US007892798B2

(12) United States Patent (10) Patent No.: US 7,892,798 B2 Pompejus et al. (45) Date of Patent: Feb. 22, 2011

(54) NUCLEICACID MOLECULES ENCODING (56) References Cited METABOLC REGULATORY U.S. PATENT DOCUMENTS FROM CORYNEBACTERIUM GLUTAMICUM, USEFUL FOR INCREASING THE 3,729,381 A 4, 1973 Nakayama et al. PRODUCTION OF METHONONE BY A 2005. O153402 A1 7/2005 Pompeius et al. MCROORGANISM FOREIGN PATENT DOCUMENTS (75) Inventors: Markus Pompeius, Freinsheim (DE); EP 1108790 A2 6, 2001 Burkhard Kröger, Limburgerhof (DE); EP 1108790 A2 * 6, 2001 Hartwig Schröder, Nussloch (DE); JP 50-31092 3, 1975 Oskar Zelder, Speyer (DE); Gregor WO WO-O2/O97096 A2 12/2002 Haberhauer, Limburgerhof (DE); OTHER PUBLICATIONS Stefan Haefner, Ludwigshafen (DE); Seffernicket al. J. Bateriol., vol. 183, pp. 2405-2410, 2001.* Corrinna Klopprogge, Ludwigshafen Branden et al. “Introduction to Structure', Garland Publish (DE) ing Inc., New York, 1991, p. 247.* Witkowski et al., Biochemistry 38: 11643-11650, 1999.* (73) Assignee: Evonik Degussa GmbH, Essen (DE) Renna et al., J Bacteriol 175:3863-3875, 1993.* Dictionary definition of “positively at Encarta.com, last viewed on (*) Notice: Subject to any disclaimer, the term of this Jan. 16, 2008, 2 pages.* patent is extended or adjusted under 35 Kolmar et al., EMBO.J. 14:3895-3904, 1995.* U.S.C. 154(b) by 239 days. Hwang et al., J. Bacteriol. 184:1277-1286, 2002.* Belfaiza, J. et al. “Direct Sulfhydrylation for Methionine (21) Appl. No.: 10/307,138 Biosynthesis in Leptospira meyeri,’ Journal of Bacteriology, vol. 180(2):250-255 (1998). (22) Filed: Nov. 29, 2002 Follettie, M.T. et al., “Organization and regulation of the Corynebacterium glutamicum hom-thrb and thr0 loci.” Molecular (65) Prior Publication Data Microbiology, vol. 2(1):53-62 (1988). Henikoff, S. et al., “A Large family of bacterial activator proteins.” US 2003/O162267 A1 Aug. 28, 2003 Proc. Natl. Acad. Sci., vol. 85: 6602-6606 (1988). Hwang, Byung-Joon et al., "Corynebacterium glutamicum Utilizes Related U.S. Application Data both Transsulfuration and Direct Sulfhydrylation Pathways for Methionine Biosynthesis,” Journal of Bacteriology, vol. (63) Continuation-in-part of application No. 09/602,874, 184(5):1277-1286 (2002). filed on Jun. 23, 2000, now abandoned. Muraoka, Shin et al., “Crystal Structure of a Full-length LysR-type Transcriptional Regulator, CnbR: Unusual Combination of Two Sub (60) Provisional application No. 60/141,031, filed on Jun. unit Forms and Molecular Bases for Causing and Changing DNA 25, 1999, provisional application No. 60/142,690, Bend.” J. Mol. Biol., vol. 328:555-566 (2003). filed on Jul. 1, 1999, provisional application No. Schell, Mark A., “Molecular Biology of the LysR Family of Tran 60/151,251, filed on Aug. 27, 1999, provisional appli scriptional Regulators.” Annu. Rev. Microbiol., vol. 47:597–626 cation No. 60/422,618, filed on Oct. 30, 2002. (1993). (30) Foreign Application Priority Data (Continued) Primary Examiner David J Steadman Jul. 1, 1999 (DE) ...... 1993O476 (74) Attorney, Agent, or Firm Nelson Mullins Riley & Jul. 8, 1999 (DE) ...... 1993.1419 Scarborough, LLP Jul. 8, 1999 (DE) ...... 199 31 420 Jul. 9, 1999 (DE) ...... 19932 122 (57) ABSTRACT Jul. 9, 1999 (DE) ...... 19932 128 Jul. 9, 1999 (DE) ...... 19932 134 Isolated nucleic acid molecules, which encode novel meta Jul. 9, 1999 (DE) ...... 199322O6 bolic regulatory proteins from Corynebacterium glutamicum are described. These nucleic acid molecules are involved in Jul. 9, 1999 (DE) ...... 19932 207 the biosynthesis of a fine chemical, e.g., methionine biosyn Jul. 14, 1999 (DE) ...... 199 33 003 thesis. The invention also provides antisense nucleic acid Aug. 31, 1999 (DE) ...... 1994.1 390 molecules, recombinant expression vectors containing meta Sep. 3, 1999 (DE) ...... 19942 088 bolic regulatory nucleic acid molecules, and host cells into Sep. 3, 1999 (DE) ...... 19942124 which the expression vectors have been introduced. The invention still further provides methods of producing (51) Int. Cl. methionine from microorganisms, e.g., C. glutamicum, CI2P 13/12 (2006.01) which involve culturing recombinant microorganisms which CI2P 2/06 (2006.01) overexpress or underexpress at least one metabolic regulatory CI2N L/20 (2006.01) molecule of the invention under conditions such that CI2N IS/00 (2006.01) methionine is produced. Also featured are methods of pro C7H 2L/04 (2006.01) ducing a fine chemical, e.g., methionine, which involve cul (52) U.S. Cl...... 435/113:435/69. 1; 435/252.3: turing recombinant microorganisms having selected meta 435/252.32:435/320.1536/23.1:536/23.4: bolic regulatory deleted or mutated under conditions 536/23.7 Such that the fine chemical, e.g., methionine, is produced. (58) Field of Classification Search ...... None See application file for complete search history. 56 Claims, 1 Drawing Sheet US 7,892,798 B2 Page 2

OTHER PUBLICATIONS Kase, Hiroshi et al., "Isolation and Characterization of S-Adenosylmethionine-requiring Mutants and Role of Database Accession No. Q9EW12, "Putative tetR-family transcrip S-Adenosylmethionine in the Regulation of Methionine tional regulator.” Streptomyces coelicolor, Mar. 1, 2004. Biosynthesis in Corynebacterium glutamicum,' Agr: Biol. Chem. Database Accession No. AP005223. “Comparative complete vol. 39(1):161-168 (1975). sequence analysis of the replacements responsible for the U.S. Appl. No. 09/602,874, Markus Pompeius et al. thermostability of Corynebacterium efficiens.” Nishio, Y. et al. Jul. Kase, Hiroshi et al. “L-Methionine Production by Methionine Ana 24, 2003. log-resistant Mutants of Corynebacterium glutamicum,’ Agr: Biol. Database Accession No. AP005281, "Complete genomic sequence of Chem., vol. 39(1): 153-160 (1975). Corynebacterium glutamicum ATCC 13032. Nakagawa, S., Aug. 8, Mondal, S. et al., “Enhancement of methionine production by 2002. methionine analogue ethionine resistant mutants of Brevibacterium Adhya, Sankar, “The LAC and GAL Operons Today.” Regulation of heali, "Acta Biotechnologica, vol. 14(2): 199-204 (1994). Expression in Escherichia coli, Chapman & Hall, New York. Moran, Charles P. "RNA Polymerase and Factors.” pp. 181-200, 1996. Bacillus subtilis and Other gram-positive bacteria, Sonenshein, A.L. Boos, Winfried et al. “The Maltose System.” Regulation of Gene etal, eds. ASM: Washington, D.C., pp. 653-667, 1993. Expression in Escherichia coli, Chapman & Hall, New York, pp. Nishio, Yousuke et al., "Comparative Complete Genome Sequence 201-229, 1996. Analysis of the Amino Acid Replacements Responsible for the Helmann, John D. et al. "Structure and Function of bacterial Sigma Thermostability of Corynebacterium efficiens,' Genome Research, Factors.” Ann. Rev. Biochem., vol. 57:839-872 (1988). vol. 13:1572-1579 (2003). Henkin, T.M. etal, “Catabolite repression of O-amylase gene expres Old, Iain G. et al., “Regulation of Methionine Biosynthesis in the sion in Bacillus subtilis involves a trans-acting gene product homolo Enterobacteriaceae.” Prog. Biophys. Molec. Biol., vol. 56: 145-185 gous to the Escherichia coli lacl and galR repressors.” Molecular (1991). Microbiology, vol. 5(3):575-584 (1991). Sharma, Sanjay et al. “Effect of Dissolved Oxygen on Continuous Lewin, B. et al., “Controlling Prokaryotic Genes by Transcription.” Production of Methionine.” Eng. Life Sci. J., vol. 2:69-73 (2001). Oxford University Press: Oxford, pp. 213-301, 1990. * cited by examiner U.S. Patent Feb. 22, 2011 US 7,892,798 B2

