EP1290178A2 - Corynebacterium glutamicum gene, welche für stress-, resistenz- und toleranzproteine kodieren - Google Patents

Corynebacterium glutamicum gene, welche für stress-, resistenz- und toleranzproteine kodieren

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Publication number
EP1290178A2
EP1290178A2 EP00944135A EP00944135A EP1290178A2 EP 1290178 A2 EP1290178 A2 EP 1290178A2 EP 00944135 A EP00944135 A EP 00944135A EP 00944135 A EP00944135 A EP 00944135A EP 1290178 A2 EP1290178 A2 EP 1290178A2
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EP
European Patent Office
Prior art keywords
nucleic acid
protein
srt
sequence
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00944135A
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English (en)
French (fr)
Inventor
Markus Pompejus
Burkhard Kröger
Hartwig Schröder
Oskar Zelder
Gregor Haberhauer
Heung-Shick Korea University LEE
Hyung-Joon Korea University KIM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Paik Kwang Industrial Co Ltd
Original Assignee
BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1290178A2 publication Critical patent/EP1290178A2/de
Priority to AU2006200801A priority Critical patent/AU2006200801A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in C. glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as stress, resistance and tolerance (SRT) proteins.
  • SRT stress, resistance and tolerance
  • C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids.
  • the SRT nucleic acid molecules of the invention can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the SRT nucleic acids of the invention, or modification of the sequence of the SRT nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species).
  • the SRT nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C.
  • the invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present.
  • Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.
  • the SRT nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms. Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.
  • the SRT proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism.
  • cloning vectors for use in Corynebacterium glutamicum such as those disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al, J Bacteriol.
  • the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals, through the ability of these proteins to permit growth and multiplication of C. glutamicum (and also continuous production of one or more fine chemicals) under circumstances which would normally impede growth of the organism, such as those conditions frequently encountered during large-scale fermentative growth.
  • a heat-shock induced protease molecule such that it is optimized in activity, one may increase the ability of the bacterium to degrade incorrectly folded proteins when the bacterium is challenged with high temperatures.
  • the cell By having fewer misfolded (and possibly misregulated or nonfunctional) proteins to interfere with normal reaction mechanisms in the cell, the cell is increased in its ability to function normally in such a culture, which should in turn provide increased viability.
  • This overall increase in number of cells having greater viability and activity in the culture should also result in an increase in yield, production, and/or efficiency of production of one or more desired fine chemicals, due at least to the relatively greater number of cells producing these chemicals in the culture.
  • This invention provides novel SRT nucleic acid molecules which encode SRT proteins which are capable of, for example, permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism.
  • Nucleic acid molecules encoding an SRT protein are referred to herein as SRT nucleic acid molecules.
  • the SRT protein participates in metabolic pathways permitting C. glutamicum to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Examples of such proteins include those encoded by the genes set forth in Table 1.
  • one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an SRT protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SRT- encoding nucleic acid (e.g., DNA or mRNA).
  • isolated nucleic acid molecules e.g., cDNAs, DNAs, or RNAs
  • nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SRT- encoding nucleic acid (e.g., DNA or mRNA).
  • the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth as the odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), or the coding region or a complement thereof of one of these nucleotide sequences.
  • the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth as an odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7....), or a portion thereof.
  • the isolated nucleic acid molecule encodes one of the amino acid sequences set forth as an even- numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8.).
  • SEQ ID NO:2 amino acid sequences set forth as an even- numbered SEQ ID NO in the Sequence Listing
  • SEQ ID NO:4 amino acid sequences set forth as an even- numbered SEQ ID NO in the Sequence Listing
  • the preferred SRT proteins of the present invention also preferably possess at least one of the SRT activities described herein.
  • the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence having an even-numbered SEQ ID NO: in the Sequence Listing), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an SRT activity.
  • the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism.
  • the protein encoded by the nucleic acid molecule is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90% and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an amino acid sequence of the invention (e.g., an entire amino acid sequence selected from those having an even-numbered SEQ ID NO in the Sequence Listing).
  • the protein is a full length C.
  • glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown the corresponding odd-numbered SEQ ID NOs in the Sequence Listing (e.g., SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7.).
