AU2002368410A1
Application number: AU2002368410A
Authority: AU
Inventors: Gregor Haberhauer; Stefan Haefner; Corinna Klopprogge; Burkhard Kroeger; Markus Pompejus; Hartwig Schroeder; Oskar Zelder
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 2002-11-29
Filing date: 2002-11-29
Publication date: 2004-06-23
Also published as: CN1694899A; BR0215958A; CN1694899B; WO2004050694A1; EP1567549A1; BRPI0215958B1; JP2006521083A

Description

WO 2004/050694 PCT/EP2002/013504 Methods for the production of methionine .. Methionine is currently produced as a racemic mixture of DL methionine by a well established chemical process. Most DL 5 methionine is being produced by variations of the same chemical procedure method involving toxic, dangerous, flammable, unsta ble, and highly odorous starting materials or intermediatesThe starting materials for the chemical synthesis are: acrolein, me thylmercaptan and hydrogen cyanide. The chemical synthesis in 10 volves the reaction of methylmercaptan and acrolein producing the intermediate 3-methylmercaptopropionaldehyde (MMP). In the further process the MMP reacts with hydrogen cyanide to form the 5-(2-methylthioethyl) hydantoin, which then can be hydrolyzed using 2 equivalents of caustics such as NaOH together with one 15 half equivalent Na 2

CO

3 to yield sodium-DL-methioninate and one equivalent Na 2

CO

3 one equivalent NH3 and one half equivalent CO 2 . In the succeeding step the sodium-DL-methioninate is neutralized with 1.5 equiv. sulfuric acid and 1 equiv.Na 2

CO

3 to yield DL methionine NaSO, and C0 2 . It is obvious that such a chemical 20 process yields a large molar excess of unused salts in compari son to the amount of methionine that is produced. This fact poses an economic and ecologic challenge. Fermentative processes are usually based on cultivating microor 25 ganisms on nutrients including carbohydrate source (e.g., sugars such as glucose fructose or saccharose), nitrogen sources (e.g., ammonia) and sulfur sources (e.g., sulfate or thiosulfate) to gether with other necessary media components. The process yields only the natural product L-methionine and only biomass as a by 30 product. Since no toxic, dangerous, flammable unstable, and highly odorous starting materials are being used and no salt is produced by a fermentative process, there is an advantage of this process over the chemical methionine synthesis. Methionine can be produced in organisms such as E. coli or Corynebacter 35 (Kase H., Nakayama K. (1975) Agric. Biol. Chem. 39 pp 153-160; Chatterjee et al. (1999) Acta Biotechnol. 14, pp199-204; Harma S. Gomes, (2001) J. Eng. Life Sci. 1 pp. 69-73, JP 50031092, DE 2105189). 40 All living cells have complex catabolic and anabolic metabolic capabilities with many interconnected pathways. In order to maintain a balance between the various parts of this extremely complex metabolic network, the cell employs a finely-tuned regu latory network. By regulating enzyme synthesis and enzyme activ- WO 2004/050694 PCT/EP2002/013504 2 ity, either independently or simultaneously, the cell is able to control the activity of disparate metabolic pathways to reflect the changing needs of the cell. The induction or repression of enzyme synthesis may occur at either the level of transcription 5 or translation, or both. Gene expression in prokaryotes is regu lated by several mechanisms at the level of transcription (for review see e.g., Lewin, B (1990) Genes IV, Part 3: "Controlling prokaryotic genes by transcription", Oxford University Press: Oxford, p. 213-301, and references therein, and Michal, G. 10 (1999) Biochemical Pathways: An Atlas of Biochemistry and Mo lecular Biology, John Wiley & Sons). All such known regulatory processes are mediated by additional genes, which themselves re spond to external influences of various kinds (e.g., tempera ture, nutrient availability, or light). Exemplary protein fac 15 tors which have been implicated in this type of regulation in clude the transcription factors. These are proteins which bind to DNA, thereby either increasing the expression of a gene (positive regulation) or decreasing gene expression (negative regulation). These expression-modulating transcription factors 20 can themselves be the subject of regulation. Their activity can, for example, be regulated by the binding of low molecular weight compounds to the DNA-binding protein, thereby stimulating or in hibiting the binding of these proteins to the appropriate bind ing site on the DNA (see, for example, Helmann, J.D. and Cham 25 berlin, M.J. (1988) "Structure and function of bacterial sigma factors." Ann. Rev. Biochem. 57: 839-872; Adhya, S. (1995) "The lac and gal operons today" and Boos, W. et al., "The maltose system.", both in: Regulation of Gene Expression in Escherichia coli (Lin, E.C.C. and Lynch, A.S., eds.) Chapman & Hall: New 30 York, p. 181-200 and 201-229; and Moran, C.P. (1993) "RNA poly merase and transcription factors." in: Bacillus subtilis and other gram-positive bacteria, Sonenshein, A.L. et al., eds. ASM: Washington, D.C., p. 653-667.) 35 The entire genome of Corynebacterium glutamicum ATCC 13032 is published under GeneBank Acc.-No.: AP005283. However, no hint is given for specific regulators of methionine biosynthesis. Up- or down-regulation of gene transcription and translation may 40 be governed by the cellular and extracellular levels of various factors, such as substrates, catabolites, and end products. Typically, the expression of genes encoding enzymes necessary for the activity of a particular pathway is induced by high lev- WO 2004/050694 PCT/EP2002/013504 3 els of substrate molecules for that pathway. Similarly, such gene expression tends to be repressed when there exist high in tracellular levels of the end product of the pathway (Snyder, L. and Champness, W. (1997) The Molecular Biology of Bacteria ASM: 5 Washington). Gene expression may also be regulated by other ex ternal and internal factors, such as environmental conditions (e.g., heat, oxidative stress, or starvation; see, for example, Lin, E.C.C. and Lynch, A.S., eds. (1995) Regulation of Gene Ex pression in Escherichia coli. Chapman & Hall: New York). 10 A thorough understanding of the regulatory networks governing cellular metabolism in microorganisms is critical for the high yield production of chemicals by fermentation. Control systems for the down-regulation of metabolic pathways could be removed 15 or lessened to improve the synthesis of desired chemicals, and similarly, those for the up-regulation of metabolic pathways for a desired product could be constitutively activated or optimized in activity (As shown in Hirose, Y. and Okada, H. (1979) "Micro bial Production of Amino Acids", in: Peppler, H.J. and Perlman, 20 D. (eds.) Microbial Technology 2 ed. Vol. 1, ch. 7 Academic Press: New York.) However, for methionine biosynthesis methods for fermentatively producing methionine based on modification of a methionine bic 25 synthesis regulatory system has not been described so far. In all reported cases methods for fermentatively producing methion ine the yields seem to be too low for an economic production. Therefore, an improved method is needed. 30 Summary of the Invention The invention provides novel methods for modulate the biosynthe sis of fine chemicals preferably sulfur-containing compounds like e.g., methionine. It has been found that the metabolic 35 regulatory ("MR") molecules of the invention are involved in biosynthesis of a fine chemical, preferably sulfur-containing compounds like e.g., methionine. In particular, it has been found that the MR molecule RKA00655 40 is a negative regulator, e.g., transcriptional regulator, of me thionine biosynthesis, cysteine biosynthesis, and/or the sulfur reduction pathway. It has also been found that the MR molecule RXNO2910 is a positive regulator, e.g., transcriptional regula- WO 2004/050694 PCT/EP2002/013504 4 tor, of methionine biosynthesis. The nucleotide sequence of RXA00655 is set forth herein as SEQ ID NO:1 and the polypeptide sequence of RXA00655 is set forth herein as SEQ ID NO:2. The nu cleotide sequence of RXN02910 is set forth herein as SEQ ID NO:5 5 and the polypeptide sequence of RXA00655 is set forth herein as SEQ ID NO:6. Homologous proteins of RXA00655 are set forth herein as SEQ ID NO: 19, 21 and 23. The nucleotide sequence of said RXNO2910 homologous proteins is set forth herein as SEQ ID NO: 18, 20 and 22. 10 Modulation of the expression of the MR nucleic acids of the in vention, e.g., increasing expression of the RXN02910 nucleic acid molecule or increasing the activity of the RXNO2910 pro tein, suppressing expression of the RXA00655 nucleic acid mole 15 cule or suppressing the activity of the RxA00655 protein, or modification of the sequence of the MR nucleic acid molecules of the invention to alter the activity of the MR nucleic acid mole cule, can be used to modulate, e.g., increase, the production of a fine chemical, e.g., methionine, cysteine, and/or other com 20 pounds of the methionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic pathway from a microorganism (e.g., to improve the yield or production of a fine chemical, e.g., methionine or other compounds of the me thionine biosynthetic pathway, from a Corynebacterium or Brevi 25 bacterium species). In another embodiment, increasing the ex pression or activity of the RXN02910 nucleic acid or protein and suppression of the expression or activity of the RXA00655 nu cleic acid or protein, in combination, can be used to modulate, e.g., increase, the production a fine chemical, e.g., methion 30 ine, cysteine, and/or other compounds of the methionine biosyn thetic pathway, the sulfur reduction pathway, and/or the cys teine biosynthetic pathway from a microorganism (e.g., Coryne bacterium or Brevibacterium species). 35 The present invention features methods of modulating production of a sulfur-containing compound by a microorganism comprising culturing a microorganism with modulated expression or activity of at least one regulator of methionine biosynthesis under con ditions such that production of a sulfur-containing compound is 40 modulated. Preferably said sulfur-containing compound is se lected from the group consisting of methionine, cysteine, S adenosylmethionine and homocycsteine. Furthermore preferably, WO 2004/050694 PCT/EP2002/013504 5 production of said sulfur-containing compound (e.g, methionine) is increased The present invention features methods of increasing production 5 of a sulfur-containing compound by a microorganism comprising culturing a microorganism which overexpresses a positive regula tor of methionine biosynthesis, e.g., RXNO2910, under conditions such that production of the sulfur-containing compound is in creased. Furthermore, the present invention features methods of 10 increasing production of a sulfur-containing compound by a mi croorganism comprising culturing a microorganism which underex presses a negative regulator of methionine biosynthesis, e.g., RXA00655, under conditions such that production of the sulfur containing compound is increased. 15 Accordingly, the present invention features methods of producing methionine as well as other compounds of the methionine biosyn thetic pathway. Such methods include culturing microorganisms overexpressing the RXNO2910 gene product under conditions such 20 that methionine, or other compounds of the methionine biosyn thetic pathway, are produced. Such methods also include cultur ing microorganisms with inhibited expression of the RXA00655 gene product such that methionine, or other compounds of the me thionine biosynthetic pathway, the sulfur reduction pathway, 25 and/or the cysteine biosynthetic pathway are produced. The pre sent invention also provides methods for producing a fine chemi cal comprising culturing microorganisms overexpressing the RXN02910 gene product in combination with microorganisms with inhibited expression of the RXA00655 gene product. 30 The MR proteins are capable of, for example, performing a func tion involved in the transcriptional, translational, or post translational regulation of proteins important for the normal metabolic functioning of cells. Given the availability of clon 35 ing vectors for use in Corynebacterium glutamicum (such as exem plified below) the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of a fine chemical, e.g., methionine. 40 This improved yield, production and/or efficiency of production of a fine chemical, e.g., methionine, may be due to a direct ef fect of manipulation of a gene of the invention, e.g., RXA00655 WO 2004/050694 PCT/EP2002/013504 6 or RXNO2910, or it may be due to an indirect effect of such ma nipulation. Specifically, alterations in C. glutamicum MR pro teins which normally regulate the yield, production and/or effi ciency of production of the methionine metabolic pathways may 5 have a direct impact on the overall production or rate of pro duction of a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sul fur reduction pathway, and/or the cysteine biosynthetic pathway from this organism. Alterations in the proteins involved in the 10 methionine metabolic pathway may also have an indirect impact on the yield, production and/or efficiency of production of a de sired fine chemical, e.g., methionine. Regulation of metabolism is necessarily complex, and the regulatory mechanisms governing different pathways may intersect at multiple points such that 15 more than one pathway can be rapidly adjusted in accordance with a particular cellular event. This enables the modification of a regulatory protein for one pathway to have an impact on the regulation of many other pathways as well, some of which may be involved in the biosynthesis or degradation of a desired fine 20 chemical, e.g., methionine. In this indirect fashion, the modu lation of action of an MR protein has an impact on the produc tion of a fine chemical produced by a pathway different from one which that MR protein directly regulates. 25 The nucleic acid and protein molecules of the invention may be utilized to directly improve the yield, production, and/or effi ciency of production of methionine from Corynebacterium glu tamicum. Using recombinant genetic techniques well known in the art, one or more of the regulatory proteins of the invention may 30 be manipulated such that its function or expression is modu lated. For example, the mutation of an MR protein, e.g., RXA00655, which is involved in the repression of transcription of a gene, e.g., the metY gene, encoding a polypeptide which is required for the biosynthesis of an amino acid, e.g., methion 35 ine, such that it no longer is able to repress transcription may result in an increase in production of that amino acid. Simi larly, the alteration of activity of an MR protein resulting in increased translation or activating posttranslational modifica tion of a C. glutamicum protein involved in the biosynthesis of 40 a desired fine chemical, e.g., methionine, may in turn increase the production of that chemical. The opposite situation may also be of benefit: by increasing the repression of transcription or translation, or by posttranslational negative modification of a WO 2004/050694 PCT/EP2002/013504 7 C. glutamicum protein involved in the regulation of a degrada tive pathway for a compound, one may increase the production of this chemical. In each case, the overall yield or rate of pro duction of the desired fine chemical may be increased. 5 It is also possible that such alterations in the protein and nu cleotide molecules of the invention may improve the yield, pro duction, and/or efficiency of production of fine chemicals, e.g., methionine, through indirect mechanisms. The metabolism of 10 any one compound is necessarily intertwined with other biosyn thetic and degradative pathways within the cell, and necessary cofactors, intermediates, or substrates in one pathway are likely supplied or limited by another such pathway. Therefore, by modulating the activity of one or more of the regulatory pro 15 teins of the invention, the production or efficiency of activity of another fine chemical biosynthetic or degradative pathway may be impacted. Further, the manipulation of one or more regulatory proteins may increase the overall ability of the cell to grow and multiply in culture, particularly in large-scale fermenta 20 tive culture, where growth conditions may be suboptimal. For example, by mutating an MR protein of the invention which would normally cause a repression in the biosynthesis of nucleotides in response to suboptimal extracellular supplies of nutrients (thereby preventing cell division) such that it is decreased in 25 repressor ability, one may increase the biosynthesis of nucleo tides and perhaps increase cell division. Changes in MR proteins which result in increased cell growth and division in culture may result in an increase in yield, production, and/or effi ciency of production of one or more desired fine chemicals from 30 the culture, due at least to the increased number of cells pro ducing the chemical in the culture. The invention provides novel transgenic expression cassettes comprising in combination with a regulatory sequence a nucleic 35 acid molecule coding for a metabolic pathway protein ((MR) pro tein] or a fragment thereof, wherein said regulatory sequence is capable of mediating expression of said nucleic acid molecule, preferably in a microorganism. Preferably said regulatory se quence is a promoter heterologous with regard to said nucleic 40 acid molecule. Preferably the regulatory sequence is a promoter sequence functional in Corynebacterium glutamicum.