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|?In6|- US 7,892,798 B2 1. 2 NUCLECACID MOLECULES ENCODING being used and no salt is produced by a fermentative process, METABOLIC REGULATORY PROTENS there is an advantage of this process over the chemical FROM CORYNEBACTERIUM GLUTAMICUM, methionine synthesis. Methionine can be produced in organ USEFUL FOR INCREASING THE isms such as E. coli or Corynebacter (Kase H., Nakayama K. PRODUCTION OF METHONONE BY A 5 (1975) Agric. Biol. Chem. 39 pp 153-160; Chatterjee et al. MICROORGANISM (1999) Acta Biotechnol. 14, pp 199-204; Harma S. Gomes, (2001) J. Eng. Life Sci. 1 pp. 69-73, JP 50031092, DE RELATED APPLICATIONS 2105189). However, in all reported cases the yields for fermentatively This application claims priority to U.S. Provisional Appli 10 produced methionine seem to be too low for an economic cation Ser. No. 60/422,618, filed on Oct. 30, 2002. This appli production. Therefore, an improved method is needed. cation also claims priority to U.S. application Ser. No. 09/602,874, filed on Jun. 23, 2000. This application also SUMMARY OF THE INVENTION claims priority to U.S. Provisional Patent Application No. 60/141,031, filed Jun. 25, 1999, U.S. Provisional Patent 15 The invention provides novel bacterial nucleic acid mol Application No. 60/142,690, filed Jul. 1, 1999, and also to ecules which modulate the biosynthesis of fine chemicals, U.S. Provisional Patent Application No. 60/151,251, filed e.g., methionine. It has been found that the metabolic regula Aug. 27, 1999. This application also claims priority to Ger tory (“MR) molecules of the invention are involved in bio man Patent Application No. 19930476.9, filed Jul. 1, 1999, synthesis of a fine chemical, e.g., methionine. In particular, it German Patent Application No. 19931419.5, filed Jul. 8, has been found that the MR molecule RXA00655 is a nega 1999, German Patent Application No. 1993.1420.9, filed Jul. tive regulator, e.g., transcriptional regulator, of methionine 8, 1999, German Patent Application No. 19932122.1, filed biosynthesis, cysteine biosynthesis, and/or the Sulfur reduc Jul. 9, 1999, German Patent Application No. 19932128.0, tion pathway. It has also been found that the MR molecule filed Jul. 9, 1999, German Patent Application No. RXNO2910 is a positive regulator, e.g., transcriptional regu 19932.134.5, filed Jul. 9, 1999, German Patent Application 25 lator, of methionine biosynthesis. The nucleotide sequence of No. 19932206.6, filed Jul. 9, 1999, German Patent Applica RXA00655 is set forth herein as SEQ ID NO:1 and the tion No. 199322074, filed Jul. 9, 1999, German Patent Appli polypeptide sequence of RXA00655 is set forth herein as cation No. 19933003.4, filed Jul. 14, 1999, German Patent SEQID NO:2. The nucleotide sequence of RXN02910 is set Application No. 1994.1390.8, filed Aug. 31, 1999, German forth hereinas SEQID NO:5 and the polypeptide sequence of Patent Application No. 19942088.2, filed Sep. 3, 1999, and 30 RXN02910 is set forth hereinas SEQID NO:6. Homologous German Patent Application No. 19942124.2, filed Sep. 3, proteins of RXA00655 are set forth herein as SEQID NOs: 1999. The entire contents of all of the aforementioned appli 19, 21 and 23. The corresponding nucleotide sequences of cations are hereby expressly incorporated herein by this ref these RXA00655 homologous proteins are set forth hereinas CCC. SEQID NOs: 18, 20 and 22. 35 Modulation of the expression of the MR nucleic acids of BACKGROUND OF THE INVENTION the invention, e.g. increasing expression of the RXNO2910 nucleic acid molecule or increasing the activity of the Methionine is currently produced as a racemic mixture of RXNO2910 protein, suppressing expression of the DL-methionine by a well established chemical process. Most RXA00655 nucleic acid molecule or suppressing the activity DL-methionine is being produced by variations of the same 40 of the RXA00655 protein, or modification of the sequence of chemical procedure method involving toxic, dangerous, the MR nucleic acid molecules of the invention to alter the flammable, unstable, and highly odorous starting materials or activity of the MR nucleic acid molecule, can be used to intermediates. The starting materials for the chemical synthe modulate, e.g., increase, the production of a fine chemical, sis are: acrolein, methylmercaptain and hydrogen cyanide. e.g., methionine, cysteine, and/or other compounds of the The chemical synthesis involves the reaction of methylmer 45 methionine biosynthetic pathway, the Sulfur reduction path captain and acrolein producing the intermediate 3-methylm way, and/or the cysteine biosynthetic pathway from a micro ercaptopropionaldehyde (MMP). In the further process the organism (e.g., to improve the yield or production of a fine MMP reacts with hydrogen cyanide to form the 5-(2-meth chemical, e.g., methionine or other compounds of the ylthioethyl) hydantoin, which then can be hydrolyzed using 2 methionine biosynthetic pathway, from a Corynebacterium equivalents of caustics such as NaOH together with one half 50 or Brevibacterium ). In another embodiment, increas equivalent NaCO to yield sodium-DL-methioninate and ing the expression or activity of the RXNO2910 nucleic acid one equivalent NaCOs one equivalent NH and one half or protein and Suppression of the expression or activity of the equivalent CO. In the Succeeding step the Sodium-DL-me RXA00655 nucleic acid or protein, in combination, can be thioninate is neutralized with 1.5 equiv. Sulfuric acid and 1 used to modulate, e.g., increase, the production of a fine equiv.NaCO to yield DL-methionine NaSO and CO. It is 55 chemical, e.g., methionine, cysteine, and/or other compounds obvious that Such a chemical process yields a large molar of the methionine biosynthetic pathway, the sulfur reduction excess of unused salts in comparison to the amount of pathway, and/or the cysteine biosynthetic pathway from a methionine that is produced. This fact poses an economic and microorganism (e.g., Corynebacterium or Brevibacterium ecologic challenge. species). Fermentative processes are usually based on cultivating 60 Accordingly, the present invention features methods of microorganisms on nutrients including carbohydrate Source producing methionine as well as other compounds of the (e.g., Sugars such as glucose fructose or saccharose), nitrogen methionine biosynthetic pathway. Such methods include cul Sources (e.g., ammonia) and Sulfur sources (e.g., Sulfate or turing microorganisms overexpressing the RXNO2910 gene thiosulfate) together with other necessary media components. product under conditions such that methionine, or other com The process yields only the natural product L-methionine and 65 pounds of the methionine biosynthetic pathway, are pro only biomass as a byproduct. Since no toxic, dangerous, duced. Such methods also include culturing microorganisms flammable unstable, and highly odorous starting materials are with inhibited expression of the RXA00655 gene product US 7,892,798 B2 3 4 Such that methionine, or other compounds of the methionine niques well known in the art, one or more of the regulatory biosynthetic pathway, the Sulfur reduction pathway, and/or proteins of the invention may be manipulated Such that its the cysteine biosynthetic pathway are produced. The present function or expression is modulated. For example, the muta invention also provides methods for producing a fine chemi tion of an MR protein, e.g., RXA00655, which is involved in cal comprising culturing microorganisms overexpressing the the repression of transcription of a gene, e.g., the metY gene, RXNO2910 gene product in combination with microorgan encoding a polypeptide which is required for the biosynthesis isms with inhibited expression of the RXA00655 gene prod of an amino acid, e.g., methionine, such that it no longer is uct. able to repress transcription may result in an increase in The present invention also features methods of increasing production of that amino acid. Similarly, the alteration of production of a Sulfur-containing compound by a microor 10 activity of an MR protein resulting in increased translation or ganism comprising culturing a microorganism which overex activating posttranslational modification of a C. glutamicum presses a positive regulator of methionine biosynthesis, e.g., protein involved in the biosynthesis of a desired fine chemi RXNO2910, under conditions such that production of the cal, e.g., methionine, may in turn increase the production of Sulfur-containing compound is increased. Furthermore, the that chemical. The opposite situation may also be of benefit: present invention features methods of increasing production 15 by increasing the repression of transcription or translation, or of a Sulfur-containing compound by a microorganism com by posttranslational negative modification of a C. glutamicum prising culturing a microorganism which underexpresses a protein involved in the regulation of a degradative pathway negative regulator of methionine biosynthesis, e.g., for a compound, one may increase the production of this RXA00655, under conditions such that production of the chemical. In each case, the overall yield or rate of production Sulfur-containing compound is increased. of the desired fine chemical may be increased. The MR proteins encoded by the novel nucleic acid mol It is also possible that Such alterations in the protein and ecules of the invention are capable of, for example, perform nucleotide molecules of the invention may improve the yield, ing a function involved in the transcriptional, translational, or production, and/or efficiency of production offine chemicals, posttranslational regulation of proteins important for the nor e.g., methionine, through indirect mechanisms. The metabo mal metabolic functioning of cells. Given the availability of 25 lism of any one compound is necessarily intertwined with cloning vectors for use in Corynebacterium glutamicum, Such other biosynthetic and degradative pathways within the cell, as those disclosed in Sinskey et al., U.S. Pat. No. 4,649,119, and necessary cofactors, intermediates, or Substrates in one and techniques for genetic manipulation of C. glutamicum pathway are likely supplied or limited by another such path and the related Brevibacterium species (e.g., lactofermentum) way. Therefore, by modulating the activity of one or more of (Yoshihama et al., J. Bacteriol. 162: 591-597 (1985); Kat 30 the regulatory proteins of the invention, the production or Sumata et al., J. Bacteriol. 159: 306–311 (1984); and San efficiency of activity of another fine chemical biosynthetic or tamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the degradative pathway may be impacted. Further, the manipu nucleic acid molecules of the invention may be utilized in the lation of one or more regulatory proteins may increase the genetic engineering of this organism to make it a better or overall ability of the cell to grow and multiply in culture, more efficient producer of a fine chemical, e.g., methionine. 35 particularly in large-scale fermentative culture, where growth This improved yield, production and/or efficiency of pro conditions may be suboptimal. For example, by mutating an duction of a fine chemical, e.g., methionine, may be due to a MR protein of the invention which would normally cause a direct effect of manipulation of a gene of the invention, e.g., repression in the biosynthesis of nucleotides in response to RXA00655 or RXN02910, or it may be due to an indirect suboptimal extracellular supplies of nutrients (thereby pre effect of such manipulation. Specifically, alterations in C. 40 venting cell division) Such that it is decreased in repressor glutamicum MR proteins which normally regulate the yield, ability, one may increase the biosynthesis of nucleotides and production and/or efficiency of production of the methionine perhaps increase cell division. Changes in MR proteins which metabolic pathways may have a direct impact on the overall result in increased cell growth and division in culture may production or rate of production of a fine chemical, e.g., result in an increase in yield, production, and/or efficiency of methionine, cysteine, and/or other compounds of the 45 production of one or more desired fine chemicals from the methionine biosynthetic pathway, the Sulfur reduction path culture, due at least to the increased number of cells produc way, and/or the cysteine biosynthetic pathway from this ing the chemical in the culture. organism. Alterations in the proteins involved in the methion The invention provides novel nucleic acid molecules which ine metabolic pathway may also have an indirect impact on encode proteins, referred to herein as metabolic pathway the yield, production and/or efficiency of production of a 50 proteins (MR), which are capable of for example, performing desired fine chemical, e.g., methionine. Regulation of an enzymatic step involved in the transcriptional, transla metabolism is necessarily complex, and the regulatory tional, or posttranslational regulation of metabolic pathways mechanisms governing different pathways may intersect at in C. glutamicum. Nucleic acid molecules encoding an MR multiple points such that more than one pathway can be protein are referred to herein as MR nucleic acid molecules. rapidly adjusted in accordance with a particular cellular 55 In a preferred embodiment, the MR protein participates in the event. This enables the modification of a regulatory protein transcriptional, translational, or posttranslational regulation for one pathway to have an impact on the regulation of many of one or more metabolic pathways. other pathways as well, some of which may be involved in the Accordingly, one aspect of the invention pertains to iso biosynthesis or degradation of a desired fine chemical, e.g., lated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) methionine. In this indirect fashion, the modulation of action 60 comprising a nucleotide sequence encoding an MR protein or of an MR protein has an impact on the production of a fine biologically active portions thereof, as well as nucleic acid chemical produced by a pathway different from one which fragments suitable as primers or hybridization probes for the that MR protein directly regulates. detection or amplification of MR-encoding nucleic acid (e.g., The nucleic acid and protein molecules of the invention DNA or mRNA). In particularly preferred embodiments, the may be utilized to directly improve the yield, production, 65 isolated nucleic acid molecule comprises one of the nucle and/or efficiency of production of methionine from Coryne otide sequences set forth in SEQID NO: 1, SEQ ID NO: 5, bacterium glutamicum. Using recombinant genetic tech SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID US 7,892,798 B2 5 6 NO: 22 or the coding region or a complement thereof of one erably, the isolated nucleic acid molecule corresponds to a of these nucleotide sequences. In other particularly preferred naturally-occurring nucleic acid molecule. More preferably, embodiments, the isolated nucleic acid molecule of the inven the isolated nucleic acid encodes a naturally-occurring C. tion comprises a nucleotide sequence which hybridizes to or glutamicum MR protein, or a biologically active portion is at least about 50%, preferably at least about 60%, more thereof. preferably at least about 70%, 80% or 90%, and even more Another aspect of the invention pertains to vectors, e.g., preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, recombinant expression vectors, containing the nucleic acid 96%, 97%, 98%, 99% or more homologous to a nucleotide molecules of the invention, and host cells into which such sequence set forth in SEQID NO: 1, SEQID NO: 5, SEQID vectors have been introduced. In one embodiment, such a host NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO:22, 10 cell is used to produce an MR protein by culturing the host or a portion thereof. In other preferred embodiments, the cell in a suitable medium. The MR protein can be then iso isolated nucleic acid molecule encodes one of the amino acid lated from the medium or the host cell. sequences set forthin SEQID NO: 2, SEQIDNO: 6, SEQID Yet another aspect of the invention pertains to a genetically NO: 17, SEQID NO: 19, SEQID NO:21, or SEQID NO. 23. altered microorganism in which an MR gene has been intro The preferred MR proteins of the present invention also pref 15 duced or altered. In one embodiment, the genome of the erably possess at least one of the MR activities described microorganism has been altered by introduction of a nucleic herein. acid molecule of the invention encoding wild-type or mutated In another embodiment, the isolated nucleic acid molecule MR sequence as a transgene. In another embodiment, an encodes a protein or portion thereof wherein the protein or endogenous MR gene within the genome of the microorgan portion thereof includes an amino acid sequence which is ism has been altered, e.g., functionally disrupted, by homolo Sufficiently homologous to an amino acid sequence of SEQ gous recombination with an altered MR gene. In another ID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, embodiment, an endogenous or introduced MR gene in a SEQID NO: 21, or SEQIDNO:23, e.g., sufficiently homolo microorganism has been altered by one or more point muta gous to an amino acid sequence of SEQID NO: 2, SEQ ID tions, deletions, or inversions, but still encodes a functional NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, or 25 MR protein. In still another embodiment, one or more of the SEQ ID NO: 23 such that the protein or portion thereof regulatory regions (e.g., a promoter, repressor, or inducer) of maintains an MR activity, e.g., modulation of biosynthesis of an MR gene in a microorganism has been altered (e.g., by a fine chemical, e.g., methionine. Preferably, the protein or deletion, truncation, inversion, or point mutation) Such that portion thereof encoded by the nucleic acid molecule main the expression of the MR gene is modulated. In a preferred tains the ability to transcriptionally, translationally, or post 30 embodiment, the microorganism belongs to the genus translationally regulate a metabolic pathway in C. Corynebacterium or Brevibacterium, with Corynebacterium glutamicum. In one embodiment, the protein encoded by the glutamicum being particularly preferred. In a preferred nucleic acid molecule is at least about 50%, preferably at least embodiment, the microorganism is also utilized for the pro about 60%, and more preferably at least about 70%, 80%, or duction of a desired compound, such as an amino acid, with 90% and most preferably at least about 90%,91%.92%.93%, 35 methionine, being particularly preferred. 94%. 95%, 96%, 97%, 98%, or 99% or more homologous to The invention also provides an isolated preparation of an an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, MR protein. In preferred embodiments, the MR protein com SEQID NO: 17, SEQIDNO: 19, SEQIDNO:21, or SEQID prises an amino acid sequence of SEQID NO: 2, SEQID NO: NO: 23 (e.g., an entire amino acid sequence selected from 6, SEQID NO: 17, SEQID NO: 19, SEQID NO: 21, or SEQ those sequences set forth in SEQID NO: 2, SEQID NO: 6, 40 ID NO. 23. In another preferred embodiment, the invention SEQID NO: 17, SEQIDNO: 19, SEQIDNO:21, or SEQID pertains to an isolated full length protein which is Substan NO. 23). In another preferred embodiment, the protein is a tially homologous to an entire amino acid sequence of SEQ full length C. glutamicum protein which is Substantially ID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, homologous to an entire amino acid sequence of SEQID NO: SEQ ID NO: 21, or SEQ ID NO: 23 (encoded by an open 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID 45 reading frame set forthin SEQID NO: 1, SEQID NO:5, SEQ NO: 21, or SEQ ID NO: 23 (encoded by an open reading ID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO: frame shown in SEQID NO: 1, SEQID NO: 5, SEQID NO: 22). In yet another embodiment, the protein is at least about 16, SEQID NO: 18, SEQID NO: 20, or SEQID NO: 22). 50%, preferably at least about 60%, and more preferably at In another preferred embodiment, the isolated nucleic acid least about 70%, 80%, or 90%, and most preferably at least molecule is derived from C. glutamicum and encodes a pro 50 about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or tein (e.g., an MR fusion protein) which includes a biologi 99% or more homologous to an entire amino acid sequence of cally active domain which is at least about 50% or more SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: homologous to one of the amino acid sequences of SEQID 19, SEQ ID NO: 21, or SEQ ID NO. 23. In other embodi NO:2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQ ments, the isolated MR protein comprises an amino acid IDNO: 21, or SEQID NO: 23 and is able to transcriptionally, 55 sequence which is at least about 50% or more homologous to translationally, or posttranslationally regulate a metabolic one of the amino acid sequences of SEQID NO: 2, SEQID pathway in C. glutamicum, or has one or more of the activities NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, or set forth herein, and which also includes heterologous nucleic SEQID NO: 23 and is able to transcriptionally, translation acid sequences encoding a heterologous polypeptide or regu ally, or posttranslationally regulate one or more metabolic latory regions. 60 pathways in C. glutamicum, or has one or more of the activi In another embodiment, the isolated nucleic acid molecule ties set forth herein. is at least 15, 20, 25, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, Alternatively, the isolated MR protein can comprise an 130, 140, 150, 160, 170, 180, 190, 200,210, 220, 225 or more amino acid sequence which is encoded by a nucleotide nucleotides in length and hybridizes under stringent condi sequence which hybridizes, e.g., hybridizes under stringent tions to a nucleic acid molecule comprising a nucleotide 65 conditions, or is at least about 50%, preferably at least about sequence of SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, 60%, more preferably at least about 70%, 80%, or 90%, and SEQID NO: 18, SEQID NO: 20, or SEQID NO: 22. Pref even more preferably at least about 90%, 91%, 92%, 93%, US 7,892,798 B2 7 8 94%. 95%, 96%, 97%,98,%, or 99% or more homologous, to particularly preferred embodiment, said fine chemical is an a nucleotide sequence of SEQID NO:2, SEQID NO: 6, SEQ amino acid. In especially preferred embodiments, said amino ID NO: 17, SEQID NO: 19, SEQID NO:21, or SEQID NO: acid is methionine. 23. It is also preferred that the preferred forms of MR proteins also have one or more of the MR activities described herein. BRIEF DESCRIPTION OF THE DRAWING The MR polypeptide, or a biologically active portion thereof, can be operatively linked to a non-MR polypeptide to FIG. 1 depicts the principal of a self-cloning technique based on homologous recombination which is used for prepa form a fusion protein. In preferred embodiments, this fusion ration of a Corynebacterium glutamicum Strain deficient in protein has an activity which differs from that of the MR 10 protein alone. In other preferred embodiments, this fusion the negative regulator of methionine biosynthesis protein transcriptionally, translationally, or posttranslation (RXA00655, set forth as SEQID NO: 1). ally regulates one or more metabolic pathways in C. glutamicum. In particularly preferred embodiments, integra DETAILED DESCRIPTION OF THE INVENTION tion of this fusion protein into a host cell modulates produc 15 tion of methionine from a cell. The present invention provides metabolic regulatory (MR) nucleic acid and protein molecules, e.g., RXA00655 and In another aspect, the invention provides methods for RXNO2910 nucleic acid and protein molecules, which are screening molecules which modulate the activity of an MR involved in the regulation of metabolism in microorganisms, protein, either by interacting with the protein itself or a sub e.g., Corynebacterium glutamicum, including regulation of strate or binding partner of the MR protein, or by modulating fine chemical metabolism, e.g., methionine biosynthesis by, the transcription or translation of an MR nucleic acid mol e.g., transcriptional regulation of genes, e.g., the metY gene, ecule of the invention. Another aspect of the invention per involved in the methionine biosynthetic pathway, the sulfur tains to a method for producing a fine chemical. This method reduction pathway, and/or the cysteine biosynthetic pathway. involves the culturing of a cell containing a vector directing 25 Accordingly, the present invention features methods based the expression of an MR nucleic acid molecule of the inven on manipulation of the methionine biosynthetic pathway, the tion, Such that a fine chemical is produced. In a preferred Sulfur reduction pathway, and/or the cysteine biosynthetic embodiment, this method further includes the step of obtain pathway in a microorganism such that a fine chemical, e.g., a ing a cell containing such a vector, in which a cell is trans Sulfur-containing compound, e.g., methionine, cysteine, and/ fected with a vector directing the expression of an MR nucleic 30 or other compounds of the methionine biosynthetic pathway, acid. In another preferred embodiment, this method further the sulfur reduction pathway, and/or the cysteine biosynthetic includes the step of recovering the fine chemical, e.g., pathway are produced. methionine, from the culture. In a particularly preferred The term “methionine biosynthetic pathway' includes a embodiment, the cell is from the genus Corynebacterium or pathway involving methionine biosynthetic enzymes (e.g., Brevibacterium, or is selected from those strains set forth in 35 polypeptides encoded by biosynthetic enzyme-encoding Table 1, below. genes), compounds (e.g., precursors, Substrates, intermedi Another aspect of the invention pertains to methods for ates or products), cofactors and the like, utilized in the for modulating production of a molecule from a microorganism. mation or synthesis of methionine. Methionine is an amino Such methods include contacting the cell with an agent which acid nutritionally required by . Bacteria synthesize 40 their own methionine from amino acids and biosynthetic modulates MR protein activity or MR nucleic acid expression intermediates thereof (Escherichia Coli and Salmonella. Cel such that a cell associated activity is altered relative to this lular and Molecular Biology, Neidhardt, Frederick C. Cur same activity in the absence of the agent. In a preferred tiss, Roy III Ingraham, John L. Eds, 2nd ed. 1996, ASM Press embodiment, the cell is modulated for one or more C. and Hwang B.J. Yeom H.J. Kim Y. Lee HS. (2002) Journal of glutamicum metabolic pathway regulatory systems, such that 45 Bacteriology 184(5):1277-86. The term "cysteine biosyn the yields or rate of production of a desired fine chemical, e.g., thetic pathway' includes a pathway involving cysteine bio methionine, by this microorganism is improved. The agent synthetic enzymes (e.g., polypeptides encoded by biosyn which modulates MR protein activity can be an agent which thetic enzyme-encoding genes), compounds (e.g., precursors, stimulates MR protein activity or MR nucleic acid expres Substrates, intermediates or products), cofactors and the like, sion. Examples of agents which stimulate MR protein activity 50 utilized in the formation or synthesis of cysteine. The term or MR nucleic acid expression include Small molecules, “sulfur reduction pathway' includes a pathway involving active MR proteins, and nucleic acids encoding MR proteins enzymes which function to metabolize inorganic compounds that have been introduced into the cell. Examples of agents such as sulfur and derivatives thereof. It has been found that which inhibit MR activity or expression include small mol RXA00655 is a negative regulator, e.g., a transcriptional ecules and antisense MR nucleic acid molecules. 55 regulator, offine chemical biosynthesis, e.g., methionine bio synthesis. Accordingly, Suppression or inhibition of the Another aspect of the invention pertains to methods for expression of the RXA00655 gene or knock-out of the modulating yields of a desired compound from a cell, involv RXA00655 gene leads to increased production of a fine ing the introduction of a wild-type or mutant MR gene into a chemical, e.g., methionine, cysteine, and/or other compounds cell, either maintained on a separate plasmid or integrated 60 of the methionine biosynthetic pathway, the sulfur reduction into the genome of the host cell. If integrated into the genome, pathway, and/or the cysteine biosynthetic pathway, in micro Such integration can be random, or it can take place by organisms, e.g., Corynebacterium glutamicum. Introduction homologous recombination Such that the native gene is of a mutation which reduces or inhibits expression of the replaced by the introduced copy, causing the production of RXA00655 gene or activity of the RXAO0655 polypeptide the desired compound from the cell to be modulated. In a 65 also leads to increased production of a fine chemical, e.g., preferred embodiment, said yields are increased. In another methionine, cysteine, and/or other compounds of the preferred embodiment, said chemical is a fine chemical. In a methionine biosynthetic pathway, the Sulfur reduction path US 7,892,798 B2 10 way, and/or the cysteine biosynthetic pathway, in microor gene and/or translation of a particular gene product, or any ganisms, e.g., Corynebacterium glutamicum. other conventional means of deregulating expression of a It has also been found that RXNO2910 is a positive regu particular gene routine in the art (including but not limited to lator, e.g., a transcriptional regulator, of fine chemical bio use of antisense nucleic acid molecules, for example, to block synthesis, e.g., methionine biosynthesis. Accordingly, over expression of repressor proteins). expression of the RXNO2910 gene leads to increased In another aspect, the present invention features a method production of methionine and other compounds of the of producing a line chemical, e.g., methionine, cysteine, and/ methionine biosynthetic pathway in microorganisms, e.g., or other compounds of the methionine biosynthetic pathway, Corynebacterium glutamicum. Introduction of a mutation the sulfur reduction pathway, and/or the cysteine biosynthetic which increases expression of the RXN02910 gene or activity 10 pathway, which includes culturing a microorganism which of the RXN02910 polypeptide also leads to increased produc has Suppressed or inhibited expression of a negative regulator tion of methionine and other compounds of the methionine of fine chemical biosynthesis, e.g., RXA00655, under condi biosynthetic pathway in microorganisms, e.g., Corynebacte tions such that a fine chemical, e.g., methionine, cysteine, rium glutamicum. and/or other compounds of the methionine biosynthetic path Furthermore, suppression or inhibition of the expression of 15 way, the Sulfur reduction pathway, and/or the cysteine bio RXA00655 or knock-out of the RXA00655 gene in combi synthetic pathway, are produced. A microorganism which has nation with overexpression of the RXNO2910 gene in a suppressed RXA00655 expression includes a microorganism microorganism also leads to increased production of a fine which has been manipulated such that RXA00655 expression chemical, e.g., methionine, cysteine, and/or other compounds is Suppressed or inhibited. of the methionine biosynthetic pathway, the sulfur reduction The term "suppression of expression.” “inhibition of pathway, and/or the cysteine biosynthetic pathway, by the expression, or “underexpression' includes expression of a microorganism. Furthermore, culture of microorganisms gene product (e.g., the RXA00655 gene product or the with suppressed or inhibited expression of RXA00655 or RXNO2910 gene product) at a level lower than that expressed microorganisms with a knock-out of the RXA00655 gene, prior to manipulation of the microorganism or in a compa together with microorganisms which overexpress 25 rable microorganism which has not been manipulated. In one RXNO2910 gene also leads to increased production of a fine embodiment, Suppression or inhibition of chemical, e.g., methionine, cysteine, and/or other compounds includes manipulation of a microorganism such that the gene of the methionine biosynthetic pathway, the sulfur reduction is no longer expressed or is knocked-out. For example, under pathway, and/or the cysteine biosynthetic pathway, by the expression of a particular gene, e.g., RXA00655, includes microorganism. 30 expression which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, Accordingly, one aspect the present invention features 80%, 90%, 100%, less than expression of the gene by an methods of producing fine chemicals, e.g., methionine or organism which has not been manipulated to underexpress other compounds of the methionine biosynthetic pathway, the particular gene. Ranges and identity values intermediate which include culturing a microorganism which overex to the above percentages are encompassed by the present presses a positive regulator of fine chemical biosynthesis, 35 invention. In one embodiment, the microorganism can be e.g., RXN02910, under conditions such that a fine chemical, genetically manipulated (e.g., genetically engineered) to e.g., methionine or other compounds of the methionine bio express a level of gene product lesser than that expressed prior synthetic pathway are produced. A microorganism which to manipulation of the microorganism or in a comparable overexpresses RXNO2910 includes a microorganism which microorganism which has not been manipulated. Genetic has been manipulated such that RXNO2910 is overexpressed. 40 manipulation can include, but is not limited to, altering or The term “overexpressed’ or “overexpression includes modifying regulatory sequences or sites associated with expression of a gene product (e.g., the RXNO2910 gene prod expression of a particular gene, modifying the chromosomal uct) at a level greater than that expressed prior to manipula location of a particular gene, altering nucleic acid sequences tion of the microorganism or in a comparable microorganism adjacent to a particular gene Such as a binding site, which has not been manipulated. For example, overexpres 45 decreasing the copy number of a particular gene, modifying sion of a particular gene, e.g., RXNO2910, includes expres proteins (e.g., regulatory proteins, suppressors, enhancers, sion which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, transcriptional activators and the like) involved in transcrip 90%, 100%, or greater than expression of the gene by an tion of a particular gene and/or translation of a particular gene organism which has not been manipulated to overexpress the product, use of antisense nucleic acid molecules, knock-out particular gene. Ranges and identity values intermediate to 50 of the target gene, or any other conventional means of deregu the above percentages are encompassed by the present inven lating expression of a particular gene routine in the art. A tion. In one embodiment, the microorganism can be geneti microorganism which is deficient in RXA00655 gene expres cally manipulated (e.g., genetically engineered) to overex sion or RXNO2910 gene expression includes a microorgan press a level of gene product greater than that expressed prior ism which has suppressed or inhibited RXA00655 or to manipulation of the microorganism or in a comparable 55 RXN02910 expression. microorganism which has not been manipulated. Genetic In another aspect, the present invention features a method manipulation can include, but is not limited to, altering or of producing a fine chemical, e.g., methionine, which modifying regulatory sequences or sites associated with includes culturing a microorganism which has increased expression of a particular gene (e.g., by adding strong pro RXNO2910 activity under conditions such that a fine chemi moters, inducible promoters or multiple promoters or by 60 cal, e.g., methionine, is produced. A "microorganism which removing regulatory sequences such that expression is con has increased RXNO2910 activity” includes a microorganism stitutive), modifying the chromosomal location of aparticular which has been manipulated such that RXN02910 activity is gene, altering nucleic acid sequences adjacent to a particular increased. gene Such as a ribosome binding site, increasing the copy In yet another aspect, the present invention features a number of a particular gene, modifying proteins (e.g., regu 65 method of producing a fine chemical, e.g., methionine, cys latory proteins, Suppressors, enhancers, transcriptional acti teine, and/or other compounds of the methionine biosynthetic vators and the like) involved in transcription of a particular pathway, the Sulfur reduction pathway, and/or the cysteine US 7,892,798 B2 11 12 biosynthetic pathway, which includes culturing a microor nucleotide sequences of these RXA00655 homologous pro ganism which has decreased RXA00655 activity under con teins are set forth herein as SEQID NOS:18, 20 and 22. ditions such that a fine chemical, e.g., methionine, cysteine, SEQ ID NOs: 16 and 17 represent mutated RXN02910 and/or other compounds of the methionine biosynthetic path nucleic acid and amino acid sequences, respectively. The way, the Sulfur reduction pathway, and/or the cysteine bio RXN02910 molecule depicted in SEQID NO: 16 contains a synthetic pathway, is produced. A "microorganism which has single nucleotide change from a guanine (G) to an adenine decreased RXA00655 activity” includes a microorganism (A) at nucleotide residue 556 in the coding region, which which has been manipulated such that RXA00655 activity is results in a change from an aspartic acid (D) to an asparagine inhibited or suppressed. (N) atamino acid residue 186 of the encoded protein, set forth The term “RXN02910 activity” includes any activity 10 as SEQID NO: 17. This polymorphism may cause modula which results in fine chemical biosynthesis, e.g., methionine tion of regulation of fine chemical biosynthesis, e.g., biosynthesis. RXNO2910 activity includes, but is not limited methionine biosynthesis by RXNO2910, e.g., decreased to, positive regulation of the methionine biosynthetic path methionine biosynthesis. way resulting in methionine biosynthesis or biosynthesis of The molecules of the invention may be utilized in the other compounds of the methionine biosynthetic pathway. 