  • the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an SRT fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and has the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism, or possesses one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.
  • a protein e.g., an SRT fusion protein
  • a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing) and has the ability to increase the survival of C. glut
  • the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention (e.g., a sequence of an odd- numbered SEQ ID NO in the Sequence Listing).
  • the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum SRT protein, or a biologically active portion thereof.
  • vectors e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, and host cells into which such vectors have been introduced.
  • a host cell is used to produce an SRT protein by culturing the host cell in a suitable medium. The SRT protein can be then isolated from the medium or the host cell.
  • Yet another aspect of the invention pertains to a genetically altered microorganism in which an SRT gene has been introduced or altered.
  • the genome of the microorganism has been altered by the introduction of a nucleic acid molecule of the invention encoding wild-type or mutated SRT sequence as a transgene.
  • an endogenous SRT gene within the genome of the microorganism has been altered, e.g., functionally disrupted, by homologous recombination with an altered SRT gene.
  • an endogenous or introduced SRT gene in a microorganism has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SRT protein.
  • one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of a SRT gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SRT gene is modulated.
  • the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred.
  • the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.
  • the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject.
  • This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in the Sequence Listing as SEQ ID NOs 1 through 304)) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
  • Still another aspect of the invention pertains to an isolated SRT protein or a portion, e.g., a biologically active portion, thereof.
  • the isolated SRT protein or portion thereof possesses the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism.
  • the isolated SRT protein or portion thereof is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing) such that the protein or portion thereof maintains the ability to increase the survival of C. glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism.
  • an amino acid sequence of the invention e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing
  • the invention also provides an isolated preparation of an SRT protein.
  • the SRT protein comprises an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
  • the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) (encoded by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO: of the Sequence Listing).).
  • the protein is at least about 50%, preferably at least about 60%, and more preferably at least about 70%, 80%, or 90%, and most preferably at least about 95%, 96%, 97%, 98%, or 99% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
  • the isolated SRT protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and is able to improve the survival rate of C glutamicum in a setting which is either chemically or environmentally hazardous to this microorganism, or has one or more of the activities set forth in Table 1.
  • the isolated SRT protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g. , hybridizes under stringent conditions, or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80%, or 90%, and even more preferably at least about 95%, 96%o, 97%, 98,%, or 99% or more homologousto a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred that the preferred forms of SRT proteins also have one or more of the SRT bioactivities described herein.
  • the SRT polypeptide, or a biologically active portion thereof, can be operatively linked to a non-SRT polypeptide to form a fusion protein.
  • this fusion protein has an activity which differs from that of the SRT protein alone.
  • this fusion protein results in increased yields, production, and/or efficiency of production of a desired fine chemical from C. glutamicum.
  • integration of this fusion protein into a host cell modulates the production of a desired compound from the cell.
  • the invention provides methods for screening molecules which modulate the activity of an SRT protein, either by interacting with the protein itself or a substrate or binding partner of the SRT protein, or by modulating the transcription or translation of an SRT nucleic acid molecule of the invention.
  • Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing a vector directing the expression of an SRT nucleic acid molecule of the invention, such that a fine chemical is produced.
  • this method further includes the step of obtaining a cell containing such a vector, in which a cell is transfected with a vector directing the expression of an SRT nucleic acid.
  • this method further includes the step of recovering the fine chemical from the culture.
  • the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3.
  • Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates SRT protein activity or SRT nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent.
  • the cell is modulated in resistance to one or more toxic chemicals or in resistance to one or more environmental stresses, such that the yields or rate of production of a desired fine chemical by this microorganism is improved.
  • the agent which modulates SRT protein activity can be an agent which ⁇ stimulates SRT protein activity or SRT nucleic acid expression.
  • agents which stimulate SRT protein activity or SRT nucleic acid expression include small molecules, active SRT proteins, and nucleic acids encoding SRT proteins that have been introduced into the cell.
  • agents which inhibit SRT activity or expression include small molecules, and antisense SRT nucleic acid molecules.
  • Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant SRT gene into a cell, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated.
  • said yields are increased.