WO 2004/050694 PCT/EP2002/013504 8 MR proteins are capable of, for example, performing an enzymatic step involved in the transcriptional, translational, or post translational regulation of metabolic pathways in C. glutamicum. Nucleic acid molecules encoding an MR protein are referred to 5 herein as MR nucleic acid molecules. In a preferred embodiment, the MR protein participates in the transcriptional, transla tional, or posttranslational regulation of one or more metabolic pathways. 10 In a especially preferred embodiment, the MR nucleic acid mole cule is selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID 15 NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide se 20 quence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a complement thereof; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% identical to the 25 amino acid sequence of SEQ ID NO:2,.SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23; and d) a nucleic acid molecule which encodes a fragment of a polypep tide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID 30 NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, 35 The transgenic expression cassette may comprise the nucleic acid molecule coding for the MR protein (e.g., RXA00655 or RXA02910) in antisense or sense orientation with regard to said promoter sequence. 40 In a preferred embodiment of the invention the nucleic acid molecule coding for the MR protein (e.g., RXA00655 or RXA02910) encodes a polypeptide comprising the amino acid sequence set WO 2004/050694 PCT/EP2002/013504 9 forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. Especially preferred, said nu cleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID 5 NO:20, or SEQ ID NO:22. The nucleic acid molecule may also code for a naturally occurring allelic variant of a polypeptide com prising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. 10 Another aspect of the invention pertains to a vector comprising at least one of the inventive transgenic expression cassettes. Preferably the vector is an expression vector. Yet another aspect of the invention pertains to a host cell 15 transfected or transformed with at least one of the transgenic expression cassettes of this invention or at least one of the inventive vectors or expressionvectors. Preferably, the host cell belongs to the genus Corynebacterium or Brevibacterium. In one embodiment, such a host cell is used to produce an MR pro 20 tein by culturing the host cell in a suitable medium. The MR protein can be then isolated from the medium or the host cell. Yet another aspect of the invention pertains to a genetically altered microorganism in which an MR gene has been introduced or 25 altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding wild-type or mutated MR sequence as a trans gene. In another embodiment, an endogenous MR gene within the genome of the microorganism has been altered, e.g., functionally 30 disrupted, by homologous recombination with an altered MR gene. In another embodiment, an endogenous or introduced MR gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional MR pro tein. In still another embodiment, one or more of the regulatory 35 regions (e.g., a promoter, repressor, or inducer) of an MR gene in a microorganism has been altered (e.g., by deletion, trunca tion, inversion, or point mutation) such that the expression of the MR gene is modulated. In a preferred embodiment, the micro organism belongs to the genus Corynebacterium or Brevibacterium, 40 with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with methionine, being particularly preferred.

WO 2004/050694 PCT/EP2002/013504 10 Another aspect of the invention pertains to a method for produc ing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an MR nu 5 cleic acid molecule of the invention, such that a fine chemical is produced. In a preferred embodiment, this method further in cludes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expres sion of an MR nucleic acid. In another preferred embodiment, 10 this method further includes the step of recovering the fine chemical, e.g., methionine, from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 1. 15 Another aspect of the invention pertains to methods for modulat ing production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates MR protein activity or MR nucleic acid expression such that a cell 20 associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C. glutamicum metabolic pathway regu latory systems, such that the yields or rate of production of a desired fine chemical, e.g., methionine, by this microorganism 25 is improved. The agent which modulates MR protein activity can be an agent which stimulates MR protein activity or MR nucleic acid expression. Examples of agents which stimulate MR protein activity or MR nucleic acid expression include small molecules, active MR proteins, and nucleic acids encoding MR proteins that 30 have been introduced into the cell. Examples of agents which in hibit MR activity or expression include small molecules and an tisense MR nucleic acid molecules. Another aspect of the invention pertains to methods for modulat 35 ing yields of a desired compound from a cell, involving the in troduction of a wild-type or mutant MR gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integra tion can be random, or it can take place by homologous recombi 40 nation such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a WO 2004/050694 PCT/EP2002/013504 11 fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid is methionine. 5 Brief Description of the Drawing Figure 1 depicts the principal of a self-cloning technique based on homologous recombination which is used for preparation of a Corynebacterium glutamicum strain deficient in the negative regu 10 lator of methionine biosynthesis (RXA00655, set forth as SEQ ID NO:1). Detailed Description of the Invention 15 The present invention provides metabolic regulatory (MR) nucleic acid and protein molecules, e.g., RXA00655 and RXNO2910 nucleic acid and protein molecules, which are involved in the regulation of metabolism in microorganisms, e.g., Corynebacterium glu tamicum, including regulation of fine chemical metabolism, e.g., 20 methionine biosynthesis by, e.g., transcriptional regulation of genes, e.g., the metY gene, involved in the methionine biosyn thetic pathway, the sulfur reduction pathway, and/or the cys teine biosynthetic pathway. 25 Accordingly, the present invention features methods based on ma nipulation of the methionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic pathway in a microorganism such that a fine chemical, e.g., a sulfur containing compound, e.g., methionine, cysteine, and/or other 30 compounds of the methionine biosynthetic pathway, the sulfur re duction pathway, and/or the cysteine biosynthetic pathway are produced. The term "methionine biosynthetic pathway" includes a pathway 35 involving methionine biosynthetic enzymes (e.g., polypeptides encoded by biosynthetic enzyme-encoding genes), compounds (e.g., precursors, substrates, intermediates or products), cofactors and the like, utilized in the formation or synthesis of methion ine. Methionine is an amino acid nutritionally required by mam 40 mals. Bacteria synthesize their own methionine from amino acids and biosynthetic intermediates thereof (Escherichia Coli and Salmonella: Cellular and Molecular Biology, Neidhardt, Frederick C. Curtiss, Roy III Ingraham, John L. Eds, 2nd ed. 1996, ASM WO 2004/050694 PCT/EP2002/013504 12 Press and Hwang BJ. Yeom HJ. Kim Y. Lee HS. Journal of Bacteri ology. 184(5):1277-86, 2002). The term "cysteine biosynthetic pathway" includes a pathway involving cysteine biosynthetic en zymes (e.g., polypeptides encoded by biosynthetic enzyme 5 encoding genes), compounds (e.g., precursors, substrates, inter mediates or products), cofactors and the like, utilized in the formation or synthesis of cysteine. The term "sulfur reduction pathway" includes a pathway involving enzymes which function to metabolize inorganic compounds such as sulfur and derivatives 10 thereof. It has been found that RXA00655 is a negative regula tor, e.g., a transcriptional regulator, of fine chemical biosyn thesis, e.g., methionine biosynthesis. Accordingly, suppression or inhibition of the expression of the RXA00655 gene or knock out of the RXA00655 gene leads to increased production of a fine 15 chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sulfur reduction path way, and/or the cysteine biosynthetic pathway, in microorgan isms, e.g., Corynebacterium glutamicum. Introduction of a muta tion which reduces or inhibits expression of the RXA00655 gene 20 or activity of the RXA00655 polypeptide also leads to increased production of a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic pathway, in microorganisms, e.g., Corynebacterium glutamicum. 25 It has also been found that RXN02910 is a positive regulator, e.g., a transcriptional regulator, of fine chemical biosynthe sis, e.g., methionine biosynthesis. Accordingly, overexpression of the RXNO2910 gene leads to increased production of methionine 30 and other compounds of the methionine biosynthetic pathway in microorganisms, e.g., Corynebacterium glutamicum. Introduction of a mutation which increases expression of the RXNO2910 gene or activity of the RXN02910 polypeptide also leads to increased production of methionine and other compounds of the methionine 35 biosynthetic pathway in microorganisms, e.g., Corynebacterium glutamicum. Furthermore, suppression or inhibition of the expression of RXA00655 or knock-out of the RXA00655 gene in combination with 40 overexpression of the RXNO2910 gene in a microorganism also leads to increased production of a fine chemical, e.g., methion ine, cysteine, and/or other compounds of the methionine biosyn thetic pathway, the sulfur reduction pathway, and/or the cys- WO 2004/050694 PCT/EP2002/013504 13 teine biosynthetic pathway, by the microorganism. Furthermore, culture of microorganisms with suppressed or inhibited expres sion of RXA00655 or microorganisms with a knock-out of the RXA00655 gene, together with microorganisms which overexpress 5 RXN02910 gene also leads to increased production of a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sulfur reduction path way, and/or the cysteine biosynthetic pathway, by the microor ganism. 10 Accordingly, one aspect the present invention features methods of producing fine chemicals, e.g., methionine or other compounds of the methionine biosynthetic pathway, which include culturing a microorganism which overexpresses RXNO2910 under conditions 15 such that a fine chemical, e.g., methionine or other compounds of the methionine biosynthetic pathway are produced. A microor ganism which overexpresses RXNO2910 includes a microorganism which has been manipulated such that RXNO2910 is overexpressed. 20 The term "overexpressed" or "overexpression" includes expression of a gene product (e.g., the RXNO2910 gene product) at a level greater than that expressed prior to manipulation of the micro organism or in a comparable microorganism which has not been ma nipulated. For example, overexpression of a particular gene, 25 e.g., RXNO2910, includes expression which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than expression of the gene by an organism which has not been manipulated to overex press the particular gene. Ranges and identity values intermedi ate to the above percentages are encompassed by the present in 30 vention. In one embodiment, the microorganism can be genetically manipulated (e.g., genetically engineered) to overexpress a level of gene product greater than that expressed prior to ma nipulation of the microorganism or in a comparable microorganism which has not been manipulated. Genetic manipulation can in 35 clude, but is not limited to, altering or modifying regulatory sequences or sites associated with expression of a particular gene (e.g., by adding strong promoters, inducible promoters or multiple promoters or by removing regulatory sequences such that expression is constitutive), modifying the chromosomal location 40 of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional ac- WO 2004/050694 PCT/EP2002/013504 14 tivators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating expression of a par ticular gene routine in the art (including but not limited to 5 use of antisense nucleic acid molecules, for example, to block expression of repressor proteins). In another aspect, the present invention features a method of producing a fine chemical, e.g., methionine, cysteine, and/or 10 other compounds of the methionine biosynthetic pathway, the sul fur reduction pathway, and/or the cysteine biosynthetic pathway, which includes culturing a microorganism which has suppressed or inhibited RXA00655 expression under conditions such that a fine chemical, e.g., methionine, cysteine, and/or other compounds of 15 the methionine biosynthetic pathway, the sulfur reduction path way, and/or the cysteine biosynthetic pathway, are produced. A microorganism which has suppressed RXA00655 expression includes a microorganism which has been manipulated such that RXA00655 expression is suppressed or inhibited. 20 The term "suppression of expression," "inhibition of expres sion," or "underexpression" includes expression of a gene prod uct (e.g., the RXA00655 gene product or the RXN02910 gene prod uct) at a level lower than that expressed prior to manipulation 25 of the microorganism or in a comparable microorganism which has not been manipulated. In one embodiment, suppression or inhibi tion of gene expression includes manipulation of a microorganism such that the gene is no longer expressed. For example, underex pression of a particular gene, e.g., RXA00655, includes expres 30 sion which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, less than expression of the gene by an organism which has not been manipulated to underexpress the particular gene. Ranges and identity values intermediate to the above percentages are encom passed by the present invention. In one embodiment, the microor 35 ganism can be genetically manipulated (e.g., genetically engi neered) to express a level of gene product lesser than that ex pressed prior to manipulation of the microorganism or in a com parable microorganism which has not been manipulated. Genetic manipulation can include, but is not limited to, altering or 40 modifying regulatory sequences or sites associated with expres sion of a particular gene, modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a ribosome binding site, decreasing the WO 2004/050694 PCT/EP2002/013504 15 copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, enhancers, transcriptional ac tivators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, use of an 5 tisense nucleic acid molecules, knock-out of the target gene, or any other conventional means of deregulating expression of a particular gene routine in the art. A microorganism which is de ficient in RXA00655 gene expression or RXNO2910 gene expression includes a microorganism which has suppressed or inhibited 10 RXA00655 or RXN02910 expression. In another aspect, the present invention features a method of producing a fine chemical, e.g., methionine, which includes cul turing a microorganism which has increased RXNO2910 activity un 15 der conditions such that a fine chemical, e.g., methionine, is produced. A "microorganism which has increased RXN02910 activ ity" includes a microorganism which has been manipulated such that RXNO2910 activity is increased. 20 In yet another aspect, the present invention features a method of producing a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sul fur reduction pathway, and/or the cysteine biosynthetic pathway, which includes culturing a microorganism which has decreased 25 RXA00655 activity under conditions such that a fine chemical, e.g., methionine, cysteine, and/or other compounds of the me thionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic pathway, is produced. A "micro organism which has decreased RXA00655 activity" includes a mi 30 croorganism which has been manipulated such that RXA00655 activ ity is inhibited or suppressed. The term "RXNO2910 activity" includes any activity which results in fine chemical biosynthesis, e.g., methionine biosynthesis. 35 RXNO2910 activity includes, but is not limited to, positive regulation of the methionine biosynthetic pathway resulting in methionine biosynthesis or biosynthesis of other compounds of the methionine biosynthetic pathway. Positive regulation of the methionine biosynthetic pathway may be by any means, including, 40 but not limited to, transcriptional and translational regulation as well as protein regulation, via, e.g., protein binding. In creased activity includes activity at a level higher than that WO 2004/050694 PCT/EP2002/013504 16 demonstrated prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. The term "RXA00655 activity" includes any activity which results in fine chemical biosynthesis, e.g., methionine biosynthesis. An 5 example of RXA00655 activity includes, but is not limited to, negative regulation of the methionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic path way as described in Escherichia Coli and Salmonella: Cellular and Molecular Biology, Neidhardt, Frederick C. Curtiss, Roy III 10 Ingraham, John L. Eds, 2nd ed. 1996, ASM Press., resulting in decreased methionine biosynthesis, decreased biosynthesis of other compounds of the methionine biosynthetic pathway, the sul fur reduction pathway , or the cysteine biosynthetic pathway. Negative regulation of the methionine biosynthetic pathway, the 15 sulfur reduction pathway, and/or the cysteine biosynthetic path way may be by any means, including, but not limited to tran scriptional and translational regulation as well as protein regulation, via, e.g., protein binding. Decreased or suppressed activity includes activity at a lower higher than that demon 20 strated prior to manipulation of the microorganism or in a com parable microorganism which has not been manipulated. The present invention features methods of increasing production of a sulfur-containing compound by a microorganism comprising 25 culturing a microorganism which overexpresses a positive regula tor of methionine biosynthesis, e.g., RXNO2910, under conditions such that production of the sulfur-containing compound is in creased. The present invention also features methods of increas ing production of a sulfur-containing compound by a microorgan 30 ism comprising culturing a microorganism which underexpresses a negative regulator of methionine biosynthesis, e.g., RXA00655, under conditions such that production of the sulfur-containing compound is increased. 35 The term "sulfur-containing compound" includes any compound which contains sulfur or a derivative thereof. Sulfur-containing compounds include amino acids, including, but not limited to, methionine, cysteine, S-adenosylmethionine, and homocycsteine. The nucleotide sequence of RXA00655 is set forth herein as SEQ 40 ID NO:1 and the polypeptide sequence of RXA00655 is set forth herein as SEQ ID NO:2. The nucleotide sequence of RXN02910 is set forth herein as SEQ ID NO:5 and the polypeptide sequence of RXA00655 is set forth herein as SEQ ID NO:6. Homologous protein WO 2004/050694 PCT/EP2002/013504 17 of RXA00655 are set forth herein as SEQ ID NO: 19, 21 and 23. The nucleotide sequence of said RXN02910 homologous proteins is set forth herein as SEQ ID NO: 18, 20 and 22. 5 SEQ ID NOs:16 and 17 represent mutated RXN02910 nucleic acid and amino acid sequences, respectively. The RKN02910 molecule de picted in SEQ ID NO:16 contains a single nucleotide change from a guanine (G) to an adenine (A) at nucleotide residue 556 in the coding region, which results in a change from an aspartic acid 10 (D) to an asparagine (N) at amino acid residue 186 of the en coded protein, set forth as SEQ ID NO:17. This polymorphism may cause modulation of regulation of fine chemical biosynthesis, e.g., methionine biosynthesis by RXN02910, e.g., decreased me thionine biosynthesis. 15 The molecules of the invention may be utilized in the modulation of production of fine chemicals, e.g., methionine, from microor ganisms, such as C. glutamicum, either directly (e.g., where modulation of the activity of a methionine biosynthesis regula tory protein has a direct impact on the yield, production, 20 and/or efficiency of production of methionine from that organ ism), or may have an indirect impact which nonetheless results in an increase in yield, production, and/or efficiency of pro duction of the desired compound (e.g., where modulation of the regulation of a nucleotide biosynthesis protein has an impact on 25 the production of an organic acid or a fatty acid from the bac terium, perhaps due to concomitant regulatory alterations in the biosynthetic or degradation pathways for these chemicals in re sponse to the altered regulation of nucleotide biosynthesis). Aspects of the invention are further explicated below. 30 The term 'fine chemical' is art-recognized and includes mole cules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, ag riculture, and cosmetics industries. Such compounds include or 35 ganic acids, such as tartaric acid, itaconic acid, and diamino pimelic acid, both proteinogenic and non-proteinogenic amino ac ids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and re lated compounds, p. 561-612, in Biotechnology vol. 6, Rehm et 40 al., eds. VCH: Weinheim, and references contained therein), lip ids, both saturated and unsaturated fatty acids (e.g., arachi donic acid), diols (e.g., propane diol, and butane diol), carbo hydrates (e.g., hyaluronic acid and trehalose), aromatic com- WO 2004/050694 PCT/EP2002/013504 18 pounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Indus trial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S., Niki, E. & 5 Packer, L. (1995) "Nutrition, Lipids, Health, and Disease" Pro ceedings of the UNESCO/Confederation of Scientific and Techno logical Associations in Malaysia, and the Society for Free Radi cal Research - Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al. (1998) 10 Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.) 15 Elements and Methods of the Invention The present invention is based, at least in part, on the func tional characterization of molecules of previous unknown func tion, referred to herein as MR nucleic acid and protein mole 20 cules, e.g., RXA00655 nucleic and protein molecules and RXN02910 nucleic acid and protein molecules, which regulate or modulate one or more metabolic pathways in C. glutamicum, e.g., the me thionine biosynthesis pathway. RXA00655 is a negative regulator of the methionine biosynthetic pathway, the sulfur reduction 25 pathway, and the cysteine biosynthetic pathway, while RXNO2910 is a positive regulator of methionine biosynthesis. Accordingly, the present invention features methods of producing a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sulfur reduction path 30 way, and/or the cysteine biosynthetic pathway and modulating production of a fine chemical, e.g., methionine, cysteine, and/or other compounds of the methionine biosynthetic pathway, the sulfur reduction pathway, and/or the cysteine biosynthetic pathway. Such methods include culturing microorganisms overex 35 pressing the RXNO2910 gene product or with increased RXN02910 activity under conditions such a fine chemical, e.g., methion ine, cysteine, and/or other compounds of the methionine biosyn thetic pathway, the sulfur reduction pathway, and/or the cys teine biosynthetic pathway is produced. Such methods also in 40 clude culturing microorganisms with inhibited expression of the RXA00655 gene product or inhibited RXA00655 activity such that a fine chemical, e.g., methionine, cysteine, and/or other com pounds of the methionine biosynthetic pathway, the sulfur reduc- WO 2004/050694 PCT/EP2002/013504 19 tion pathway, and/or the cysteine biosynthetic pathway, is pro duced. The present invention also provides methods for producing a fine chemical, e.g., methionine, comprising culturing microor ganisms overexpressing the RXNO2910 gene product and simultane 5 ously exhibiting an inhibited expression of the RKA00655 gene product or inhibited RXA00655 activity. Furthermore, the present invention also provides methods for producing a fine chemical, e.g., methionine, comprising culturing microorganisms overex pressing the RXNO2910 gene product in combination with microor 10 ganisms with inhibited expression of the RXA00655 gene product or inhibited RXA00655 activity. In one embodiment, the MR molecules of the invention transcrip tionally, translationally, or posttranslationally regulate a 15 metabolic pathway in C. glutamicum. In a preferred embodiment, the activity of the MR molecules of the present invention to regulate one or more C. glutamicum metabolic pathways has an im pact on the production of a desired fine chemical, e.g., me thionine, by this organism. In a particularly preferred embodi 20 ment, the MR molecules of the invention are modulated in activ ity, such that the C. glutamicum metabolic pathways which the MR proteins of the invention regulate are modulated in efficiency or output, which either directly or indirectly modulates the yield, production, and/or efficiency of production of a desired 25 fine chemical, e.g., methionine, by C. glutamicum. The language, "MR protein" or "MR polypeptide" includes proteins which transcriptionally, translationally, or posttranslationally regulate a metabolic pathway in C. glutamicum. The terms "MR 30 gene" or "MR nucleic acid sequence" include nucleic acid se quences encoding an MR protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. The terms "production" or "productivity" are art-recognized and include the concentration of the fermentation product (for exam 35 ple, the desired fine chemical) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter). The term "efficiency of production" includes the time required for a particular level of production to be achieved (for exam ple, how long it takes for the cell to attain a particular rate 40 of output of a fine chemical). The term "yield", "product/carbon yield", or "production" is art-recognized and includes the effi ciency of the conversion of the carbon source into the product (i.e., methionine). This 'is generally written as, for example, WO 2004/050694 PCT/EP2002/013504 20 kg product per kg carbon source. By increasing the yield or pro duction of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. 5 The terms "degradation" or a "degradation pathway" are art recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (gener ally speaking, smaller or less complex molecules) in what may be 10 a multistep and highly regulated process. The language "metabo lism" is art-recognized and includes the totality of the bio chemical reactions that take place in an organism. The metabo lism of a particular compound, then, (e.g., the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, 15 modification, and degradation pathways in the cell related to this compound. The term, "regulation" is art-recognized and in cludes the activity of a protein to govern or modulate the ac tivity of another protein. The term, "transcriptional regula tion" is art-recognized and includes the activity of a protein 20 to impede or activate the conversion of a DNA encoding a target protein to mRNA. The term, "translational regulation" is art recognized and includes the activity of a protein to impede or activate the conversion of an mRNA encoding a target protein to a protein molecule. The term, "posttranslational regulation" is 25 art-recognized and includes the activity of a protein to impede or improve the activity of a target protein by covalently modi fying the target protein (e.g., by methylation, glucosylation, or phosphorylation, or by binding the target protein). 30 In another embodiment, the MR molecules of the invention are ca pable of modulating the production of a desired molecule, such as a fine chemical, e.g., methionine, in a microorganism such as C. glutamicum. Using recombinant genetic techniques, one or more of the regulatory proteins of the invention may be manipulated 35 such that its function is modulated. For example, a biosynthetic enzyme may be improved in efficiency, or its allosteric control region destroyed such that feedback inhibition of production of the compound is prevented. Similarly, a degradative enzyme may be deleted or modified by substitution, deletion, or- addition 40 such that its degradative activity is lessened for the desired compound without impairing the viability of the cell. In each case, the overall yield or rate of production of one of these desired fine chemicals may be increased.