15 modulation of production offine chemicals, e.g., methionine, Positive regulation of the methionine biosynthetic pathway from microorganisms, such as C. glutamicum, either directly may be by any means, including, but not limited to, transcrip (e.g., where modulation of the activity of a methionine bio tional and translational regulation as well as protein regula synthesis regulatory protein has a direct impact on the yield, tion, Via, e.g., protein binding. Increased activity includes production, and/or efficiency of production of methionine activity at a level higher than that demonstrated prior to from that organism), or may have an indirect impact which manipulation of the microorganism or in a comparable micro nonetheless results in an increase in yield, production, and/or organism which has not been manipulated. efficiency of production of the desired compound (e.g., where The term “RXA00655 activity” includes any activity modulation of the regulation of a nucleotide biosynthesis which results in fine chemical biosynthesis, e.g., methionine protein has an impact on the production of an organic acid or biosynthesis. An example of RXA00655 activity includes, 25 a fatty acid from the bacterium, perhaps due to concomitant but is not limited to, negative regulation of the methionine regulatory alterations in the biosynthetic or degradation path biosynthetic pathway, the Sulfur reduction pathway, and/or ways for these chemicals in response to the altered regulation the cysteine biosynthetic pathway as described in Escherichia of nucleotide biosynthesis). Aspects of the invention are fur Coli and Salmonella. Cellular and Molecular Biology, ther explicated below. Neidhardt, Frederick C. Curtiss, Roy III Ingraham, John L. 30 I. Fine Chemicals Eds, 2nd ed. 1996, ASM Press., resulting in decreased The term fine chemical is art-recognized and includes methionine biosynthesis, decreased biosynthesis of other molecules produced by an organism which have applications compounds of the methionine biosynthetic pathway, the Sul in various industries, such as, but not limited to, the pharma fur reduction pathway, or the cysteine biosynthetic pathway. ceutical, agriculture, and cosmetics industries. Such com Negative regulation of the methionine biosynthetic pathway, 35 pounds include organic acids, such as tartaric acid, itaconic the sulfur reduction pathway, and/or the cysteine biosynthetic acid, and diaminopimelic acid, Sulfur-containing com pathway may be by any means, including, but not limited to pounds, both proteinogenic and non-proteinogenic amino transcriptional and translational regulation as well as protein acids, purine and pyrimidine bases, nucleosides, and nucle regulation, via, e.g., protein binding. Decreased or Sup otides (as described e.g. in Kuninaka, A. (1996) Nucleotides pressed activity includes activity at a lower higher than that 40 and related compounds, p. 561-612, in Biotechnology Vol. 6, demonstrated prior to manipulation of the microorganism or Rehm et al., eds. VCH: Weinheim, and references contained in a comparable microorganism which has not been manipu therein), lipids, both Saturated and unsaturated fatty acids lated. (e.g., arachidonic acid), diols (e.g., propane diol, and butane The present invention features methods of increasing pro diol), carbohydrates (e.g., hyaluronic acid and trehalose), duction of a Sulfur-containing compound by a microorganism 45 comprising culturing a microorganism which overexpresses a aromatic compounds (e.g., aromatic amines, Vanillin, and positive regulator of methionine biosynthesis, e.g., indigo), vitamins and cofactors (as described in Ullmann's RXNO2910, under conditions such that production of the Encyclopedia of Industrial Chemistry, vol. A27. “Vitamins’. Sulfur-containing compound is increased. The present inven p. 443-613 (1996) VCH: Weinheim and references therein; tion also features methods of increasing production of a Sul 50 and Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, fur-containing compound by a microorganism comprising Lipids, Health, and Disease” Proceedings of the UNESCO/ Confederation of Scientific and Technological Associations culturing a microorganism which underexpresses a negative in Malaysia, and the Society for Free Radical Research— regulator of methionine biosynthesis, e.g., RXA00655, under Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press, conditions such that production of the Sulfur-containing com (1995)), enzymes, polyketides (Cane et al. (1998) Science pound is increased. 55 282: 63-68), and all other chemicals described in Gutcho The term "sulfur-containing compound includes any (1983) Chemicals by Fermentation, Noyes Data Corporation, compound which contains sulfur or a derivative thereof. Sul ISBN: 0818805086 and references therein. The metabolism fur-containing compounds include amino acids, including, and uses of certain of these fine chemicals are further expli but not limited to, methionine, cysteine, S-adenosylmethion cated below. ine, and homocycsteine. 60 The nucleotide sequence of RXA00655 is set forth herein A. Amino Acid Metabolism and Uses as SEQID NO:1 and the polypeptide sequence of RXA00655 Amino acids comprise the basic structural units of all pro is set forth herein as SEQID NO:2. The nucleotide sequence teins, and as Such are essential for normal cellular functioning of RXN02910 is set forth herein as SEQ ID NO:5 and the in all organisms. The term "amino acid is art-recognized. polypeptide sequence of RXN02910 is set forth herein as 65 The proteinogenic amino acids, of which there are 20 species, SEQID NO:6. Proteins whicharehomologous to RXA00655 serve as structural units for proteins, in which they are linked are set forth herein as SEQ ID NOS:19, 21 and 23. The by peptide bonds, while the nonproteinogenic amino acids US 7,892,798 B2 13 14 (hundreds of which are known) are not normally found in Biology, John Wiley & Sons). All such known regulatory proteins (see Ulmann's Encyclopedia of Industrial Chemis processes are mediated by additional genes, which them try, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids selves respond to external influences of various kinds (e.g., may be in the D- or L-optical configuration, though L-amino temperature, nutrient availability, or light). Exemplary pro acids are generally the only type found in naturally-occurring 5 tein factors which have been implicated in this type of regu proteins. Biosynthetic and degradative pathways of each of lation include the transcription factors. These are proteins the 20 proteinogenic amino acids have been well character which bind to DNA, thereby either increasing the expression ized in both prokaryotic and eukaryotic cells (see, for of a gene (positive regulation, as in the case of e.g. the ara example, Stryer, L. Biochemistry, 3' edition, pages 578-590 operon from E. coli) or decreasing gene expression (negative (1988)). The essential amino acids (methionine, histidine, 10 regulation, as in the case of the lac operon from E. coli). These isoleucine, leucine, , phenylalanine, , tryp expression-modulating transcription factors can themselves tophan, and valine), so named because they are generally a be the Subject of regulation. Their activity can, for example, nutritional requirement due to the complexity of their biosyn be regulated by the binding of low molecular weight com theses, are readily converted by simple biosynthetic pathways pounds to the DNA-binding protein, thereby stimulating (as to the remaining 11 nonessential amino acids (alanine, argi 15 in the case of arabinose for the ara operon) or inhibiting (as in nine, asparagine, aspartate, cysteine, glutamate, glutamine, the case of the lactose for the lac operon) the binding of these , proline, , and ). Higher animals do proteins to the appropriate binding site on the DNA (see, for retain the ability to synthesize some of these amino acids, but example, Helmann, J. D. and Chamberlin, M. J. (1988) the essential amino acids must be supplied from the diet in “Structure and function of bacterial sigma factors.” Ann. Rev. order for normal protein synthesis to occur. Biochem. 57: 839-872: Adhya, S. (1995) “The lac and gal Aside from their function in protein biosynthesis, many operons today' and Boos, W. et al., “The maltose system.”. amino acids have been found to have various applications in both in: Regulation of Gene Expression in Escherichia coli the food, feed, chemical, cosmetics, agriculture, and pharma (Lin, E. C. C. and Lynch, A. S., eds.) Chapman & Hall: New ceutical industries. Methionine is an importantamino acid in York, p. 181-200 and 201-229; and Moran, C. P. (1993)“RNA the nutrition not only of , but also of other animals. 25 polymerase and transcription factors.” in: Bacillus subtilis The biosynthesis of these natural amino acids in organisms and other gram-positive bacteria, Sonenshein, A. L. et al., eds. capable of producing them, Such as bacteria, has been well ASM: Washington, D.C., p. 653-667.) characterized (for review of bacterial amino acid biosynthesis Aside from the transcriptional level, protein synthesis is and regulation thereof, see Umbarger, H. E. (1978) Ann. Rev. also often regulated at the level of translation. There are Biochem. 47: 533-606). Methionine is produced by the con 30 multiple mechanisms by which Such regulation may occur, version of aspartate, which is also the common precursor of including alteration of the ability of the ribosome to bind to lysine, threonine, and isoleucine. Cysteine is produced from one or more mRNAs, binding of the ribosome to the mRNA, serine. Amino acids in excess of the protein synthesis needs of the maintenance or removal of mRNA secondary structure, the cell cannot be stored, and are instead degraded to provide the utilization of common or less common codons for a par intermediates for the major metabolic pathways of the cell 35 ticular gene, the degree of abundance of one or more tRNAS, (for review see Stryer, L. Biochemistry 3' ed. Ch. 21 “Amino and special regulation mechanisms, such as attenuation (see Acid Degradation and the Urea Cycle” p. 495-516 (1988)). Vellanoweth, R. I. (1993) Translation and its regulation in Although the cell is able to convertunwanted amino acids into Bacillus subtilis and other gram-positive bacteria, Sonen useful metabolic intermediates, amino acid production is shein, A. L. et al., eds. ASM: Washington, D.C., p. 699-711 costly in terms of energy, precursor molecules, and the 40 and references cited therein). enzymes necessary to synthesize them. Thus it is not surpris Transcriptional and translational regulation may be tar ing that amino acid biosynthesis is regulated by feedback geted to a single protein (sequential regulation) or simulta inhibition, in which the presence of a particular amino acid neously to several proteins in different metabolic pathways serves to slow or entirely stop its own production (for over (coordinate regulation). Often, genes whose expression is view of feedback mechanisms in amino acid biosynthetic 45 coordinately regulated are physically located near one pathways, see Stryer, L. Biochemistry, 3" ed. Ch. 24: “Bio another in the genome, in an operon or regulon. Such up- or synthesis of Amino Acids and Heme” p. 575-600 (1988)). down-regulation of gene transcription and translation is gov Thus, the output of any particular amino acid is limited by the erned by the cellular and extracellular levels of various fac amount of that amino acid present in the cell. tors. Such as Substrates (precursor and intermediate mol 50 ecules used in one or more metabolic pathways), catabolites II. Mechanisms of Metabolic Regulation (molecules produced by biochemical pathways concerned All living cells have complex catabolic and anabolic meta with the production of energy from the breakdown of com bolic capabilities with many interconnected pathways. In plex organic molecules Such as Sugars), and end products (the order to maintain a balance between the various parts of this molecules resulting at the end of a metabolic pathway). Typi extremely complex metabolic network, the cell employs a 55 cally, the expression of genes encoding enzymes necessary finely-tuned regulatory network. By regulating enzyme Syn for the activity of a particular pathway is induced by high thesis and enzyme activity, either independently or simulta levels of substrate molecules for that pathway. Similarly, such neously, the cell is able to control the activity of disparate gene expression tends to be repressed when there exist high metabolic pathways to reflect the changing needs of the cell. intracellular levels of the end product of the pathway (Snyder, The induction or repression of enzyme synthesis may 60 L. and Champness, W. (1997) The Molecular Biology of occurat either the level of transcription or translation, or both. Bacteria ASM: Washington). Gene expression may also be Gene expression in prokaryotes is regulated by several regulated by other external and internal factors, such as envi mechanisms at the level of transcription (for review see e.g., ronmental conditions (e.g., heat, oxidative stress, or starva Lewin, B (1990) Genes IV, Part 3: “Controlling prokaryotic tion). These global environmental changes cause alterations genes by transcription'. Oxford University Press: Oxford, p. 65 in the expression of specialized modulating genes, which 213-301, and references therein, and Michal, G. (1999) Bio directly or indirectly (via additional genes or proteins) trigger chemical Pathways: An Atlas of Biochemistry and Molecular the expression of genes by means of binding to DNA and US 7,892,798 B2 15 16 thereby inducing or repressing transcription (see, for way, and/or the cysteine biosynthetic pathway. Such methods example, Lin, E. C. C. and Lynch, A. S., eds. (1995) Regula include culturing microorganisms overexpressing the tion of Gene Expression in Escherichia coli. Chapman & RXN02910 gene productor with increased RXN02910 activ Hall: New York). ity under conditions such a fine chemical, e.g., methionine, Yet another mechanism by which cellular metabolism may cysteine, and/or other compounds of the methionine biosyn be regulated is at the level of the protein. Such regulation is thetic pathway, the Sulfur reduction pathway, and/or the cys accomplished either by the activities of other proteins, or by teine biosynthetic pathway is produced. Such methods also binding of low-molecular-weight components which either include culturing microorganisms with inhibited expression impede or enable the normal functioning of the protein. of the RXA00655 gene product or inhibited RXA00655 Examples of protein regulation by the binding of low-mo 10 activity Such that a fine chemical, e.g., methionine, cysteine, lecular-weight compounds include the binding of GTP or and/or other compounds of the methionine biosynthetic path NAD. The binding of a low-molecular-weight chemical is way, the Sulfur reduction pathway, and/or the cysteine bio typically reversible, as is the case with the GTP-binding pro synthetic pathway, is produced. The present invention also teins. These proteins exist in two stages (with bound GTP or provides methods for producing a fine chemical, e.g. GDP), one stage being the activated form of the protein, and 15 methionine, comprising culturing microorganisms overex one stage being inactive. pressing the RXN02910 gene product and simultaneously Regulation of protein activity by the action of other exhibiting an inhibited expression of the RXA00655 gene enzymes typically takes the form of covalent modification of product or inhibited RXA00655 activity. Furthermore, the the protein (i.e., of amino acid residues such present invention also provides methods for producing a fine as histidine or aspartate, or methylation). Such covalent chemical, e.g., methionine, comprising culturing microor modification is typically reversible, as mediated by an ganisms overexpressing the RXNO2910 gene product incom enzyme of the opposite activity. An example of this is the bination with microorganisms with inhibited expression of opposite activities of kinases and phosphorylases in protein the RXAO0655 gene productor inhibited RXAO0655 activity. phosphorylation; protein kinases phosphorylate specific resi In one embodiment, the MR molecules of the invention dues on a target protein (e.g., serine or threonine), while 25 transcriptionally, translationally, or posttranslationally regu protein phosphorylases remove phosphate groups from Such late a metabolic pathway in C. glutamicum. In a preferred proteins. Typically, enzymes which modulate the activity of embodiment, the activity of the MR molecules of the present other proteins are themselves modulated by external stimuli. invention to regulate one or more C. glutamicum metabolic These stimuli are mediated through proteins which function pathways has an impact on the production of a desired fine as sensors. A well known mechanism by which Such sensor 30 chemical, e.g., methionine, by this organism. In a particularly proteins may mediate these external signals is by dimeriza preferred embodiment, the MR molecules of the invention are tion, but others are also known (see, for example, Msadek, T. modulated in activity, such that the C. glutamicum metabolic et al. (1993) “Two-Component Regulatory Systems', in: pathways which the MR proteins of the invention regulate are Bacillus subtilis and Other Gram-Positive Bacteria, Sonen modulated in efficiency or output, which either directly or shein, A. L. et al., eds. ASM: Washington p. 729-745 and 35 indirectly modulates the yield, production, and/or efficiency references cited therein). of production of a desired fine chemical, e.g., methionine, by A thorough understanding of the regulatory networks gov C. glutamicum. erning cellular metabolism in microorganisms is critical for The language, “MR protein' or “MR polypeptide' the high-yield production of chemicals by fermentation. Con includes proteins which transcriptionally, translationally, or trol systems for the down-regulation of metabolic pathways 40 posttranslationally regulate a metabolic pathway in C. could be removed or lessened to improve the synthesis of glutamicum. The terms “MR gene' or “MR nucleic acid desired chemicals, and similarly, those for the up-regulation sequence' include nucleic acid sequences encoding an MR of metabolic pathways for a desired product could be consti protein, which consist of a coding region and also corre tutively activated or optimized in activity (As shown in sponding untranslated 5' and 3' sequence regions. The terms Hirose, Y. and Okada, H. (1979) “Microbial Production of 45 “production' or “productivity” are art-recognized and Amino Acids, in: Peppler, H. J. and Perlman, D. (eds.) include the concentration of the fermentation product (for Microbial Technology 2" ed. Vol. 1, ch. 7 Academic Press: example, the desired fine chemical) formed within a given time and a given fermentation Volume (e.g., kg product per New York.) hour per liter). The term “efficiency of production' includes III. Elements and Methods of the Invention 50 the time required for a particular level of production to be The present invention is based, at least in part, on the achieved (for example, how long it takes for the cell to attain discovery of novel molecules, referred to herein as MR a particular rate of output of a fine chemical). The term nucleic acid and protein molecules, e.g., RXA00655 nucleic "yield”, “product/carbon yield', or “production' is art-rec and protein molecules and RXN02910 nucleic acid and pro ognized and includes the efficiency of the conversion of the tein molecules, which regulate or modulate one or more meta 55 carbon Source into the product (i.e., methionine). This is bolic pathways in C. glutamicum, e.g., the methionine bio generally written as, for example, kg product per kg carbon synthesis pathway. RXA00655 is a negative regulator of the Source. By increasing the yield or production of the com methionine biosynthetic pathway, the Sulfur reduction path pound, the quantity of recovered molecules, or of useful way, and the cysteine biosynthetic pathway, while recovered molecules of that compound in a given amount of RXNO2910 is a positive regulator of methionine biosynthe 60 culture over a given amount of time is increased. sis. Accordingly, the present invention features methods of The terms “degradation' or a “degradation pathway' are producing a fine chemical, e.g., methionine, cysteine, and/or art-recognized and include the breakdown of a compound, other compounds of the methionine biosynthetic pathway, the preferably an organic compound, by a cell to degradation Sulfur reduction pathway, and/or the cysteine biosynthetic products (generally speaking, Smaller or less complex mol pathway and modulating production of a fine chemical, e.g., 65 ecules) in what may be a multistep and highly regulated methionine, cysteine, and/or other compounds of the process. The language “metabolism’ is art-recognized and methionine biosynthetic pathway, the Sulfur reduction path includes the totality of the biochemical reactions that take US 7,892,798 B2 17 18 place in an organism. The metabolism of a particular com tively. SEQID NOs: 16 and 17 represent mutated RXN02910 pound, then, (e.g., the metabolism of an amino acid such as nucleic acid and amino acid sequences, respectively. The glycine) comprises the overall biosynthetic, modification, RXN02910 molecule depicted in SEQID NO: 16 contains a and degradation pathways in the cell related to this com single nucleotide change from a guanine (G) to an adenine pound. The term, “regulation' is art-recognized and includes (A) at nucleotide residue 556 in the coding region, which the activity of a protein to governor modulate the activity of results in a change from an aspartic acid (D) to an asparagine another protein. The term, “transcriptional regulation' is art (N) atamino acid residue 186 of the encoded protein, set forth recognized and includes the activity of a protein to impede or as SEQID NO: 17. This polymorphism may cause modula activate the conversion of a DNA encoding a target protein to tion of regulation of methionine biosynthesis or other fine mRNA. The term, “translational regulation' is art-recognized 10 and includes the activity of a protein to impede or activate the chemicals by RXNO2910, e.g., decreased methionine biosyn conversion of an mRNA encoding a target protein to a protein thesis. molecule. The term, 'posttranslational regulation' is art-rec The present invention also pertains to proteins which have ognized and includes the activity of a protein to impede or an amino acid sequence which is substantially homologous to improve the activity of a target protein by covalently modi 15 an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6, fying the target protein (e.g., by methylation, glucosylation, SEQID NO: 17, SEQIDNO: 19, SEQID NO:21, or SEQID or phosphorylation, or by binding the target protein). NO. 23. As used herein, a protein which has an amino acid In another embodiment, the MR molecules of the invention sequence which is substantially homologous to a selected are capable of modulating the production of a desired mol amino acid sequence is least about 50% homologous to the ecule. Such as a fine chemical, e.g., methionine, in a micro selected amino acid sequence, e.g., the entire selected amino organism such as C. glutamicum. Using recombinant genetic acid sequence. A protein which has an amino acid sequence techniques, one or more of the regulatory proteins of the which is Substantially homologous to a selected amino acid invention may be manipulated Such that its function is modu sequence can also be least about 50-60%, preferably at least lated. For example, a biosynthetic enzyme may be improved about 60-70%, and more preferably at least about 70-80%, in efficiency, or its allosteric control region destroyed Such 25 80-90%, or 90-95%, and most preferably at least about 90%, that feedback inhibition of production of the compound is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more prevented. Similarly, a degradative enzyme may be deleted or homologous to the selected amino acid sequence. modified by substitution, deletion, or addition such that its The MR protein or a biologically active portion or frag degradative activity is lessened for the desired compound ment thereof of the invention can transcriptionally, transla without impairing the viability of the cell. In each case, the 30 tionally, or posttranslationally regulate a metabolic pathway overall yield or rate of production of one of these desired fine in C. glutamicum, e.g., the methionine metabolic pathway, or chemicals may be increased. have one or more of the activities set forth herein. It is also possible that Such alterations in the protein and Various aspects of the invention are described in further nucleotide molecules of the invention may improve the pro detail in the following subsections: duction of fine chemicals, e.g. methionine, in an indirect 35 fashion. The regulatory mechanisms of metabolic pathways A. Isolated Nucleic Acid Molecules in the cell are necessarily intertwined, and the activation of One aspect of the invention pertains to isolated nucleic acid one pathway may lead to the repression or activation of molecules that encode MR polypeptides or biologically another in a concomitant fashion. Therefore, by modulating active portions thereof, as well as nucleic acid fragments the activity of one or more of the proteins of the invention, the 40 sufficient for use as hybridization probes or primers for the production or efficiency of activity of another fine chemical identification or amplification of MR-encoding nucleic acid biosynthetic or degradative pathway may be impacted. For (e.g., MR DNA). As used herein, the term “nucleic acid example, by decreasing the ability of an MR protein to repress molecule' is intended to include DNA molecules (e.g. cDNA the transcription of a gene encoding a particular amino acid or genomic DNA) and RNA molecules (e.g., mRNA) and biosynthetic protein, one may concomitantly derepress other 45 analogs of the DNA or RNA generated using nucleotide ana amino acid biosynthetic pathways, since these pathways are logs. This term also encompasses untranslated sequence interrelated. Further, by modifying the MR proteins of the located at both the 3' and 5' ends of the coding region of the invention, one may uncouple the growth and division of cells gene: at least about 100 nucleotides of sequence upstream from their extracellular Surroundings to a certain degree; by from the 5' end of the coding region and at least about 20 impairing an MR protein which normally represses biosyn 50 nucleotides of sequence downstream from the 3' end of the thesis of a nucleotide when the extracellular conditions are coding region of the gene. The nucleic acid molecule can be suboptimal for growth and cell division such that it now lacks single-stranded or double-stranded, but preferably is double this function, one may permit growth to occur even when the stranded DNA. An "isolated nucleic acid molecule is one extracellular conditions are poor. This is of particular rel which is separated from other nucleic acid molecules which evance in large-scale fermentative growth, where conditions 55 are present in the natural source of the nucleic acid. Prefer within the culture are often suboptimal in terms of tempera ably, an "isolated nucleic acid is free of sequences which ture, nutrient Supply or aeration, but would still Support naturally flank the nucleic acid (i.e., sequences located at the growth and cell division if the cellular regulatory systems for 5' and 3' ends of the nucleic acid) in the genomic DNA of the these factors were eliminated. organism from which the nucleic acid is derived. For In one embodiment, the isolated nucleic acid sequences of 60 example, in various embodiments, the isolated MR nucleic the invention are contained within the genome of a Coryne acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 bacterium glutamicum Strain available through the American kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which Type Culture Collection, given designation ATCC 13032. naturally flank the nucleic acid molecule in genomic DNA of The nucleotide and amino acid sequences of RXA00655 are the cell from which the nucleic acid is derived (e.g., a C. depicted in SEQ ID NOs: 1, 18, 20, 22 and 2, 19, 21, 23, 65 glutamicum cell). Moreover, an "isolated nucleic acid mol respectively, and the nucleotide and amino acid sequences of ecule, such as a DNA molecule, can be substantially free of RXN02910 are depicted in SEQ ID NOs: 5 and 6, respec other cellular material, or culture medium when produced by US 7,892,798 B2 19 20 recombinant techniques, or chemical precursors or other which is a complement of one of the nucleotide sequences chemicals when chemically synthesized. shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 16, A nucleic acid molecule of the present invention, e.g., a SEQID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, or a nucleic acid molecule having a nucleotide sequence of SEQ portion thereof. A nucleic acid molecule which is comple ID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: 18, 5 mentary to one of the nucleotide sequences shown in SEQID SEQID NO:20, or SEQID NO: 22, or a portion thereof, can NO: 1, SEQID NO:5, SEQIDNO: 16, SEQIDNO: 18, SEQ be isolated using standard molecular biology techniques and ID NO: 20, or SEQ ID NO: 22 is one which is sufficiently the sequence information provided herein. For example, a complementary to one of the nucleotide sequences shown in MR DNA can be isolated from a C. glutamicum library using SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: all or portion of one of the sequences of SEQID NO: 1, SEQ 10 18, SEQ ID NO: 20, or SEQ ID NO: 22 such that it can ID NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, hybridize to one of the nucleotide sequences shown in SEQ or SEQ ID NO: 22 as a hybridization probe and standard ID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: 18, hybridization techniques (e.g., as described in Sambrook, J., SEQID NO:20, or SEQID NO:22, thereby forming a stable Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Labora duplex. tory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold 15 In still another preferred embodiment, an isolated nucleic Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., acid molecule of the invention comprises a nucleotide 1989). Moreover, a nucleic acid molecule encompassing all sequence which is at least about 50%, 51%, 52%, 53%, 54%, or a portion of one of the sequences of SEQID NO: 1, SEQID 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, or 70%, SEQ ID NO: 22 can be isolated by the polymerase chain more preferably at least about 71%, 72%, 73%, 74%, 75%, reaction using oligonucleotide primers designed based upon 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, this sequence (e.g., a nucleic acid molecule encompassing all 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and or a portion of one of the sequences of SEQID NO: 1, SEQID even more preferably at least about 95%, 96%, 97%, 98%, NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or 99% or more homologous to a nucleotide sequence shown in SEQ ID NO: 22 can be isolated by the polymerase chain 25 SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: reaction using oligonucleotide primers designed based upon 18, SEQID NO: 20, or SEQID NO:22, or a portion thereof. this same sequence of SEQID NO: 1, SEQID NO:5, SEQID Ranges and identity values intermediate to the above-recited NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO:22). ranges, (e.g., 70-90% identical or 80-95% identical) are also For example, mRNA can be isolated from normal bacterial intended to be encompassed by the present invention. For cells (e.g., by the guanidinium-thiocyanate extraction proce 30 example, ranges of identity values using a combination of any dure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) of the above values recited as upper and/or lower limits are and DNA can be prepared using reverse transcriptase (e.g., intended to be included. In an additional preferred embodi Moloney MLV reverse transcriptase, available from Gibco/ ment, an isolated nucleic acid molecule of the invention com BRL. Bethesda, Md.; or AMV reverse transcriptase, available prises a nucleotide sequence which hybridizes, e.g., hybrid from Seikagaku America, Inc., St. Petersburg, Fla.). Syn 35 izes under stringent conditions, to one of the nucleotide thetic oligonucleotide primers for polymerase chain reaction sequences shown in SEQID NO: 1, SEQID NO: 5, SEQID amplification can be designed based upon one of the nucle NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO:22, otide sequences shown in SEQID NO: 1, SEQIDNO: 5, SEQ or a portion thereof. ID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO: Moreover, the nucleic acid molecule of the invention can 22. A nucleic acid of the invention can be amplified using 40 comprise only a portion of the coding region of one of the cDNA or, alternatively, genomic DNA, as a template and sequences in SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, appropriate oligonucleotide primers according to standard SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22, for PCR amplification techniques. The nucleic acid so amplified example afragment which can be used as a probe or primer or can be cloned into an appropriate vector and characterized by a fragment encoding a biologically active portion of an MR DNA sequence analysis. Furthermore, oligonucleotides cor 45 protein. The nucleotide sequences determined from the clon responding to an MR nucleotide sequence can be prepared by ing of the MR genes from C. glutamicum allows for the standard synthetic techniques, e.g., using an automated DNA generation of probes and primers designed for use in identi synthesizer. fying and/or cloning MR homologues in other cell types and In a preferred embodiment, an isolated nucleic acid mol organisms, as well as MR homologues from other Coryne ecule of the invention comprises one of the nucleotide 50 bacteria or related species. The probe?primer typically com sequences shown in SEQID NO: 1, SEQID NO: 5, SEQID prises Substantially purified oligonucleotide. The oligonucle NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO: 22. otide typically comprines a region of nucleotide sequence The sequences of SEQID NO: 1, SEQID NO: 5, SEQID NO: that hybridizes under Stringent conditions to at least about 12, 16, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22 preferably about 25, more preferably about 40, 50 or 75 correspond to the MR DNAs of the invention. This DNA 55 consecutive nucleotides of a sense Strand of one of the comprises sequences encoding MR proteins (i.e., the “coding sequences set forthin SEQID NO: 1, SEQID NO: 5, SEQID region', indicated in each sequence in SEQIDNO: 1, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO:22, NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or an anti-sense sequence of one of the sequences set forth in SEQID NO: 22), as well as 5' untranslated sequences and 3' SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: untranslated sequences, also indicated in SEQID NO: 1, SEQ 60 18, SEQIDNO:20, or SEQID NO:22, or naturally occurring ID NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, mutants thereof. Primers based on a nucleotide sequence of or SEQID NO: 22. Alternatively, the nucleic acid molecule SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: can comprise only the coding region of any of the sequences 18, SEQID NO: 20, or SEQID NO: 22 can be used in PCR in SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, SEQID reactions to clone MR homologues. Probes based on the MR NO: 18, SEQID NO: 20, or SEQID NO: 22. 65 nucleotide sequences can be used to detect transcripts or In another preferred embodiment, an isolated nucleic acid genomic sequences encoding the same or homologous pro molecule of the invention comprises a nucleic-acid molecule teins. In preferred embodiments, the probe further comprises US 7,892,798 B2 21 22 a label group attached thereto, e.g. the label group can be a erably described by the amino acid sequence comprising the radioisotope, a fluorescent compound, an enzyme, or an sequence of SEQ ID NO: 19; the RXA00655 protein from enzyme co-factor. Such probes can be used as a part of a Corynebacterium efficiens (YS-314 Genbank Accession No. diagnostic test kit for identifying cells which misexpress an AP005223), preferably described by the amino acid sequence MR protein, Such as by measuring a level of an MR-encoding 5 comprising the sequence of SEQ ID NO: 21; and the nucleic acid in a sample of cells, e.g., detecting MR mRNA RXA00655 protein from Streptomyces coelicolor (Genbank levels or determining whether a genomic MR gene has been accession No. NC 003888), preferably described by the mutated or deleted. amino acid sequence comprising the sequence of SEQ ID In one embodiment, the nucleic acid molecule of the inven NO: 23. tion encodes a protein or portion thereof which includes an 10 RXNO2910 is intended to describe polypeptides encoded amino acid sequence which is sufficiently homologous to an by a nucleic acid molecule comprising a nucleic acid mol amino acid sequence of SEQID NO: 2, SEQID NO: 6, SEQ ecule selected from the group consisting of a nucleic acid ID NO: 17, SEQID NO: 19, SEQID NO:21, or SEQID NO: molecule comprising a nucleotide sequence which is at least 23 such that the protein or portion thereof maintains the 60% identical to the nucleotide sequence of SEQID NO: 5 or ability to transcriptionally, translationally, or posttranslation 15 16; a nucleic acid molecule comprising a fragment of at least ally regulate a metabolic pathway, e.g., methionine biosyn 30 nucleotides of a nucleic acid comprising the nucleotide thetic pathway, the cysteine biosynthetic pathway, or the Sul sequence of SEQ ID NO: 5 or 16; a nucleic acid molecule fur reduction pathway, in a microorganism, e.g., C. which encodes a polypeptide comprising an amino acid glutamicum. As used herein, the language “sufficiently sequence at least about 60% identical to the amino acid homologous' refers to proteins or portions thereof which sequence of SEQID NO: 6 or 17; and a nucleic acid molecule have amino acid sequences which include a minimum num which encodes a fragment of a polypeptide comprising the ber of identical or equivalent (e.g., an amino acid residue amino acid sequence of SEQID NO: 6 wherein the fragment which has a similar side chain as an amino acid residue in one comprises at least 10 contiguous amino acid residues of the of the sequences of SEQID NO: 2, SEQID NO: 6, SEQID amino acid sequence of SEQID NO: 6 or 17. NO: 17, SEQID NO:19, SEQIDNO:21, or SEQID NO. 23) 25 amino acid residues to an amino acid sequence of SEQ ID RXA00655 is intended to describe polypeptides encoded NO:2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQ by a nucleic acid molecule comprising a nucleic acid mol IDNO: 21, or SEQID NO: 23 such that the protein orportion ecule selected from the group consisting of a nucleic acid thereof is able to transcriptionally, translationally, or post molecule comprising a nucleotide sequence which is at least translationally regulate a metabolic pathway, e.g., methionine 30 60% identical to the nucleotide sequence of SEQID NO: 1. biosynthesis, in C. glutamicum. Protein members of Such 18, 20 or 22; a nucleic acid molecule comprising a fragment metabolic pathways, as described herein, may function to of at least 30 nucleotides of a nucleic acid comprising the regulate the biosynthesis or degradation of one or more fine nucleotide sequence of SEQID NO: 1, 18, 20 or 22; a nucleic chemicals. Examples of Such activities are also described acid molecule which encodes a polypeptide comprising an herein. Thus, “the function of an MR protein’ contributes to 35 amino acid sequence at least about 60% identical to the amino the overall regulation of one or more fine chemical metabolic acid sequence of SEQID NO: 2, 19, 21 or 23; and a nucleic pathway, or contributes, either directly or indirectly, to the acid molecule which encodes a fragment of a polypeptide yield, production, and/or efficiency of production of one or comprising the amino acid sequence of SEQID NO: 2, 19, 21 more fine chemicals e.g., methionine. or 23 wherein the fragment comprises at least 10 contiguous In another embodiment, the protein is at least about 40 amino acid residues of the amino acid sequence of SEQ ID 50-60%, preferably at least about 60-70%, and more prefer NO: 2, 19, 21 or 23. ably at least about 70-80%, 80-90%,90-95%, and most pref Portions of proteins encoded by the MR nucleic acid mol erably at least about 96%, 97%, 98%, 99% or more homolo ecules of the invention are preferably biologically active por gous to an entire amino acid sequence of SEQID NO: 2, SEQ tions of one of the MR proteins. As used herein, the term ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, 45 “biologically active portion of an MR protein’ is intended to or SEQID NO. 23. include a portion, e.g., a domain/motif, of an MR protein that As described above, homologous MR proteins, preferably transcriptionally, translationally, or posttranslationally regu homologous RXA00655 or RXN02910 proteins may be lates a metabolic pathway, e.g., methionine biosynthetic path derived from other microorganisms, preferably from way, the cysteine biosynthetic pathway, or the Sulfur reduc prokaryotic microorganisms. 