  • said chemical is a fine chemical.
  • said fine chemical is an amino acid.
  • said amino acid is L-lysine.
  • the present invention provides SRT nucleic acid and protein molecules which are involved in the survival of C. glutamicum upon exposure of this microorganism to chemical or environmental hazards.
  • the molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, since these SRT proteins provide a means for continued growth and multiplication of C. glutamicum in the presence of toxic chemicals or hazardous environmental conditions, such as may be encountered during large-scale fermentative growth.
  • toxic chemicals or hazardous environmental conditions such as may be encountered during large-scale fermentative growth.
  • the term 'fine chemical' is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries.
  • Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds.
  • VCH Weinheim, and references contained therein
  • lipids both saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol), carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in UUmann's Encyclopedia of Industrial Chemistry, vol. A27, "Vitamins", p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A.S., Niki, E. & Packer, L.
  • Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms.
  • amino acid is art- recognized.
  • the proteinogenic amino acids of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)).
  • Amino acids may be in the D- or L- optical configuration, though L- amino acids are generally the only type found in naturally-occurring proteins.
  • the 'essential' amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 11 'nonessential' amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine). Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for normal protein synthesis to occur.
  • Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine.
  • Glutamate is most commonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine. Glycine, L- methionine and tryptophan are all utilized in the pharmaceutical industry.
  • Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/ L- methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids - technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim).
  • amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as N- acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH: Weinheim, 1985.
  • cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain ⁇ -carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase.
  • Phenylalanine, and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate. Tryptophan is also produced from these two initial molecules, but its synthesis is an 11- step pathway.
  • Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase.
  • Alanine, valine, and leucine are all biosynthetic products of pyruvate, the final product of glycolysis.
  • Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle.
  • Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate.
  • Isoleucine is formed from threonine.
  • a complex 9-step pathway results in the production of histidine from 5-phosphoribosyl-l-pyrophosphate, an activated sugar.
  • Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3 rd ed. Ch. 21 "Amino Acid Degradation and the Urea Cycle” p. 495-516 (1988)).
  • the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesize them.
  • amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3 rd ed. Ch. 24: "Biosynthesis of Amino Acids and Heme” p. 575-600 (1988)).
  • the output of any particular amino acid is limited by the amount of that amino acid present in the cell.
  • Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms, such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins" vol. A27, p.
  • vitamin is art- recognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself.
  • the group of vitamins may encompass cofactors and nutraceutical compounds.
  • cofactor includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic.
  • nutraceutical includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g. , polyunsaturated fatty acids).
  • Thiamin (vitamin Bi) is produced by the chemical coupling of pyrimidine and thiazole moieties.
  • Riboflavin (vitamin B ) is synthesized from guanosine-5 '-triphosphate (GTP) and ribose-5' -phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
  • 'vitamin B 6 ' e.g., pyridoxine, pyridoxamine, pyridoxa- 5 '-phosphate, and the commercially used pyridoxin hydrochloride
  • Pantothenate pantothenic acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)- ⁇ -alanine
  • pantothenate biosynthesis consist of the ATP-driven condensation of ⁇ -alanine and pantoic acid.
  • pantothenate The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to ⁇ - alanine and for the condensation to panthotenic acid are known.
  • the metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in 5 enzymatic steps.
  • Pantothenate, pyridoxal-5' -phosphate, cysteine and ATP are the precursors of Coenzyme A.
  • These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, (R)- panthenol (provitamin B 5 ), pantetheine (and its derivatives) and coenzyme A.
  • Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins.
  • Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the ⁇ -ketoglutarate dehydrogenase complex.
  • the folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6- methylpterin.
  • guanosine-5 '-triphosphate GTP
  • L-glutamic acid L-glutamic acid
  • p- amino-benzoic acid has been studied in detail in certain microorganisms.
  • Corrinoids such as the cobalamines and particularly vitamin B ⁇
  • porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system.
  • the biosynthesis of vitamin B 12 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known.
  • Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed 'niacin'.
  • Niacin is the precursor of the important coenzymes NAD
  • NADP nicotinamide adenine dinucleotide phosphate
  • purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections.