WO 2004/050694 PCT/EP2002/013504 21 It is also possible that such alterations in the protein and nu cleotide molecules of the invention may improve the production of fine chemicals, e.g., methionine, in an indirect fashion. The 5 regulatory mechanisms of metabolic pathways in the cell are necessarily intertwined, and the activation of one pathway may lead to the repression or activation of another in a concomitant fashion. Therefore, by modulating the activity of one or more of the proteins of the invention, the production or efficiency of 10 activity of another fine chemical biosynthetic or degradative pathway may be impacted. For example, by decreasing the ability of an MR protein to repress the transcription of a gene encoding a particular amino acid biosynthetic protein, one may concomi tantly derepress other amino acid biosynthetic pathways, since 15 these pathways are interrelated. Further, by modifying the MR proteins of the invention, one may uncouple the growth and divi sion of cells from their extracellular surroundings to a certain degree; by impairing an MR protein which normally represses bio synthesis of a nucleotide when the extracellular conditions are 20 suboptimal for growth and cell division such that it now lacks this function, one may permit growth to occur even when the ex tracellular conditions are poor. This is of particular relevance in large-scale fermentative growth, where conditions within the culture are often suboptimal in terms of temperature, nutrient 25 supply or aeration, but would still support growth and cell di vision if the cellular regulatory systems for these factors were eliminated. The isolated nucleic acid sequences of the invention are con 30 tained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide and amino acid sequences of RXA00655 are depicted in SEQ ID NOs:l, 18, 20, 22 and 2, 19, 21, 23 respectively, and the nucleotide and amino acid sequences 35 of RXN02910 are depicted in SEQ ID NOs:5 and 6, respectively. SEQ ID NOs:16 and 17 represent mutated RXN02910 nucleic acid and amino acid sequences, respectively. The RXNO2910 molecule de picted in SEQ ID NO:16 contains a single nucleotide change from a guanine (G) to an adenine (A) at nucleotide residue 556 in the 40 coding region, which results in a change from an aspartic acid (D) to an asparagine (N) at amino acid residue 186 of the en coded protein, set forth as SEQ ID NO:17. This polymorphism may cause modulation of regulation of methionine biosynthesis or WO 2004/050694 PCT/EP2002/013504 22 other fine chemicals by RXNO2910, e.g., decreased methionine biosynthesis . The present invention also pertains to proteins which have an 5 amino acid sequence which is substantially homologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% 10 homologous to the selected amino acid sequence, e.g., the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 15 80-90%, or 90-95%, and most preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence. The MR protein or a biologically active portion or fragment 20 thereof of the invention can transcriptionally, translationally, or posttranslationally regulate a metabolic pathway in C. glu tamicum, e.g., the methionine metabolic pathway, or have one or more of the activities set forth herein. 25 Various aspects of the invention are described in further detail in the following subsections: A. Transgenic Expression Cassettes, Vectors and Host Cells 30 The invention provides novel transgenic expression cassettes comprising in combination with a regulatory sequence a nucleic acid molecule coding for a metabolic pathway protein ((MR) pro tein] or a fragment thereof, wherein said regulatory sequence is 35 capable of mediating expression of said nucleic acid molecule. Preferably, said regulatory sequence is a promoter heterologous with regard to said nucleic acid molecule. Preferably the regu latory sequence is a promoter sequence functional in Corynebac terium glutamicum. 40 MR proteins are capable of, for example, performing an enzymatic step involved in the transcriptional, translational, or post translational regulation of metabolic pathways in C. glutamicum.