50 tion pathway, in a microorganism, e.g., C. glutamicum, or has The term “prokaryotic microorganism' is intended to an activity as set forth herein. To determine whether an MR include gram-positive and gram-negative bacteria. Prefer protein or a biologically active portion thereof can transcrip ably, the term includes all genera and species of the Entero tionally, translationally, or posttranslationally regulate a bacteriaceae or Nocardiaceae family, e.g., the Enterobacteri metabolic pathway, e.g., methionine biosynthetic pathway, aceae species Escherichia, Serratia, Proteus, Enterobacter, 55 the cysteine biosynthetic pathway, or the sulfur reduction Klebsiella, Salmonella, Shigella, Edwardsielle, Citrobacter, pathway, in a microorganism, e.g., C. glutamicum, an assay of Morganella, Providencia and Yersinia. Furthermore pre enzymatic activity may be performed. Such assay methods ferred are all Pseudomonas, Burkholderia, Nocardia, Aceto are well known to those of ordinary skill in the art, as detailed bacter, Streptomyces, Gluconobacter; Corynebacterium, in Example 8 of the Exemplification. Brevibacterium, Bacillus, Clostridium, Cyanobacter; Staphy 60 Additional nucleic acid fragments encoding biologically lococcus, Aerobacter; Alcaligenes, Rhodococcus and Penicil active portions of an MR protein can be prepared by isolating lium species. Most preferred are Corynebacterium and Strep a portion of one of the sequences in SEQID NO: 2, SEQID tomyces species such as, e.g., the Corynebacterium species NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, or exemplified in Table 1, Corynebacterium diphtheriae, SEQID NO: 23, expressing the encoded portion of the MR Corynebacterium efficiens and Streptomyces coelicolor: 65 protein or peptide (e.g., by recombinant expression in vitro) Examples of homologous MR proteins include the and assessing the activity of the encoded portion of the MR RXA00655 protein from Corynebacterium diphtheriae, pref protein or peptide. US 7,892,798 B2 23 24 The invention further encompasses nucleic acid molecules ecule of the invention is at least 15 nucleotides in length and that differ from one of the nucleotide sequences shown in hybridizes understringent conditions to the nucleic acid mol SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: ecule comprising a nucleotide sequence of SEQ ID NO: 1. 18, SEQID NO:20, or SEQID NO: 22 (and portions thereof) SEQ ID NO. 5, SEQ ID NO: 16, SEQ ID NO: 18, SEQID due to degeneracy of the genetic code and thus encode the NO: 20, or SEQ ID NO: 22. In other embodiments, the same MR proteinas that encoded by the nucleotide sequences nucleic acid is at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 16, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. In 220, 225 or more nucleotides in length. As used herein, the another embodiment, an isolated nucleic acid molecule of the term “hybridizes under stringent conditions” is intended to invention has a nucleotide sequence encoding a protein hav 10 describe conditions for hybridization and washing under ing an amino acid sequence shown in SEQID NO: 2, SEQID which nucleotide sequences at least 60% homologous to each NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, or other typically remain hybridized to each other. Preferably, SEQ ID NO. 23. In a still further embodiment, the nucleic the conditions are Such that sequences at least about 65%, acid molecule of the invention encodes a full length C. more preferably at least about 70%, and even more preferably glutamicum protein which is Substantially homologous to an 15 at least about 75% or more homologous to each other typi amino acid sequence of SEQID NO: 2, SEQID NO: 6, SEQ cally remain hybridized to each other. Such stringent condi ID NO: 17, SEQID NO: 19, SEQID NO:21, or SEQID NO: tions are known to those of ordinary skill in the art and can be 23 (encoded by an open reading frame shown in SEQID NO: found in Current Protocols in Molecular Biology, John 1, SEQID NO:5, SEQID NO: 16, SEQID NO: 18, SEQID Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non NO: 20, or SEQID NO: 22). limiting example of stringent hybridization conditions are In one embodiment, the invention includes nucleotide and hybridization in 6x sodium chloride/sodium citrate (SSC) at amino acid sequences having a percent identity to a nucle about 45° C., followed by one or more washes in 0.2xSSC, otide oramino acid sequence of the invention which is greater 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acid than that of a sequence of the prior art (e.g., a Genbank molecule of the invention that hybridizes under stringent sequence (or the protein encoded by Such a sequence). One of 25 conditions to a sequence of SEQID NO: 1, SEQ ID NO: 5, ordinary skill in the art would be able to calculate the lower SEQID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID threshold of percent identity for any given sequence of the NO: 22 corresponds to a naturally-occurring nucleic acid invention by examining the GAP-calculated percent identity molecule. As used herein, a “naturally-occurring nucleic scores for each of the three top hits for a given sequence, and acid molecule refers to an RNA or DNA molecule having a by subtracting the highest GAP-calculated percent identity 30 nucleotide sequence that occurs in nature (e.g., encodes a from 100 percent. One of ordinary skill in the art will also natural protein). In one embodiment, the nucleic acid encodes appreciate that nucleic acid and amino acid sequences having a natural C. glutamicum MR protein. percent identities greater than the lower threshold so calcu In addition to naturally-occurring variants of the MR lated (e.g., at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, sequence that may exist in the population, one of ordinary 57%, 58%, 59%, or 60%, preferably at least about 61%. 62%, 35 skill in the art will further appreciate that changes can be 63%, 64%. 65%, 66%, 67%, 68%, 69%, or 70%, more pref introduced by mutation into a nucleotide sequence of SEQID erably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, NO: 1, SEQID NO:5, SEQIDNO: 16, SEQIDNO: 18, SEQ 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, ID NO:20, or SEQID NO: 22, thereby leading to changes in 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more the amino acid sequence of the encoded MR protein, without preferably at least about 95%, 96%, 97%, 98%, 99% or more 40 altering the functional ability of the MR protein. For example, identical) are also encompassed by the invention. nucleotide Substitutions leading to amino acid Substitutions at In addition to the C. glutamicum MR nucleotide sequences “non-essential amino acid residues can be made in a shown in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 16, sequence of SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, SEQID NO: 18, SEQID NO: 20, or SEQID NO: 22, it will SEQ ID NO: 18, SEQ ID NO: 20, or SEQ ID NO: 22. A be appreciated by those of ordinary skill in the art that DNA 45 “non-essential amino acid residue is a residue that can be sequence polymorphisms that lead to changes in the amino altered from the wild-type sequence of one of the MR proteins acid sequences of MR proteins may exist within a population (SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQIDNO: (e.g., the C. glutamicum population). Such genetic polymor 19, SEQID NO: 21, or SEQID NO. 23) without altering the phism in the MR gene may exist among individuals within a activity of said MR protein, whereas an “essential amino population due to natural variation. As used herein, the terms 50 acid residue is required for MR protein activity. Other amino “gene' and “recombinant gene’ refer to nucleic acid mol acid residues, however, (e.g., those that are not conserved or ecules comprising an open reading frame encoding an MR only semi-conserved in the domain having MR activity) may protein, preferably a C. glutamicum MR protein. Such natural not be essential for activity and thus are likely to be amenable variations can typically result in 1-5% Variance in the nucle to alteration without altering MR activity. otide sequence of the MR gene. Any and all Such nucleotide 55 Accordingly, another aspect of the invention pertains to variations and resulting amino acid polymorphisms in MR nucleic acid molecules encoding MR proteins that contain that are the result of natural variation and that do not alter the changes in amino acid residues that are not essential for MR functional activity of MR proteins are intended to be within activity. Such MR proteins differ in amino acid sequence the scope of the invention. from a sequence contained in SEQID NO: 2, SEQID NO: 6, Nucleic acid molecules corresponding to natural variants 60 SEQID NO: 17, SEQIDNO: 19, SEQID NO:21, or SEQID and non-C. glutamicum homologues of the C. glutamicum NO: 23 yet retain at least one of the MR activities described MR DNA of the invention can be isolated based on their herein. In one embodiment, the isolated nucleic acid mol homology to the C. glutamicum MR nucleic acid disclosed ecule comprises a nucleotide sequence encoding a protein, herein using the C. glutamicum DNA, or a portion thereof, as wherein the protein comprises an amino acid sequence at a hybridization probe according to standard hybridization 65 least about 50% homologous to an amino acid sequence of techniques under Stringent hybridization conditions. Accord SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: ingly, in another embodiment, an isolated nucleic acid mol 19, SEQ ID NO: 21, or SEQ ID NO. 23 and is capable of US 7,892,798 B2 25 26 transcriptionally, translationally, or posttranslationally regu tophan, histidine). Thus, a predicted nonessential amino acid lating a metabolic pathway in C. glutamicum, or has one or residue in an MR protein is preferably replaced with another more activities set forth herein. Preferably, the protein amino acid residue from the same side chain family. Alterna encoded by the nucleic acid molecule is at least about 50-60% tively, in another embodiment, mutations can be introduced homologous to one of the sequences in SEQID NO: 2, SEQ randomly along all or part of an MR coding sequence, Such as ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, by Saturation mutagenesis, and the resultant mutants can be or SEQ ID NO: 23, more preferably at least about 60-70% screened for an MR activity described herein to identify homologous to one of the sequences in SEQID NO: 2, SEQ mutants that retain MR activity. Following mutagenesis of ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, one of the sequences of SEQID NO: 1, SEQID NO: 5, SEQ or SEQ ID NO: 23, even more preferably at least about 10 ID NO: 16, SEQID NO: 18, SEQID NO:20, or SEQID NO: 70-80%, 80-90% homologous to one of the sequences in SEQ 22, the encoded protein can be expressed recombinantly and ID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, the activity of the protein can be determined using, for SEQ ID NO: 21, or SEQID NO: 23, and most preferably at example, assays described herein (see Example 8 of the least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Exemplification). 98%, or 99% homologous to one of the sequences in SEQID 15 In addition to the nucleic acid molecules encoding MR NO:2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQ proteins described above, another aspect of the invention ID NO: 21, or SEQID NO. 23. pertains to isolated nucleic acid molecules which are anti To determine the percent homology of two amino acid sense thereto. An 'antisense' nucleic acid comprises a nucle sequences (e.g., one of the sequences of SEQID NO: 2, SEQ otide sequence which is complementary to a “sense' nucleic ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, acid encoding a protein, e.g., complementary to the coding or SEQ ID NO: 23 and a mutant form thereof) or of two strand of a double-stranded DNA molecule or complemen nucleic acids, the sequences are aligned for optimal compari tary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense son purposes (e.g., gaps can be introduced in the sequence of nucleic acid can be complementary to an entire MR coding one protein or nucleic acid for optimal alignment with the Strand, or to only a portion thereof. In one embodiment, an other protein or nucleic acid). The amino acid residues or 25 antisense nucleic acid molecule is antisense to a "coding nucleotides at corresponding amino acid positions or nucle region' of the coding strand of a nucleotide sequence encod otide positions are then compared. When a position in one ing an MR protein. The term “coding region” refers to the sequence (e.g., one of the sequences of SEQID NO: 2, SEQ region of the nucleotide sequence comprising codons which ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, are translated into amino acid residues. In another embodi or SEQ ID NO. 23) is occupied by the same amino acid 30 ment, the antisense nucleic acid molecule is antisense to a residue or nucleotide as the corresponding position in the “noncoding region' of the coding strand of a nucleotide other sequence (e.g., a mutant form of the sequence selected sequence encoding MR. The term “noncoding region” refers from SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID to 5' and 3' sequences which flank the coding region that are NO: 19, SEQ ID NO: 21, or SEQ ID NO. 23), then the not translated into amino acids (i.e., also referred to as 5' and molecules are homologous at that position (i.e., as used herein 35 3' untranslated regions). amino acid or nucleic acid “homology is equivalent to amino Given the coding strand sequences encoding MR mol acid or nucleic acid “identity”). The percent homology ecules disclosed herein (e.g., the sequences set forth in SEQ ID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: 18, between the two sequences is a function of the number of SEQID NO: 20, or SEQID NO: 22), antisense nucleic acids identical positions shared by the sequences (i.e., '% homol of the invention can be designed according to the rules of ogy—it of identical positions/total # of positionsx100). 40 Watson and Crick base pairing. The antisense nucleic acid Anisolated nucleic acid molecule encoding an MR protein molecule can be complementary to the entire coding region of homologous to a protein sequence of SEQID NO: 2, SEQID MR mRNA, but more preferably is an oligonucleotide which NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, or is antisense to only a portion of the coding or noncoding SEQ ID NO: 23 can be created by introducing one or more region of MR mRNA. For example, the antisense oligonucle nucleotide Substitutions, additions or deletions into a nucle 45 otide can be complementary to the region Surrounding the otide sequence of SEQID NO: 1, SEQIDNO:5, SEQID NO: translation start site of MR mRNA. An antisense oligonucle 16, SEQID NO: 18, SEQID NO:20, or SEQID NO: 22 such otide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of that one or more amino acid substitutions, additions or dele the invention can be constructed using chemical synthesis and tions are introduced into the encoded protein. Mutations can enzymatic ligation reactions using procedures known in the be introduced into one of the sequences of SEQ ID NO: 1. 50 art. For example, an antisense nucleic acid (e.g., an antisense SEQ ID NO. 5, SEQ ID NO: 16, SEQ ID NO: 18, SEQID oligonucleotide) can be chemically synthesized using natu NO: 20, or SEQID NO: 22 by standard techniques, such as rally occurring nucleotides or variously modified nucleotides site-directed mutagenesis and PCR-mediated mutagenesis. designed to increase the biological stability of the molecules Preferably, conservative amino acid substitutions are made at or to increase the physical stability of the duplex formed one or more predicted non-essential amino acid residues. A 55 between the antisense and sense nucleic acids, e.g., phospho “conservative amino acid substitution' is one in which the rothioate derivatives and acridine substituted nucleotides can amino acid residue is replaced with an amino acid residue be used. Examples of modified nucleotides which can be used having a similar side chain. Families of amino acid residues to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, having similar side chains have been defined in the art. These Xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) families include amino acids with basic side chains (e.g., 60 uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-car lysine, arginine, histidine), acidic side chains (e.g., aspartic boxymethylaminomethyluracil, dihydrouracil, beta-D-galac acid, ), uncharged polar side chains (e.g., gly tosylqueosine, inosine, N6-isopentenyladenine, 1-meth cine, asparagine, glutamine, serine, threonine, tyrosine, cys ylguanine, 1-methylinosine, 2,2-dimethylguanine, teine), nonpolar side chains (e.g., alanine, Valine, leucine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-me isoleucine, proline, phenylalanine, methionine, tryptophan), 65 thylcytosine, N6-adenine, 7-methylguanine, 5-methylami beta-branched side chains (e.g., threonine, Valine, isoleucine) nomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta and aromatic side chains (e.g., tyrosine, phenylalanine, tryp D-mannosylqueosine, 5'-methoxycarboxymethyluracil, US 7,892,798 B2 27 28 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, prevent transcription of an MR gene in target cells. Seegen uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, erally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl Helene, C. et al. (1992) Ann. N.Y Acad. Sci. 660:27-36; and ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, Maher, L.J. (1992) Bioassays 14(12):807-15. 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6- Instill another embodiment, additional or alternative meth diaminopurine. Alternatively, the antisense nucleic acid can ods known to the person skilled in the art can be used to be produced biologically using an expression vector into modulate, e.g., suppress or increase, expression from a gene which a nucleic acid has been subcloned in an antisense (e.g., a gene coding for a negative regulator of methionine orientation (i.e., RNA transcribed from the inserted nucleic biosynthesis) or to modulate, e.g., increase or suppress or acid will be of an antisense orientation to a target nucleic acid 10 inhibit activity of the corresponding gene product. of interest, described further in the following subsection). One method which may be used is the expression of a The antisense nucleic acid molecules of the invention are dominant-negative variant of the gene product (e.g., a gene typically administered to a cell or generated in situ such that coding for a negative regulator of methionine biosynthesis) to they hybridize with orbind to cellular mRNA and/or genomic Suppress activity of e.g., a negative regulator of methionine DNA encoding an MR proteinto thereby inhibit expression of 15 biosynthesis. the protein, e.g., by inhibiting transcription and/or transla A negative regulator of methionine biosynthesis (e.g., tion. The hybridization can be by conventional nucleotide RXA00655 as exemplified by SEQID NO: 1) is preferably a complementarity to form a stable duplex, or, for example, in transcriptional regulator. Transcriptional regulators are the case of an antisense nucleic acid molecule which binds to known to exists as dimers which bind to opposite parts of the DNA duplexes, through specific interactions in the major DNA to structures that might contain repeating sequences in groove of the double helix. The antisense molecule can be so called dyad symmetry. The activity of DNA binding is modified such that it specifically binds to a receptor or an determined within the amino acid sequence of the transcrip antigen expressed on a selected cell surface, e.g., by linking tional regulator. Examples of the structural basis of the the antisense nucleic acid molecule to a peptide or an anti dimeric binding of such transcriptional regulators can be body which binds to a cell surface receptor or antigen. The 25 found in Schumacher M. A. and Brennan R. G. (2002) antisense nucleic acid molecule can also be delivered to cells Molecular Microbiology. 45:885-93. For example, expres using the vectors described herein. To achieve sufficient intra sion of different alleles of the same protein in one cell by cellular concentrations of the antisense molecules, vector producing hetero-dimeric proteins consisting of an unmu constructs in which the antisense nucleic acid molecule is tated and of a mutated allele of a regulatory protein confer a placed under the control of a strong prokaryotic, viral, or 30 dominant negative phenotype for such an organism. eukaryotic promoter are preferred. Examples of methods for constructing a mutant dominant In yet another embodiment, the antisense nucleic acid mol negative repressor protein in organisms also expressing ecule of the invention is an O-anomeric nucleic acid molecule. unmutated alleles of the same gene can be found in Journal of An O-anomeric nucleic acid molecule forms specific double Molecular Biology (2002) 322(2):311-24, and in Journal of stranded hybrids with complementary RNA in which, con 35 Biological Chemistry (1994).269(11):8246-54. For example, trary to the usual B-units, the strands run parallel to each other the DNA-binding domain of said negative regulator of (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). methionine biosynthesis may be inactivated. In another The antisense nucleic acid molecule can also comprise a embodiment, mRNA stabilisation and destabilisation can be 2'-O-methylribonycleotide (Inoue et al. (1987) Nucleic Acids used as a method to either increase or decrease the lifetime of Res. 15:6131-6148) or a chimeric RNA-DNA analogue (In 40 a given mRNA molecule. Methods to influence the lifetime of oue et al. (1987) FEBS Lett. 215:327-330). a given mRNA can be found in Smolke C. D. and Keasling J. In still another embodiment, an antisense nucleic acid of D. (2002) Biotechnology & Bioengineering. 78(4):412-24, in the invention is a ribozyme. Ribozymes are catalytic RNA Smolke C. D. et al. (2001) Metabolic Engineering 3(4):313 molecules with ribonuclease activity which are capable of 21, and in Carrier T. A. and Keasling J. D. (1999) Biotech cleaving a single-stranded nucleic acid, such as an mRNA, to 45 nology Progress 15(1):58-64. Another method which may be which they have a complementary region. Thus, ribozymes used to modulate expression of a gene includes expression of (e.g., hammerhead ribozymes (described in Haselhoff and DNA-binding proteins increasing or blocking or reducing Gerlach (1988) Nature 334:585-591)) can be used to catalyti expression from the gene, e.g., a gene coding for a regulatory cally cleave MR mRNA transcripts to thereby inhibit trans protein of methionine biosynthesis. lation of MR mRNA. A ribozyme having specificity for an 50 Blocking or reducing expression from the gene, e.g., a gene MR-encoding nucleic acid can be designed based upon the coding for a negative regulatory protein of methionine bio nucleotide sequence of an MR DNA disclosed herein (i.e., synthesis can be realized by utilizing specific DNA-binding SEQID NO: 1, SEQID NO:5, SEQID NO: 16, SEQID NO: proteins, e.g., of the zinc-finger protein class of transcription 18, SEQ ID NO: 20, or SEQ ID NO: 22). For example, a factors. Said factors may be directed to e.g., regulatory derivative of a Tetrahymena L-19 IVS RNA can be con 55 regions of the gene to be suppressed. Utilization of said fac structed in which the nucleotide sequence of the active site is tors allows suppression of expression without altering the complementary to the nucleotide sequence to be cleaved in an corresponding gene sequence. Methods are known to the MR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. person skilled in the art to construct artificial DNA-binding 4.987,071 and Cech et al. U.S. Pat. No. 5,116,742. Alterna factors capable of binding to a specific target sequence. tively, MR mRNA can be used to select a catalytic RNA 60 Increasing gene expression, e.g., coding for a positive regu having a specific ribonuclease activity from a pool of RNA lator of methionine biosynthesis, can be realized by using the molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) above described artificial DNA-binding factors by fusing Science 261:141 1-1418. them to a transcription activation domain, thereby creating an Alternatively, MR gene expression can be inhibited by artificial initiator of transcription. (Dreier B et al. (2001) J targeting nucleotide sequences complementary to the regula 65 Biol Chem 276(31):294.66-78; Dreier B et al. (2000) J Mol tory region of an MR nucleotide sequence (e.g., an MR pro Biol 303(4):489-502; Beerli R Retal. (2000) Proc Natl Acad moter and/or enhancers) to form triple helical structures that Sci USA 97 (4): 1495-1500; Beerli R. Ret al. (2000) J Biol US 7,892,798 B2 29 30 Chem 275(42):32617-32627; Segal DJ and Barbas C F 3rd. The recombinant expression vectors of the invention com (2000) Curr Opin Chem Biol 4(1):34–39; Kang J S and Kim J prise a nucleic acid of the invention in a form suitable for S (2000) J Biol Chem 275(12):8742-8748; Beerli R. Ret al. expression of the nucleic acid in a host cell, which means that (1998) Proc Natl AcadSci USA 95(25): 14628-14633; Kim J the recombinant expression vectors include one or more regu Set al. (1997) Proc Natl Acad Sci USA 94(8):3616-3620; latory sequences, selected on the basis of the host cells to be Klug A (1999) J Mol Biol 293(2):215-218: Tsai SY et al. used for expression, which is operatively linked to the nucleic (1998) Adv. Drug Deliv Rev. 30(1-3):23-31; Mapp A K et al. acid sequence to be expressed. Within a recombinant expres (2000) Proc Natl AcadSci USA 97(8):3930-3935; Sharrocks sion vector, “operably linked' is intended to mean that the A D et al. (1997) Int J Biochem Cell Biol 29(12): 1371-1387: nucleotide sequence of interest is linked to the regulatory Zhang L et al. (2000) J Biol Chem 275(43):33850-33860). 10 sequence(s) in a manner which allows for expression of the Suppression of gene expression can also be realized by using nucleotide sequence (e.g., in an in vitro transcription/transla customized low-molecular weight synthetic compounds, e.g., tion system or in a host cell when the vector is introduced into of the polyamide type (Dervan P B and Birli RW (1999) the host cell). The term “regulatory sequence' is intended to Current Opinion in Chemical Biology 3:688-693; Gottesfeld include promoters, enhancers and other expression control JMetal. (2000) Gene Expr 9(1-2):77-91). These compounds 15 elements (e.g., polyadenylation signals). Such regulatory can be adopted on a rational basis to any specific DNA target sequences are described, for example, in Goeddel; Gene sequence allowing Suppression or, if the compound is used in Expression Technology. Methods in Enzymology 185, Aca fusion with a transcription activation domain, initiation of demic Press, San Diego, Calif. (1990) and in Vasicova P. gene expression. Methods are known to the person skilled in Patek M. Nesvera J. Sahm H. Eikmanns B. (1999) Journal of the art to construct the artificial DNA-binding factors capable Bacteriology 181 (19):6188-91, in Patek M. et al. (1996) of binding to a specific target sequence (Bremer R E et al. Microbiology 142:1297-309, and in Mateos et al. (1994) (2001) Bioorg Med Chem. 9(8):2093-103; Ansari A. Z. et al. Journal of Bacteriology 176:7362-71. Regulatory sequences (2001) Chem Biol. 8(6):583-92: Gottesfeld J. M. etal. (2001) include those which direct constitutive expression of a nucle J Mol Biol. 309(3):615-29; Wurtz.N. R. et al. (2001) Org Lett otide sequence in many types of host cell and those which 3(8): 1201-3: Wang C. C. et al. (2001) Bioorg Med Chem 25 direct expression of the nucleotide sequence only in certain 9(3):653-7: Urbach A. R. and Dervan P. B. (2001) Proc Natl host cells. Preferred regulatory sequences are, for example, AcadSci USA 98(8):4343-8; Chiang S.Y. et al. (2000).J. Biol promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, Chem. 275(32):24246-54). In another embodiment, expres lpp-lac-, lacI7- T7- T5-, T3-, gal-, trc-, ara-, SP6-, arny, sion of protein-binding factors activating or blocking or SPO2, w-P- or w P, which are used preferably in bacteria. reducing expression or activity of the gene coding for a regu 30 Additional regulatory sequences are, for example, promoters latory protein of methionine biosynthesis may be utilized. from and fungi, such as ADC1, MFC, AC, P-60, CYC1, Protein-binding factors suitable for binding regulators of GAPDH, TEF, rp28, ADH, promoters from plants such as methionine biosynthesis and thereby suppressing activity CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiq may be based on RNA Such as, e.g. aptamers (Famulok Mund uitin- or phaseolin-promoters. It is also possible to use artifi Mayer G (1999) Curr Top Microbiol Immunol 243:123-36), 35 cial promoters. It will be appreciated by one of ordinary skill antibodies, antibody fragments, or single-chain antibodies. in the art that the design of the expression vector can depend Methods for construction and utilization of these protein on such factors as the choice of the host cell to be transformed, binding factors are known to the person skilled in the art. the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to B. Recombinant Expression Vectors and Host Cells 40 thereby produce proteins or peptides, including fusion pro Another aspect of the invention pertains to vectors, prefer teins or peptides, encoded by nucleic acids as described ably expression vectors, containing a nucleic acid encoding herein (e.g., MR proteins, mutant forms of MR proteins, an MR protein (or a portion thereof). As used herein, the term fusion proteins, etc.). “vector” refers to a nucleic acid molecule capable of trans The recombinant expression vectors of the invention can be porting another nucleic acid to which it has been linked. One 45 designed for expression of MR proteins in prokaryotic or type of vector is a “plasmid', which refers to a circular double eukaryotic cells. For example, MR genes can be expressed in stranded DNA loop into which additional DNA segments can bacterial cells such as C. glutamicum, cells (using be ligated. Another type of vector is a viral vector, wherein baculovirus expression vectors), and other fungal cells additional DNA segments can be ligated into the viral (see Romanos, M. A. et al. (1992) “Foreign gene expression genome. Certain vectors are capable of autonomous replica 50 in yeast: a review'', Yeast 8:423-488; Van den Hondel, C. A. tion in a host cell into which they are introduced (e.g., bacte M. J. J. et al. (1991) “Heterologous gene expression in fila rial vectors having a bacterial origin of replication and episo mentous fungi in: More Gene Manipulations in Fungi, J. W. mal mammalian vectors). Other vectors (e.g., non-episomal Bennet & L. L. Lasure, eds., p. 396-428: Academic Press: San mammalian vectors) are integrated into the genome of a host Diego; and van den Hondel, C. A. M. J. J. & Punt, P.J. (1991) cell upon introduction into the host cell, and thereby are 55 “Gene transfer systems and vector development for filamen replicated along with the host genome. Moreover, certain tous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, vectors are capable of directing the expression of genes to J. F. et al., eds., p. 1-28, Cambridge University Press: Cam which they are operatively linked. Such vectors are referred to bridge), algae and multicellular plant cells (see Schmidt, R. herein as “expression vectors'. In general, expression vectors and Willimitzer, L. (1988) High efficiency Agrobacterium of utility in recombinant DNA techniques are often in the 60 tumefaciens—mediated transformation of Arabidopsis form of plasmids. In the present specification, "plasmid' and thaliana leaf and cotyledon explants' Plant Cell Rep. 583 “vector” can be used interchangeably as the plasmid is the 586), or mammalian cells. Suitable host cells are discussed most commonly used form of vector. However, the invention further in Goeddel, Gene Expression Technology: Methods in is intended to include Such other forms of expression vectors, Enzymology 185, Academic Press, San Diego, Calif. (1990). Such as viral vectors (e.g., replication defective retroviruses, 65 Alternatively, the recombinant expression vector can be tran adenoviruses and adeno-associated viruses), which serve scribed and translated in vitro, for example using T7 promoter equivalent functions. regulatory sequences and T7 polymerase. US 7,892,798 B2 31 32 Expression of proteins in prokaryotes is most often carried expression, such as C. glutamicum (Wada et al. (1992) out with vectors containing constitutive or inducible promot Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic ers directing the expression of either fusion or non-fusion acid sequences of the invention can be carried out by standard proteins. Fusion vectors add a number of amino acids to a DNA synthesis techniques. protein encoded therein, usually to the amino terminus of the 5 In another embodiment, the MR protein expression vector recombinant protein. Such fusion vectors typically serve is a yeast expression vector. Examples of vectors for expres three purposes: 1) to increase expression of recombinant pro sion in yeast S. cerevisiae include pYepSec1 (Baldari, et al., tein; 2) to increase the solubility of the recombinant protein; (1987) Embo J. 6:229-234), 2, p.AG-1, Yepé, Yep 13, pEM and 3) to aid in the purification of the recombinant protein by BLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933 acting as a ligand in affinity purification. Often, in fusion 10 943), p.RY88 (Schultz et al., (1987) Gene 54:113-123), and expression vectors, a proteolytic cleavage site is introduced at pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors the junction of the fusion moiety and the recombinant protein and methods for the construction of vectors appropriate for to enable separation of the recombinant protein from the use in other fungi, Such as the filamentous fungi, include those fusion moiety Subsequent to purification of the fusion protein. detailed in: van den Hondel, C. A. M. J. J. & Punt, P.J. (1991) Such enzymes, and their cognate recognition sequences, 15 “Gene transfer systems and vector development for filamen include Factor Xa, thrombin and enterokinase. tous fungi, in: Applied Molecular Genetics of Fungi, J. F. Typical fusion expression vectors include pGEX (Pharma Peberdy, et al., eds., p. 1-28, Cambridge University Press: cia Biotech Inc.; Smith, D. B. and Johnson, K. S. (1988) Gene Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) Elsevier: N.Y. (IBSN 0444904018). and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glu Alternatively, the MR proteins of the invention can be tathione S-transferase (GST), maltose E binding protein, or expressed in insect cells using baculovirus expression vec protein A, respectively, to the target recombinant protein. In tors. Baculovirus vectors available for expression of proteins one embodiment, the coding sequence of the MR protein is in cultured insect cells (e.g., Sf9 cells) include the pac series cloned into a pCEX expression vector to create a vector (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL encoding a fusion protein comprising, from the N-terminus to 25 series (Lucklow and Summers (1989) Virology 170:31-39). the C-terminus, GST-thrombin cleavage site-X protein. The In another embodiment, the MR proteins of the invention fusion protein can be purified by affinity chromatography may be expressed in unicellular plant cells (such as algae) or using glutathione-agarose resin. Recombinant MR protein in plant cells from higher plants (e.g., the spermatophytes, unfused to GST can be recovered by cleavage of the fusion Such as crop plants). Examples of plant expression vectors protein with thrombin. 30 include those detailed in: Becker, D., Kemper, E., Schell, J. Examples of suitable inducible non-fusion E. coli expres and Masterson, R. (1992) "New plant binary vectors with sion vectors include pTrc (Amann et al., (1988) Gene 69:301 selectable markers located proximal to the left border”, Plant 315) plG338, p.ACYC184, pBR322, p UC18, puC19, Mol. Biol. 20: 1195-1197: and Bevan, M. W. (1984) “Binary pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pIG200, Agrobacterium vectors for plant transformation’. Nucl. Acid. pUR290, pIN-III 113-B1, gt11, pBdC1, and pET 11d 35 Res. 12: 8711-8721, and include plGV23, pGHlac+, (Studier et al., Gene Expression Technology: Methods in pBIN19, p.AK2004, and pPH51 (Pouwels et al., eds. (1985) Enzymology 185, Academic Press, San Diego, Calif. (1990) Cloning Vectors. Elsevier: N.Y. IBSN 0444904018). 60-89; and Pouwels et al., eds. (1985) Cloning Vectors. In yet another embodiment, a nucleic acid of the invention Elsevier: N.Y. ISBN 0444 904018). Other examples for is expressed in mammalian cells using a mammalian expres inducible E. coli C. glutamicum shuttle expression vectors 40 sion vector. Examples of mammalian expression vectors can be found in Eikmanns B.J., et al. (1991) Gene 102:93-8. include pCDM8 (Seed, B. (1987) Nature 329:840) and Target gene expression from the pTrc Vector relies on host pMT2PC (Kaufman et al. (1987) EMBO.J. 6:187-195). When RNA polymerase transcription from a hybrid trp-lac fusion used in mammalian cells, the expression vector's control promoter. Target gene expression from the pET 11d vector functions are often provided by viral regulatory elements. For relies on transcription from a T7 gn10-lac fusion promoter 45 example, commonly used promoters are derived from mediated by a coexpressed viral RNA polymerase (T7 gn1). polyoma, Adenovirus 2, cytomegalovirus and Simian Virus This viral polymerase is supplied by host strains BL21 (DE3) 40. For other suitable expression systems for both prokaryotic or HMS174(DE3) from a resident prophage harboring a T7 and eukaryotic cells see chapters 16 and 17 of Sambrook, J., gn1 gene under the transcriptional control of the lacUV 5 Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Labora promoter. For transformation of other varieties of bacteria, 50 tory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold appropriate vectors may be selected. For example, the plas Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., mids plJ100, pIJ364, pIJ702 and plJ361 are known to be 1989. useful in transforming Streptomyces, while plasmids In another embodiment, the recombinant mammalian pUB110, pC194, or pBD214 are suited for transformation of expression vector is capable of directing expression of the Bacillus species. Several plasmids of use in the transfer of 55 nucleic acid preferentially in a particular cell type (e.g., tis genetic information into Corynebacterium include Sue-specific regulatory elements are used to express the pHM1519, pBL1, pSAT7, or pAJ667 (Pouwels et al., eds. nucleic acid). Tissue-specific regulatory elements are known (1985) Cloning Vectors. Elsevier: N.Y. IBSN 0444904018). in the art. Non-limiting examples of Suitable tissue-specific One strategy to maximize recombinant protein expression is promoters include the albumin promoter (liver-specific: Pink to express the protein in a host bacteria with an impaired 60 ert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific capacity to proteolytically cleave the recombinant protein promoters (Calame and Eaton (1988) Adv. Immunol. 