  • the language “purine” or “pyrimidine” includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides.
  • the term “nucleotide” includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose), and phosphoric acid.
  • the language “nucleoside” includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess.
  • nucleic acid molecules By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).
  • energy stores i.e., AMP
  • coenzymes i.e., FAD and NAD
  • purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p. 561- 612).
  • fine chemicals e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin
  • energy carriers for the cell e.g., ATP or GTP
  • chemicals themselves commonly used as flavor enhancers (e.g., IMP or GMP
  • Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout.
  • Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound inosine-5'- phosphate (IMP), resulting in the production of guanosine-5 '-monophosphate (GMP) or adenosine-5' -monophosphate (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell.
  • IMP inosine-5'- phosphate
  • GMP guanosine-5 '-monophosphate
  • AMP adenosine-5' -monophosphate
  • Pyrimidine biosynthesis proceeds by the formation of uridine-5' -monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5'-triphosphate (CTP).
  • UMP uridine-5' -monophosphate
  • CTP cytidine-5'-triphosphate
  • the deoxy- forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.
  • Trehalose consists of two glucose molecules, bound in ⁇ , ⁇ -1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al, (1998) U.S.
  • Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.
  • Examples of environmental stresses typically encountered in large-scale fermentative culture include mechanical stress, heat stress, stress due to limited oxygen, stress due to oxygen radicals, pH stress, and osmotic stress.
  • the stirring mechanism used in most large-scale fermentors to ensure aeration of the culture produces heat, thus increasing the temperature of the culture. Increases in temperature induce the well- characterized heat shock response, in which a set of proteins are expressed which not only aid in the survival of the bacterium in the face of high temperatures, but also increase survival in response to a number of other environmental stresses (see Neidhardt, F.C., et al., eds. (1996) E. coli and Salmonella. ASM Press: Washington, D.C., p. 1382-1399; Wosten, M. M.
  • chaperones proteins which assist in the folding or unfolding of other proteins - see, e.g., Fink, A.L. (1999) Physiol. Rev. 79(2): 425-449
  • proteases which can destroy any improperly folded proteins.
  • chaperones expressed during the heat shock response include GroEL and DNAK; proteases known to be expressed during this cellular reaction to heat shock include Lon, FtsH, and ClpB.
  • fermentor stirring process is meant to introduce oxygen into the culture, oxygen may remain in limited supply, particularly when the culture is advanced in growth and the oxygen needs of the culture are thereby increased; an insufficient supply of oxygen is another stress for the microorganism.
  • Cells in fermentor cultures are also subjected to a number of osmotic stresses, particularly when nutrients are added to the culture, resulting in a high extracellular and low intracellular concentration of these molecules. Further, the large quantities of the desired molecules produced by these organisms in culture may contribute to osmotic stress of the bacteria.
  • bacteria have elegant gene systems which are expressed upon exposure to one or more stresses, such as the aforementioned heat shock system.
  • Genes expressed in response to osmotic stress for example, encode proteins capable of transporting or synthesizing compatible solutes such that osmotic intake or export of a particular molecule is slowed to manageable levels.
  • Other examples of stress-induced bacterial proteins are those involved in trehalose biosynthesis, those encoding enzymes involved in ppGpp metabolism, those involved in signal transduction, particularly those encoding two-component systems which are sensitive to osmotic pressure, and those encoding transcription factors which are responsive to a variety of stress factors (e.g., RssB analogues and/or sigma factors). Many other such genes and their protein products are known in the art.
  • cells may also experience a number of chemical stresses. These may fall into two categories. The first are natural waste products of metabolism and other cellular processes which are secreted by the cell to the surrounding medium. The second are chemicals present in the extracellular medium which do not originate from the cell. Generally, when cells excrete toxic waste products from the concentrated intracellular cytoplasm into the relatively much more dilute extracellular medium, these products dissipate such that extracellular levels of the possibly toxic compound are quite low. However, in large-scale fermentative culture of the bacterium, this may not be the case: so many bacteria are grown in a relatively small environment and at such a high metabolic rate that waste products may accumulate in the medium to nearly toxic levels.