WO 2004/050694 PCT/EP2002/013504 23 Nucleic acid molecules encoding an MR protein are referred to herein as MR nucleic acid molecules. In a preferred embodiment, the MR protein participates in the transcriptional, transla tional, or posttranslational regulation of one or more metabolic 5 pathways. In a especially preferred embodiment, the MR nucleic acid mole cule is selected from the group consisting of: 10 a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a complement thereof; 15 b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide se quence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a complement thereof; 20 c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23; and 25 d) a nucleic acid molecule which encodes a fragment of a polypep tide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2, 30 SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, The transgenic expression cassette may comprise the nucleic acid molecule coding for the MR protein (e.g., RXA00655 or RXA02910) 35 in antisense or sense orientation with regard to said promoter sequence. In a preferred embodiment of the invention the nucleic acid molecule coding for the MR protein (e.g., RXA00655 or RXA02910) 40 encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. Especially preferred, said nu cleic acid molecule comprises the nucleotide sequence set forth WO 2004/050694 PCT/EP2002/013504 24 in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. The nucleic acid molecule may also code for a naturally occurring allelic variant of a polypeptide com prising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID 5 NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA mole cules (e.g., mRNA) and analogs of the DNA or RNA generated using 10 nucleotide analogs. This term also encompasses untranslated se quence located at both the 3' and 5' ends of the coding region of the gene: at least about 100 nucleotides of sequence upstream from the 5' end of the coding region and at least about 20 nu cleotides of sequence downstream from the 3'end of the coding 15 region of the gene. The nucleic acid molecule can be single stranded or double-stranded, but preferably is double-stranded DNA. As used herein, the term "transgenic expression cassette" is in 20 tended to include all kind of nucleic acids constructs obtained by methods of molecularbiology, wherein in said constructs ei ther a) the MR nucleic acid or part thereof (e.g., a nucleic compris 25 ing the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ TD NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a complement thereof; or part of the before mentioned), or b) the regulatory sequence (e.g. the promoter sequence) combined 30 with (a), or c) both(a) und (b) are not localized within their natural, genetic environment or 35 are modified by mean of molecular biology or gene techniques. Such modification may include substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. "Natural genetic environment" is intended to describe the natu ral genetic of a specific gene. For example, a transgenic ex 40 pression construct may include the combination of an MR nucleic acid in combination with its natural promoter sequence, as long as this combination is isolated from its natural genomic context and/or placed in a different genomic context. Preferably, the WO 2004/050694 PCT/EP2002/013504 25 promoter sequence is heterologous with regard to said MR nucleic acid. "Heterologous" is intended to describe the combination of a MR 5 nucleic acid with a promoter sequence, wherein said promoter se quence is naturally not regulating expression of the very same MR nucleic acid. "Transgenic" with regard to a vector or a host organism intents 10 to describe vector or host organisms which comprise a transgenic expression cassette as defined above. An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natu 15 ral source of the nucleic acid. A nucleic acid molecule of the present invention, e.g., a nu cleic acid molecule having a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 20 NO:22, or a portion thereof, can be isolated using standard mo lecular biology techniques and the sequence information provided herein. For example, a C. glutamicum MR DNA can be isolated from a C. glutamicum library using all or portion of one of the se quences of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, 25 SEQ ID NO:20, or SEQ ID NO:22 as a hybridization probe and stan dard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Labora tory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). 30 Moreover, a nucleic acid molecule encompassing all or a portion of one of the sequences of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 can be iso lated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid 35 molecule encompassing all or a portion of one of the sequences of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO':16, SEQ ID 40 NO:18, SEQ ID NO:20, or SEQ ID NO:22). For example, mRNA can be isolated from normal bacterial cells (e.g., by the guanidinium thiocyanate extraction procedure of Chirgwin et al. (1979) Bio chemistry 18: 5294-5299) and DNA can be prepared using reverse WO 2004/050694 PCT/EP2002/013504 26 transcriptase (e.g., Moloney MLV reverse transcriptase, avail able from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for polymerase chain reaction 5 amplification can be designed based upon one of the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers ac 10 cording to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonu cleotides corresponding to an MR nucleotide sequence can be pre pared by standard synthetic techniques, e.g., using an automated 15 DNA synthesizer. In a preferred embodiment, a transgenic expression cassette com prises one of the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 20 NO:22. The sequences of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 correspond to the Corynebacterium glutamicum MR DNAs of the invention. This DNA comprises sequences encoding MR proteins (i.e., the "coding re gion", indicated in each sequence in SEQ ID NO:l, SEQ ID NO:5, 25 SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the 30 sequences in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In another preferred embodiment, a transgenic expression cas sette of the invention comprises a nucleic acid molecule which 35 is a complement of one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO~:18, SEQ ID 40 NO:20, or SEQ ID NO:22 is one which is sufficiently complemen tary to one of the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 such that it can hybridize to one of the nucleotide se- WO 2004/050694 PCT/EP2002/013504 27 quences shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, thereby forming a stable duplex. 5 In still another preferred embodiment, a transgenic expression cassette of the invention comprises a nucleotide sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 10 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 15 NO:22, or a portion thereof. Ranges and identity values interme diate to the above-recited ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or 20 lower limits are intended to be included. In an additional pre ferred embodiment, a transgenic expression cassette of the in vention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences shown in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ 25 ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, or a portion thereof. Moreover, the transgenic expression cassette of the invention can comprise only a portion of the coding region of one of the sequences in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID 30 NO:18, SEQ ID NO:20, or SEQ ID NO:22, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an MR protein. The nucleotide sequences determined from the cloning of the MR 35 genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning MR homo logues in other cell types and organisms, as well as MR homo logues from other Corynebacteria or related species. The probe/primer typically comprises substantially purified oligonu 40 cleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of WO 2004/050694 PCT/EP2002/013504 28 the sequences set forth in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, an anti sense sequence of one of the sequences set forth in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 5 NO:22, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 can be used in PCR reactions to clone MR homologues. Probes based on the MR nucleo tide sequences can be used to detect transcripts or genomic se 10 quences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluores cent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying 15 cells which misexpress an MR protein, such as by measuring a level of an MR-encoding nucleic acid in a sample of cells, e.g., detecting MR mRNA levels or determining whether a genomic MR gene has been mutated or deleted. 20 In one embodiment, the transgenic expression cassette comprises a nucleic acid molecule encoding a protein or portion thereof which includes an amino acid sequence which is sufficiently ho mologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 such 25 that the protein or portion thereof maintains the ability to transcriptionally, translationally, or posttranslationally regu late a metabolic pathway in C. glutamicum. As used herein, the language "sufficiently homologous" refers to 30 proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in one of the sequences of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23) 35 amino acid residues to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 such that the protein or portion thereof is able to tran scriptionally, translationally, or posttranslationally regulate a metabolic pathway, e.g., methionine biosynthesis, 'in C. glu 40 tamicum. Protein members of such metabolic pathways, as de scribed herein, may function to regulate the biosynthesis or degradation of one or more fine chemicals. Examples of such ac tivities are also described herein. Thus, "the function of an MR WO 2004/050694 PCT/EP2002/013504 29 protein" contributes to the overall regulation of one or more fine chemical metabolic pathway, or contributes, either directly or indirectly, to the yield, production, and/or efficiency of production of one or more fine chemicals e.g., methionine. 5 In another embodiment, the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid 10 sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. As described above, homologous MR proteins, preferably homolo gous RXA00655 or RXN02910 proteins may be derived from other mi 15 croorganisms, preferably from procaryotic microorganisms. The term "Procaryotic microorganism" is intended to include gram-positive and gram-negative bacteria. Preferably ther term includes all genera and species of the Enterobacteriaceae or No 20 cardiaceae family, e.g. the Enterobacteriaceae species Es cherichia, Serratia, Proteus, Enterobacter, Klebsiella, Salmo nella, Shigella, Edwardsielle, Citrobacter, Morganella, Provi dencia and Yersinia. Furthermore preferred are all Pseudomonas, Burkholderia, Nocardia, Acetobacter, Streptomyces, Gluconobac 25 ter, Corynebacterium, Brevibacterium, Bacillus, Clostridium, Cyanobacter, Staphylococcus, Aerobacter, Alcaligenes, Rhodococ cus and Penicillium species. Most preferred are Corynebacterium and Streptomyces species like e.g., the Corynebacterium species exemplified in Table 1, Corynebacterium diphtheriae, Corynebac 30 terium efficiens and Streptomyces-coelicolor. Examples for homologous MR proteins may include: a) The RXA00655 protein from Corynebacterium diphtheriae, pref 35 erably described by a amino acid sequence comprising the se quence of SEQ ID NO: 19 b) The RXA00655 protein from Corynebacterium efficiens YS-314 Genbank Accession Nr. AP005223, preferably described by a 40 amino acid sequence comprising the sequence of SEQ ID NO: 21 WO 2004/050694 PCT/EP2002/013504 30 c) The RXA00655 protein.from Streptomyces-coelicolor Genbank ac cession NC_003888, preferably described by a amino acid se quence comprising the sequence of SEQ ID NO: 23 5 Preferably, RXN02910 is intended to describe polypeptides en coded by a nucleic acid molecule comprising a nucleic acid mole cule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which 10 is at least 60% identical to the nucleotide sequence of SEQ ID NO:5 or 16; b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide se 15 quence of SEQ ID NO:5 or 16; c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO:6 or 17 and 20 d) a nucleic acid molecule which encodes a fragment of a polypep tide comprising the amino acid sequence of SEQ ID NO:6 wherein the fragment comprises at least 10 contiguous amino acid resi dues of the amino acid sequence of SEQ ID NO:6 or 17. 25 Preferably, RXA00655 is intended to describe polypeptides en coded by a nucleic acid molecule comprising a nucleic acid mole cule selected from the group consisting of: 30 a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide sequence of SEQ ID NO:1, 18, 20 or 22; b) a nucleic acid molecule comprising a fragment of at least 35 30 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, 18, 20 or 22; c) a nucleic acid molecule which encodes a polypeptide com prising an amino acid sequence at least about 60% identical 40 to the amino acid sequence of SEQ ID NO:2, 19, 21 or 23; and d) a nucleic acid molecule which encodes a fragment of a poly- WO 2004/050694 PCT/EP2002/013504 31 peptide comprising the amino acid sequence of SEQ ID NO:2, 19, 21 or 23 wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2, 19, 21 or 23, 5 Portions of proteins encoded by the MR nucleic acid molecules of the invention are preferably biologically active portions of one of the MR proteins. As used herein, the term "biologically ac tive portion of an MR protein" is intended to include a portion, 10 e.g., a domain/motif, of an MR protein that transcriptionally, translationally, or posttranslationally regulates a metabolic pathway in C. glutamicum, or has an activity as set forth herein. To determine whether an MR protein or a biologically ac tive portion thereof can transcriptionally, translationally, or 15 posttranslationally regulate a metabolic pathway in C. glu tamicum, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification. 20 Additional nucleic acid fragments encoding biologically active portions of an MR protein can be prepared by isolating a portion of one of the sequences in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 expressing the encoded por 25 tion of the MR protein or peptide (e.g., by recombinant expres sion in vitro) and assessing the activity of the encoded portion of the MR protein or peptide. The invention further encompasses nucleic acid molecules that 30 differ from one of the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 (and por tions thereof) due to degeneracy of the genetic code and thus encode the same MR protein as that encoded by the nucleotide se 35 quences shown in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In another embodiment, an isolated nu cleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID 40 NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. In a still further embodiment, the nucleic acid molecule of the in vention encodes a full length C. glutamicum protein which is WO 2004/050694 PCT/EP2002/013504 32 substantially homologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 (en coded by an open reading frame shown in SEQ ID NO:l, SEQ ID 5 NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22). In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or 10 amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence). One of ordinary skill in the art would be able to calculate the lower threshold of per cent identity for any given sequence of the invention by examin 15 ing the GAP-calculated percent identity scores for each of the three top hits for a given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than 20 the lower threshold so calculated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 25 or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more identical) are also encom passed by the invention. In addition to the C. glutamicum MR nucleotide sequences shown 30 in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 it will be appreciated by those of ordinary skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of MR proteins may exist within a popula 35 tion (e.g., the C. glutamicum population). Such genetic polymor phism in the MR gene may exist among individuals within a popu lation due to natural variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an MR protein, pref 40 erably a C. glutamicum MR protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the MR gene. Any and all such nucleotide variations and result ing amino acid polymorphisms in MR that are the result of natu- WO 2004/050694 PCT/EP2002/013504 33 ral variation and that do not alter the functional activity of MR proteins are intended to be within the scope of the inven tion. 5 Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum MR DNA of the invention can be isolated based on their homology to the C. glu tamicum MR nucleic acid disclosed herein using the C. glutamicum DNA, or a portion thereof, as a hybridization probe according to 10 standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nu cleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nu cleic acid molecule comprising a nucleotide sequence of SEQ ID 15 NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In other embodiments, the nucleic acid is at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 225 or more nucleotides in length. As 20 used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more 25 preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hy bridized to each other. Such stringent conditions are known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 30 6.3.1-6.3.6. A preferred, non-limiting example of stringent hy bridization conditions are hybridization in 6X sodium chlo ride/sodium citrate (SSC) at about 45*C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65'C. Preferably, an iso lated nucleic acid molecule of the invention that hybridizes un 35 der stringent conditions to a sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or 40 DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nu cleic acid encodes a natural C. glutamicum MR protein.

WO 2004/050694 PCT/EP2002/013504 34 In addition to naturally-occurring variants of the MR sequence that may exist in the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, 5 SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 thereby leading to changes in the amino acid sequence of the encoded MR protein, without altering the functional ability of the MR protein. For example, nucleotide substitutions leading to amino acid substi 10 tutions at "non-essential" amino acid residues can be made in a sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of one 15 of the MR proteins (SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23) without altering the activity of said MR protein, whereas an "essential" amino acid residue is required for MR protein activity. Other amino acid residues, however, 20 (e.g., those that are not conserved or only semi-conserved in the domain having MR activity) may not be essential for activity and thus are likely to be amenable to alteration without alter ing MR activity. 25 Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding MR proteins that contain changes in amino acid residues that are not essential for MR activity. Such MR proteins differ in amino acid sequence from a sequence con tained in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, 30 SEQ ID NO:21, or SEQ ID NO:23, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 yet retain at least one of the MR activities de scribed herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 35 about 50% homologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 and is capable of transcriptionally, translationally, or posttranslationally regulating a metabolic pathway in C. glu tamicum, or has one or more activities set forth herein. Pref 40 erably, the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to one of the sequences in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, more preferably at least about 60-70% homologous WO 2004/050694 PCT/EP2002/013504 35 to one of the sequences in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23, even more preferably at least about 70-80%, 80-90% homologous to one of the sequences in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID 5 NO:19, SEQ ID NO:21, or SEQ ID NO:23, and most preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to one of the sequences in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. 10 To determine the percent homology of two amino acid sequences (e.g., one of the sequences of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in 15 the sequence of one protein or nucleic acid for optimal align ment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the sequences of SEQ ID NO:2, SEQ ID 20 NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the sequence selected from SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23), then the 25 molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical posi tions shared by the sequences (i.e., % homology = # of identical 30 positions/total # of positions x 100). An isolated nucleic acid molecule encoding an MR protein homolo gous to a protein sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 can be cre 35 ated by introducing one or more nucleotide substitutions, addi tions or deletions into a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein'. Mutations 40 can be introduced into one of the sequences of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino WO 2004/050694 PCT/EP2002/013504 36 acid substitutions are made at one or more predicted non essential amino acid residues. A "conservative amino acid sub stitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families 5 of amino acid residues having similar side chains have been de fined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, 10 threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, praline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a pre 15 dicted nonessential amino acid residue in an MR protein is pref erably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, muta tions can be introduced randomly along all or part of an MR cod ing sequence, such as by saturation mutagenesis, and the resul 20 tant mutants can be screened for an MR activity described herein to identify mutants that retain MR activity. Following mutagene sis of one of the sequences of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22, the encoded protein can be expressed recombinantly and the activity of the 25 protein can be determined using, for example, assays described herein (see Example 6 of the Exemplification). The invention also provides MR chimeric or fusion proteins. As used herein, an MR "chimeric protein" or "fusion protein" com 30 prises an MR polypeptide operatively linked to a non-MR polypep tide. An "MR polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an MR protein, whereas a "non-MR polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substan 35 tially homologous to the MR protein, e.g., a protein which is different from the MR protein and which is derived from the same or a different organism. Within the fusion protein, the term "operatively linked" is intended to indicate that the MR poly peptide and the non-MR polypeptide are fused in-frame to each 40 other. The non-MR polypeptide can be fused to the N-terminus or C-terminus of the MR polypeptide. For example, in one embodiment the fusion protein is a GST-MR fusion protein in which the MR sequences are fused to the C-terminus of the GST sequences. Such WO 2004/050694 PCT/EP2002/013504 37 fusion proteins can facilitate the purification of recombinant MR proteins. In another embodiment, the fusion protein is an MR protein containing a heterologous signal sequence at its N terminus. In certain host cells (e.g., mammalian host cells), 5 expression and/or secretion of an MR protein can be increased through use of a heterologous signal sequence. Preferably, an MR chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, 10 DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional tech niques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appro 15 priate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fu sion gene can be synthesized by conventional techniques includ ing automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which 20 give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reampli fied to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are com 25 mercially available that already encode a fusion moiety (e.g., a GST polypeptide). An MR-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the MR protein. 30 Homologues of the MR protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the MR protein. As used herein, the term "homologue" refers to a variant form of the MR protein which acts as an agonist or antagonist of the ac tivity of the MR protein. An agonist of the MR protein can re 35 tain substantially the same, or a subset, of the biological ac tivities of the MR protein. An antagonist of the MR protein can inhibit one or more of the activities of the naturally occurring form of the MR protein, by, for example, competitively binding to a downstream or upstream member of the MR regulatory cascade 40 which includes the MR protein. Thus, the C. glutamicum MR pro tein and homologues thereof of the present invention may modu late the activity of one or more metabolic pathways which MR proteins regulate in this microorganism.

WO 2004/050694 PCT/EP2002/013504 38 In an alternative embodiment, homologues of the MR protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the MR protein for MR protein ago 5 nist or antagonist activity. In one embodiment, a variegated li brary of MR variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of MR variants can be produced by, for example, enzymatically ligating a mixture of synthetic oli 10 gonucleotides into gene sequences such that a degenerate set of potential MR sequences is expressible as individual polypep tides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of MR sequences therein. There are a variety of methods which can be used to 15 produce libraries of potential MR homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expres sion vector. Use of a degenerate set of genes allows for the 20 provision, in one mixture, of all of the sequences encoding the desired set of potential MR sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Na rang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; 25 Ike et al. (1983) Nucleic Acid Res. 11:477. In addition, libraries of fragments of the MR protein coding can be used to generate a variegated population of MR fragments for screening and subsequent selection of homologues of an MR pro 30 tein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an MR coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the dou ble stranded DNA, renaturing the DNA to form double stranded DNA 35 which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed du plexes by treatment with Sl nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C 40 terminal and internal fragments of various sizes of the MR pro tein.