43:235 (Gottesman, S. Gene Expression Technology. Methods in 275), in particular promoters of T cell receptors (Winoto and Enzymology 185, Academic Press, San Diego, Calif. (1990) Baltimore (1989) EMBO.J. 8:729-733) and immunoglobulins 119-128). Another strategy is to alter the nucleic acid (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore sequence of the nucleic acid to be inserted into an expression 65 (1983) Cell 33:741-748), neuron-specific promoters (e.g., the vector so that the individual codons for each amino acid are neurofilament promoter; Byrne and Ruddle (1989) PNAS those preferentially utilized in the bacterium chosen for 86:5473-5477), pancreas-specific promoters (Edlund et al. US 7,892,798 B2 33 34 (1985) Science 230:912-916), and mammary gland-specific the foreign DNA into their genome. In order to identify and promoters (e.g., milk whey promoter, U.S. Pat. No. 4,873.316 select these integrants, a gene that encodes a selectable and European Application Publication No. 264,166). Devel marker (e.g., resistance to antibiotics) is generally introduced opmentally-regulated promoters are also encompassed, for into the host cells along with the gene of interest. Preferred example the murine hox promoters (Kessel and Gruss (1990) selectable markers include those which confer resistance to Science 249:374-379) and the a-fetoprotein promoter drugs, such as G418, hygromycin and methotrexate. Nucleic (Campes and Tilghman (1989) Genes Dev. 3:537-546). acid encoding a selectable marker can be introduced into a The invention further provides a recombinant expression host cell on the same vector as that encoding an MR protein or vector comprising a DNA molecule of the invention cloned can be introduced on a separate vector. Cells stably trans into the expression vector in an antisense orientation. That is, 10 fected with the introduced nucleic acid can be identified by, the DNA molecule is operatively linked to a regulatory for example, drug selection (e.g., cells that have incorporated sequence in a manner which allows for expression (by tran the selectable marker gene will survive, while the other cells scription of the DNA molecule) of an RNA molecule which is die). antisense to MR mRNA. Regulatory sequences operatively For transformation of microorganisms, it is known that, linked to a nucleic acid cloned in the antisense orientation can 15 depending upon the expression vector and transformation be chosen which direct the continuous expression of the anti technique used, only a small fraction of cells may incorporate sense RNA molecule in a variety of cell types, for instance the foreign DNA either with an episomal localisation or into viral promoters and/or enhancers, or regulatory sequences their genome by processes involving recombination. In order can be chosen which direct constitutive, tissue specific or cell to identify and select these transformants, a gene that encodes type specific expression of antisense RNA. The antisense a selectable marker (e.g., resistance to antibiotics) is gener expression vector can be in the form of a recombinant plas ally introduced into the host cells along with the gene of mid, phagemidor attenuated virus in which antisense nucleic interest. Preferred selectable markers include those which acids are produced under the control of a high efficiency confer resistance to drugs, such as kanamycin, tetracyclin, regulatory region, the activity of which can be determined by bleomycin, chloramphenicol, lincomycin. Nucleic acid the cell type into which the vector is introduced. For a dis 25 encoding a selectable marker can be introduced into a host cussion of the regulation of gene expression using antisense cell on the same vector as that encoding an MR protein or can genes see Weintraub, H. et al., Antisense RNA as a molecular beintroduced on a separate vector. Cells transformed with the tool for genetic analysis; Reviews—Trends in Genetics, Vol. introduced nucleic acid can be identified by, for example, 1(1) 1986. drug selection (e.g., cells that have incorporated the select Another aspect of the invention pertains to host cells into 30 able marker gene will survive, while the other cells die). which a recombinant expression vector of the invention has Examples for antibiotic resistance genes that can be used in been introduced. The terms "host cell' and "recombinant host C. glutamicum can be found in, for example, Tauch A. et al. cell are used interchangeably herein. It is understood that (1992) Applied Microbiology & Biotechnology 36(6):759-62; such terms refer not only to the particular subject cell but to Eikmanns B.J. et al. (1991) Gene 102(1): 93-8:Yoshihama M. the progeny or potential progeny of Such a cell. Because 35 et al. (1985) Journal of Bacteriology 162(2):591-7; Tauch A. certain modifications may occur in Succeeding generations et al. (1998) Plasmid 40(2):126-39; Cadenas RFetal. (1991) due to either mutation or environmental influences, such Gene 98(1): 117-21. progeny may not, in fact, be identical to the parent cell, but are To create a homologous recombinant microorganism, a still included within the scope of the term as used herein. vector is prepared which contains at least a portion of an MR A host cell can be any prokaryotic or eukaryotic cell. For 40 gene into which a deletion, addition or Substitution has been example, an MR protein can be expressed in bacterial cells introduced to thereby alter, e.g., functionally disrupt, the MR Such as C. glutamicum, insect cells, yeast or mammalian cells gene. Preferably, this MR gene is a Corynebacterium (such as Chinese hamster ovary cells (CHO) or COS cells). glutamicum MR gene, but it can be a homologue from a Other suitable host cells are known to one of ordinary skill in related bacterium or even from a mammalian, yeast, or insect the art. Microorganisms related to Corynebacterium 45 Source. In a preferred embodiment, the vector is designed glutamicum which may be conveniently used as host cells for Such that, upon homologous recombination, the endogenous the nucleic acid and protein molecules of the invention are set MR gene is functionally disrupted (i.e., no longer encodes a forth in Table 1, below. functional protein; also referred to as a “knock-out vector). Vector DNA can be introduced into prokaryotic or eukary Alternatively, the vector can be designed such that, upon otic cells via conventional transformation or transfection 50 homologous recombination, the endogenous MR gene is techniques. As used herein, the terms “transformation' and mutated or otherwise altered but still encodes functional pro “transfection' are intended to refer to a variety of art-recog tein (e.g., the upstream regulatory region can be altered to nized techniques for introducing foreign nucleic acid (e.g., thereby alter the expression of the endogenous MR protein). linear DNA or RNA (e.g., a linearized vector or a gene con In the homologous recombination vector, the altered portion struct alone without a vector) or nucleic acid in the form of a 55 of the MR gene is flanked at its 5' and 3' ends by additional Vector (e.g., a plasmid, phage, phasmid, phagemid, transpo nucleic acid of the MR gene to allow for homologous recom Son or other DNA) into a host cell, including calcium phos bination to occur between the exogenous MR gene carried by phate or calcium chloride co-precipitation, DEAE-dextran the vector and an endogenous MR gene in a microorganism. mediated transfection, lipofection, or electroporation. The additional flanking MR nucleic acid is of sufficient length Suitable methods for transforming or transfecting host cells 60 for Successful homologous recombination with the endog can be found in Sambrook, et al. (Molecular Cloning. A enous gene. Typically, the flanking DNA should have lengths Laboratory Manual. 2nd, ed., Cold Spring Harbor Labora between 100 basepairs and a few kilobases (both at the 5' and tory, Cold Spring Harbor Laboratory Press, Cold Spring Har 3' ends) are included in the vector (see e.g., Thomas, K. R. bor, N.Y., 1989), and other laboratory manuals. and Capecchi, M. R. (1987) Cell 51:503 for a description of For stable transfection of mammalian cells, it is known 65 homologous recombination vectors). In addition, methods that, depending upon the expression vector and transfection are known in which another selection can be used to construct technique used, only a small fraction of cells may integrate chromosomal recombinations in which the selection marker US 7,892,798 B2 35 36 is retrieved by a method of positive selection (Schafer A. et al. tions of MR protein in which the protein is separated from (1994) Gene 145(1):69-73) The vector is introduced into a chemical precursors or other chemicals which are involved in microorganism (e.g., by electroporation) and cells in which the synthesis of the protein. In one embodiment, the language the introduced MR gene has homologously recombined with “substantially free of chemical precursors or other chemi the endogenous MR gene are selected, using art-known tech 5 cals' includes preparations of MR protein having less than niques. about 30% (by dry weight) of chemical precursors or non-MR In another embodiment, recombinant microorganisms can chemicals, more preferably less than about 20% chemical be produced which contain selected systems which allow for precursors or non-MR chemicals, still more preferably less regulated expression of the introduced gene. For example, than about 10% chemical precursors or non-MR chemicals, inclusion of an MR gene on a vector placing it under control 10 and most preferably less than about 5% chemical precursors of the lac operon permits expression of the MR gene only in or non-MR chemicals. In preferred embodiments, isolated the presence of IPTG. Such regulatory systems are well proteins or biologically active portions thereof lack contami known in the art and are described in e.g., Eikmanns B.J. etal. nating proteins from the same organism from which the MR (1991) Gene 102:93-8. protein is derived. Typically, such proteins are produced by In another embodiment, an endogenous MR gene in a host 15 recombinant expression of for example, a C. glutamicum MR cell is disrupted (e.g., by homologous recombination or other protein in a microorganism Such as C. glutamicum. genetic means known in the art) Such that expression of its An isolated MR protein or a portion thereof of the invention protein product does not occur. In another embodiment, an can transcriptionally, translationally, or posttranslationally endogenous or introduced MR gene in a host cell has been regulate a metabolic pathway in C. glutamicum, or has one or altered by one or more point mutations, deletions, or inver more of the activities set forth herein. In preferred embodi sions, but still encodes a functional MR protein. In still ments, the protein orportion thereof comprises an amino acid another embodiment, one or more of the regulatory regions sequence which is sufficiently homologous to an amino acid (e.g., a promoter, repressor, or inducer) of an MR gene in a sequence of SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, microorganism has been altered (e.g., by deletion, truncation, SEQID NO: 19, SEQID NO: 21, or SEQID NO:23 such that inversion, or point mutation) Such that the expression of the 25 the protein or portion thereof maintains the ability to tran MR gene is modulated. One of ordinary skill in the art will Scriptionally, translationally, or posttranslationally regulate a appreciate that host cells containing more than one of the metabolic pathway in C. glutamicum. The portion of the described MR gene and protein modifications may be readily protein is preferably a biologically active portion as described produced using the methods of the invention, and are meant to herein. In another preferred embodiment, an MR protein of be included in the present invention. 30 the invention has an amino acid sequence shown in SEQID A host cell of the invention, Such as a prokaryotic or NO:2, SEQID NO: 6, SEQIDNO: 17, SEQIDNO: 19, SEQ eukaryotic host cell in culture, can be used to produce (i.e. ID NO: 21, or SEQ ID NO. 23. In yet another preferred express) an MR protein. Accordingly, the invention further embodiment, the MR protein has an amino acid sequence provides methods for producing MR proteins using the host which is encoded by a nucleotide sequence which hybridizes, cells of the invention. In one embodiment, the method com 35 e.g., hybridizes under Stringent conditions, to a nucleotide prises culturing the host cell of invention (into which a recom sequence of SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, binant expression vector encoding an MR protein has been SEQID NO: 18, SEQID NO: 20, or SEQID NO: 22. In still introduced, or into which genome has been introduced a gene another preferred embodiment, the MR protein has an amino encoding a wild-type or altered MR protein) in a suitable acid sequence which is encoded by a nucleotide sequence that medium until MR protein is produced. In another embodi 40 is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, ment, the method further comprises isolating MR proteins 58%, 59%, or 60%, preferably at least about 61%. 62%. 63%, from the medium or the host cell. 64%. 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, C. Isolated MR Proteins 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, Another aspect of the invention pertains to isolated MR 45 89%, or 90%, or 91%, 92%, 93%, 94%, and even more proteins, and biologically active portions thereof. An "iso preferably at least about 95%, 96%, 97%, 98%, 99% or more lated' or “purified protein or biologically active portion homologous to one of the nucleic acid sequences of SEQID thereof is substantially free of cellular material when pro NO: 1, SEQID NO:5, SEQIDNO: 16, SEQIDNO: 18, SEQ duced by recombinant DNA techniques, or chemical precur ID NO: 20, or SEQID NO: 22, or a portion thereof. Ranges sors or other chemicals when chemically synthesized. The 50 and identity values intermediate to the above-recited values, language “substantially free of cellular material' includes (e.g., 70-90% identical or 80-95% identical) are also intended preparations of MR protein in which the protein is separated to be encompassed by the present invention. For example, from cellular components of the cells in which it is naturally ranges of identity values using a combination of any of the or recombinantly produced. In one embodiment, the language above values recited as upper and/or lower limits are intended “substantially free of cellular material includes preparations 55 to be included. The preferred MR proteins of the present of MR protein having less than about 30% (by dry weight) of invention also preferably possess at least one of the MR non-MR protein (also referred to herein as a “contaminating activities described herein. For example, a preferred MR pro protein'), more preferably less than about 20% of non-MR tein of the present invention includes an amino acid sequence protein, still more preferably less than about 10% of non-MR encoded by a nucleotide sequence which hybridizes, e.g., protein, and most preferably less than about 5% non-MR 60 hybridizes under stringent conditions, to a nucleotide protein. When the MR protein or biologically active portion sequence of SEQID NO: 1, SEQID NO: 5, SEQID NO: 16, thereof is recombinantly produced, it is also preferably sub SEQID NO: 18, SEQID NO: 20, or SEQID NO: 22, and stantially free of culture medium, i.e., culture medium repre which can transcriptionally, translationally, or posttransla sents less than about 20%, more preferably less than about tionally regulate a metabolic pathway in C. glutamicum, or 10%, and most preferably less than about 5% of the volume of 65 which has one or more of the activities set forth herein. the protein preparation. The language 'substantially free of In other embodiments, the MR protein is substantially chemical precursors or other chemicals' includes prepara homologous to an amino acid sequence of SEQ ID NO: 2, US 7,892,798 B2 37 38 SEQ ID NO: 6, SEQ ID NO: 17, SEQ ID NO: 19, SEQID an MR protein, whereas a “non-MR polypeptide' refers to a NO: 21, or SEQID NO: 23 and retains the functional activity polypeptide having an amino acid sequence corresponding to of the protein of one of the sequences of SEQID NO: 2, SEQ a protein which is not substantially homologous to the MR ID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID NO:21, protein, e.g., a protein which is different from the MR protein or SEQID NO: 23 yet differs in amino acid sequence due to 5 and which is derived from the same or a different organism. natural variation or mutagenesis, as described in detail in Within the fusion protein, the term “operatively linked' is Subsection I above. Accordingly, in another embodiment, the intended to indicate that the MR polypeptide and the non-MR MR protein is a protein which comprises an amino acid polypeptide are fused in-frame to each other. The non-MR sequence which is at least about 50%, 51%, 52%, 53%, 54%, polypeptide can be fused to the N-terminus or C-terminus of 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 10 the MR polypeptide. For example, in one embodiment the 61%. 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, or 70%, fusion protein is a GST-MR fusion protein in which the MR more preferably at least about 71%, 72%, 73%, 74%, 75%, sequences arefused to the C-terminus of the GST sequences. 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, Such fusion proteins can facilitate the purification of recom 86%, 87%, 88%, 89%, or 90%, 91%, 92%, 93%, 94%, and binant MR proteins. In another embodiment, the fusion pro even more preferably at least about 95%, 96%, 97%, 98%, 15 tein is an MR protein containing a heterologous signal 99% or more homologous to an entire amino acid sequence of sequence at its N-terminus. In certain host cells (e.g., mam SEQID NO: 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: malian host cells), expression and/or secretion of an MR 19, SEQID NO: 21, or SEQID NO: 23 and which has at least protein can be increased through use of a heterologous signal one of the MR activities described herein. Ranges and iden Sequence. tity values intermediate to the above-recited values, (e.g., Preferably, an MR chimeric or fusion protein of the inven 70-90% identical or 80-95% identical) are also intended to be tion is produced by standard recombinant DNA techniques. encompassed by the present invention. For example, ranges For example, DNA fragments coding for the different of identity values using a combination of any of the above polypeptide sequences are ligated together in-frame in accor values recited as upper and/or lower limits are intended to be dance with conventional techniques, for example by employ included. In another embodiment, the invention pertains to a 25 ing blunt-ended or stagger-ended termini for ligation, restric full length C. glutamicum protein which is Substantially tion enzyme digestion to provide for appropriate termini, homologous to an entire amino acid sequence of SEQID NO: filling-in of cohesive ends as appropriate, alkaline phos 2, SEQID NO: 6, SEQID NO: 17, SEQID NO: 19, SEQID phatase treatment to avoid undesirable joining, and enzy NO: 21, or SEQID NO. 23. matic ligation. In another embodiment, the fusion gene can be Biologically active portions of an MR protein include pep 30 synthesized by conventional techniques including automated tides comprising amino acid sequences derived from the DNA synthesizers. Alternatively, PCR amplification of gene amino acid sequence of an MR protein, e.g., the an amino acid fragments can be carried out using anchor primers which give sequence shown in SEQID NO: 2, SEQID NO: 6, SEQID rise to complementary overhangs between two consecutive NO: 17, SEQID NO: 19, SEQID NO:21, or SEQID NO:23 gene fragments which can Subsequently be annealed and or the amino acid sequence of a proteinhomologous to an MR 35 reamplified to generate a chimeric gene sequence (see, for protein, which include fewer amino acids than a full length example, Current Protocols in Molecular Biology, eds. MR protein or the full length protein which is homologous to Ausubel et al. John Wiley & Sons: 1992). Moreover, many an MR protein, and exhibit at least one activity of an MR expression vectors are commercially available that already protein. Typically, biologically active portions (peptides, e.g., encode a fusion moiety (e.g., a GST polypeptide). An MR peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 40 encoding nucleic acid can be cloned into Such an expression 38, 39, 40, 50, 100, 150, 200 or more amino acids in length) vector such that the fusion moiety is linked in-frame to the comprise a domain or motif with at least one activity of an MR MR protein. flomologues of the MR protein can be generated protein. Moreover, other biologically active portions, in by mutagenesis, e.g., discrete point mutation or truncation of which other regions of the protein are deleted, can be prepared the MR protein. As used herein, the term “homologue' refers by recombinant techniques and evaluated for one or more of 45 to a variant form of the MR protein which acts as an agonist the activities described herein. Preferably, the biologically or antagonist of the activity of the MR protein. An agonist of active portions of an MR protein include one or more selected the MR protein can retain substantially the same, or a subset, domains/motifs orportions thereofhaving biological activity. of the biological activities of the MR protein. Anantagonist of MR proteins are preferably produced by recombinant the MR protein can inhibit one or more of the activities of the DNA techniques. For example, a nucleic acid molecule 50 naturally occurring form of the MR protein, by, for example, encoding the protein is cloned into an expression vector (as competitively binding to a downstream or upstream member described above), the expression vector is introduced into a of the MR regulatory cascade which includes the MR protein. host cell (as described above) and the MR protein is expressed Thus, the C. glutamicum MR protein and homologues thereof in the host cell. The MR protein can then be isolated from the of the present invention may modulate the activity of one or cells by an appropriate purification scheme using standard 55 more metabolic pathways which MR proteins regulate in this protein purification techniques. Alternative to recombinant microorganism. expression, an MR protein, polypeptide, or peptide can be In an alternative embodiment, homologues of the MR pro synthesized chemically using standard peptide synthesis tein can be identified by screening combinatorial libraries of techniques. Moreover, native MR protein can be isolated mutants, e.g., truncation mutants, of the MR protein for MR from cells (e.g., endothelial cells), for example using an anti 60 protein agonist or antagonist activity. In one embodiment, a MR antibody, which can be produced by standard techniques variegated library of MR variants is generated by combina utilizing an MR protein or fragment thereof of this invention. torial mutagenesis at the nucleic acid level and is encoded by The invention also provides MR chimeric or fusion pro a variegated gene library. A variegated library of MR variants teins. As used herein, an MR "chimeric protein’ or “fusion can be produced by, for example, enzymatically ligating a protein’ comprises an MR polypeptide operatively linked to 65 mixture of synthetic oligonucleotides into gene sequences a non-MR polypeptide. An “MR polypeptide' refers to a Such that a degenerate set of potential MR sequences is polypeptide having an amino acid sequence corresponding to expressible as individual polypeptides, or alternatively, as a US 7,892,798 B2 39 40 set of larger fusion proteins (e.g., for phage display) contain ecules of the invention, e.g., Suppressing or increasing ing the set of MR sequences therein. There are a variety of expression or activity of the nucleic acid or protein molecules, methods which can be used to produce libraries of potential respectively, results in the modulation of methionine biosyn MR homologues from a degenerate oligonucleotide thesis. For example, a Corynebacterium glutamicum Strain sequence. Chemical synthesis of a degenerate gene sequence which overexpresses or has increased expression of can be performed in an automatic DNA synthesizer, and the RXNO2910, a positive regulator of methionine biosynthesis, synthetic gene then ligated into an appropriate expression displays a significant increase in the production of methion vector. Use of a degenerate set of genes allows for the provi ine or other compounds of the methionine biosynthetic path Sion, in one mixture, of all of the sequences encoding the way. Furthermore, a Corynebacterium glutamicum Strain desired set of potential MR sequences. Methods for synthe 10 which is deficient in or has suppressed expression of sizing degenerate oligonucleotides are known in the art (see, RXA00655, a negative regulator of methionine biosynthesis, e.g., Narang, S. A. (1983) Tetrahedron 39:3: Itakura et al. also displays a significant increase in the production of (1984) Annu. Rev. Biochem, 53:323: Itakura et al. (1984) methionine or other compounds of the methionine biosyn Science 198: 1056; Ike etal. (1983) Nucleic Acid Res. 11:477. thetic pathway. In addition, libraries offragments of the MR protein coding 15 Furthermore, manipulation of the MR nucleic acid mol can be used to generate a variegated population of MR frag ecules of the invention may lead to production of MR proteins ments for Screening and Subsequent selection of homologues having functional differences from the wild-type MR pro of an MR protein. In one embodiment, a library of coding teins. These proteins may be improved in efficiency or activ sequence fragments can be generated by treating a double ity, may be present in greater numbers in the cell than is usual, stranded PCR fragment of an MR coding sequence with a or may be decreased in efficiency or activity. nuclease under conditions wherein nicking occurs only about The invention provides methods for screening molecules once per molecule, denaturing the double stranded DNA, which modulate the activity of an MR protein, either by renaturing the DNA to form double stranded DNA which can interacting with the protein itself or a substrate or binding include sense/antisense pairs from different nicked products, partner of the MR protein, or by modulating the transcription removing single stranded portions from reformed duplexes 25 or translation of an MR nucleic acid molecule of the inven by treatment with S1 nuclease, and ligating the resulting tion. In Such methods, a microorganism expressing one or fragment library into an expression vector. By this method, an more MR proteins of the invention is contacted with one or expression library can be derived which encodes N-terminal, more test compounds, and the effect of each test compound on C-terminal and internal fragments of various sizes of the MR the activity or level of expression of the MR protein is protein. 30 assessed. Several techniques are known in the art for screening gene Such changes in activity may directly modulate the yield, products of combinatorial libraries made by point mutations production, and/or efficiency of production of one or more or truncation, and for Screening cDNA libraries for gene fine chemicals from C. glutamicum, e.g., methionine. For products having a selected property. Such techniques are example, by optimizing the activity of an MR protein, e.g., a adaptable for rapid screening of the gene libraries generated 35 positive regulator of methionine biosynthesis, e.g., by the combinatorial mutagenesis of MR homologues. The RXNO2910, which activates the transcription or translation of most widely used techniques, which are amenable to high a gene encoding a biosynthetic protein for a desired fine through-put analysis, for Screening large gene libraries typi chemical, or by impairing or abrogating the activity of an MR cally include cloning the gene library into replicable expres protein, e.g., a negative regulator of methionine biosynthesis, sion vectors, transforming appropriate cells with the resulting 40 e.g., RXA00655, which represses the transcription or trans library of vectors, and expressing the combinatorial genes lation of such a gene, one may also increase the activity or rate under conditions in which detection of a desired activity ofactivity of that biosynthetic pathway. Similarly, by altering facilitates isolation of the vector encoding the gene whose the activity of an MR protein such that it constitutively post product was detected. Recursive ensemble mutagenesis translationally inactivates a protein involved in a degradation (REM), a new technique which enhances the frequency of 45 pathway for a desired fine chemical, or by altering the activity functional mutants in the libraries, can be used in combina of an MR protein such that it constitutively represses the tion with the Screening assays to identify MR homologues transcription or translation of Such a gene, one may increase (Arkin and Yourvan (1992) PNAS89:7811-7815; Delgrave et the yield and/or rate of production of the fine chemical from al. (1993) Protein Engineering 6(3):327-331). the cell, due to decreased degradation of the compound. In another embodiment, cell based assays can be exploited 50 Further, by modulating the activity of one or more MR to analyze a variegated MR library, using methods well proteins, one may indirectly stimulate the production or known in the art. improve the rate of production of one or more fine chemicals from the cell due to the interrelatedness of disparate meta D. Uses and Methods of the Invention bolic pathways. For example, by increasing the yield, produc The nucleic acid molecules, proteins, proteinhomologues, 55 tion, and/or efficiency of production by activating the expres fusion proteins, primers, vectors, and host cells described sion of one or more lysine biosynthetic enzymes, one may herein can be used in one or more of the following methods: concomitantly increase the expression of other compounds, modulation of cellular production of a desired compound, Such as other amino acids, which the cell would naturally Such as a fine chemical, e.g., methionine; identification of C. require in greater quantities when lysine is required in greater glutamicum and related organisms; mapping of of 60 quantities. Also, regulation of metabolism throughout the cell organisms related to C. glutamicum; identification and local may be altered such that the cell is better able to grow or ization of C. glutamicum sequences of interest, evolutionary replicate under the environmental conditions of fermentative studies; determination of MR protein regions required for culture (where nutrient and oxygen Supplies may be poor and function; modulation of an MR protein activity; and modula possibly toxic waste products in the environment may be at tion of the activity of one or more metabolic pathways. 65 high levels). For example, by mutagenizing an MR protein The MR nucleic acid molecules of the invention have a which represses the synthesis of molecules necessary for cell variety of uses. Manipulation of the MR nucleic acid mol membrane production in response to high levels of waste US 7,892,798 B2 41 42 products in the extracellular medium (in order to block cell homologous to C. diphtheriae nucleic acid and protein mol growth and division in Suboptimal growth conditions) Such ecules, and can therefore be used to detect C. diphtheriae in a that it no longer is able to repress Such synthesis, one may Subject. increase the growth and multiplication of the cell in cultures The nucleic acid and protein molecules of the invention even when the growth conditions are suboptimal. Such may also serve as markers for specific regions of the genome. enhanced growth or viability should also increase the yields This has utility not only in the mapping of the genome, but and/or rate of production of a desired fine chemical from also for functional studies of C. glutamicum proteins. For fermentative culture, due to the relatively greater number of example, to identify the region of the genome to which a cells producing this compound in the culture. particular C. glutamicum DNA-binding protein binds, the C. The aforementioned mutagenesis strategies for MR pro 10 glutamicum genome could be digested, and the fragments teins to result in increased yields of a fine chemical, e.g., incubated with the DNA-binding protein. Those which bind methionine, from C. glutamicum are not meant to be limiting: the protein may be additionally probed with the nucleic acid variations on these strategies will be readily apparent to one molecules of the invention, preferably with readily detectable of ordinary skill in the art. Using Such strategies, and incor labels; binding of Such a nucleic acid molecule to the genome porating the mechanisms disclosed herein, the nucleic acid 15 fragment enables the localization of the fragment to the and protein molecules of the invention may be utilized to genome map of C. glutamicum, and, when performed mul generate C. glutamicum or related Strains of bacteria express tiple times with different enzymes, facilitates a rapid deter ing mutated MR nucleic acid and protein molecules such that mination of the nucleic acid sequence to which the protein the yield and/or efficiency of production of a desired com binds. Further, the nucleic acid molecules of the invention pound is improved. This desired compound may be any natu may be sufficiently homologous to the sequences of related ral product of C. glutamicum, which includes the final prod species such that these nucleic acid molecules may serve as ucts of biosynthesis pathways and intermediates of naturally markers for the construction of a genomic map in related occurring metabolic pathways, as well as molecules which do bacteria, Such as Brevibacterium lactofermentum. not naturally occur in the metabolism of C. glutamicum, but The MR nucleic acid molecules of the invention are also which are produced by a C. glutamicum Strain of the inven 25 useful for evolutionary and protein structural studies. The metabolic processes in which the molecules of the invention tion. participate are utilized by a wide variety of prokaryotic and The MR molecules of the invention may also be used to eukaryotic cells; by comparing the sequences of the nucleic identify an organism as being Corynebacterium glutamicum acid molecules of the present invention to those encoding or a close relative thereof. Also, they may be used to identify 30 similar enzymes from other organisms, the evolutionary relat the presence of C. glutamicum or a relative thereof in a mixed edness of the organisms can be assessed. Similarly, such a population of microorganisms. The invention provides the comparison permits an assessment of which regions of the nucleic acid sequences of a number of C. glutamicum genes; sequence are conserved and which are not, which may aid in by probing the extracted genomic DNA of a culture of a determining those regions of the protein which are essential unique or mixed population of microorganisms under strin 35 for the functioning of the enzyme. This type of determination gent conditions with a probe spanning a region of a C. is of value for protein engineering studies and may give an glutamicum gene which is unique to this organism, one can indication of what the protein can tolerate in terms of ascertain whether this organism is present. mutagenesis without losing function. Although Corynebacterium glutamicum itself is nonpatho This invention is further illustrated by the following genic, it is related to pathogenic species, such as Corynebac 40 examples which should not be construed as limiting. The terium diphtheriae. Corynebacterium diphtheriae is the caus contents of all references, Figure, patent applications, pat ative agent of diphtheria, a rapidly developing, acute, febrile ents, published patent applications, Tables, and the Sequence infection which involves both local and systemic pathology. Listing cited throughout this application are hereby incorpo In this disease, a local lesion develops in the upper respiratory rated by reference. tract and involves necrotic injury to epithelial cells; the bacilli 45 secrete toxin which is disseminated through this lesion to EXAMPLES distal Susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these Example 1 tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic 50 Preparation of Total Genomic DNA of pathology of the disease. Diphtheria continues to have high Corynebacterium Glutamicum ATCC 13032 incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former A culture of Corynebacterium glutamicum (ATCC 13032) Soviet Union. An ongoing epidemic of diphtheria in the latter was grown overnight at 30°C. with vigorous shaking in BHI two regions has resulted in at least 5,000 deaths since 1990. 55 medium (Difico). The cells were harvested by centrifugation, In one embodiment, the invention provides a method of the Supernatant was discarded and the cells were resuspended identifying the presence or activity of Cornyebacterium diph in 5 ml buffer-I (5% of the original volume of the culture—all theriae in a subject. This method includes detection of one or indicated volumes have been calculated for 100 ml of culture more of the nucleic acid or amino acid sequences of the volume). Composition of buffer-I: 140.34 g/1 sucrose, 2.46g/1 invention (e.g., the sequences set forth in SEQID NO: 1, SEQ 60 MgSO4x7H2O, 10 ml/l KHPO solution (100 g/l, adjusted to ID NO:5, SEQID NO: 16, SEQID NO: 18, SEQID NO:20, pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/1 or SEQID NO: 22 or SEQID NO: 2, SEQID NO: 6, SEQID (NH4)2SO 1 g/l NaCl, 2 g/l MgSOx7H2O, 0.2 g/l CaCl, NO: 17, SEQID NO:19, SEQIDNO:21, or SEQID NO. 23) 0.5 g/l yeast extract (Difico), 10 ml/l trace-elements-mix (200 in a subject, thereby detecting the presence or activity of mg/l FeSOXHO, 10 mg/l ZnSOax7 HO, 3 mg/l MnClx4 Corynebacterium diphtheriae in the Subject. C. glutamicum 65 H2O, 30 mg/l HBO 20 mg/l CoClax6 HO, 1 mg/l NiClax6 and C. diphtheriae are related bacteria, and many of the H2O, 3 mg/l NaMoOx2 H2O, 500 mg/l complexing agent nucleic acid and protein molecules in C. glutamicum are (EDTA or critic acid), 100 ml/l vitamins-mix (0.2 mg/l biotin, US 7,892,798 B2 43 44 0.2 mg/l folic acid, 20 mg/l p-amino benzoic acid, 20 mg/1 5'-GGAAACAGTATGACCATG-3' (SEQ ID NO: 24) or riboflavin, 40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 5'-GTAAAACGACGGCCAGT3' (SEQID NO:25). 40 mg/l pyridoxole hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the Suspension to a final concentra Example 4 tion of 2.5 mg/ml. After an approximately 4h incubation at 37° C., the cell wall was degraded and the resulting proto In Vivo Mutagenesis plasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM In vivo mutagenesis of Corynebacterium glutamicum can Tris-HCl, 1 mM EDTA, pH8). The pellet was resuspended in be performed by passage of plasmid (or other vector) DNA 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml 10 through E. coli or other microorganisms (e.g. Bacillus spp. or NaCl solution (5 M) are added. After adding of proteinase K yeasts Such as ) which are impaired to a final concentration of 200 ug/ml, the Suspension is incu in their capabilities to maintain the integrity of their genetic bated for ca. 18h at 37°C. The DNA was purified by extrac information. Typical mutator Strains have mutations in the tion with phenol, phenol-chloroform-isoamylalcohol and genes for the DNA repair system (e.g., mutHLS, mutD, mutT. chloroform-isoamylalcohol using standard procedures. 