  • wastes are carbon dioxide, metal ions, and reactive oxygen species such as hydrogen peroxide. These compounds may interfere with the activity or structure of cell surface molecules, or may re-enter the cell, where they can seriously damage proteins and nucleic acids alike. Certain other chemicals hazardous to the normal functioning of cells may be naturally found in the extracellular medium. For example, metal ions such as mercury, cadmium, nickel or copper are frequently found in water sources, and may form tight complexes with cellular enzymes which prevent the normal functioning of these proteins.
  • Bacteriocidal proteins or antibiotics may also be found in the extracellular milieu, either through the intervention of the researcher, or as a natural product from another organism, utilized to gain a competitive advantage.
  • Microorganisms have several art-known mechanisms to protect themselves against antimicrobial chemicals. Degradation, modification, and export of compounds toxic to the cell are common methods by which microorganisms eliminate or detoxify antibiotics. Cytoplasmic
  • 'efflux-pumps' are known in several prokaryotes and show similarities to the so-called 'multidrug resistance' proteins from higher eukaryotes (Neyfakh, A. A. , et al. (1991) Proc. Natl. Acad. Sci. USA 88: 4781-4785).
  • Examples of such proteins include emrAB from E. coli (Lomovskaya, O. and K. Lewis (1992) Proc. Natl. Acad. Sci. USA 89: 8938-8942), ImrB from B. subtilis (Kumano, M. et al. (1997) Microbiology 143: 2775- 2782), smr from S.
  • Lincosamides are recognized as effective antibiotics against Corynebacterium species (Soriano, F. et al. (1995) Antimicrob. Agents Chemother. 39: 208-214).
  • An unexpected result of the present invention was the identification of a gene encoding a lincosamide-resistance protein (in particular, a lincomycin-resistance protein).
  • the LMRB protein from C. glutamicum shows 40% homology to the product of the ImrB gene from B. subtilis (see Genbank accession no. AL009126), as calculated using version 1.7 of the program CLUSTALW (Thompson, J.D., Higgins, D.G., Gibson, T. J. (1994) Nucl. Acids Res. 22: 4673-4680) using standard parameters (PAIRWISE
  • Environmental stress, chemical stress, and antibiotic or other antimicrobial stress may influence the behavior of the microorganisms during fermentor culture, and may have an impact on the production of the desired compound from these organisms.
  • osmotic stress of a microorganism may cause inappropriate or inappropriately rapid uptake of one or more compounds which can ultimately lead to cellular damage or death due to osmotic shock.
  • chemicals present in the culture either exogenously added (e.g., antimicrobial compounds intended to eliminate unwanted microbes) or generated by the bacteria themselves (e.g., waste compounds such as heavy metals or oxygen radicals, or even antimicrobial compounds) may result in inhibition of fine chemical production or even death of the organism.
  • the genes of the invention encode C. glutamicum proteins which act to prevent cell damage or death, by specifically counteracting the source or effect of the environmental or chemical stress.
  • the present invention is based, at least in part, on the discovery of novel molecules, referred to herein as SRT nucleic acid and protein molecules, which increase the ability of C. glutamicum to survive in chemically or environmentally hazardous settings.
  • the SRT molecules function to confer resistance to one or more environmental or chemical stresses to C. glutamicum.
  • the activity of the SRT molecules of the present invention has an impact on the production of a desired fine chemical by this organism.
  • the SRT molecules of the invention are modulated in activity, such that the yield, production, and/or efficiency of production of one or more fine chemicals from C. glutamicum is also modulated.
  • SRT protein or "SRT polypeptide” includes proteins which participate in the resistance of C.
  • SRT proteins include those encoded by the SRT genes set forth in Table 1 and by the odd-numbered SEQ ID NOs.
  • SRT gene or "SRT nucleic acid sequence” include nucleic acid sequences encoding an SRT protein, which consist of a coding region and also corresponding untranslated 5' and 3' sequence regions. Examples of SRT genes include those set forth in Table 1.
  • production or “productivity” are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume (e.g. , kg product per hour per liter). The term
  • efficiency of production includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical).