WO 2004/050694 PCT/EP2002/013504 39 Several techniques are known in the art for screening gene prod ucts of combinatorial libraries made by point mutations or trun cation, and for screening cDNA libraries for gene products hav ing a selected property. Such techniques are adaptable for rapid 5 screening of the gene libraries generated by the combinatorial mutagenesis of MR homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate 10 cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a de sired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of func 15 tional mutants in the libraries, can be used in combination with the screening assays to identify MR homologues (Arkin and Your van (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein En gineering 6(3):327-331). 20 In another embodiment, cell based assays can be exploited to analyze a variegated MR library, using methods well known in the art. In addition to the nucleic acid molecules encoding MR proteins 25 described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded 30 DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an en tire MR coding strand, or to only a portion thereof. In one em bodiment, an antisense nucleic acid molecule is antisense to a 35 "coding region" of the coding strand of a nucleotide sequence encoding an MR protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding re 40 gion" of the coding strand of a nucleotide sequence encoding MR. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).

WO 2004/050694 PCT/EP2002/013504 40 Given the coding strand sequences encoding MR molecules dis closed herein (e.g., the sequences set forth in SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID 5 NO:22), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The an tisense nucleic acid molecule can be complementary to the entire coding region of MR mRNA, but more preferably is an oligonucleo tide which is antisense to only a portion of the coding or non 10 coding region of MR mRNA. For example, the antisense oligonu cleotide can be complementary to the region surrounding the translation start site of MR mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the in 15 vention can be constructed using chemical synthesis and enzy matic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonu cleotide) can be chemically synthesized using naturally occur ring nucleotides or variously modified nucleotides designed to 20 increase the biological stability of the molecules or to in crease the physical stability of the duplex formed between the antisense and sense nucleic acids. Alternatively and preferably, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned 25 in an antisense orientation (i.e., RNA transcribed from the in serted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the fol lowing subsection). 30 Another aspect of the invention pertains to vectors, preferably expression vectors, containing at least one transgenic expres sion cassette of the invention comprising a nucleic acid encod ing an MR protein (or a portion thereof). 35 As used herein, the term "vector" refers to a nucleic acid mole cule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vec 40 tor, wherein additional DNA segments can be ligated into the vi ral genome. Certain vectors are capable of autonomous replica tion in a host cell into which they are introduced (e.g., bacte rial vectors having a bacterial origin of replication). Other WO 2004/050694 PCT/EP2002/013504 41 vectors are integrated into the genome of a host cell upon in troduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively 5 linked. Such vectors are referred to herein as "expression vec tors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangea bly as the plasmid is the most commonly used form of vector. 10 However, the invention is intended to include such other forms of expression vectors, such as phage vectors which serve equiva lent functions. The transgenic expression vectors of the invention comprise a 15 transgenic expression cassette of the invention in a form suit able for expression of the MR nucleic acid in a host cell. The transgenic expression cassette includes one or more regula tory sequences (promoters), selected on the basis of the host 20 cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a transgenic expression cassette, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nu 25 cleotide sequence (e.g., in an in vitro transcrip tion/translation system or in a host cell when the vector is in troduced into the host cell). The term "regulatory sequence" is intended to include promoters, 30 enhancers and other expression control elements (e.g., polyade nylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in En zymology 185, Academic Press, San Diego, CA (1990) and in Vasi cova P. Patek M. Nesvera J. Sahm H. Eikmanns B. Journal of Bac 35 teriology (1999) 181(19):6188-91, in Patek M. et al. (1996) Mi crobiology 142:1297-309, and in Mateos et al. (1994) Journal of Bacteriology 176:7362-71. Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the 40 nucleotide sequence only in certain host cells. Preferred regu latory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacI4-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, A,-P,- or k-Ps, which are WO 2004/050694 PCT/EP2002/013504 42 used preferably in bacteria. It is also possible to use artifi cial promoters. It will be appreciated by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be trans 5 formed, the level of expression of protein desired, etc. The ex pression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., MR proteins, mutant forms of MR proteins, fusion 10 proteins, etc.). The transgenic expression cassettes or expression vectors of the invention can be designed for expression of MR proteins in pro karyotic or eukaryotic cells. For example, MR genes can be ex 15 pressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells, algae and multicellular plant cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Ex pression Technology: Methods in Enzymology 185, Academic Press, 20 San Diego, CA (1990). Alternatively, the transgenic expression cassette or expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. 25 Expression of proteins in prokaryotes.is most often carried out with vectors containing constitutive or inducible promoters di recting the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded 30 therein, usually to the amino terminus of the recombinant pro tein. Typical fusion expression vectors may fuse glutathione S transferase (GST), maltose E binding protein, or protein A, re spectively, to the target recombinant protein. 35 Examples of suitable inducible E. coli expression vectors in clude pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pA CYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-Bl, Xgtll, pBdCl, and pET 1ld (Studier et al., Gene Expression Technology: Methods in Enzymol 40 ogy 185, Academic Press, San Diego, California (1990) 60-89; and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York ISBN 0 444 904018).

WO 2004/050694 PCT/EP2002/013504 43 Other examples for inducible E. coli C. glutamicum shuttle ex pression vectors can be found in Eikmanns B.J., et al. (1991) Gene 102:93-8. Suitable cloning vectors for use in Corynebacte rium glutamicum, are for example those disclosed in Sinskey et 5 al., U.S. Patent No. 4,649,119, and techniques for genetic ma nipulation of C. glutamicum and the related Brevibacterium spe cies (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 10 (1984)). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. For transformation of other varieties of bacteria, appropriate 15 vectors may be selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB110, pC194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium in 20 clude pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Got 25 tesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). An other strategy is to alter the nucleic acid sequence of the nu cleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially 30 utilized in the bacterium chosen for expression, such as C. glu tamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. 35 Another aspect of the invention pertains to transgenic host cells into which a transgenic expression cassette or expression vector of the invention has been introduced. The terms "host cell" and "transgenic host cell" are used interchangeably herein. It is understood that such terms refer not only to the 40 particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in suc ceeding generations due to either mutation or environmental in fluences, such progeny may not, in fact, be identical to the WO 2004/050694 PCT/EP2002/013504 44 parent cell, but are still included within theiscope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For exam 5 ple, an MR-protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells. Other suitable host cells are known to one of ordinary skill in the art. Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and 10 protein molecules of the invention are set forth in Table 1. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection tech niques. As used herein, the terms "transformation" and "trans 15 fection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) 20 into a host cell, including calcium phosphate or calcium chlo ride co-precipitation, DEAE-dextran-mediated transfection, li pofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Mo lecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Har 25 bor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals. For transformation of microorganisms, it is known that, depend ing upon the expression vector and transformation technique 30 used, only a small fraction-of cells may incorporate the foreign DNA either with an episomal localisation or into their genome by processes involving recombination. In order to identify and se lect these transformants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced 35 into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as kanamycin, tetracyclin, bleomycin, chlorampheni col, lincomycin. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encod 40 ing an MR protein or can be introduced on a separate vector. Cells transformed with the introduced nucleic acid can be iden tified by, for example, drug selection (e.g., cells that have WO 2004/050694 PCT/EP2002/013504 45 incorporated the selectable marker gene will survive, while the other cells die). Examples for antibiotic resistence genes that can be used in C. 5 glutamicum can be found in: Tauch A. Puhler A. Kalinowski J. Thierbach G. Plasmid. 44(3):285-91, 2000, Kim HJ. Kim Y. Lee MS. Lee HS. Molecules & Cells. 12(l):112-6, 2001 Guerrero C. Mateos LM. Malumbres M. Martin JF. Applied Microbiology & Biotechnol ogy. 36(6):759-62, 1992, Eikmanns BJ. Kleinertz E. Liebl W. Sahm 10 H. Gene. 102(l):93-8, 1991, Yoshihama M. Higashiro K. Rao EA. Akedo M. Shanabruch WG. Follettie MT. Walker GC. Sinskey AJ. Journal of Bacteriology. 162(2):591-7, 1985. Tauch A. Zheng Z. Puhler A. Kalinowski J. Plasmid. 40(2):126-39, 1998.Cadenas RF. Martin JF. Gil JA. Gene. 98(l):117-21, 1991 15 In another embodiment, transgenic host organisms (preferably mi croorganisms) can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of an MR gene on a vector placing it under 20 control of the lac operon permits expression of the MR gene only in the presence of IPTG. Such regulatory systems are well known in the art and are described in e.g., Eikmanns B.J. et al. (1991) Gene 102:93-8. 25 A host cell of the invention, such as a prokaryotic or eu karyotic host cell in culture, can be used to produce (i.e., ex press) an MR protein. Accordingly, the invention further pro vides methods for producing MR proteins using the host cells of the invention. In one embodiment, the method comprises culturing 30 the host cell of invention (into which a transgenic expression cassette or expression vector encoding an MR protein has been introduced, or into which genome has been introduced a gene en coding a wild-type or altered MR protein) in a suitable medium until MR protein is produced. In another embodiment, the method 35 further comprises isolating MR proteins from the medium or the host cell. B. Methods for suppressing expression of a MR protein 40 There are numerous methods known to the person skilled in the art how to induce deficiency with regard to a specific gene in an organism or how to suppress or reduce expression or activity of said gene or the corresponding gene product. It is an pre- WO 2004/050694 PCT/EP2002/013504 46 ferred embodiment within this invention to apply said methods to a negative regulator of methionine biosynthesis (e.g., RXA00655), thereby increasing methionine biosynthesis. 5 Said methods may include one or more of the methods selected from the following group of methods consisting of: a) knock-out of the gene encoding said MR protein (e.g. the negative regulatory protein) 10 b) mutagenesis of the gene encoding said MR protein (e.g., nega tive regulatory protein), wherein said mutation can be in duced in the coding, non-coding, or regulatory regions of said gene; 15 c) expression of an anti-sense RNA, wherein said anti-sense RNA is complementary to at least part of the RNA encoding said MR protein (e.g., negative regulatory protein); 20 d) expression of DNA-binding proteins blocking or reducing ex pression from the gene encoding said MR protein (e.g., nega tive regulatory protein); e) expression of protein-binding factors blocking or reducing 25 expression from the gene encoding said MR protein (e.g., negative regulatory protein); f) expression of a dominant-negative variant of said MR protein (e.g., negative regulatory protein); and 30 g) destabilization of the mRNA encoding said MR protein (e.g., negative regulatory protein). In a preferred embodiment, an endogenous MR gene in a host cell 35 is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced MR gene in a host cell has been altered by one or more point mutations, deletions, or inversions. Said MR gene may 40 still encodes a functional MR protein but may also encode a non funtional MR protein or a MR protein with modified (e.g., in creased or decreased) actrivity. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repres- WO 2004/050694 PCT/EP2002/013504 47 sor, or inducer) of an MR gene in a microorganism has been al tered (e.g., by deletion, truncation, inversion, or point muta tion) such that the expression of the MR gene is modulated. One of ordinary skill in the art will appreciate that host cells 5 containing more than one of the described MR gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present inven tion. 10 To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an MR gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the MR gene. Pref erably, this MR gene is a Corynebacterium glutamicum MR gene, 15 but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous MR gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a 20 "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous MR gene is mutated or otherwise altered (e.g., the upstream regulatory region can be altered to thereby alter the expression of the en dogenous MR protein). In the homologous recombination vector, 25 the altered portion of the MR gene is flanked at its 5' and 3' ends by additional nucleic acid of the MR gene to allow for ho mologous recombination to occur between the exogenous MR gene carried by the vector and an endogenous MR gene in a microorgan ism. The additional flanking MR nucleic acid is of sufficient 30 length for successful homologous recombination with the endoge nous gene. Typically, the flanking DNA should have lengths be tween 100basepairs and a few kilobases (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R., and Capecchi, M.R. (1987) Cell 51: 503 for a description of homolo 35 gous recombination vectors). In addition methods are known in which another selection can be used to construct chromosomal re combinations in which the selection marker is retrieved by a method of postive selection Schafer A. Tauch A. Jager W. Kali nowski J. Thierbach G. Puhler A. Gene. 145(l):69-73, 1994. . The 40 vector is introduced into a microorganism (e.g., by electropora tion) and cells in which the introduced MR gene has homologously recombined with the endogenous MR gene are selected, using art known techniques.