15 etc.; for reference, see Rupp, W. D. (1996) DNA repair Then, the DNA was precipitated by adding 1/50 volume of 3 mechanisms, in: Escherichia coli and Salmonella, p. 2277 M sodium acetate and 2 volumes of ethanol, followed by a 30 2294, ASM: Washington.) Such strains are well known to one min incubation at -20° C. and a 30 min centrifugation at of ordinary skill in the art. The use of such strains is illus 12,000 rpm in a high speed centrifuge using a SS34 rotor trated, for example, in Greener, A. and Callahan, M. (1994) (Sorvall). The DNA was dissolved in 1 ml TE-buffer contain Strategies 7: 32-34. ing 20 ug/ml RNaseA and dialysed at 4°C. against 1000 ml TE-buffer for at least 3 hours. During this time, the buffer was Example 5 exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2M LiCl and 0.8 ml of ethanol are added. DNA Transfer Between Escherichia Coli and After a 30 min incubation at -20°C., the DNA was collected 25 Corynebacterium Glutamicum by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE Several Corynebacterium and Brevibacterium species con buffer. DNA prepared by this procedure could be used for all tain endogenous plasmids (as e.g., pHM1519 or plBL1) which purposes, including Southern blotting or construction of replicate autonomously (for review see, e.g., Martin, J. F. et genomic libraries. 30 al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be Example 2 readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), “Molecular Cloning: A Labora Construction of Genomic Libraries in Escherichia tory Manual', Cold Spring Harbor Laboratory Press or Coli of Corynebacterium Glutamicum ATCC13032 35 Ausubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley & Sons) to whicha origin or replication Using DNA prepared as described in Example 1, cosmid for and a Suitable marker from Corynebacterium glutamicum and plasmid libraries were constructed according to known is added. Such origins of replication are preferably taken from and well established methods (see e.g., Sambrook, J. et al. endogenous plasmids isolated from Corynebacterium and (1989) “Molecular Cloning: A Laboratory Manual, Cold 40 Brevibacterium species. Of particular use as transformation Spring Harbor Laboratory Press, or Ausubel, F. M. et al. markers for these species are genes for kanamycin resistance (1994) “Current Protocols in Molecular Biology”, John (such as those derived from the Tn5 or Tn903 transposons) or Wiley & Sons.) chloramphenicol (Winnacker, E. L. (1987) “From Genes to Any plasmid or cosmid could be used. Of particular use Clones—Introduction to Gene Technology, VCH, Wein were the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. 45 heim). There are numerous examples in the literature of the Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & construction of a wide variety of shuttle vectors which repli Cohen (1978).J. Bacteriol 134:1141-1156), plasmids of the cate in both E. coli and C. glutamicum, and which can be used pBS series (p3SSK+, pBSSK- and others; Stratagene, for several purposes, including gene over-expression (for ref LaJolla, USA), or cosmids as SuperCoS1 (Stratagene, erence, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol. LaJolla, USA) or Lorist6 (Gibson, T. J., Rosenthal A. and 50 162:591-597, Martin J. F. et al. (1987) Biotechnology, 5:137 Waterson, R. H. (1987) Gene 53:283-286. Gene libraries 146 and Eikmanns, B.J. et al. (1991) Gene, 102:93-98). specifically for use in C. glutamicum may be constructed Using standard methods, it is possible to clone a gene of using plasmid pSL109 (Lee, H.-S. and A.J. Sinskey (1994).J. interest into one of the shuttle vectors described above and to Microbiol. Biotechnol. 4: 256-263). introduce such a hybrid vectors into strains of Corynebacte 55 rium glutamicum. Transformation of C. glutamicum can be Example 3 achieved by protoplast transformation (KastSumata, R. et al. (1984).J. Bacteriol. 159306–311), electroporation (Liebl, E. DNA Sequencing and Computational Functional et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in Analysis cases where special vectors are used, also by conjugation (as 60 described e.g. in Schäfer, A et al. (1990).J. Bacteriol. 172: Genomic libraries as described in Example 2 were used for 1663-1666). It is also possible to transfer the shuttle vectors DNA sequencing according to standard methods, in particu for C. glutamicum to E. coli by preparing plasmid DNA from lar by the chain termination method using ABI377 sequenc C. glutamicum (using standard methods well-known in the ing machines (see e.g., Fleischman, R. D. et al. (1995) art) and transforming it into E. coli. This transformation step “Whole-genome Random Sequencing and Assembly of Hae 65 can be performed using standard methods, but it is advanta mophilus Influenzae Rd., Science, 269:496-512). Sequencing geous to use an Mcr-deficient E. coli strain, such as NM522 primers with the following nucleotide sequences were used: (Gough & Murray (1983).J. Mol. Biol. 166:1-19). US 7,892,798 B2 45 46 Genes may be overexpressed in C. glutamicum Strains tity of label observed indicates the presence and quantity of using plasmids which comprise pCO1 (U.S. Pat. No. 4,617, the desired mutant protein present in the cell. 267) or fragments thereof, and optionally the gene for kana mycin resistance from TN903 (Grindley, N. D. and Joyce, C. Example 7 M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In Growth of Corynebacterium Glutamicum—Media addition, genes may be overexpressed in C. glutamicum and Culture Conditions strains using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994).J. Microbiol. Biotechnol. 4: 256-263). Corynebacteria are cultured in synthetic or natural growth Aside from the use of replicative plasmids, gene overex 10 media. A number of different growth media for Corynebac pression can also be achieved by integration into the genome. teria are both well-known and readily available (Lieb et al. Genomic integration in C. glutamicum or other Corynebac (1989) Appl. Microbiol. Biotechnol., 32:205-210; von der terium or Brevibacterium species may be accomplished by Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent well-known methods, such as homologous recombination DE 4,120,867; Liebl (1992) “The Genus Corynebacterium, 15 in: The Procaryotes, Volume II, Balows, A. et al., eds. with genomic region(s), restriction endonuclease mediated Springer-Verlag). These media consist of one or more carbon integration (REMI) (see, e.g., DE Patent 19823834), or Sources, nitrogen Sources, inorganic salts, vitamins and trace through the use of transposons. It is also possible to modulate elements. Preferred carbon Sources are Sugars, such as mono-, the activity of a gene of interest by modifying the regulatory di, or . For example, glucose, fructose, man regions (e.g., a promoter, a repressor, and/or an enhancer) by nose, galactose, ribose, Sorbose, ribulose, lactose, maltose, sequence modification, insertion, or deletion using site-di Sucrose, raffinose, starch or cellulose serve as very good rected methods (such as homologous recombination) or carbon Sources. It is also possible to Supply Sugar to the media methods based on random events (such as transposon via complex compounds such as molasses or other by-prod mutagenesis or REMI). Nucleic acid sequences which func ucts from Sugar refinement. It can also be advantageous to 25 supply mixtures of different carbon sources. Other possible tion as transcriptional terminators may also be inserted 3' to carbon sources are alcohols and organic acids, such as metha the coding region of one or more genes of the invention. Such nol, ethanol, acetic acid or lactic acid. Nitrogen Sources arc terminators are well-known in the art and are described, for usually organic or inorganic nitrogen compounds, or materi example, in Winnacker, E. L. (1987) From Genes to Clones— als which contain these compounds. Exemplary nitrogen Introduction to Gene Technology. VCH: Weinheim. 30 Sources include ammonia gas or ammonia salts, such as NHCl or (NH4)2SO. NH-OH, nitrates urea, amino acids or Example 6 complex nitrogen Sources like corn steep liquor, Soy bean flour, soybean protein, yeast extract, meat extract and others. Assessment of the Expression of the Mutant Protein Inorganic salt compounds which may be included in the 35 media include the chloride-, phosphorous- or Sulfate-salts of calcium, magnesium, Sodium, cobalt, molybdenum, potas Observations of the activity of a mutated protein in a trans sium, manganese, Zinc, copper and iron. Chelating com formed host cell rely on the fact that the mutant protein is pounds can be added to the medium to keep the metal ions in expressed in a similar fashion and in a similar quantity to that Solution. Particularly useful chelating compounds include of the wild-type protein. A useful method to ascertain the 40 dihydroxyphenols, like catechol or protocatechuate, or level of transcription of the mutant gene (an indicator of the organic acids, such as citric acid. It is typical for the media to amount of mRNA available for translation to the gene prod also contain other growth factors, such as vitamins or growth uct) is to perform a Northern blot (for reference see, for promoters, examples of which include biotin, riboflavin, thia example, Ausubel et al. (1988) Current Protocols in Molecu min, folic acid, nicotinic acid, pantothenate and pyridoxin. lar Biology, Wiley: N.Y.), in which a primer designed to bind 45 Growth factors and salts frequently originate from complex to the gene of interest is labeled with a detectable tag (usually media components such as yeast extract, molasses, corn steep radioactive or chemiluminescent). Such that when the total liquor and others. The exact composition of the media com RNA of a culture of the organism is extracted, run on gel. pounds depends strongly on the immediate experiment and is transferred to a stable matrix and incubated with this probe, individually decided for each specific case. Information about the binding and quantity of binding of the probe indicates the 50 media optimization is available in the textbook “Applied presence and also the quantity of mRNA for this gene. This Microbiol. Physiology, A Practical Approach (eds. P. M. information is evidence of the degree of transcription of the Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 mutant gene. Total cellular RNA can be prepared from 199635773). It is also possible to select growth media from Corynebacterium glutamicum by several methods, all well commercial suppliers, like standard 1 (Merck) or BHI (brain known in the art, such as that described in Bormann, E. R. et 55 heart infusion, DIFCO) or others. al. (1992) Mol. Microbiol. 6:317-326. All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121°C.) or by sterile filtration. The To assess the presence or relative quantity of protein trans components can either be sterilized together or, if necessary, lated from this mRNA, standard techniques, such as a West separately. All media components can be present at the begin ern blot, may be employed (see, for example, Ausubel et al. 60 ning of growth, or they can optionally be added continuously (1988) Current Protocols in Molecular Biology, Wiley: N.Y.). or batchwise. In this process, total cellular proteins are extracted, separated Culture conditions are defined separately for each experi by gel electrophoresis, transferred to a matrix Such as nitro ment. The temperature should be in a range between 15° C. cellulose, and incubated with a probe, such as an antibody, and 45° C. The temperature can be kept constant or can be which specifically binds to the desired protein. This probe is 65 altered during the experiment. The pH of the medium should generally tagged with a chemiluminescent or calorimetric be in the range of 5 to 8.5, preferably around 7.0, and can be label which may be readily detected. The presence and quan maintained by the addition of buffers to the media. An exem US 7,892,798 B2 47 48 plary buffer for this purpose is a potassium phosphate buffer. effect of such proteins on the expression of other molecules Synthetic buffers such as MOPS, HEPES, ACES and others can be measured using reporter gene assays (such as that can alternatively or simultaneously be used. It is also possible described in Kolmar, H. et al. (1995) EMBO.J. 14:3895-3904 to maintain a constant culture pH through the addition of and references cited therein). Reporter gene test systems are NaOH or NHOH during growth. If complex medium com 5 well known and established for applications in both pro- and ponents such as yeast extract are utilized, the necessity for eukaryotic cells, using enzymes Such as beta-galactosidase, additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermen green fluorescent protein, and several others. tor is utilized for culturing the micro-organisms, the pH can The determination of activity of membrane-transport pro also be controlled using gaseous ammonia. 10 teins can be performed according to techniques such as those The incubation time is usually in a range from several described in Gennis, R. B. (1989) “Pores, Channels and hours to several days. This time is selected in order to permit Transporters', in Biomembranes, Molecular Structure and the maximal amount of product to accumulate in the broth. Function, Springer: Heidelberg, p. 85-137; 199-234; and 270 The disclosed growth experiments can be carried out in a 322. variety of vessels, such as microtiter plates, glass tubes, glass 15 flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms Example 9 should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml shake Analysis of Impact of Mutant Protein on the flasks are used, filled with 10% (by volume) of the required Production of the Desired Product growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100-300 The effect of the genetic modification in C. glutamicum on rpm. Evaporation losses can be diminished by the mainte production of a desired compound (such as an amino acid) nance of a humid atmosphere; alternatively, a mathematical can be assessed by growing the modified microorganism correction for evaporation losses should be performed. 25 If genetically modified clones are tested, an unmodified under suitable conditions (such as those described above) and control clone or a control clone containing the basic plasmid analyzing the medium and/or the cellular component for without any insert should also be tested. The medium is increased production of the desired product (i.e., an amino inoculated to an ODoo of O0.5-1.5 using cells grown on agar acid). Such analysis techniques are well known to one of plates, such as CM plates (10 g/l glucose, 2.5 g/l NaCl, 2 g/1 30 ordinary skill in the art, and include spectroscopy, thin layer urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, chromatography, staining methods of various kinds, enzy 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, matic and microbiological methods, and analytical chroma 5 g/l meat extract, 22 g/1 agar, pH 6.8 with 2MNaOH) that had tography Such as high performance liquid chromatography been incubated at 30°C. Inoculation of the media is accom (see, for example, Ullman, Encyclopedia of Industrial Chem plished by either introduction of a saline suspension of C. 35 glutamicum cells from CM plates or addition of a liquid istry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) “Applications of HPLC in preculture of this bacterium. Biochemistry” in: Laboratory Techniques in Biochemistry Example 8 and Molecular Biology, Vol. 17: Rehmet al. (1993) Biotech 40 nology, Vol. 3, Chapter III: “Product recovery and purifica In Vitro Analysis of the Function of Mutant Proteins tion”, page 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstream processing for biotech The determination of activities and kinetic parameters of nology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. enzymes is well established in the art. Experiments to deter S. (1992) Recovery processes for biological materials, John mine the activity of any given altered enzyme must be tailored 45 to the specific activity of the wild-type enzyme, which is well Wiley and Sons: Shaeiwitz, J. A. and Henry, J. D. (1988) within the ability of one of ordinary skill in the art. Overviews Biochemical separations, in: Ulmann's Encyclopedia of about enzymes in general, as well as specific details concern Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: ing structure, kinetics, principles, methods, applications and Weinheim; and Dechow, F. J. (1989) Separation and purifi examples for the determination of many enzyme activities 50 cation techniques in biotechnology, Noyes Publications.) may be found, for example, in the following references: In addition to the measurement of the final product of Dixon, M., and Webb, E. C., (1979) Enzymes. Longmans: fermentation, it is also possible to analyze other components London: Fersht, (1985) Enzyme Structure and Mechanism. of the metabolic pathways utilized for the production of the Freeman: N.Y.; Walsh, (1979) Enzymatic Reaction Mecha desired compound, such as intermediates and side-products, nisms. Freeman: San Francisco; Price, N. C., Stevens, L. 55 (1982) Fundamentals of Enzymology. Oxford Univ. Press: to determine the overall yield, production, and/or efficiency Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3" ed. Aca of production of the compound. Analysis methods include demic Press: New York; Bisswanger, H., (1994) Enzymkine measurements of nutrient levels in the medium (e.g., Sugars, tik, 2" ed. VCH: Weinheim (ISBN 3527300325); Bergm hydrocarbons, nitrogen sources, phosphate, and other ions), eyer, H. U. Bergmeyer, J., Graf31, M., eds. (1983-1986) 60 measurements of biomass composition and growth, analysis Methods of Enzymatic Analysis, 3" ed., vol. I-XII, Verlag of the production of common metabolites of biosynthetic Chemie: Weinheim; and Ullmann's Encyclopedia of Indus pathways, and measurement of gasses produced during fer trial Chemistry (1987) vol. A9, “Enzymes”. VCH: Weinheim, mentation. Standard methods for these measurements are p. 352-363. outlined in Applied Microbial Physiology, A Practical The activity of proteins which bind to DNA can be mea 65 Approach, P. M. Rhodes and P. F. Stanbury, eds. IRL Press, p. sured by several well-established methods, such as DNA 103-129; 131-163; and 165-192 (ISBN: 0199635773) and band-shift assays (also called gel retardation assays). The references cited therein. US 7,892,798 B2 49 50 Example 10 obtain nucleotide sequences homologous to MR nucleic acid molecules of the invention. BLAST protein searches can be Purification of the Desired Product from C. performed with the XBLAST program, score-50, Glutamicum Culture wordlength 3 to obtain amino acid sequences homologous to MR protein molecules of the invention. To obtain gapped Recovery of the desired product from the C. glutamicum alignments for comparison purposes, Gapped BLAST can be cells or supernatant of the above-described culture can be utilized as described in Altschul et al., (1997) Nucleic Acids performed by various methods well known in the art. If the Res. 25(17):3389-3402. When utilizing BLAST and Gapped desired product is not secreted from the cells, the cells can be BLAST programs, one of ordinary skill in the art will know harvested from the culture by low-speed centrifugation, the 10 how to optimize the parameters of the program (e.g., cells can be lysed by Standard techniques. Such as mechanical XBLAST and NBLAST) for the specific sequence being force or sonication. The cellular debris is removed by cen analyzed. trifugation, and the Supernatant fraction containing the Another example of a mathematical algorithm utilized for soluble proteins is retained for further purification of the the comparison of sequences is the algorithm of Meyers and desired compound. If the product is secreted from the C. 15 Miller (1988) Comput. Appl. Biosci. 4: 11-17). Such an glutamicum cells, then the cells are removed from the culture algorithm is incorporated into the ALIGN program (version by low-speed centrifugation, and the Supernate fraction is 2.0) which is part of the GCG sequence alignment software retained for further purification. package. When utilizing the ALIGN program for comparing The supernatant fraction from either purification method is amino acid sequences, a PAM 120 weight residue table, a gap Subjected to chromatography with a suitable resin, in which length penalty of 12, and a gap penalty of 4 can be used. the desired molecule is either retained on a chromatography Additional algorithms for sequence analysis are known in the resin while many of the impurities in the sample are not, or art, and include ADVANCE and ADAM. described in Torelli where the impurities are retained by the resin while the and Robotti (1994) Comput. Appl. Biosci. 10:3-5; and sample is not. Such chromatography steps may be repeated as FASTA, described in Pearson and Lipman (1988) PN.A.S. necessary, using the same or different chromatography resins. 25 85:2444-8. One of ordinary skill in the art would be well-versed in the The percent homology between two amino acid sequences selection of appropriate chromatography resins and in their can also be accomplished using the GAP program in the GCG most efficacious application for a particular molecule to be Software package (available at www.gcg.com), using either a purified. The purified product may be concentrated by filtra Blosum 62 matrix or a PAM250 matrix, and a gap weight of tion or ultrafiltration, and stored at a temperature at which the 30 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent stability of the product is maximized. homology between two nucleic acid sequences can be accom There are a wide array of purification methods known to plished using the GAP program in the GCG software pack the art and the preceding method of purification is not meant age, using standard parameters, such as a gap weight of 50 to be limiting. Such purification techniques are described, for and a length weight of 3. example, in Bailey, J. E. & Ollis, D. F. Biochemical Engineer 35 ing Fundamentals, McGraw-Hill: New York (1986). Example 12 The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include Construction and Operation of DNA Microarrays high-performance liquid chromatography (HPLC), spectro scopic methods, staining methods, thin layer chromatogra 40 The sequences of the invention may additionally be used in phy, NIRS, enzymatic assay, or microbiologically. Such the construction and application of DNA microarrays (the analysis methods are reviewed in: Patek et al. (1994) Appl. design, methodology, and uses of DNA arrays are well known Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) in the art, and are described, for example, in Schena, M. et al. Biotekhnologiya 11:27-32; and Schmidt etal. (1998) Biopro (1995) Science 270: 467-470; Wodicka, L. et al. (1997) cess Engineer. 19: 67-70. Ulmann's Encyclopedia of Indus 45 Nature Biotechnology 15: 1359–1367; DeSaizieu, A. et al. trial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, (1998) Nature Biotechnology 16: 45-48; and DeRisi, J. L. et p. 521-540, p. 540-547, p. 559-566,575-581 and p. 581-587; al. (1997) Science 278: 680-686). Michal, G. (1999) Biochemical Pathways: An Atlas of Bio DNA microarrays are solid or flexible supports consisting chemistry and Molecular Biology, John Wiley and Sons: Fal of nitrocellulose, nylon, glass, silicone, or other materials. lon, A. etal. (1987) Applications of HPLC in Biochemistry in: 50 Nucleic acid molecules may be attached to the surface in an Laboratory Techniques in Biochemistry and Molecular Biol ordered manner. After appropriate labeling, other nucleic ogy, Vol. 17. acids or nucleic acid mixtures can be hybridized to the immo bilized nucleic acid molecules, and the label may be used to Example 11 monitor and measure the individual signal intensities of the 55 hybridized molecules at defined regions. This methodology Analysis of the Gene Sequences of the Invention allows the simultaneous quantification of the relative or abso lute amount of all or selected nucleic acids in the applied The comparison of sequences and determination of percent nucleic acid sample or mixture. DNA microarrays, therefore, homology between two sequences are art-known techniques, permit an analysis of the expression of multiple (as many as and can be accomplished using a mathematical algorithm, 60 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. such as the algorithm of Karlin and Altschul (1990) Proc. (1996) BioEssays 18(5): 427-431). Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and The sequences of the invention may be used to design Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-77. Such oligonucleotide primers which are able to amplify defined an algorithm is incorporated into the NBLAST and XBLAST regions of one or more C. glutamicum genes by a nucleic acid programs (version 2.0) of Altschul, et al. (1990).J. Mol Biol. 65 amplification reaction Such as the polymerase chain reaction. 215:403-10. BLAST nucleotide searches can be performed The choice and design of the 5' or 3' oligonucleotide primers with the NBLAST program, score=100, wordlength=12 to or of appropriate linkers allows the covalent attachment of the US 7,892,798 B2 51 52 resulting PCR products to the surface of a support medium not only the amino acid sequence but also posttranslational described above (and also described, for example, Schena, M. modifications of the protein). Another, more preferred et al. (1995) Science 270: 467-470). method of protein analysis is the consecutive combination of Nucleic acid microarrays may also be constructed by in both IEF-PAGE and SDS-PAGE, known as 2-D-gel electro situ oligonucleotide synthesis as described by Wodicka, L. et 5 phoresis (described, for example, in Hermann et al. (1998) al. (1997) Nature Biotechnology 15: 1359–1367. By photo Electrophoresis 19: 3217-3221: Fountoulakis et al. (1998) lithographic methods, precisely defined regions of the matrix Electrophoresis 19: 1193-1202: Langen et al. (1997) Electro are exposed to light. Protective groups which are photolabile phoresis 18: 1184-1192; Antelmann et al. (1997) Electro are thereby activated and undergo nucleotide addition, phoresis 18: 1451-1463). Other separation techniques may whereas regions that are masked from light do not undergo 10 also be utilized for protein separation, such as capillary gel any modification. Subsequent cycles of protection and light electrophoresis; such techniques are well known in the art. activation permit the synthesis of different oligonucleotides Proteins separated by these methodologies can be visual at defined positions. Small, defined regions of the genes of the ized by standard techniques, such as by staining or labeling. invention may be synthesized on microarrays by Solid phase Suitable stains are known in the art, and include Coomassie oligonucleotide synthesis. 15 Brilliant Blue, silver stain, or fluorescent dyes such as Sypro The nucleic acid molecules of the invention present in a Ruby (Molecular Probes). The inclusion of radioactively sample or mixture of nucleotides may be hybridized to the labeled amino acids or other protein precursors (e.g., S microarrays. These nucleic acid molecules can be labeled methionine, S-cysteine, C-labelled amino acids, N according to standard methods. In brief nucleic acid mol amino acids, "NO or 'NH" or 'C-labelled amino acids) ecules (e.g., mRNA molecules or DNA molecules) are in the medium of C. glutamicum permits the labeling of labeled by the incorporation of isotopically or fluorescently proteins from these cells prior to their separation. Similarly, labeled nucleotides, e.g., during reverse transcription or DNA fluorescent labels may be employed. These labeled proteins synthesis. Hybridization of labeled nucleic acids to microar can be extracted, isolated and separated according to the rays is described (e.g., in Schena, M. et al. (1995) supra; previously described techniques. Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. 25 Proteins visualized by these techniques can be furtherana (1998), supra). The detection and quantification of the hybrid lyzed by measuring the amount of dye or label used. The ized molecule are tailored to the specific incorporated label. amount of a given protein can be determined quantitatively Radioactive labels can be detected, for example, as described using, for example, optical methods and can be compared to in Schena, M. et al. (1995) supra) and fluorescent labels may the amount of other proteins in the same gel or in other gels. be detected, for example, by the method of Shalon et al. 30 Comparisons of proteins on gels can be made, for example, by (1996) Genome Research 6: 639-645). optical comparison, by spectroscopy, by image scanning and The application of the sequences of the invention to DNA analysis of gels, or through the use of photographic films and microarray technology, as described above, permits compara screens. Such techniques are well-known in the art. tive analyses of different strains of C. glutamicum or other To determine the identity of any given protein, direct Corynebacteria. For example, studies of inter-strain varia 35 sequencing or other standard techniques may be employed. tions based on individual transcript profiles and the identifi For example, N- and/or C-terminal amino acid sequencing cation of genes that are important for specific and/or desired (such as Edman degradation) may be used, as may mass strain properties Such as pathogenicity, productivity and spectrometry (in particular MALDI or ESI techniques (see, stress tolerance are facilitated by nucleic acid array method e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). ologies. Also, comparisons of the profile of expression of 40 The protein sequences provided herein can be used for the genes of the invention during the course of a fermentation identification of C. glutamicum proteins by these techniques. reaction are possible using nucleic acid array technology. The information obtained by these methods can be used to compare patterns of protein presence, activity, or modifica Example 13 tion between different samples from various biological con 45 ditions (e.g., different organisms, time points offermentation, Analysis of the Dynamics of Cellular Protein media conditions, or different biotopes, among others). Data Population (Proteomics) obtained from Such experiments alone, or in combination with other techniques, can be used for various applications, The genes, compositions, and methods of the invention Such as to compare the behavior of various organisms in a may be applied to study the interactions and dynamics of 50 given (e.g., metabolic) situation, to increase the productivity populations of proteins, termed proteomics. Protein popu of strains which produce fine chemicals or to increase the lations of interest include, but are not limited to, the total efficiency of the production of fine chemicals. protein population of C. glutamicum (e.g., in comparison with the protein populations of other organisms), those pro Example 14 teins which are active under specific environmental or meta 55 bolic conditions (e.g., during fermentation, at high or low Preparation of a Corynebacterium Glutamicum temperature, or at high or low pH), or those proteins which are Strain Deficient in the Negative Regulator of active during specific phases of growth and development. Methionine Biosynthesis (RXA00655) Protein populations can be analyzed by various well known techniques, such as gel electrophoresis. Cellular pro 60 Preparation of a Corynebacterium glutamicum Strain defi teins may be obtained, for example, by lysis or extraction, and cient in the negative regulator of methionine biosynthesis may be separated from one another using a variety of elec (RXA00655) is carried out by using a self-cloning technique trophoretic techniques. Sodium dodecyl sulfate polyacryla based on homologous recombination. The principle of said mide gel clectrophoresis (SDS-PAGE) separates proteins technique is visualized in FIG. 1. largely on the basis of their molecular weight. Isoelectric 65 The plasmid named pS deltaG55 (SEQ ID NO: 3) for focusing polyacrylamide gel electrophoresis (IEF-PAGE) preparation of a C. glutamicum Strain deficient in the negative separates proteins by their isoelectric point (which reflects regulator of methionine biosynthesis (RXA00655) is con US 7,892,798 B2 53 54 structed by ligating PCR amplified fragments of the 5’- and 3' regions of RXA00655 (SEQ ID NO: 1) into the vector pCLiK5MCS integrativ sacB (SEQ ID NO: 4) using stan dard methods (Sambrook et al. (1989), Molecular Cloning. A Medium II: Laboratory Manual, Cold Spring Harbor; Innis et al. (1990) 40 g/l Sucrose 60 g/l molasse (calculated on 100% Sugar content) PCR Protocols. A Guide to Methods and Applications, Aca 25 g/l (NH4)2SO4 demic Press). The PCR amplified 5'-fragment is flanked at its 0.4 g/l MgSO4*7H2O 0.6 g/l KH2PO4 5'-end by a primer-introduced XhoI site and at its 3'-end by an 0.3 mg/l thiamin HCl endogenous Nhe site. The PCR amplified 3'-fragment is 10 1 mg/l biotin (from a 1 mg/ml sterile filtered stock solution flanked at its 5'-end by an Nhe site followed by anTAA-stopp adjusted to pH 8.0 with NH4OH) 2 mg/l FeSO4 codon, which are introduced by the primer, and at its 3'-end by 2 mg/l MnSO4 a primer-introduced Mlul site. The pH is brought to pH 7.8 with NHOH. Thereafter, the pS. deltaG55 (SEQID NO:3) is electroporated into the C. medium is autoclaved (121°C., 20 min). Additional vitamin 15 B12 from a stock solution (200 g/ml, sterile filtered) is added glutamicum strain ATCC13032 as described (Liebl et al. to a final concentration of 200 g/l. (1989) FEMS Microbiol Let 53:299-303). Transformands with via intermolecular homologous recombination inte The amount of formed methionine was determined with an grated plasmid are selected on CMagar plates Supplemented Agilent 1100 series LC system HPLC according to a method with 50 mg/l kanamycin. 2O from Agilent. The with ortho-pthalaldehyde pre-column deri vated amino acids are separated on a Hypersil AA-column (Agilent). The strain ATCC13032 delta rxa(00655 produces CM-agar: significantly more methionine than ATCC13032. 10.0 g/L D-glucose 25 Example 16 2.5 g/L NaCl 2.0 g/L urea Preparation of a Corynebacterium Glutamicum 10.0 g/L bacto pepton (Difico) 5.0 g/L yeast extract (Difco) Strain Overexpressing the Positive Regulator of 5.0 g/L beef extract (Difco) Methionine Biosynthesis (RXN02910) 22.0 g/Lagar (Difco) 30 autoclaved (20 min. 121° C.) The plasmid named pGrxn2910 (SEQID NO:13) for over expressing the positive regulator of methionine biosynthesis (RXN02910; SEQID NO:5 or 6) is constructed by ligating a Kanamycin-resistant clones are incubated unselectively PCR amplified fragment of RXN02910 (SEQ ID NO:9), (without Kanamycin) overnight in CM medium to achieve which encodes the RXN02910 polypeptide (SEQID NO:10), excision of the plasmid together with the chromosomal copy 35 into the vector pG (SEQID NO:12) using standard methods (Sambrook et al. (1989), Molecular Cloning. A Laboratory of RXA00655. The cultures are plated on CMagar containing Manual, Cold Spring Harbor; Innis et al. (1990) PCR Proto 10% sucrose. Only those clones in which the integrated cols. A Guide to Methods and Applications, Academic Press). pS. deltað55 plasmid is excised can grow on CMagar con The following pair of oligonucleotide primers was used for taining Sucrose, because the sacB gene in pS deltað55 con- 40 the amplification: verts Sucrose into levan Sucrase, which is toxic for C. sense primer: (SEO ID NO: 7) glutamicum. In the excision either the deletion construct of 5 " - GAGAGACTCGAGTGGTTTAGGGGATGAGAAACCG-3' RXA00655 or the chromosomal copy of RXAO0655 is elimi nated together with the plasmid. antisense primer: (SEQ ID NO: 8) 45 5 - CTCTCACTAGTCTACCGCGCCAACAACAGCG-3 To identify a clone which has eliminated the chromosomal The PCR amplified fragment is flanked at its 5'-end by a copy of RXA00655, chromosomal DNA from all potential primer-introduced XhoI site and at its 3'-end by a primer clones are prepared (Tauchet al. (1995) Plasmid 33:168-179 introduced Bcul site. The amplified fragments contains the or Eikmanns et al. (1994) Microbiology 140:1817-1828) and open reading frame (ORF) of RXN02910 with additional 5' controlled with a Southern Blot analysis according to Sam- 50 regions of the corresponding gene including the promotor. brook et al. (1989), Molecular Cloning. A Laboratory The resulting plasmid pGrxn02910 (SEQ ID NO: 13) is Manual, Cold Spring Harbor. The knock-out strain is called electroporated into the C. glutamicum strain ATCC13032 as ATCC13032 delta rxaO0655. described (Liebl, et al. (1989) FEMS Microbiology Letters 53:299-303). Transformands are selected on CMagar plates Example 15 55 supplemented with 50 mg/l kanamycin (see above). The resulting strain is named ATCC 13032/pGrxn02910. Preparation of Methionine with Example 17 ATCC13032 DELTA RXAO0655 Preparation of Methionine with 60 The strains ATCC13032 and ATCC13032 delta rxa(00655 ATCC13032APGRXNO2910 are incubated on a CM-agar plate for 2d at 30°C. The cells are The strains ATCC13032 and ATCC13032/pGrxn02910 are scraped from the plate and Suspended in Saline. For the main incubated on a CM-agarplate for 2 days at 30°C. The cells are culture 10 ml medium II and 0.5g autoclaved CaCO (Riedel scraped from the plate and Suspended in Saline. For the main de Haen) in a 100 ml conical flaskare inoculated with the cell 65 culture 10 ml medium II (see above) and 0.5 g autoclaved suspension to a final OD600 nm of 1.5. Culturing is carried CaCO (Riedel de Haen) in a 100 ml conical flask are inocu out at 30° C. for 72 h. lated with the cell suspension to a final OD600 nm of 1.5. US 7,892,798 B2 55 56 Culturing is carried out at 30° C. for 72 h. In the case of PCR Protocols. A Guide to Methods and Applications, Aca ATCC13032/pGrxn02910 all plates and cultures contain demic Press). The PCR amplified fragment (SEQID NO: 11) additional 50 ug/l kanamycin. The amount of formed is flanked at its 5'-end by a primer-introduced XhoI site and at methionine was determined with an Agilent 1100 series LC its 3'-end by a primer-introduced EcoRV site. system HPLC according to a method from Agilent. The with pIntegrativ delta2910 (SEQID NO: 15) is electroporated ortho-phtalaldehyde pre-column derivated amino acids are into the C. glutamicum strain ATCC13032 as described (Liebl separated on a Hypersil AA-column (Agilent). The strain et al. (1989). FEMS Microbiol Let 53:299-303). Transfor mands with via intermolecular homologous recombination ATCC13032/pGrxn02910 produces significantly more integrated plasmid are selected on CM agar plates Supple methionine than ATCC13032. mented with 50 mg/l kanamycin. To identify a clone which Example 18 10 has an integrated plasmid and thus disrupted the chromo somal copy of RXNO2910, chromosomal DNA from poten tial clones are prepared (Tauch et al. (1995) Plasmid 33:168 Preparation of a Corynebacterium Glutamicum 179 or Eikmanns et al. (1994) Microbiology 140:1817-1828) Strain Deficient in the Positive Regulator of and controlled with a Southern Blot analysis according to Methionine Biosynthesis (RXN02910) 15 Sambrook et al. (1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. Preparation of a Corynebacterium glutamicum Strain defi The knock-out strain is called cient in the positive regulator of methionine biosynthesis ATCC13032 delta rXn02910. The amount of formed (RXNO2910) is carried out by insertion of a kanamycin methionine was determined with an Agilent 1100 series LC selectable vector. system HPLC according to a method from Agilent. The with The plasmid named pintegrativ delta2910 (SEQ ID NO: ortho-phtalaldehyde pre-column derivated amino acids are 15) for preparation of a C. glutamicum Strain deficient in the regulator of methionine biosynthesis (rxn02910) is con separated on a Hypersil AA-column (Agilent). structed by ligating a PCR amplified fragment of rxn02910 The strain ATCC13032 delta rxn02910 produces signifi into the vector pintegrativ (SEQ ID NO: 14) using standard cant less methionine than ATCC13032, thereby demonstrat methods (Sambrook et al. (1989), Molecular Cloning. A 25 ing that rxn02910 is indeed a positive regulator of methionine Laboratory Manual, Cold Spring Harbor; Innis et al. (1990) biosynthesis.