  • yield or “product/carbon yield” is art-recognized and includes the efficiency of the conversion of the carbon source into the product (i.e., fine chemical). This is generally written as, for example, kg product per kg carbon source.
  • biosynthesis or a “biosynthetic pathway” are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process.
  • degradation or a “degradation pathway” are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process.
  • the language “metabolism” is art-recognized and includes the totality of the biochemical reactions that take place in an organism.
  • the metabolism of a particular compound comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.
  • the terms “resistance” and “tolerance” are art-known and include the ability of a cell to not be affected by exposure to a chemical or an environment which would otherwise be detrimental to the normal functioning of these organisms.
  • the terms “stress” or “hazard” include factors which are detrimental to the normal functioning of cells such as C. glutamicum. Examples of stresses include “chemical stress", in which a cell is exposed to one or more chemicals which are detrimental to the cell, and “environmental stress” where a cell is exposed to an environmental condition outside of those to which it is adapted.
  • Chemical stresses may be either natural metabolic waste products such as, but not limited to reactive oxygen species or carbon dioxide, or chemicals otherwise present in the environment, including, but not limited to heavy metal ions or bacteriocidal proteins such as antibiotics.
  • Environmental stresses may be, but are not limited to temperatures outside of the normal range, suboptimal oxygen availability, osmotic pressures, or extremes of pH, for example.
  • the SRT molecules of the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. Using recombinant genetic techniques, one or more of the SRT proteins of the invention may be manipulated such that its function is modulated.
  • the alteration of activity of stress response, resistance or tolerance genes such that the cell is increased in tolerance to one or more stresses may improve the ability of that cell to grow and multiply in the relatively stressful conditions of large- scale fermentor culture.
  • a heat-shock induced chaperone molecule such that it is optimized in activity
  • one may increase the ability of the bacterium to correctly fold proteins in the face of nonoptimal temperature conditions.
  • the cell is increased in its ability to function normally in such a culture, which should in turn provide increased viability.
  • This overall increase in number of cells having greater viability and activity in the culture should also result in an increase in the yield, production, and/or efficiency of production of one or more desired fine chemicals, due at least to the relatively greater number of cells producing these chemicals in the culture.
  • the isolated nucleic acid sequences of the invention are contained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032.
  • the nucleotide sequence of the isolated C. glutamicum SRT DNAs and the predicted amino acid sequences of the C. glutamicum SRT proteins are shown the Sequence Listing as odd-numbered SEQ ID NOs and even-numbered SEQ ID NOs, respectively.,. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode chemical and environmental stress, resistance, and tolerance proteins.
  • the present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention (e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing).
  • a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, e.g., the entire selected amino acid sequence.
  • a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to the selected amino acid sequence.
  • Ranges and identity values intermediate to the above-recited values, e.g., 75%-80% identical, 85-87% identical, 91-92% identical
  • ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included.
  • SRT proteins or biologically active portions or fragments thereof of the invention can confer resistance or tolerance to one or more chemical or environmental stresses, or may have one or more of the activities set forth in Table 1.
  • nucleic acid molecules that encode SRT polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of SRT-encoding nucleic acid (e.g., SRT DNA).
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated SRT nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g, a C. glutamicum cell).
  • an "isolated" nucleic acid molecule such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a C. glutamicum SRT DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T ' . Molecular Cloning: A Laboratory Manual.
  • nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence).
  • mRNA can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to an SRT nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing.
  • the nucleic acid sequences of the invention correspond to the Corynebacterium glutamicum SRT DNAs of the invention.
  • This DNA comprises sequences encoding SRT proteins (i.e., the "coding region", indicated in each odd- numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5' untranslated sequences and 3' untranslated sequences, also indicated in each odd-numbered SEQ ID NO: in the Sequence Listing.
  • the nucleic acid molecule can comprise only the coding region of any of the nucleic acid sequences of the Sequence Listing.
  • each of the nucleic acid and amino acid sequences set forth in the Sequence Listing has an identifying RXA, RXN, or RXS number having the designation "RXA”, “RXN”, or “RXS” followed by 5 digits (i.e., RXA01524, RXN00493, or RXS01027).