WO 2004/050694 PCT/EP2002/013504 48 A decrease in expression of a MR protein may be achieved by ex pressing antisense RNA directed against the MR encoding mRNA. Said antisense RNA is described above. Methods for regulation of 5 gene expression using antisense genes are well known to the per son skilled in the art (see e.g., Weintraub, H. et al., An tisense RNA as a molecular tool for genetic analysis, Reviews Trends in Genetics, Vol. 1(1) 1986). The antisense nucleic acid molecules of the invention are typically administered to a cell 10 or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an MR protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, 15 or, for example, in the case of an antisense nucleic acid mole cule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by link 20 ing the antisense nucleic acid molecule to a peptide or an anti body which binds to a cell surface receptor or antigen. The an tisense nucleic acid molecule can also be delivered to cells us ing the vectors described herein. To achieve sufficient intra cellular concentrations of the antisense molecules, vector con 25 structs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred. In yet another embodiment, the antisense nucleic acid molecule 30 of the invention is an a-anomeric nucleic acid molecule. An a anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual p-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic 35 acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chi meric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327 330). 40 In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a sin gle-stranded nucleic acid, such as an mRNA, to which they have a WO 2004/050694 PCT/EP2002/013504 49 complementary region. Thus, ribozymes (e.g., hammerhead ri bozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave MR mRNA tran scripts to thereby inhibit translation of MR mRNA. A ribozyme 5 having specificity for an MR-encoding nucleic acid can be de signed based upon the nucleotide sequence of an MR DNA disclosed herein (i.e., SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the 10 nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an MR-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742. Alternatively, MR mRNA can be used to se lect a catalytic RNA having a specific ribonuclease activity 15 from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418. Alternatively, MR gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of 20 an MR nucleotide sequence (e.g., an MR promoter and/or enhan cers) to form triple helical structures that prevent transcrip tion of an MR gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6) :569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) 25 Bioassays 14(12):807-15. In still another embodiment, additional or alternative methods known to the person skilled in the art can be used to modulate, e.g., suppress or increase, expression from a gene (e.g., a gene 30 coding for a negative regulator of methionine biosynthesis) or to modulate, e.g., increase or suppress or inhibit activity of the corresponding gene product. One method which may be used is the expression of a dominant 35 negative variant of the gene product (e.g., a gene coding for a negative regulator of methionine biosynthesis) to suppress ac tivity of, e.g., a negative regulator of methionine biosynthe sis. 40 A negative regulator of methionine biosynthesis (e.g., RXA00655 as exemplified by SEQ ID NO:1, 18, 20 or 22) is preferably a tran scriptional regulator. Transcriptional regulators are known to ex ists as dimers which bind to opposite parts of the DNA to struc- WO 2004/050694 PCT/EP2002/013504 50 tures that might contain repeating sequences in so called dyad sym metry. The activity of DNA binding is determined within the amino acid sequence of the transcriptional regulator. Examples of the structural basis of the dimeric binding of such transcriptional 5 regulators can be found in Schumacher M.A. and Brennan R.G. (2002) Molecular Microbiology. 45:885-93. For example, expression of dif ferent alleles of the same protein in one cell by producing hetero dimeric proteins consisting of an unmutated and of a mutated allele of a regulatory protein confer a dominant negative phenotype for 10 such an organism. Examples of methods for constructing a mutant dominant negative repressor protein in organisms also expressing unmutated alleles of the same gene can be found in Journal of Mo lecular Biology (2002) 322(2):311-24, and in Journal of Biological Chemistry (1994). 269(11):8246-54. For example, the DNA-binding 15 domain of said negative regulator of methionine biosynthesis may be inactivated.In another embodiment, mRNA stabilisation and destabi lisation can be used as a method to either increase or decrease the life time of a given mRNA molecule. Methods to influence the life time of a given mRNA can be found in Smolke C.D. and Keasling J.D. 20 (2002) Biotechnology & Bioengineering. 78(4):412-24, in Smolke C.D. et al. (2001) Metabolic Engineering 3(4):313-21, and in Carrier T.A. and Keasling J.D. (1999) Biotechnology Progress 15(1):58 64.Another method which may be used to modulate expression of a gene includes expression of DNA-binding proteins increasing or 25 blocking or reducing expression from the gene, e.g., a gene coding for a regulatory protein of methionine biosynthesis. Blocking or reducing expression from the gene, e.g., a gene coding for a negative regulatory protein of methionine biosynthesis can be 30 realized by utilizing specific DNA-binding proteins, e.g., of the zinc-finger protein class of transcription factors. Said factors may be directed to .g., regulatory regions of the gene to be sup pressed. Utilization of said factors allows suppression of expres sion without altering the corresponding gene sequence. Methods are 35 known to the person skilled in the art to construct artificial DNA binding factors capable of binding to a specific target sequence. Increasing gene expression, e.g., coding for a positive regulator of methionine biosynthesis, can be realized by using the above de scribed artificial DNA-binding factors by fusing them to a tran 40 scription activation domain, thereby creating an artificial initia tor of transcription. (Dreier B et al. (2001) J Biol Chem 276(31) :29466-78; Dreier B et al. (2000) J Mol Biol 303(4) :489-502; Beerli RR et al. (2000) Proc Natl Acad Sci USA 97 (4):1495-1500; WO 2004/050694 PCT/EP2002/013504 51 Beerli RR et al. (2000) J Biol Chem 275(42):32617-32627; Segal DJ and Barbas CF 3rd. (2000) Curr Opin Chem Biol 4(1):34-39; Kang JS and Kim JS (2000) J Biol Chem 275(12):8742-8748; Beerli RR et al. (1998) Proc Natl Acad Sci USA 95(25):14628- 14633; Kim JS et al. 5 (1997) Proc Natl Acad Sci USA 94(8):3616 -3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai SY et al. (1998) Adv Drug Deliv Rev 30(1 3):23-31; Mapp AK et al. (2000) Proc Natl Acad Sci USA 97(8):3930 3935; Sharrocks AD et al. (1997) Int J Biochem Cell Biol 29(12):1371-1387; Zhang L et al. (2000) J Biol Chem 275(43):33850 10 33860). Suppression of gene expression can also be realized by us ing customized low-molecular weight synthetic compounds, e.g., of the polyamide type (Dervan PB and B rli RW (1999) Current Opinion in Chemical Biology 3:688-693; Gottesfeld JM et al. (2000) Gene Expr 9(1-2):77-91). These compounds can be adopted on a rational 15 basis to any specific DNA target sequence allowing suppression or, if the compound is used in fusion with a transcription activation domain, initiation of gene expression. Methods are known to the person skilled in the art to construct the artificial DNA-binding factors capable of binding to a specific target sequence (Bremer RE 20 et al. (2001) Bioorg Med Chem. 9(8).:2093-103; Ansari A.Z. et al. (2001) Chem Biol. 8(6):583-92; Gottesfeld J.M. et al. (2001) J Mol Biol. 309(3):615-29; Wurtz N.R. et al. (2001) Org Lett 3(8):1201-3; Wang C.C. et al. (2001) Bioorg Med Chem 9(3):653-7; Urbach A.R. and Dervan P.B. (2001) Proc Natl Acad Sci USA 98(8):4343-8; Chiang S.Y. 25 et al. (2000) J Biol Chem. 275(32):24246-54).In another embodiment, expression of protein-binding factors activating or blocking or reducing expression or activity of the gene coding for a regulatory protein of methionine biosynthesis may be utilized. 30 Protein-binding factors suitable for binding regulators of methion ine biosynthesis and thereby suppressing activity may be based on RNA such as, e.g. aptamers (Famulok M und Mayer G (1999) Curr Top Microbiol Immunol 243:123-36), antibodies, antibody fragments, or single-chain antibodies. Methods for construction and utilization 35 of these protein-binding factors are known to the person skilled in the art. C. Uses and Methods of the Invention 40 The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, transgenic expression cassettes, expression vectors, and host cells described herein can be used in one or more of the following methods: modulation of cellular production WO 2004/050694 PCT/EP2002/013504 52 of a desired compound, such as a fine chemical, e.g., methion ine; identification of C. glutamicum and related organisms; map ping of genomes of organisms related to C. glutamicum; identifi cation and localization of C. glutamicum sequences of interest; 5 evolutionary studies; determination of MR protein regions re quired for function; modulation of an MR protein activity; and modulation of the activity of one or more metabolic pathways. The MR nucleic acid molecules of the invention have a variety of 10 uses. Manipulation of the MR nucleic acid molecules of the in vention, e.g., suppressing or increasing expression or activity of the nucleic acid or protein molecules, respectively, results in the modulation of methionine biosynthesis. For example, a Corynebacterium glutamicum strain which overexpresses or has in 15 creased expression of RXN02910, a positive regulator of methio ine biosynthesis, displays a significant increase in the produc tion of methionine or other compounds of the methionine biosyn thetic pathway. Furthermore, a Corynebacterium glutamicum strain which is deficient in or has suppressed expression of RXN00655, 20 a negative regulator of methioine biosynthesis, also displays a significant increase in the production of methionine or other compounds of the methionine biosynthetic pathway. Furthermore, manipulation of the MR nucleic acid molecules of 25 the invention may lead to production of MR proteins having func tional differences from the wild-type MR proteins. These pro teins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be de creased in efficiency or activity. 30 The invention provides methods for screening molecules which modulate the activity of an MR protein, either by interacting with the protein itself or a substrate or binding partner of the MR protein, or by modulating the transcription or translation of 35 an MR nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more MR proteins of the inven tion is contacted with one or more test compounds, and the ef fect of each test compound on the activity or level of expres sion of the MR protein is assessed. 40 Such changes in activity may directly modulate the yield, pro duction, and/or efficiency of production of one or more fine chemicals from C. glutamicum, e.g., methionine. For example, by WO 2004/050694 PCT/EP2002/013504 53 optimizing the activity of an MR protein, e.g., a positive regu lator of methionine biosynthesis, e.g., RXNO2910, which acti vates the transcription or translation of a gene encoding a bio synthetic protein for a desired fine chemical, or by impairing 5 or abrogating the activity of an MR protein, e.g., a negative regulator of methionine biosynthesis, e.g., RXA00655, which re presses the transcription or translation of such a gene, one may also increase the activity or rate of activity of that biosyn thetic pathway. Similarly, by altering the activity of an MR 10 protein such that it constitutively posttranslationally inacti vates a protein involved in a degradation pathway for a desired fine chemical, or by altering the activity of an MR protein such that it constitutively represses the transcription or transla tion of such a gene, one may increase the yield and/or rate of 15 production of the fine chemical from the cell, due to decreased degradation of the compound. Further, by modulating the activity of one or more MR proteins, one may indirectly stimulate the production or improve the rate 20 of production of one or more fine chemicals from the cell due to the interrelatedness of disparate metabolic pathways. For exam ple, by increasing the yield, production, and/or efficiency of production by activating the expression of one or more lysine biosynthetic enzymes, one may concomitantly increase the expres 25 sion of other compounds, such as other amino acids, which the cell would naturally require in greater quantities when lysine is required in greater quantities. Also, regulation of metabo lism throughout the cell may be altered such that the cell is better able to grow or replicate under the environmental condi 30 tions of fermentative culture (where nutrient and oxygen sup plies may be poor and possibly toxic waste products in the envi ronment may be at high levels). For example, by mutagenizing an MR protein which represses the synthesis of molecules necessary for cell membrane production in response to high levels of waste 35 products in the extracellular medium (in order to block cell growth and division in suboptimal growth conditions) such that it no longer is able to repress such synthesis, one may increase the growth and multiplication of the cell in cultures even when the growth conditions are suboptimal. Such enhanced growth or 40 viability should also increase the yields and/or rate of produc tion of a desired fine chemical from fermentative culture, due to the relatively greater number of cells producing this com pound in the culture.

WO 2004/050694 PCT/EP2002/013504 54 The aforementioned mutagenesis strategies for MR proteins to re sult in increased yields of a fine chemical, e.g., methionine, from C. glutamicum are not meant to be limiting; variations on 5 these strategies will be readily apparent to one of ordinary skill in the art. Using such strategies, and incorporating the mechanisms disclosed herein, the nucleic acid and protein mole cules of the invention may be utilized to generate C. glutamicum or related strains of bacteria expressing mutated MR nucleic 10 acid and protein molecules such that the yield and/or efficiency of production of a desired compound is improved. This desired compound may be any natural product of C. glutamicum, which in cludes the final products of biosynthesis pathways and interme diates of naturally-occurring metabolic pathways, as well as 15 molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C. glutamicum strain of the invention. The MR molecules of the invention may also be used to identify 20 an organism as being Corynebacterium glutamicum or a close rela tive thereof. Also, they may be used to identify the presence of C. glutamicum or a relative thereof in a mixed population of mi croorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted ge 25 nomic DNA of a culture of a unique or mixed population of micro organisms under stringent conditions with a probe spanning a re gion of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. 30 The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glu 35 tamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA binding protein. Those which bind the protein may be addition ally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nu 40 cleic acid molecule to the genome fragment enables the localiza tion of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facili tates a rapid determination of the nucleic acid sequence to WO 2004/050694 PCT/EP2002/013504 55 which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bac 5 teria, such as Brevibacterium lactofermentum. This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, Figure, patent applications, patents, published pat 10 ent applications, Tables, and the Sequence Listing cited throughout this application are hereby incorporated by refer ence. EXAMPLES 15 EXAMPLE 1: PREPARATION OF TOTAL GENOMIC DNA OF CORYNEBACTERIUM GLUTAMICUM ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown 20 overnight at 30 0 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 buffer-I (5% of the original volume of the culture - all indicated volumes have been calculated for 100 ml of culture volume). Composition of 25 buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO, x 7H 2 0, 10 ml/l

KH

2 PO solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/l (NH 4

)

2

SO

4 , 1 g/l NaCl, 2 g/l MgSO, x 7H 2 0, 0.2 g/l CaCl 2 , 0.5 g/l yeast extract (Difco), 10 ml/l trace elements-mix (200 mg/l FeSO 4 x H 2 0, 10 mg/l ZnSO, x 7 H 2 0, 3 mg/l 30 MnCl 2 x 4 H 2 0, 30 mg/l H 3 BO, 20 mg/l CoCl 2 x 6 HO, 1 mg/l NiCl 2 x 6 H 2 0, 3 mg/l Na 2 MoO, x 2 H 2 0, 500 mg/l complexing agent (EDTA or critic acid), 100 ml/l vitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, 20 mg/l p-amino benzoic acid, 20 mg/l riboflavin, 40 mg/i ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/l pyri 35 doxole hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37 0 C, the cell wall was de graded and the resulting protoplasts are harvested by centrifu gation. The pellet was washed once with 5 ml buffer-I and once 40 with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pel let was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) are added. After adding of proteinase K to a final concentration of 200 pg/ml, the suspen- WO 2004/050694 PCT/EP2002/013504 56 sion is incubated for ca.18 h at 37 0 C. The DNA was purified by extraction with phenol, phenol-chloroform-isOamylalcohol and chloroform-isoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate 5 and 2 volumes of ethanol, followed by a 30 min incubation at 20 0 C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing 20 ptg/ml RNaseA and dialysed at 4 0 C against 1000 ml TE-buffer for at least 3 hours. During this 10 time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. After a 30 min incubation at -20 0 C, the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE 15 buffer. DNA prepared by this procedure could be used for all purposes, including southern blotting or construction of genomic libraries. EXAMPLE 2: CONSTRUCTION OF GENOMIC LIBRARIES IN ESCHERICHIA COLI OF 20 CORYNEBACTERIUM GLUTAMICUM ATCC13032. Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al. (1989) "Molecular Cloning 25 A Laboratory Manual", Cold Spring Harbor Laboratory Press, or Ausubel, F.M. et al. (1994) "Current Protocols in Molecular Biol ogy", John Wiley & Sons.) Any plasmid or cosmid could be used. Of particular use were the plas 30 mids pBR322 (Sutcliffe, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacterial 134:1141 1156), plasmids of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla, USA), or cosmids as SuperCosl (Stratagene, La Jolla, USA) or Lorist6 (Gibson, T.J., Rosenthal A. and Waterson, R.H. 35 (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263). EXAMPLE 3: DNA SEQUENCING AND COMPUTATIONAL FUNCTIONAL ANALYSIS 40 Genomic libraries as described in Example 2 were used for DNA se quencing according to standard methods, in particular by the WO 2004/050694 PCT/EP2002/013504 57 chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R.D. et al. (1995) "Whole-genome Random Se quencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512) . Sequencing primers with the following nucleotide 5 sequences were used: 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO:18) or 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO:19). EXAMPLE 4: IN VIVO MUTAGENESIS In vivo mutagenesis of Corynebacterium glutamicum can be performed by 10 passage of plasmid (or other vector) DNA through E. coli or other mi croorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cer evisiae) which are impaired in their capabilities to maintain the in tegrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, 15 mutT, etc.; for reference, see Rupp, W.D. (1996) DNA repair mecha nisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Wash ington.) Such strains are well known to one of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34. 20 EXAMPLE 5: GROWTH OF CORYNEBACTERIUM GLUTAMICUM - MEDIA AND CULTURE CONDITIONS Corynebacteria are cultured in synthetic or natural growth media. 25 A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al. (1989) Appl. Micro biol. Biotechnol., 32:205-210; von der Osten et al. (1998) Bio technology Letters, 11:11-16; Patent DE 4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume IT, Ba 30 lows, A. et al., eds. 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. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, 35 maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mix tures of different carbon sources. Other possible carbon sources 40 are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or in organic nitrogen compounds, or materials which contain these com- WO 2004/050694 PCT/EP2002/013504 58 pounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NHCl or (NH 4