TABLE 1. Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention

Genus species ATCC FERM NRRL CECT NCIMB CBS NCTC DSMZ

Brevibacterium ammoniagenes 2 1054 Brevibacterium ammoniagenes 1 9350 Brevibacterium ammoniagenes 1 9351 Brevibacterium ammoniagenes 1 9352 Brevibacterium ammoniagenes 1 9353 Brevibacterium ammoniagenes 1 9354 Brevibacterium ammoniagenes 1 9355 Brevibacterium ammoniagenes 1 9356 Brevibacterium ammoniagenes 2 055 Brevibacterium ammoniagenes O77 Brevibacterium ammoniagenes 553 Brevibacterium ammoniagenes S8O Brevibacterium ammoniagenes 9101 Brevibacterium butanicum 196 Brevibacterium divaricatum 792 P928 Brevibacterium flavum 474 Brevibacterium flavum 129 Brevibacterium flavum 518 Brevibacterium flavum B11474 Brevibacterium flavum B11472 Brevibacterium flavum 127 Brevibacterium flavum 128 Brevibacterium flavum 427 Brevibacterium flavum 475 Brevibacterium flavum 517 Brevibacterium flavum 528 Brevibacterium flavum 529 Brevibacterium flavum B11477 Brevibacterium flavum B11478 Brevibacterium flavum 127 Brevibacterium flavum B11474 Brevibacterium healii 1 5527 Brevibacterium ketoglutamictim OO4 Brevibacterium ketoglutamictim O89 Brevibacterium ketosoreductim 914 Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74 Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 798 Brevibacterium lactofermentum 799 US 7,892,798 B2 57 58

TABLE 1-continued Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention

Genus species ATCC FERM NRRL CECT NCIMB CBS NCTC DSMZ Brevibacterium lactofermentum 21800 Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B11470 Brevibacterium lactofermentum B11471 Brevibacterium lactofermentum 21086 Brevibacterium lactofermentum 2142O Brevibacterium lactofermentum 21086 Brevibacterium lactofermentum 31269 Brevibacterium inens 91.74 Brevibacterium inens 19391 Brevibacterium inens 8377 Brevibacterium parafinolytictim 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 acetoglutamictim B11473 Corynebacterium acetoglutamictim B11475 Corynebacterium acetoglutamictim 15806 Corynebacterium acetoglutamictim 21491 Corynebacterium acetoglutamictim 31270 Corynebacterium acetophilum B3671 Corynebacterium ammoniagenes 6872 2399 Corynebacterium ammoniagenes 15511 Corynebacterium filiiokense 21496 Corynebacterium glutamictim 14067 Corynebacterium glutamictim 391.37 Corynebacterium glutamictim 21254 Corynebacterium glutamictim 21255 Corynebacterium glutamictim 31830 Corynebacterium glutamictim 13032 Corynebacterium glutamictim 1430S Corynebacterium glutamictim 15455 Corynebacterium glutamictim 13058 Corynebacterium glutamictim 13059 Corynebacterium glutamictim 13060 Corynebacterium glutamictim 21492 Corynebacterium glutamictim 21513 Corynebacterium glutamictim 21526 Corynebacterium glutamictim 21543 Corynebacterium glutamictim 13287 Corynebacterium glutamictim 2 851 Corynebacterium glutamictim 253 Corynebacterium glutamictim S1.4 Corynebacterium glutamictim S16 Corynebacterium glutamictim 299 Corynebacterium glutamictim 3OO Corynebacterium glutamictim 9684 Corynebacterium glutamictim 488 Corynebacterium glutamictim 649 Corynebacterium glutamictim 6SO Corynebacterium glutamictim 9223 Corynebacterium glutamictim 3869 Corynebacterium glutamictim 157 Corynebacterium glutamictim 158 Corynebacterium glutamictim 159 Corynebacterium glutamictim 355 Corynebacterium glutamictim 808 Corynebacterium glutamictim 674 Corynebacterium glutamictim S62 Corynebacterium glutamictim 563 Corynebacterium glutamictim S64 Corynebacterium glutamictim 565 Corynebacterium glutamictim 566 Corynebacterium glutamictim 567 Corynebacterium glutamictim 568 Corynebacterium glutamictim 569 Corynebacterium glutamictim 570 US 7,892,798 B2 59 60

TABLE 1-continued Corynebacterium and Brevibacterium Strains Which May be Used in the Practice of the Invention

Genus species ATCC FERM NRRL CECT NCIMB CBS NCTC DSMZ Corynebacterium glutamicum 21571 Corynebacterium glutamicum 21572 Corynebacterium glutamicum 21573 Corynebacterium glutamicum 21579 Corynebacterium glutamicum 19049 Corynebacterium glutamicum 190SO Corynebacterium glutamicum 19051 Corynebacterium glutamicum 19052 Corynebacterium glutamicum 19053 Corynebacterium glutamicum 19054 Corynebacterium glutamicum 19055 Corynebacterium glutamicum 190S6 Corynebacterium glutamicum 19057 Corynebacterium glutamicum 19058 Corynebacterium glutamicum 19059 Corynebacterium glutamicum 1906O Corynebacterium glutamicum 1918S 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 Corynebacterium glutamicum B12418 Corynebacterium glutamicum B11476 Corynebacterium glutamicum 21608 Corynebacterium ilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacterium spec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 3.1088 Corynebacterium spec. 3.1089 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 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 DSMZ; Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (4th edn), World federation for culture collections world data center on microorganisms, Saimata, Japen, 50 EQUIVALENTS many equivalents to the specific embodiments of the inven Those of ordinary skill in the art will recognize, or will be tion described herein. Such equivalents are intended to be able to ascertain using no more than routine experimentation, encompassed by the following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 25

<21 Os SEQ ID NO 1 &211s LENGTH: 762 &212s. TYPE: DNA <213> ORGANISM: Corynebacterium glutamicum 22 Os. FEATURE: <221s NAME/KEY: CDS

US 7,892,798 B2 63

- Continued

Thr Asn Lieu Phe Thr Thr Glu Gly Ile Arg Val Ile Gly Ile Asp Arg 35 4 O 45 Ile Lieu. Arg Glu Ala Asp Val Ala Lys Ala Ser Lieu. Tyr Ser Lieu. Phe SO 55 60 Gly Ser Lys Asp Ala Lieu Val Ile Ala Tyr Lieu. Glu Asn Lieu. Asp Glin 65 70 7s 8O Lieu. Trp Arg Glu Ala Trp Arg Glu Arg Thr Val Gly Met Lys Asp Pro 85 90 95 Glu Asp Llys Ile Ile Ala Phe Phe Asp Glin Cys Ile Glu Glu Glu Pro 1OO 105 11 O Glu Lys Asp Phe Arg Gly Ser His Phe Glin Asn Ala Ala Ser Glu Tyr 115 12 O 125 Pro Arg Pro Glu Thir Asp Ser Glu Lys Gly Ile Val Ala Ala Val Lieu. 13 O 135 14 O Glu. His Arg Glu Trp Cys His Llys Thr Lieu. Thir Asp Lieu. Lieu. Thr Glu 145 150 155 160 Lys Asn Gly Tyr Pro Gly. Thir Thr Glin Ala Asn Gln Lieu. Leu Val Phe 1.65 17O 17s Lieu. Asp Gly Gly Lieu Ala Gly Ser Arg Lieu Val His Asn. Ile Ser Pro 18O 185 19 O Lieu. Glu Thir Ala Arg Asp Lieu Ala Arg Glin Lieu Lleu Ser Ala Pro Pro 195 2OO 2O5 Ala Asp Tyr Ser Ile 21 O

<210s, SEQ ID NO 3 &211s LENGTH: 49 OO &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: knock-out vector construct pS delta655 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (14) . . (295 <223> OTHER INFORMATION: 5' - fragment of RXAO0655 sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (305) . . (614) <223> OTHER INFORMATION: 3'-fragment of RXAO655 sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (3003) . . (44.24) <223> OTHER INFORMATION: coding for levan sucrase (sacB) from Bacillus subtilis 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (4.425) . . (4887 <223> OTHER INFORMATION: promoter region for sacB 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1042) . . (1833) <223> OTHER INFORMATION: coding for kanamycin resistance 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1042) . . (1833) <223> OTHER INFORMATION: kanamycin resistance from Tn5 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2100) ... (2960 <223> OTHER INFORMATION: Ori from plMB for replication in E. coli <4 OOs, SEQUENCE: 3 tittaaatcto gagtgc.cggt ggcggcttga t ctitcagctt cat cacatgt Ctgattggtg 6 O

US 7,892,798 B2 75

- Continued titt to gtg gtg ttg ccg aag gat cat cqt ctit gcg ggg gala gga gtg 643 Phe Cys Val Val Lieu Pro Lys Asp His Arg Lieu Ala Gly Glu Gly Val 17O 17s 18O gtg gat ttg gtg gat Ctg gct aaa gat ggg ttt gtg acg acg ccg gag 691 Val Asp Lieu Val Asp Lieu Ala Lys Asp Gly Phe Val Thir Thr Pro Glu 185 19 O 195 titt gcg ggg tot gtg titt agg aat tcc acc titt Cag titg tdt gct gag 739 Phe Ala Gly Ser Val Phe Arg Asn Ser Thr Phe Gln Lieu. Cys Ala Glu 2OO 2O5 210 gct ggit ttt gtg ccg agg atc agc cag caa gtt aat gat cot tac atg 787 Ala Gly Phe Val Pro Arg Ile Ser Glin Glin Val Asn Asp Pro Tyr Met 215 22O 225 gcg Ctg ttgttg gcg C9g tagt caatca togggagta t cc 828 Ala Lieu. Lieu. Lieu Ala Arg 23 O 235

<210s, SEQ ID NO 6 &211s LENGTH: 235 212. TYPE: PRT <213> ORGANISM: Corynebacterium glutamicum

<4 OOs, SEQUENCE: 6 Val Glu Ile Arg Trp Lieu. Glu Gly Phe Ile Ala Val Ala Glu Glu Lieu 1. 5 1O 15 His Phe Ser Asn Ala Ala Ile Arg Lieu. Gly Met Pro Glin Ser Pro Leu 2O 25 3 O Ser Glin Lieu. Ile Arg Arg Lieu. Glu Ser Glu Lieu. Gly Glin Llys Lieu. Phe 35 4 O 45 Asp Arg Ser Thr Arg Ser Val Glu Lieu. Thir Ala Ala Gly Arg Ala Phe SO 55 60 Lieu Pro His Ala Arg Gly Ile Val Ala Ser Ala Ala Val Ala Arg Glu 65 70 7s 8O Ala Val Asn Ala Ala Glu Gly Glu Ile Val Gly Val Val Arg Ile Gly 85 90 95 Phe Ser Gly Val Lieu. Asn Tyr Ser Thr Lieu Pro Leu Lleu. Thir Ser Glu 1OO 105 11 O Val His Lys Arg Lieu Pro Asn Val Glu Lieu. Glu Lieu Val Gly Glin Lys 115 12 O 125 Lieu. Thir Arg Glu Ala Val Ser Lieu. Lieu. Arg Lieu. Gly Ala Lieu. Asp Ile 13 O 135 14 O Thr Lieu Met Gly Lieu Pro Ile Glu Asp Pro Glu Ile Glu Thr Arg Lieu. 145 150 155 160 Ile Ser Lieu. Glu Glu Phe Cys Val Val Lieu Pro Lys Asp His Arg Lieu. 1.65 17O 17s Ala Gly Glu Gly Val Val Asp Lieu Val Asp Lieu Ala Lys Asp Gly Phe 18O 185 19 O Val Thir Thr Pro Glu Phe Ala Gly Ser Val Phe Arg Asn Ser Thr Phe 195 2OO 2O5 Glin Lieu. Cys Ala Glu Ala Gly Phe Val Pro Arg Ile Ser Glin Glin Val 21 O 215 22O Asn Asp Pro Tyr Met Ala Lieu. Lieu. Lieu Ala Arg 225 23 O 235

<210s, SEQ ID NO 7 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 7,892,798 B2 77 78

- Continued <223> OTHER INFORMATION: Description of Artificial Sequence: oligonucleotide primer 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (34) <223> OTHER INFORMATION: sense primer <4 OO > SEQUENCE: 7 gagagactic agtggtttag gggatgagaa accg 34

<210s, SEQ ID NO 8 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: oligonucleotide primer 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) ... (31) <223> OTHER INFORMATION: antisense primer <4 OOs, SEQUENCE: 8 citct cactag totaccgc.gc caacaa.ca.gc g 31

<210s, SEQ ID NO 9 &211s LENGTH: 990 &212s. TYPE: DNA <213> ORGANISM: Corynebacterium glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (12) <223> OTHER INFORMATION: synthetic linker attached by PCR 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (980) ... (990) <223> OTHER INFORMATION: synthetic linker attached by PCR 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (13) . . (271 <223> OTHER INFORMATION: promoter and 5'-untranslated region of rxao2910 gene 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (272) ... (976) <223> OTHER INFORMATION: coding for positive regulator of methionine biosynthesis (rxaO2910

<4 OOs, SEQUENCE: 9 gagagactic agtggtttag gggatgagaa accggacaca C9tgccaaaa citt.cggctitt 6 O titcgc.caatc ttgtcacgcc ttctggittt gcct cqgatg aggtgattt C atggccalaga 12 O

Cttctaaaag titcgacct cq Caggat.cgct tctaagggcc tittagcggac Caacctaggc 18O cgat acccat gtggaaat ct cacgt.ctta aatggacgat tagctaala accacgaaca 24 O gctgggattt t cc acgatag gattgggit ct c gtg gag att cqt tdg ttg gaa 292 Val Glu Ile Arg Trp Lieu. Glu 1. 5 ggc titt at C gcg gtc gcg gaa gala ttg cac titt agt aat gct gcg att 34 O Gly Phe Ile Ala Val Ala Glu Glu Lieu. His Phe Ser Asn Ala Ala Ile 10 15 2O cgt ttgggg atg cc.g caa ticg ccg ttg agt cag titg atc. c99 cqg ttg 388 Arg Lieu. Gly Met Pro Glin Ser Pro Lieu. Ser Glin Lieu. Ile Arg Arg Lieu 25 3O 35 gag tog gag ttgggg cag aag ctt titt gat cqc agt acc cqg tcg gtg 436 Glu Ser Glu Lieu. Gly Glin Llys Lieu. Phe Asp Arg Ser Thr Arg Ser Val 4 O 45 SO 55 gag tta act gcc gcg ggit cqg gCd ttt ttg cca Cat gcc agg ggg att 484 US 7,892,798 B2 79 80

- Continued

Glu Luell Thir Ala Ala Gly Arg Ala Phe Lieu. Pro His Ala Arg Gly Ile 60 65 70 gtg gcg agc gct gcg gtg gcg agg gala gct gtg aat gct gcc gag 999 532 Wall Ala Ser Ala Ala Wall Ala Arg Glu Ala Wall Asn Ala Ala Glu Gly 7s 8O 85 gag at C gtt ggt gtt gtt cgc att ggit titt tot ggit gtg Ctg aac tat 58 O Glu Ile Wall Gly Wall Wall Arg Ile Gly Phe Ser Gly Wall Luell Asn Tyr 9 O 95 1OO t cc acg Ctg cc.g citt ttg a CC agt gag gtg Cat a.a.a. cgg citt cott aat 628 Ser Thir Luell Pro Lell Lell Thir Ser Glu Wall His Lys Arg Luell Pro Asn 105 11O 115 gtg gag ttg gag citc. gtt ggit cag aag ttg acg agg gaa gcg gta agt 676 Wall Glu Luell Glu Lell Wall Gly Glin Llys Lieu. Thir Arg Glu Ala Wall Ser 12O 125 13 O 135 ttg Ctg cgc ttg 999 gcg ttg gat att acg ttg atg ggit ttg cc c att 724 Lell Luell Arg Luell Gly Ala Lell Asp Ile Thr Luell Met Gly Luell Pro Ile 14 O 145 15 O gag gat CC a gag att gag act cgg ct g att agt ttg gaa gag titt tgc 772 Glu Asp Pro Glu Ile Glu Thir Arg Lieu. Ile Ser Lell Glu Glu Phe Cys 155 16 O 1.65 gtg gtg ttg cc.g aag gat Cat cgt Ctt gcg 999 gaa gga gtg gtg gat Wall Wall Luell Pro Lys Asp His Arg Lieu Ala Gly Glu Gly Wall Wall Asp 17 O 17s 18O ttg gtg gat Ctg gct a.a.a. gat 999 titt gtg acg acg gag titt gcg 868 Lell Wall Asp Luell Ala Asp Gly Phe Wall Thir Thir Pro Glu Phe Ala 185 190 195

999 tot gtg titt agg aat t cc acc ttt cag ttg gct gag gct ggit 916 Gly Ser Wall Phe Arg Asn Ser Thir Phe Glin Luell Cys Ala Glu Ala Gly 2 OO 2O5 210 215 titt gtg cc.g agg atc. agc Cag Cala gtt aat gat cott tac atg gcg Ctg 964 Phe Wall Pro Arg Ile Ser Glin Glin Wall Asn Asp Pro Met Ala Luell 22O 225 23 O ttg ttg gcg cgg tag act agtg agag 990 Lell Luell Ala Arg 235

SEQ ID NO 10 LENGTH: 235 TYPE : PRT ORGANISM: Corynebacterium glutamicum

< 4 OOs SEQUENCE: 10

Wall Glu Ile Arg Trp Lell Glu Gly Phe Ile Ala Wall Ala Glu Glu Luell 1. 5 1O 15

His Phe Ser Asn Ala Ala Ile Arg Lieu. Gly Met Pro Glin Ser Pro Luell 25 3 O

Ser Glin Luell Ile Arg Arg Lell Glu Ser Glu Luell Gly Glin Luell Phe 35 4 O 45

Asp Arg Ser Thir Ser Wall Glu Lieu. Thir Ala Ala Gly Arg Ala Phe SO 55 60

Lell Pro His Ala Arg Gly Ile Wall Ala Ser Ala Ala Wall Ala Arg Glu 65 70

Ala Wall Asn Ala Ala Glu Gly Glu Ile Wall Gly Wall Wall Arg Ile Gly 85 90 95

Phe Ser Gly Wall Lell Asn Tyr Ser Thir Lieu. Pro Lell Lell Thir Ser Glu 105 11 O

Wall His Lys Arg Lell Pro Asn Wall Glu Lieu. Glu Lell Wall Gly Glin 115 12 O 125 US 7,892,798 B2 81 82

- Continued Lell Thir Arg Glu Ala W al Ser Lieu. Lieu. Arg Lieu. Gly Ala Lieu. Asp Ile 13 O 135 14 O

Thir Luell Met Gly Lieu P ro Ile Glu Asp Pro Glu Ile Glu Thir Arg Lieu. 145 1. SO 155 160

Ile Ser Luell Glu Glul P he Cys Val Wall Leu Pro Lys Asp His Arg Lieu. 1.65 17O 17s

Ala Gly Glu Gly Val V al Asp Lieu Val Asp Lieu. Ala Asp Gly Phe 18O 185 19 O

Wall Thir Thir Pro Glul P he Ala Gly Ser Wall Phe Arg Asn Ser Thir Phe 195 2OO 2O5

Glin Luell Ala Glu Ala Gly Phe Val Pro Arg Ile Ser Glin Glin Wall 21 O 215 22O

Asn Asp Pro Tyr Met Ala Lieu. Lieu Lieu Ala Arg 225 2 235

SEQ ID NO 11 LENGTH: 439 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: PCR fragment for rxn02910 knock-out

<4 OOs, SEQUENCE: 11 tatact cag ttgatcgcag taccc.ggit cq gtggagittaa tcgggcgttt 6 O ttgccacatg cCagggggat cgagggaagic tgttgaatgct 12 O gcc.gaggggg agat.cgttgg tgttgttcgc attggitttitt gaact attcc 18O acgctg.ccgc ttittgaccag tgaggtgcat aaacggcttic Ctaatgtgga gttggagctic 24 O gttggit Caga agttgacgag ggaag.cggta agtttgctgc gCttgggggc gttggatatt 3OO acgttgatgg gtttgcc cat tgaggat.cca gagattgaga Ctcggctgat tagtttggaa 360 gagttittgcg gaaggat cat cgt. Cttgcgg gggaaggagt ggtggatttg gtggatctgg at at cagag 439

<210s, SEQ ID NO 12 &211s LENGTH: 51.56 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: &223s OTHER INFORMATION: Description of Artificial Sequence: vector for Corynebacterium glutamicum po 22 Os. FEATURE: <221 > NAMEAKEY: terminator <222s. LOCATION: (125) . . (184) 223 OTHER INFORMATION: GroEL t erminator 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (534) . . (1325) 223 OTHER INFORMAT ON: kanamycin resistance gene from Tn5 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (36.06) ... (4727) 223 OTHER INFORMAT ON: Rep gene for replication in C. glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2598) ... (3272) 223 OTHER INFORMAT ON: ORF1 for replication in C. glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1592) ... (2452) 223 OTHER INFORMAT ON: Ori from plMB for replication in E. coli

<4 OOs, SEQUENCE: 12

US 7,892,798 B2 87 88

- Continued ggacgc.cgaa agctt.cccag taaatgtgcc atctogtagg Cagaaaacgg ttc.ccc.cgta 48OO gggit ct ct ct cttggcct Co tttctagg to gggctgattg Ctcttgaagc tict ct agggg 486 O ggct cacacic at aggcagat aacgttcCCC accggct cqc Ctcgtaagcg cacaaggact 492 O gctic ccaaag atc.ttcaaag ccactg.ccgc gactgcc titc gcgaa.gc.ctt gcc.ccgcgga 498O aatttic ct co accoagttcg togcacacc cc tatgccaagc ttctitt cacc ctaaatt cqa 5040 gagattggat t cittaccgtg gaaattctitc gcaaaaatcg tcc cctdatc gcc cttgcga cgttggcgtc. g.gtgcc.gctg gttgcgcttg gcttgaccga Cttgat cagc ggcc.gc 51.56

<210s, SEQ ID NO 13 &211s LENGTH: 6087 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: vector pGrxb2O10 for overexpression of ositive regulator of methionine biosynthesis (rxn2910) 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1465) . . (2256) <223> OTHER INFORMATION: kanamycin resistance from Tn5 22 Os. FEATURE: <221 > NAMEAKEY: misc difference <222s. LOCATION: (3529).. (42O3) 223 OTHER INFORMAT ON: Orf1 for replication in C. glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (4537) . . (5658 223 OTHER INFORMAT ON: Rep gene for replication in C. glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (2523) . . (3383) 223 OTHER INFORMAT ON: Ori from plMB for replication in E. coli 22 Os. FEATURE: <221 > NAMEAKEY: terminator <222s. LOCATION: (1056) . . (1115) 223 OTHER INFORMAT ON: GroEl terminator from C. glutamicum 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (268) ... (984) <223> OTHER INFORMATION: coding for rxino 2910 22 Os. FEATURE: <221> NAME/KEY: promoter <222s. LOCATION: (19) . . (267) 223 OTHER INFORMAT ON: promoter of rxnO2910 gene

<4 OOs, SEQUENCE: 13 tcgatttalaa tict cagtgg tittaggggat gagaalaccgg acacacgtgc caaaactitcg 6 O gctttitt.cgc caatcttgtc acgc.ctgtct ggtttgcctic ggatgaggtg attt catggc 12 O caag acttct aaaagttcga Cct coagga tcqcttctaa gggcctt tag cigaccalacc 18O taggc.cgata cc catgtgga aatct cqacg t cittaaatgg acgattggag ctaaaac cac 24 O gaac agctgg gattitt CCaC gataggattg ggtCtcgtgg agatt.cgttg gttggaaggc 3OO tittatcgcgg togcggaaga attgcactitt agtaatgctg cgatt.cgttt ggggatgcc.g 360 Caatcgc.cgt tagt cagtt gatcc.ggcgg agttggggca gaagctttitt gatcgcagta Cccggit cqgt ggagittaact gggcgtttitt gcc acatgcc agggggattig tigcgagcgc agggaagctg tgaatgctgc C9agggggag 54 O atcgttggtg ttgttcgcat tggitttitt ct ggtgttgctga act attccac gotgcc.gctt ttgaccagtg aggtgcataa acggct tcct aatgtggagt tggagct cqt tdgtcagaag 660 ttgacgaggg aag.cgg talag tittgctg.cgc ttggggg.cgt. tggat attac gttgatgggit 72 O ttgcc cattg aggat.ccaga gattgagact cggctgatta gtttggalaga gttittgcgtg

US 7,892,798 B2 101 102

- Continued

215 22 O caa gtt aat gat cot tac atg gog Ctg ttgttg gcg C9g tag 717 Glin Val Asn Asp Pro Tyr Met Ala Lieu. Lieu. Lieu Ala Arg 225 23 O 235

SEO ID NO 17 LENGTH: 235 TYPE PRT ORGANISM: Corynebacterium glutamicum

< 4 OOs SEQUENCE: 17

Val Glu Ile Arg Trp Lieu. Glu Gly Phe Ile Ala Wall Ala Glu Glu Lieu. 1. 5 1O 15

His Phe Ser Asn Ala Ala Ile Arg Leu Gly Met Pro Glin Ser Pro Leu 25 3 O

Ser Glin Lieu. Ile Arg Arg Lieu. Glu Ser Glu Lieu Gly Glin Lieu. Phe 35 4 O 45

Asp Arg Ser Thr Arg Ser Val Glu Lieu. Thir Ala Ala Gly Arg Ala Phe SO 55 60

Lieu Pro His Ala Arg Gly Ile Val Ala Ser Ala Ala Wall Ala Arg Glu 65 70 7s 8O

Ala Val Asn Ala Ala Glu Gly Glu Ile Val Gly Wall Wall Arg Ile Gly 85 90 95

Phe Ser Gly Val Lieu. Asn Tyr Ser Thir Lieu Pro Lell Lell Thir Ser Glu 105 11 O

Val His Lys Arg Lieu. Pro ASn Val Glu Lieu. Glu Lell Wall Gly Gln Lys 115 12 O 125

Lieu. Thir Arg Glu Ala Val Ser Lieu Lieu. Arg Lieu. Gly Ala Luell Asp Ile 13 O 135 14 O

Thr Lieu Met Gly Lieu Pro Ile Glu Asp Pro Glu Ile Glu Thir Arg Lieu. 145 150 155 160

Ile Ser Lieu. Glu Glu Phe Cys Val Wall Leu Pro Asp His Arg Lieu. 1.65 17O 17s

Ala Gly Glu Gly Val Val Asn Lieu. Val Asp Lieu. Ala Asp Gly Phe 18O 185 19 O

Val Thir Thr Pro Glu Phe Ala Gly Ser Wall Phe Arg Asn Ser Thir Phe 195 2OO 2O5

Glin Lieu. Cys Ala Glu Ala Gly Phe Val Pro Arg Ile Ser Glin Glin Wall 21 O 215 22O Asn Asp Pro Tyr Met Ala Lieu. Lieu. Lieu Ala Arg 225 23 O 235

SEQ ID NO 18 LENGTH: 627 TYPE: DNA ORGANISM: Corynebacterium diphtheriae FEATURE: NAME/KEY: CDS LOCATION: (1) . . (624) OTHER INFORMATION: RXAO0655 protein homologue

SEQUENCE: 18 ttg gcc gag gC9 aaa agc acg aag acg acg agt cgt cga cgt. aac cqt 48 Lieu Ala Glu Ala Lys Ser Thr Lys Thir Thir Ser Arg Arg Arg Asn Arg 1. 5 1O 15 ccg agc cct cqt cag cqc cta ttg gat gc gca acg Cag citt titt acc 96 Pro Ser Pro Arg Glin Arg Lieu. Lieu Asp Gly Ala Thir Glin Luell Phe Thr 25 3 O US 7,892,798 B2 103 104

- Continued a CC gag gga att cgg gtg atc. ggc att gat cgc att ttg cgt. gag gct 144 Thir Glu Gly Ile Arg Wall Ile Gly Ile Asp Arg Ile Lell Arg Glu Ala 35 4 O 45 gat gta gcc aag gcg agt ttg tac to c Ctg titc gga t cc aag gat gcg 192 Asp Wall Ala Lys Ala Ser Lell Ser Luell Phe Gly Ser Lys Asp Ala SO 55 60

Ctg gtt att gcc tat ttg Cag aac ttg gat gaa aag tgg cgt. gag cag 24 O Lell Wall Ile Ala Tyr Lell Glin Asn Luell Asp Glu Lys Trp Arg Glu Glin 65 70 7s 8O tat tac gag cgc act gct gag atg ggt tog CCa agc gag a.a.a. att citc. 288 Glu Arg Thir Ala Glu Met Gly Ser Pro Ser Glu Ile Luell 85 90 95 gcg titt titt gat Cag tgt att gat gag gag cc.g Ctg aag gat tat cgc 336 Ala Phe Phe Asp Glin Cys Ile Asp Glu Glu Pro Lell Lys Asp Arg 1OO 105 11 O ggit tog CaC ttic Cag aat gct gct aac gag tac cc.g cgc CC a gag acg 384 Gly Ser His Phe Glin Asn Ala Ala Asn Glu Tyr Pro Arg Pro Glu Thir 115 12 O 125 gat agt gag cgc gag atc. gtg tog gtt gtg atg gaa Cat cgc cgg tgg 432 Asp Ser Glu Arg Glu Ile Wall Ser Wall Wall Met Glu His Arg Arg Trp 13 O 135 14 O tgt gag acg tta act Cag ttg ttg acg gag aag aac 999 tat cc c Cys Luell Glu Thir Lell Thir Glin Luell Luell Thir Glu Lys Asn Gly Pro 145 150 155 160 ggc act gtt cag gct aat Cag ttg atg gtt titt Ctg gat ggt ggt citt 528 Gly Thir Wall Glin Ala Asn Glin Luell Met Wall Phe Lell Asp Gly Gly Luell 1.65 17O 17s gcg ggt tgt cgt Ctg aat cgc tog gtg gag tog ttg aag act gct cgg 576 Ala Gly Cys Arg Lell Asn Arg Ser Wall Glu Ser Lell Lys Thir Ala Arg 18O 185 19 O gat citt gcg gtt Cag ttg Ctg tot gct cott cott gct gat tat tog att 624 Asp Luell Ala Wall Glin Lell Lell Ser Ala Pro Pro Ala Asp Ser Ile 195 2OO 2O5 tag 627

<210s, SEQ ID NO 19 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Corynebacterium diphtheriae

<4 OOs, SEQUENCE: 19

Lell Ala Glu Ala Lys Ser Thir Thir Thir Ser Arg Arg Arg Asn Arg 1. 5 15

Pro Ser Pro Arg Glin Arg Lell Luell Asp Gly Ala Thir Glin Luell Phe Thir 25 3 O

Thir Glu Gly Ile Arg Wall Ile Gly Ile Asp Arg Ile Lell Arg Glu Ala 35 4 O 45

Asp Wall Ala Lys Ala Ser Lell Ser Luell Phe Gly Ser Asp Ala SO 55 60

Lell Wall Ile Ala Tyr Lell Glin Asn Luell Asp Glu Trp Glu Glin 65 70

Glu Arg Thir Ala Glu Met Gly Ser Pro Ser Glu Ile Luell 85 90 95

Ala Phe Phe Asp Glin Ile Asp Glu Glu Pro Lell Asp Arg 105 11 O

Gly Ser His Phe Glin Asn Ala Ala Asn Glu Tyr Pro Arg Pro Glu Thir 115 12 O 125

Asp Ser Glu Arg Glu Ile Wall Ser Wall Wall Met Glu His Arg Arg Trp

US 7,892,798 B2 107 108

- Continued Asp Tyr Ser Ile 21 O

<210s, SEQ ID NO 21 &211s LENGTH: 212 212. TYPE : PRT &213s ORGANISM: Corynebacterium efficiens YS-314

<4 OOs, SEQUENCE: 21

Wall Ala Wal Ser Ala Ser Gly Ser Arg Thir Ser Thir Gly Gly Arg 1. 5 15

Arg Arg Asp Arg Pro Ser Pro Arg Glin Arg Luell Lell Asp Ser Ala Thir 25 3 O

Asn Luell Phe Thir Thir Glu Gly Ile Arg Wall Ile Gly Ile Asp Arg Ile 35 4 O 45

Lell Arg Glu Ala Asp Wall Ala Ala Ser Luell Tyr Ser Luell Phe Gly SO 55 60

Ser Asp Ala Lell Wall Ile Ala Luell Glu Asn Lell Asp Glin Glin 65 70

Trp Asp Ala Trp His Glu Arg Thir Asp Glin Lell Asp Pro Glu 85 90 95

Asp Ile Ile Ala Phe Phe Asp Glin Ile Glu Glu Glu Pro Lys 105 11 O

Gly Phe Arg Gly Ser His Phe Glin Asn Ala Ala Asn Glu Pro 115 12 O 125

Arg Pro Glu Thir Glu Ser Glu Gly Ile Wall Ala Ala Wall Met Glu 13 O 135 14 O

His Arg Arg Trp Cys His Glin Thir Luell Thir Asp Lell Lell Thir Glu Lys 145 150 155 160

Asn Gly Pro Gly Thir Thir Glin Ala Asn Glin Lell Lell Wall Phe Luell 1.65 17O 17s

Asp Gly Gly Luell Ala Gly Ser Arg Luell Wall Glin Asn Ile Gly Pro Luell 18O 185 19 O

Glu Thir Ala Arg Asp Lell Ala Arg Glin Luell Luell Ser Ala Pro Pro Ala 195 2OO 2O5

Asp Tyr Ser Ile 21 O

SEQ ID NO 22 LENGTH: 588 TYPE: DNA ORGANISM: Streptomyces coelicolor FEATURE: NAME/KEY: CDS LOCATION: (1) . . (585) OTHER INFORMATION: coding for RXAO0655 protein homologue

<4 OOs, SEQUENCE: 22 atg aca tog acc gcc gcc aga cc.g ggc aga gtg gcg aag Ctg cc.g cc c 48 Met Thir Ser Thir Ala Ala Arg Pro Gly Arg Wall Ala Lys Luell Pro Pro 1. 5 1O 15 cgc gag cgc at C citc. gac gcg gcc gag gag citc. tto Cag ggc gag ggc 96 Arg Glu Arg Ile Lell Asp Ala Ala Glu Glu Luell Phe Glin Gly Glu Gly 2O 25 3 O atc. cga cgc gtg 999 gtc Cag gcg at C gcc gag cgg gcc gag acc acc 144 Ile Arg Arg Wall Gly Wall Glin Ala Ile Ala Glu Arg Ala Glu Thir Thir 35 4 O 45 aag atg gcg at C tac cgg CaC ttic gag acc aag gac gca citc. gt C gcc 192 Lys Met Ala Ile Tyr Arg His Phe Glu Thir Lys Asp Ala Luell Wall Ala US 7,892,798 B2 109 110

- Continued

SO 55 60 gaa tgg Ctg cgg atc. Ctg gcc gcc gag tac cag gcg gcc ttic gac 24 O Glu Trp Luell Arg Ile Lell Ala Ala Glu Tyr Glin Ala Ala Phe Asp 65 70 7s gtc gag gcc gala Cat c cc ggc cgg cc c cgg gag Cag atc. Ctg ggc citc. 288 Wall Glu Ala Glu His Pro Gly Arg Pro Arg Glu Glin Ile Luell Gly Luell 85 90 95 gcc cgc ttic at C gcc gac 999 Ctg cc.g 999 citc. tcg CaC cgg ggc tgc 336 Ala Arg Phe Ile Ala Asp Gly Luell Pro Gly Luell Ser His Arg Gly Cys 1OO 105 11 O c cc ttic at C aac t cc citc. gcc gag Ctg cc c gac cgc t cc CaC cc c gcg 384 Pro Phe Ile Asn Ser Lell Ala Glu Luell Pro Asp Arg Ser His Pro Ala 115 12 O 125 cga cgg gtg at C gag gag CaC aag gcc cgc cag a CC agg Ctg gt C 432 Arg Arg Wall Ile Glu Glu His Lys Ala Arg Glin Thir Arg Arg Luell Wall 13 O 135 14 O ggc atg tgt gcc gag gcg 999 atg cc c gac coc gaa Cag gtC gcg gcc Gly Met Cys Ala Glu Ala Gly Met Pro Asp Pro Glu Glin Wall Ala Ala 145 150 155 160

Cag at C acc ttic gtc citc. gaa 999 gcg cag gtc agc acg cag aac gca 528 Glin Ile Thir Phe Wall Lell Glu Gly Ala Glin Wall Ser Thir Glin Asn Ala 1.65 17O 17s agc at C gac cgg gcg 999 gag cgg ttg atg cgc atc. gtc gag gcg at C 576 Ser Ile Asp Arg Ala Gly Glu Arg Luell Met Arg Ile Wall Glu Ala Ile 18O 185 19 O gtc gac cag tag 588 Wall Asp Glin 195

<210s, SEQ ID NO 23 &211s LENGTH: 195 212. TYPE : PRT &213s ORGANISM: Streptomyces coelicolor

<4 OOs, SEQUENCE: 23

Met Thir Ser Thr Ala Ala Arg Pro Gly Arg Wall Ala Luell Pro Pro 1. 5 15

Arg Glu Arg Ile Lell Asp Ala Ala Glu Glu Luell Phe Glin Gly Glu Gly 25 3 O

Ile Arg Arg Wall Gly Wall Glin Ala Ile Ala Glu Arg Ala Glu Thir Thir 35 4 O 45

Met Ala Ile Tyr Arg His Phe Glu Thir Asp Ala Luell Wall Ala SO 55 60

Glu Trp Luell Arg Ile Lell Ala Ala Glu Tyr Glin Ala Ala Phe Asp Arg 65 70

Wall Glu Ala Glu His Pro Gly Arg Pro Arg Glu Glin Ile Luell Gly Luell 85 90 95

Ala Arg Phe Ile Ala Asp Gly Luell Pro Gly Luell Ser His Arg Gly 105 11 O

Pro Phe Ile Asn Ser Lell Ala Glu Luell Pro Asp Arg Ser His Pro Ala 115 12 O 125

Arg Arg Wall Ile Glu Glu His Ala Arg Glin Thir Arg Arg Luell Wall 13 O 135 14 O

Gly Met Ala Glu Ala Gly Met Pro Asp Pro Glu Glin Wall Ala Ala 145 150 155 160

Glin Ile Thir Phe Wall Lell Glu Gly Ala Glin Wall Ser Thir Glin Asn Ala 1.65 17O 17s US 7,892,798 B2 111 112

- Continued Ser Ile Asp Arg Ala Gly Glu Arg Lieu Met Arg Ile Val Glu Ala Ile 18O 185 19 O Val Asp Glin 195

SEQ ID NO 24 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: primer

<4 OOs, SEQUENCE: 24 ggaalacagta taccatg 18

SEO ID NO 25 LENGTH: 17 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: primer

<4 OOs, SEQUENCE: 25 gtaaaacgac ggc.cagt 17

What is claimed: 11. The method of claim 7, wherein expression of the 1. An isolated nucleic acid molecule which encodes a 30 nucleic acid molecule from said vector results in increased polypeptide comprising the amino acid sequence set forth in production of said methionine. SEQID NO:6. 12. An isolated nucleic acid molecule comprising the full 2. An isolated nucleic acid molecule comprising the complement of the nucleic acid molecule of claim 1. nucleic acid molecule of claim 1, and a nucleotide sequence 13. A vector comprising the isolated nucleic acid molecule encoding a heterologous polypeptide. 35 of claim 12. 3. A vector comprising the isolated nucleic acid molecule 14. A host cell comprising the vector of claim 13. of claim 1. 15. An isolated nucleic acid molecule comprising the 4. The vector of claim 3, which is an expression vector. nucleotide sequence set forth in SEQID NO:5. 16. An isolated nucleic acid molecule comprising the 5. A host cell comprising the expression vector of claim 4. 40 nucleic acid molecule of claim 15, and a nucleotide sequence 6. A method of producing a polypeptide comprising the encoding a heterologous polypeptide. amino acid sequence of SEQ ID NO:6 or SEQ ID NO:17, comprising culturing the host cell of claim 5 in an appropriate 17. A vector comprising the isolated nucleic acid molecule culture medium to thereby produce the polypeptide. of claim 15. 45 18. The vector of claim 17, which is an expression vector. 7. A method for producing methionine, comprising cultur 19. A host cell comprising the expression vector of claim ing a microorganism belonging to the genus Corynebacte 18. rium or Brevibacterium transformed with the vector of claim 20. A method of producing a polypeptide comprising the 3 such that methionine is produced. amino acid sequence of SEQ ID NO:6 or SEQ ID NO:17, 8. The method of claim 7, wherein said method further 50 comprising culturing the host cell of claim 19 in an appropri comprises the step of recovering methionine from said cul ate culture medium to thereby produce the polypeptide. ture. 21. A method for producing methionine, comprising cul 9. The method of claim 7, wherein said microorganism turing a microorganism belonging to the genus Corynebac belongs to the genus Corynebacterium. terium or Brevibacterium transformed with the vector of 10. The method of claim 7, wherein said microorganism is 55 claim 18 such that methionine is produced. selected from the group consisting of Corynebacterium 22. The method of claim 21, wherein said method further glutamicum, Corynebacterium efficiens, Corynebacterium comprises the step of recovering methionine from said cul herculis, Corynebacterium lilium, Corynebacterium ture. acetoacidophilum, Corynebacterium acetoglutamicum, 23. The method of claim 21, wherein said microorganism Corynebacterium acetophilum, Corynebacterium ammoni 60 belongs to the genus Corynebacterium. agenes, Corynebacterium fijiokense, Corynebacterium 24. The method of claim 21, wherein said microorganism is nitrilophilus, Brevibacterium ammoniagenes, Brevibacte selected from the group consisting of Corynebacterium rium butanicum, Brevibacterium divaricatum, Brevibacte glutamicum, Corynebacterium efficiens, Corynebacterium rium flavum, Brevibacterium healii, Brevibacterium keto herculis, Corynebacterium lilium, Corynebacterium glutamicum, Brevibacterium ketosoreductum, 65 acetoacidophilum, Corynebacterium acetoglutamicum, Brevibacterium lactofermentum, Brevibacterium linens, and Corynebacterium acetophilum, Corynebacterium ammoni Brevibacterium parafinolyticum. agenes, Corynebacterium fijiokense, Corynebacterium US 7,892,798 B2 113 114 nitrilophilus, Brevibacterium ammoniagenes, Brevibacte 39. The method of claim 35, wherein expression of the rium butanicum, Brevibacterium divaricatum, Brevibacte nucleic acid molecule from said vector results in increased rium flavum, Brevibacterium healii, Brevibacterium keto production of said methionine. glutamicum, Brevibacterium ketosoreductum, 40. An isolated nucleic acid molecule comprising the full Brevibacterium lactofermentum, Brevibacterium linens, and 5 complement of the nucleic acid molecule of claim 29. Brevibacterium parafinolyticum. 41. A vector comprising the isolated nucleic acid molecule 25. The method of claim 21, wherein expression of the of claim 40. nucleic acid molecule from said vector results in increased 42. A host cell comprising the vector of claim 41. production of said methionine. 43. An isolated nucleic acid molecule comprising the 26. An isolated nucleic acid molecule comprising the full 10 nucleotide sequence set forth in SEQID NO:16. complement of the nucleic acid molecule of claim 15. 44. An isolated nucleic acid molecule comprising the nucleic acid molecule of claim 43, and a nucleotide sequence 27. A vector comprising the isolated nucleic acid molecule encoding a heterologous polypeptide. of claim 26. 45. A vector comprising the isolated nucleic acid molecule 28. A host cell comprising the vector of claim 27. 15 of claim 43. 29. An isolated nucleic acid molecule which encodes a 46. The vector of claim 45, which is an expression vector. polypeptide comprising the amino acid sequence set forth in 47. A host cell comprising the expression vector of claim SEQID NO:17. 46. 30. An isolated nucleic acid molecule comprising the 48. A method of producing a polypeptide comprising the nucleic acid molecule of claim 29, and a nucleotide sequence amino acid sequence of SEQID NO:17, comprising culturing encoding a heterologous polypeptide. the host cell of claim 47 in an appropriate culture medium to 31. A vector comprising the isolated nucleic acid molecule thereby produce the polypeptide. of claim 29. 49. A method for producing methionine, comprising cul 32. The vector of claim 31, which is an expression vector. turing a microorganism belonging to the genus Corynebac 25 terium or Brevibacterium transformed with the vector of 33. A host cell comprising the expression vector of claim claim 46 Such that methionine is produced. 32. 50. The method of claim 49, wherein said method further 34. A method of producing a polypeptide comprising the comprises the step of recovering methionine from said cul amino acid sequence of SEQID NO:17, comprising culturing ture. the host cell of claim 33 in an appropriate culture medium to 30 51. The method of claim 49, wherein said microorganism thereby produce the polypeptide. belongs to the genus Corynebacterium. 35. A method for producing methionine, comprising cul 52. The method of claim 49, wherein said microorganism is turing a microorganism belonging to the genus Corynebac selected from the group consisting of Corynebacterium terium or Brevibacterium transformed with the vector of glutamicum, Corynebacterium efficiens, Corynebacterium claim 31 such that methionine is produced. 35 herculis, Corynebacterium lilium, Corynebacterium 36. The method of claim 35, wherein said method further acetoacidophilum, Corynebacterium acetoglutamicum, comprises the step of recovering methionine from said cul Corynebacterium acetophilum, Corynebacterium ammoni ture. agenes, Corynebacterium fijiokense, Corynebacterium 37. The method of claim 35, wherein said microorganism nitrilophilus, Brevibacterium ammoniagenes, Brevibacte belongs to the genus Corynebacterium. 40 rium butanicum, Brevibacterium divaricatum, Brevibacte 38. The method of claim35, wherein said microorganism is rium flavum, Brevibacterium healii, Brevibacterium keto selected from the group consisting of Corynebacterium glutamicum, Brevibacterium ketosoreductum, glutamicum, Corynebacterium efficiens, Corynebacterium Brevibacterium lactofermentum, Brevibacterium linens, and herculis, Corynebacterium lilium, Corynebacterium Brevibacterium parafinolyticum. acetoacidophilum, Corynebacterium acetoglutamicum, 45 53. The method of claim 49, wherein expression of the Corynebacterium acetophilum, Corynebacterium ammoni nucleic acid molecule from said vector results in increased agenes, Corynebacterium fijiokense, Corynebacterium production of said methionine. nitrilophilus, Brevibacterium ammoniagenes, Brevibacte 54. An isolated nucleic acid molecule comprising the full rium butanicum, Brevibacterium divaricatum, Brevibacte complement of the nucleic acid molecule of claim 43. rium flavum, Brevibacterium healii, Brevibacterium keto 50 55. A vector comprising the isolated nucleic acid molecule glutamicum, Brevibacterium ketosoreductum, of claim 54. Brevibacterium lactofermentum, Brevibacterium linens, and 56. A host cell comprising the vector of claim 55. Brevibacterium parafinolyticum. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,892,798 B2 Page 1 of 1 APPLICATIONNO. : 10/307138 DATED : February 22, 2011 INVENTOR(S) : Markus Pompeius et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the title page please correct the title to read:

-- NUCLEIC ACID MOLECULES ENCODING METABOLIC REGULATORY PROTEINS FROM CORYNEBACTERIUM GLUTAMICUM, USEFUL FOR INCREASING THE PRODUCTION OF METHIONINE BY A MICROORGANISM --

At Page 1, Column 1 please correct the title to read:

-- NUCLEIC ACID MOLECULES ENCODING METABOLIC REGULATORY PROTEINS FROM CORYNEBACTERIUM GLUTAMICUM, USEFUL FOR INCREASING THE PRODUCTION OF METHIONINE BY A MICROORGANISM --

Signed and Sealed this Nineteenth Day of April, 2011

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David J. Kappos Director of the United States Patent and Trademark Office