)

2

SO

4 , NH 4 OH, nitrates, urea, amino ac ids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others. 5 Inorganic salt compounds which may be included in the media in clude the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the 10 medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include bio 15 tin, riboflavin, thiamin, folic acid, nicotinic acid, pantothen ate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is in 20 dividually decided for each specific case. Information about me dia optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P.M. Rhodes, P.F. Stan bury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like 25 standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or oth ers. All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121 0 C) or by sterile filtration. The components 30 can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise. Culture conditions are defined separately for each experiment. 35 The temperature should be in a range between 15 0 C and 45 0 C. The temperature can be kept constant or can be altered during the ex periment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media. An exemplary buffer for this purpose is 40 a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH 4 OH during growth. If complex WO 2004/050694 PCT/EP2002/013504 59 medium components such as yeast extract are utilized, the neces sity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermen tor is utilized for culturing the micro-organisms, the pH can 5 also be controlled using gaseous ammonia. The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maxi mal amount of product to accumulate in the broth. The disclosed 10 growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baf 15 fles. Preferably 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical 20 correction for evaporation losses should be performed. If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD 0 25 of 0.5 - 1.5 using cells grown on agar plates, such as CM plates (10 g/l glucose, 2,5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30 0 C. 30 Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium. EXAMPLE 6: IN VITRO ANALYSIS OF THE FUNCTION OF MUTANT PROTEINS 35 The activity of proteins which bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such pro teins on the expression of other molecules can be measured using 40 reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes WO 2004/050694 PCT/EP2002/013504 60 such as beta-galactosidase, green fluorescent protein, and sev eral others. EXAMPLE 7: ANALYSIS OF IMPACT OF MUTANT PROTEIN ON THE PRODUC 5 TION OF THE DESIRED PRODUCT The effect of the genetic modification in C. glutamicum on pro duction of a desired compound (such as an amino acid) can be as sessed by growing the modified microorganism under suitable con 10 ditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product (i.e., an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of 15 various kinds, enzymatic and microbiological methods, and ana lytical chromatography such as high performance liquid chroma tography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) "Applications of HPLC in Bio 20 chemistry" in: Laboratory Techniques in Biochemistry and Molecu lar Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III: "Product recovery and purification", page 469-714, VCH: Weinheim; Belter, P.A. et al. (1988) Bioseparations: down stream processing for biotechnology, John Wiley and Sons; Ken 25 nedy, J.F. and Cabral, J.M.S. (1992) Recovery processes for bio logical materials, John Wiley and Sons; Shaeiwitz, J.A. and Henry, J.D. (1988) Biochemical separations, in: Ulmann's Ency clopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1 27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and puri 30 fication techniques in biotechnology, Noyes Publications.) In addition to the measurement of the final product of fermenta tion, it is also possible to analyze other components of the metabolic pathways utilized for the production of the desired 35 compound, such as intermediates and side-products, to determine the overall yield, production, and/or efficiency of production of the compound. Analysis methods include measurements of nutri ent levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass 40 composition and growth, analysis of the production of common me tabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these meas urements are outlined in Applied Microbial Physiology, A Practi- WO 2004/050694 PCT/EP2002/013504 61 cal Approach, P.M. Rhodes and P.F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references cited therein. 5 EXAMPLE 8: PURIFICATION OF THE DESIRED PRODUCT FROM C. GLU TAMICUM CULTURE Recovery of the desired product from the C. glutamicum cells or 10 supernatant of the above-described culture can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The 15 cellular debris is removed by centrifugation, and the super natant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C. glutamicum cells, then the cells are re moved from the culture by low-speed centrifugation, and the 20 supernate fraction is retained for further purification. The supernatant fraction from either purification method is sub jected to chromatography with a suitable resin, in which the de sired molecule is either retained on a chromatography resin 25 while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appro 30 priate chromatography resins and in their most efficacious ap plication for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the prod uct is maximized. 35 There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be lim iting. Such purification techniques are described, for example, in Bailey, J.E. & Ollis, D.F. Biochemical Engineering Fundamen 40 tals, McGraw-Hill: New York (1986). The identity and purity of the isolated compounds may be as sessed by techniques standard in the art. These include high- WO 2004/050694 PCT/EP2002/013504 62 performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic as say, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; 5 Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclo pedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of 10 Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Labo ratory Techniques in Biochemistry and Molecular Biology, vol. 17. 15 EXAMPLE 9: ANALYSIS OF THE GENE SEQUENCES OF THE INVENTION The comparison of sequences and determination of percent homol ogy between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algo 20 rithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorpo rated into the NBLAST and XBLAST programs (version 2.0) of Alt schul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide 25 searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to MR nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to MR protein 30 molecules of the invention. To obtain gapped alignments for com parison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the 35 program (e.g., XBLAST and NBLAST) for the specific sequence be ing analyzed. Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller 40 ((1988) Comput. Apple. Biosci. 4: 11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 WO 2004/050694 PCT/EP2002/013504 63. weight residue table, a gap length penalty of 12, and a gap pen alty of 4 can be used. Additional algorithms for sequence analy sis are known in the art, and include ADVANCE and ADAM. de scribed in Torelli and Robotti (1994) Comput. Apple. Biosci. 5 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8. The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software pack 10 age (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent homology be tween two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parame 15 ters, such as a gap weight of 50 and a length weight of 3. EXAMPLE 10: CONSTRUCTION AND OPERATION OF DNA MICROARRAYS The sequences of the invention may additionally be used in the 20 construction and application of DNA microarrays (the design, methodology, and uses of DNA arrays are well known in the art, and are described, for example, in Schena, M. et al. (1995) Sci ence 270: 467-470; Wodicka, L. et al. (1997) Nature Biotechnol ogy 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotech 25 nology 16: 45-48; and DeRisi, J.L. et al. (1997) Science 278: 680-686) DNA microarrays are solid or flexible supports consisting of ni trocellulose, nylon, glass, silicone, or other materials. Nu 30 cleic acid molecules may be attached to the surface in an or dered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nu cleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized 35 molecules at defined regions. This methodology allows the simul taneous quantification of the relative or absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids 40 in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427 431).

WO 2004/050694 PCT/EP2002/013504 64 The sequences of the invention may be used to design oligonu cleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and 5 design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470). 10 Nucleic acid microarrays may also be constructed by in situ oli gonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367. By photolithographic meth ods, precisely defined regions of the matrix are exposed to 15 light. Protective groups which are photolabile are thereby acti vated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, de 20 fined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis. The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. 25 These nucleic acid molecules can be labeled according to stan dard methods. In brief, nucleic acid molecules (e.g., mRNA mole cules or DNA molecules) are labeled by the incorporation of iso topically or fluorescently labeled nucleotides, e.g., during re verse transcription or DNA synthesis. Hybridization of labeled 30 nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantifi cation of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for ex 35 ample, as described in Schena, M. et al. (1995) supra) and fluo rescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645). The application of the sequences of the invention to DNA mi 40 croarray technology, as described above, permits comparative analyses of different strains of C. glutamicum or other Coryne bacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of WO 2004/050694 PCT/EP2002/013504 65 genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress toler ance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the inven 5 tion during the course of a fermentation reaction are possible using nucleic acid array technology. EXAMPLE 11: ANALYSIS OF THE DYNAMICS OF CELLULAR PROTEIN POPU LATIONS (PROTEOMICS) 10 The genes, compositions, and methods of the invention may be ap plied to study the interactions and dynamics of populations of proteins, termed 'proteomics'. Protein populations of interest include, but are not limited to, the total protein population of 15 C. glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under spe cific environmental or metabolic conditions (e.g., during fer mentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth 20 and development. Protein populations can be analyzed by various well-known tech niques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be sepa 25 rated from one another using a variety of electrophoretic tech niques. Sodium dodecyl sulfate polyacrylamide gel electrophore sis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel elec trophoresis (IEF-PAGE) separates proteins by their isoelectric 30 point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combina tion of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electropho resis (described, for example, in Hermann et al. (1998) Electro 35 phoresis 19: 3217-3221; Fountoulakis et al. (1998) Electrophore sis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451 1463). Other separation techniques may also be utilized for pro tein separation, such as capillary gel electrophoresis; such 40 techniques are well known in the art. Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable WO 2004/050694 PCT/EP2002/013504 66 stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Mo lecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., "S-methionine, "S 5 cysteine, "C-labelled amino acids, "N-amino acids, "NO, or "NH,* or " 3 C-labelled amino acids) in the medium of C. glutamicum per mits the labeling of proteins from these cells prior to their separation. Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated ac 10 cording to the previously described techniques. Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for exam 15 ple, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of pro teins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such tech 20 niques are well-known in the art. To determine the identity of any given protein, direct sequenc ing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degra 25 dation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Elec trophoresis 18: 1184-1192)). The protein sequences provided herein can be used for the identification of C. glutamicum pro teins by these techniques. 30 The information obtained by these methods can be used to compare patterns of protein presence, activity, or modification between different samples from various biological conditions (e.g., dif ferent organisms, time points of fermentation, media conditions, 35 or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behav ior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine 40 chemicals or to increase the efficiency of the production of fine chemicals.

WO 2004/050694 PCT/EP2002/013504 67 EXAMPLE 12: PREPARATION OF A CORYNEBACTERIUM GLUTAMICUM STRAIN DEFICIENT IN THE NEGATIVE REGULATOR OF METHIONINE BIOSYNTHESIS (RXA00655) 5 Preparation of a Corynebacterium glutamicum strain deficient in the negative regulator of methionine biosynthesis (RXA00655) is carried out by using a self-cloning technique based on homologous recombination. The principle of said technique is visualized in Figure 1. 10 The plasmid named pSdelta655 (SEQ ID NO:3) for preparation of a C. glutamicum strain deficient in the negative regulator of me thionine biosynthesis (RXA00655) is constructed by ligating PCR amplified fragments of the 5'- and 3' regions of RXA00655 (SEQ ID 15 NO:1) into the vector pCLiK5MCSintegrativ-sacB (SEQ ID NO:4) us ing standard methods (Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor; Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press). The PCR amplified 5'-fragment is flanked at its 5'-end by a 20 primer-introduced XhoI site and at its 3'-end by an endogenous NheI site. The PCR amplified 3'-fragment is flanked at its 5'-end by an NheI site followed by an TAA-stopp codon, which are intro duced by the primer, and at its 3'-end by a primer-introduced MluI site. 25 pS-delta655 (SEQ ID NO:3) is electroporated into the C. glu tamicum strain ATCC13032 as described (Liebl et al. (1989) FEMS Microbiol Let 53:299-303). Transformands with via intermolecular homologous recombination integrated plasmid are selected on CM 30 agar plates supplemented with 50 mg/l kanamycin. CM-agar: 10.0 g/L D-glucose 2.5 g/L NaCl 35 2.0 g/L urea 10.0 g/L bacto pepton (Difco) 5.0 g/L yeast extract (Difco) 5.0 g/L beef extract (Difco) 22.0 g/L agar (Difco) 40 autoclaved (20 min. 121 0 C) Kanamycin-resistant clones are incubated unselectively (without Kanamycin) overnight in CM medium to achieve excision of the WO 2004/050694 PCT/EP2002/013504 68 plasmid together with the chromosomal copy of RXA00655. The cul tures are plated on CM agar containing 10% sucrose. Only those clones in which the integrated pS-delta655 plasmid is excised can grow on CM agar containing sucrose, because the sacB gene in 5 pS-delta655 converts sucrose into levan sucrase, which is toxic for C. glutamicum. In the excision either the deletion construct of RXA00655 or the chromosomal copy of RXA00655 is eliminated together with the plasmid. 10 To identify a clone which has eliminated the chromosomal copy of RXA00655, chromosomal DNA from all potential clones are prepared (Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994) Microbiology 140:1817-1828) and controlled with a South ern Blot analysis according to Sambrook et al. (1989), Molecular 15 Cloning. A Laboratory Manual, Cold Spring Harbor. The knock-out strain is called ATCC13032_deltarxaOO655. EXAMPLE 15: PREPARATION OF METHIONINE WITH ATCC13032_DELTARXA00655 20 The strains ATCCl3032 and ATCC13032 deltarxa00655 are incubated on a CM-agar plate for 2 d at 30 0 C. The cells are scraped from the plate and suspended in saline. For the main culture 10 ml medium II and 0.5 g autoclaved CaCO, (Riedel de Haen) in a 100 25 ml conical flask are inoculated with the cell suspension to a final OD600nm of 1.5. Culturing is carried out at 30 0 C for 72 h. Medium II: 40 g/l sucrose 30 60 g/l molasse (calculated on 100% sugar content) 25 g/l (NH4)2SO4 0.4 g/l MgSO4*7H20 0.6 g/l KH2PO4 0.3 mg/l thiamin*HCl 35 1 mg/l biotin (from a 1 mg/ml sterile filtered stock solution adjusted to pH 8.0 with NH40H) 2 mg/l FeSO4 2 mg/l MnSO4 The pH is brought to pH 7.8 with NH 4 OH. Thereaf ter, the medium is autoclaved (121 0 C, 20 min). Additional vita 40 min B12 from a stock solution (200 pg/ml, sterile filtered) is added to a final concentration of 200 pg/l.

WO 2004/050694 PCT/EP2002/013504 69 The amount of formed methionine was determined with an Agilent 1100 series LC system HPLC according to a method from Agilent. The with ortho-pthalaldehyde pre-column derivated amino acids are separated on a Hypersil AA-column (Agilent). The strain 5 ATCC13032_deltarxa00655 produces significantly more methionine than ATCCl3032. EXAMPLE 16: PREPARATION OF A CORYNEBACTERIUM GLUTAMICUM STRAIN 10 OVEREXPRESSING THE POSITIVE REGULATOR OF METHIONINE BIOSYNTHESIS (RXN02910) The plasmid named pGrxn2910 (SEQ ID NO:13) for overexpressing the positive regulator of methionine biosynthesis (RXN02910; SEQ 15 ID NO:5 or 6) is constructed by ligating a PCR amplified frag ment of RXNO2910 (SEQ ID NO:9) into the vector pG (SEQ ID NO:12) using standard methods (Sambrook et al. (1989), Molecular Clon ing. A Laboratory Manual, Cold Spring Harbor; Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Aca 20 demic Press). The following pair of oligonucleotide primers was used for the amplification: sense primer (SEQ ID NO: 7): 5'-GAGAGACTCGAGTGGTTTAGGGGATGAGAAACCG-3' 25 antisense primer (SEQ ID NO: 8): 5'-CTCTCACTAGTCTACCGCGCCAACAACAGCG-3' The PCR amplified fragment is flanked at its 5'-end by a primer 30 introduced XhoI site and at its 3'-end by a primer-introduced BcuI site. The amplified fragments contains the open reading frame (ORF) of RXNO2910 with additional 5' regions of the corre sponding gene including the promotor. 35 The resulting plasmid pGrxnO2910 (SEQ ID NO:13) is electropo rated into the C. glutamicum strain ATCC13032 as described (Liebl, et al. (1989) FEMS Microbiology Letters 53:299-303). Transformands are selected on CM agar plates supplemented with 50 mg/l kanamycin (see above). The resulting strain is named 40 ATCC13032/pGrxnO2910.

WO 2004/050694 PCT/EP2002/013504 70 EXAMPLE 17: PREPARATION OF METHIONINE WITH ATCC13032/PGRXNO2910 The strains ATCC13032 and ATCC13032/pGrxnO2910 are incubated on a CM-agar plate for 2 days at 300C. The cells are scraped from 5 the plate and suspended in saline. For the main culture 10 ml medium II (see above) and 0.5 g autoclaved CaCO, (Riedel de Haen) in a 100 ml conical flask are inoculated with the cell suspension to a final OD600nm of 1.5. Culturing is carried out at 300C for 72 h. In the case of ATCC13032/pGrxnO2910 all plates 10 and cultures contain additional 50 pg/l kanamycin. The amount of formed methionine was determined with an Agilent 1100 series LC system HPLC according to a method from Agilent. The with ortho phtalaldehyde pre-column derivated amino acids are separated on a Hypersil AA-column (Agilent). The strain ATCC13032/pGrxnO2910 15 produces significantly more methionine than ATCCl3032. EXAMPLE 18: PREPARATION OF A CORYNEBACTERIUM GLUTAMICUM STRAIN DEFICIENT IN THE POSITIVE REGULATOR OF ME THIONINE BIOSYNTHESIS (RXN02910) 20 Preparation of a Corynebacterium glutamicum strain deficient in the positive regulator of methionine biosynthesis (RXNO2910) is carried out by insertion of a kanamycin selectable vector. 25 The plasmid named pIntegrativdelta2910 (SEQ ID NO:15) for prepa ration of a C. glutamicum strain deficient in the regulator of methionine biosynthesis (rxnO2910) is constructed by ligating a PCR amplified fragment of rxnO2910 into the vector pIntegrativ (SEQ ID NO:14) using standard methods (Sambrook et al. (1989), 30 Molecular Cloning. A Laboratory Manual, Cold Spring Harbor; Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press). The PCR amplified fragment (SEQ ID NO:ll) is flanked at its 5'-end by a primer-introduced XhoI site and at its 3'-end by a primer-introduced EcoRV site. 35 pIntegrativ-delta2910 (SEQ ID NO:15) is electroporated into the C. glutamicum strain ATCC13032 as described (Liebl et al. (1989) FEMS Microbiol Let 53:299-303). Transformands with via intermo lecular homologous recombination integrated plasmid are selected 40 on CM agar plates supplemented with 50 mg/l kanamycin. To iden tify a clone which has an integrated plasmid and thus disrupted the chromosomal copy of RXNO2910, chromosomal DNA from potential clones are prepared (Tauch et al. (1995) Plasmid 33:168-179 or WO 2004/050694 PCT/EP2002/013504 71 Eikmanns et al. (1994) Microbiology 140:1817-1828) and con trolled with a Southern Blot analysis according to Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. 5 The knock-out strain is called ATCCl3032_deltarxnO2910. The amount of formed methionine was determined with an Agilent 1100 series LC system HPLC according to a method from Agilent. The with ortho-phtalaldehyde pre-column derivated amino acids are 10 separated on a Hypersil AA-column (Agilent). The strain ATCCl3032_deltarxnO2910 produces significant less me thionine than ATCC13032, thereby demonstrating that rxnO2910 is indeed a positive regulator of methionine biosynthesis. 15 Equivalents Those of ordinary skill in the art will recognize, or will be able to ascertain using no more than routine experimentation, 20 many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encom passed by the following claims.

WO 2004/050694 PCT/EP2002/013504 72 TABLE 1: Corynebacterium and Brevibacterium Strains Which May be Used in the Prac tice of the Invention AC FERM NRRL CECT NCIMB .CBS NCTC DSMZ Brevibacterium ammoniagenes 21054 Brevibacterium ammoniagenes 19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes 19352 Brevibacterium ammoniagenes 19353 Brevibacterium ammoniagenes 19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes 19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes 21077 Brevibacterium ammoniagenes 21553 Brevibacterium ammoniagenes 21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196 Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474 Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacterium flavum B11474 Brevibacterium flavum B1 1472 Brevibacterium flavum 21127 Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacterium flavum 21475 Brevibacterium flavum 21517 Brevibacterium flavum 21528 Brevibacterium flavum 21529 Brevibacterium flavum B1 1477 Brevibacterium flavum BI 1478 Brevibacterium flavum 21127 Brevibacterium flavum B1 1474 Brevibacterium healii 15527 WO 2004/050694 PCT/EP2002/013504 73 Brevibacterium ketoglutamicum 21004 Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914 Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 21798 Brevibacterium lactofermentum 21799 Brevibacterium lactofermentum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B1 1470 Brevibacterium lactofermentum B1 1471 Brevibacterium lactofermentum 21086 Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum 21086 Brevibacterium lactofermentum 31269 Brevibacterium linens 9174 Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacterium paraffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec. 717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860 Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacterium spec. 21866 Brevibacterium spec. 19240 Corynebacterium acetoacidophilum 21476 Corynebacterium acetoacidophilum 13870 Corynebacterium acetoglutamicum B11473 Corynebacterium acetoglutamicum B1 1475 Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum 21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilum B3671 WO 2004/050694 PCT/EP2002/013504 74 Corynebacterium ammoniagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacterium glutamicum 14067 Corynebacterium glutamicum 39137 Corynebacterium glutamicum 21254 Corynebacterium glutamicum 21255 Corynebacterium glutamicum 31830 Corynebacterium glutamicum 13032 Corynebacterium glutamicum 14305 Corynebacterium glutamicum 15455 Corynebacterium glutamicum 13058 Corynebacterium glutamicum 13059 Corynebacterium glutamicum 13060 Corynebacterium glutamicum 21492 Corynebacterium glutamicum 21513 Corynebacterium glutamicum 21526 Corynebacterium glutamicum 21543 Corynebacterium glutamicum 13287 Corynebacterium glutamicum 21851 Corynebacterium glutamicum 21253 Corynebacterium glutamicum 21514 Corynebacterium glutamicum 21516 Corynebacterium glutamicum 21299 Corynebacterium glutamicum 21300 Corynebacterium glutamicum 39684 Corynebacterium glutamicum 21488 Corynebacterium glutamicum 21649 Corynebacterium glutamicum 21650 Corynebacterium glutamicum 19223 Corynebacterium glutamicum 13869 Corynebacterium glutamicum 21157 Corynebacterium glutamicum 21158 Corynebacterium glutamicum 21159 Corynebacterium glutamicum 21355 Corynebacterium glutamicum 31808 WO 2004/050694 PCT/EP2002/013504 75 Corynebacterium glutamicum 21674 Corynebacterium giutamicum 21562 Corynebacterium glutamicum 21563 Corynebacterium glutamicum 21564 Corynebacterium glutamicum 21565 Corynebacterium glutamicum 21566 Corynebacterium glutamicum 21567 Corynebacterium glutamicum 21568 Corynebacterium glutamicum 21569 Corynebacterium glutamicum 21570 Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 19050 Corynebacterium glutamicum 19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 19056 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum 19059 Corynebacterium glutamicum 19060 Corynebacterium glutamicum 19185 Corynebacterium glutamicum 13286 Corynebacterium glutamicum 21515 Corynebacterium glutamicum 21527 Corynebacterium glutamicum 21544 Corynebacterium glutamicum 21492 Corynebacterium glutamicum B8183 Corynebacterium glutamicum B8182 Corynebacterium glutamicum B12416 Corynebacterium glutamicum B12417 WO 2004/050694 PCT/EP2002/013504 76 Corynebacterium glutamicum B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum 21608 Corynebacterium lilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088 Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 15954 20145 Corynebacterium spec. 21857 Corynebacterium spec. 21862 Corynebacterium spec. 21863 ATCC: American Type Culture Collection, Rockville, MD, USA FERM: Fermentation Research Institute, Chiba, Japan 5 NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK CBS: Centraalbureau voor Schimmelcultures, Baarn, NL NCTC: National Collection of Type Cultures, London, UK 10 DSMZ: Deutsche Sammlung von Mikroorganismen und Zelikulturen, Braunschweig, Germany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of micro organisms: Bacteria, fungi and yeasts ( 4 h edn), World federation for culture collections world data center on microorganisms, Saimata, Japen. 15

Claims

1. A method of modulating production of a sulfur-containing compound by a microorganism comprising culturing a micro 5 organism with modulated expression or activity of at least one regulator of methionine biosynthesis under conditions such that production of a sulfur-containing compound is modulated. 10

2. The method of claim 1, wherein said sulfur-containing com pound is selected from the group consisting of methionine, cysteine, S-adenosylmethionine and homocycsteine.

3. The method as claimed in either claim 1 or 2, wherein pro 15 duction of methionine is increased

4. The method as claimed in any one of claims 1 to 3, wherein said regulator of methionine biosynthesis is a positive or negative transcriptional regulatory protein. 20

5. The method as claimed in any one of claims 1 to 4, wherein said method comprises overexpression of a positive regula tor of methionine biosynthesis 25

6. The method as claimed in any one of claims 1 to 5, wherein said positive regulator of methionine biosynthesis is RXN02910.

7. The method as claimed in any one of claims 1 to 6, wherein 30 RXNO2910 is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence 35 which is at least 60% identical to the nucleotide se quence of SEQ ID NO:5 or 16; b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the 40 nucleotide sequence of SEQ ID NO:5 or 16; c) a nucleic acid molecule which encodes a polypeptide com prising an amino acid sequence at least about 60% iden- WO 2004/050694 PCT/EP2002/013504 78 tical to the amino acid sequence of SEQ ID NO:6 or 17 and d) a nucleic acid molecule which encodes a fragment of a 5 polypeptide comprising the amino acid sequence of SEQ ID NO:6 or 17 wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid se quence of SEQ ID NO:6 or 17, 10

8. The method as claimed in any one of claims 1 to 7, wherein RXNO2910 is encoded by a nucleic acid molecule comprising the nucleotide sequence set forth as SEQ ID NO:5 or SEQ ID NO:16. 15

9. A method as claimed in any one of claims 1 to 4, comprising culturing a microorganism which is deficient in a negative regulator of methionine biosynthesis or a microorganism which exhibits a decreased activity of a negative regulator of methionine biosynthesis under conditions such that pro 20 duction of methionine is increased.

10. The method as claimed in any one of claims 1 to 4 or 9, wherein said negative regulator of methionine biosynthesis is RXA00655. 25

11. The method as claimed in any one of claims 1 to 4 or 9 or 10, wherein RXA00655 is encoded by a nucleic acid molecule comprising a nucleic acid molecule selected from the group consisting of: 30 a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% identical to the nucleotide se quence of SEQ ID NO:1, 18, 20 or 22; 35 b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, 18, 20 or 22; c) a nucleic acid molecule which encodes a polypeptide com 40 prising an amino acid sequence at least about 60% iden tical to the amino acid sequence of SEQ ID NO:2, 19, 21 or 23; and WO 2004/050694 PCT/EP2002/013504 79 d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 19, 21, or 23, wherein the fragment comprises at least 10 contiguous amino acid residues of the amino 5 acid sequence of SEQ ID NO:2, 19, 21, or 23,

12. The method as claimed in any one of claims 1 to 4 or 9 to 12, wherein RXA00655 is encoded by a nucleic acid molecule comprising the nucleotide sequence set forth as SEQ ID 10 NO:l.

13. The method as claimed in any one of claims 1 to 4 or 9 to 12, wherein deficiency or decreased activity is achieved by a method selected from the group consisting of: 15 a) knock-out of the gene encoding said negative regulatory protein; b) mutagenesis of the gene encoding said negative regula 20 tory protein, wherein said mutation can be induced in the coding, non-coding, or regulatory regions of said gene; c) expression of an anti-sense RNA, wherein said anti-sense 25 RNA is complementary to at least part of the RNA encod ing said negative regulatory protein; d) expression of DNA-binding proteins blocking or reducing expression from the gene encoding said negative regula 30 tory protein; e) expression of protein-binding factors blocking or reduc ing expression from the gene encoding said negative regulatory protein; 35 f) expression of a dominant-negative variant of said nega tive regulatory protein; and g) destabilization of the mRNA encoding said negative regu 40 latory protein.

14. A method of producing methionine comprising culturing a microorganism which overexpresses RXNO2910 or has increased WO 2004/050694 PCT/EP2002/013504 80 RXN02910 activity under conditions such that methionine is produced.

15. A method of producing methionine comprising culturing a 5 microorganism which has suppressed expression or decreased activity of RXA00655 under conditions such that methionine is produced.

16. A method of producing methionine comprising culturing a 10 microorganism which a) overexpresses RXN02910 or has increased RXNO2910 activi ty and which 15 b) has suppressed expression or decreased activity of RXA00655 under conditions such that methionine is produced. 20

17. The method of any of the preceeding claims, wherein said microorganism belongs to the genus Corynebacterium or Bre vibacterium.

18. The method of any of the preceeding claims, wherein said 25 microorganism is selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium herculis, Corynebacterium, lilium, Coryne bacterium acetoacidophilum, Corynebacterium acetoglu tamicum, Corynebacterium acetophilum, Corynebacterium ammo 30 niagenes, Corynebacterium fujiokense, Corynebacterium ni trilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium fla vum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermen 35 tum, Brevibacterium linens, Brevibacterium paraffinolyti cum, and those strains set forth in Table 1.

19. The method of any of the preceeding claims, wherein said microorganism is Corynebacterium glutamicum. 40

20. The method of any one of preceeding claims, further com prising recovering methionine. WO 2004/050694 PCT/EP2002/013504 81

21. A transgenic expression cassette comprising in combination with a regulatory sequence a nucleic acid molecule selected from the group consisting of: 5 a) a nucleic acid molecule comprising a nucleotide se quence which is at least 60% identical to the nucleo tide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 or a complement thereof; 10 b) a nucleic acid molecule comprising a fragment of at least 30 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22 or a 15 complement thereof; c) a nucleic acid molecule which encodes a polypeptide com prising an amino acid sequence at least about 60% iden tical to the amino acid sequence of SEQ ID NO:2, SEQ ID 20 NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23; and d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID 25 NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23 wherein the fragment comprises at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. 30 wherein said regulatory sequence is capable of mediating expression of said nucleic acid molecule in a microorgan ism. 35

22. A transgenic expression cassette of claim 21, wherein said regulatory sequence is a promoter sequence heterologous with regard to said nucleic acid molecule.

23. A transgenic expression cassette of either claim 21 or 22, 40 wherein said nucleic acid molecule is arranged in antisense or sense orientation with regard to said promoter sequence.

24. A transgenic expression cassette of any of the claims 21 WO 2004/050694 PCT/EP2002/013504 82 to 23, wherein said nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23. 5

25. A transgenic expression cassette of any of the claims 21 to 24, wherein said nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. 10

26. A transgenic expression cassette of any of the claims 21 to 25, wherein said nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:6, 15 SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23.

27. A vector comprising a transgenic expression cassette of any one of claims 21 to 26. 20

28. The vector of claim 27, which is an expression vector.

29. A transgenic host cell transfected with a transgenic ex pression cassette of any of the claims 21 to 26, or a vec tor of claim 27 or 28. 25

30. The transgenic host cell of claim 29, wherein said host cell belongs to the genus Corynebacterium or Brevibacte ri um.