  • Each of the nucleic acid sequences comprises up to three parts: a 5' upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, or RXS designation to eliminate confusion.
  • the coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also et forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence.
  • the coding region for RXAOl 524 is set forth in SEQ ID NO: 1
  • the amino acid sequence which it encodes is set forth as SEQ ID NO:2.
  • sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, or RXS designations as the amino acid molecules which they encode, such that they can be readily correlated.
  • the amino acid sequence designated RXAOl 524 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXAOl 524
  • the amino acid sequence designated RXN00034 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXN00034
  • the amino acid sequence in designated RXS00568 is a translation of the coding region of the nucleotide sequence of nucleic acid molecule RXS00568.
  • Table 1 The correspondence between the RXA, RXN, and RXS nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1.
  • F-designated genes include those genes set forth in Table 1 which have an 'F' in front of the RXA, RXN, or RXS designation.
  • SEQ ID NO: 7, designated, as indicated on Table 1, as “F RXA00498” is an F-designated gene, as are SEQ ID NOs: 25, 33, and 37 (designated on Table 1 as "F RXAOl 345", "F RXA02543", and “F RXA02282", respectively).
  • the nucleic acid molecules of the present invention are not intended to include those compiled in Table 2.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing, or a portion thereof.
  • a nucleic acid molecule which is complementary to one of the nucleotide sequences of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention,, or a portion thereof.
  • the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an SRT protein.
  • the nucleotide sequences determined from the cloning of the SRT genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning SRT homologues in other cell types and organisms, as well as SRT homologues from other Corynebacteria or related species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the nucleotide sequences of the invention (e.g., a sequence of one of the odd- numbered SEQ ID NOs of the Sequence Listing),, an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof.
  • Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone SRT homologues.
  • Probes based on the SRT nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress an SRT protein, such as by measuring a level of an SRT-encoding nucleic acid in a sample of cells, e.g., detecting SRT mRNA levels or determining whether a genomic SRT gene has been mutated or deleted.
  • the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even- numbered SEQ ID NO of the Sequence Listing) such that the protein or portion thereof maintains the ability to confer resistance or tolerance of C. glutamicum to one or more chemical or environmental stresses.
  • the language "sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., ' an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the even- numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is capable of participating in the resistance of C. glutamicum to one or more chemical or environmental stresses. Protein members of such metabolic pathways, as described herein, function to increase the resistance or tolerance of C. glutamicum to one or more environmental or chemical hazards or stresses. Examples of such activities are also described herein. Thus, “the function of an SRT protein” contributes to the overall resistance of C. glutamicum to elements of its surroundings which may impede its normal growth or functioning, and/or contributes, either directly or indirectly, to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of SRT protein activities are set forth in Table 1.
  • the protein is at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-80%, 80-90%, 90-95%, and most preferably at least about 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
  • Ranges and identity values intermediate to the above-recited values, are also intended to be encompassed by the present invention.
  • ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included.
  • Portions of proteins encoded by the SRT nucleic acid molecules of the invention are preferably biologically active portions of one of the SRT proteins.
  • biologically active portion of an SRT protein is intended to include a portion, e.g., a domain/motif, of an SRT protein that is capable of imparting resistance or tolerance to one or more environmental or chemical stresses or hazards, or has an activity as set forth in Table 1.
  • an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification.
  • Additional nucleic acid fragments encoding biologically active portions of an SRT protein can be prepared by isolating a portion of one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the SRT protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the SRT protein or peptide.
  • the invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same SRT protein as that encoded by the nucleotide sequences of the invention.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:).
  • the nucleic acid molecule of the invention encodes a full length C.
  • glutamicum protein which is substantially homologous to an amino acid sequence of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing). It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Tables 2 or 4 which were available prior to the present invention.
  • the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence) set forth in Tables 2 or 4).
  • the invention includes a nucleotide sequence which is greater than and/or at least 39% identical to the nucleotide sequence designated RXA00084 (SEQ ID NO: 189), a nucleotide sequence which is greater than and/or at least 56% identical to the nucleotide sequence designated RXA00605 (SEQ ID NO: 11), and a nucleotide sequence which is greater than and/or at least 50% identical to the nucleotide sequence designated RXA00886 (SEQ ID NO:39).
  • nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated are also encompassed by the invention.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of SRT proteins may exist within a population (e.g., the C. glutamicum population). Such genetic polymo ⁇ hism in the SRT gene may exist among individuals within a population due to natural variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an SRT protein, preferably a C. glutamicum SRT protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the SRT gene.
  • Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum SRT DNA of the invention can be isolated based on their homology to the C. glutamicum SRT nucleic acid disclosed herein using the C glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the Sequence Listing.
  • the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those of ordinary skill in the art in the art and can be found in Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • the nucleic acid encodes a natural C. glutamicum SRT protein.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of one of the SRT proteins (e.g., an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said SRT protein, whereas an "essential" amino acid residue is required for SRT protein activity.
  • Other amino acid residues e.g., those that are not conserved or only semi-conserved in the domain having SRT activity
  • nucleic acid molecules encoding SRT proteins that contain changes in amino acid residues that are not essential for SRT activity.
  • SRT proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the SRT activities described herein.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of increasing the resistance or tolerance of C. glutamicum to one or more environmental or chemical stresses, or has one or more of the activities set forth in Table 1.
  • the protein encoded by the nucleic acid molecule is at least about 50-60% homologous to the amino acid sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, more preferably at least about 60-70% homologous to one of these sequences, even more preferably at least about 70-80%, 80-90%, 90-95% homologous to one of these sequences in, and most preferably at least about 96%, 97%, 98%, or 99% homologous to one of the amino acid sequences of the invention.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid).
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity”
  • An isolated nucleic acid molecule encoding an SRT protein homologous to a protein sequence of the invention can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a predicted nonessential amino acid residue in an SRT protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an SRT coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an SRT activity described herein to identify mutants that retain SRT activity.
  • the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire SRT coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an SRT protein.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the entire coding region of SEQ ID NO.: 120 (RXA00600) comprises nucleotides 1 tol098).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding SRT.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (/ ' . e. , also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of SRT mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of SRT mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of SRT mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an SRT protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • the antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g.
  • the antisense nucleic acid molecule by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave SRT mRNA transcripts to thereby inhibit translation of SRT mRNA.
  • a ribozyme having specificity for an SRT-encoding nucleic acid can be designed based upon the nucleotide sequence of an SRT cDNA disclosed herein (i.e., SEQ ID NO:l 19 (RXA00600)).
  • SEQ ID NO:l 19 RXA00600
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an SRT-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No. 5,116,742.
  • SRT mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel, D. and Szostak, J.W. (1993) Science 261 :1411-1418.
  • SRT gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an SRT nucleotide sequence (e.g., an SRT promoter and/or enhancers) to form triple helical structures that prevent transcription of an SRT gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N Y. Acad. Sci. 660:27- 36; and Maher, L.J. (1992) Bioassays 14(12):807-15.
  • vectors preferably expression vectors, containing a nucleic acid encoding an SRT protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells.
  • Preferred regulatory sequences are, for example, promoters such as cos-, tac-, t ⁇ -, tet-, t ⁇ -tet-, lpp-, lac-, lpp-lac-, lacl q -, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, ⁇ -P R - or ⁇ P L , which are used preferably in bacteria.
  • promoters such as cos-, tac-, t ⁇ -, tet-, t ⁇ -tet-, lpp-, lac-, lpp-lac-, lacl q -, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, ⁇ -P R - or ⁇ P L , which are used preferably in bacteria.
  • Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MF ⁇ , AC, P-60, CYC1, GAPDH, TEF, ⁇ 28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. , SRT proteins, mutant forms of SRT proteins, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of SRT proteins in prokaryotic or eukaryotic cells.
  • SRT genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M.A. et al. (1992) "Foreign gene expression in yeast: a review", Yeast 8: 423-488; van den Hondel,
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione S-transferase
  • the coding sequence of the SRT protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant SRT protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN- III 113-B 1 , ⁇ gtl 1 , pBdCl, and pET 11 d (Studier et al. , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89; and Pouwels et al, eds.
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid t ⁇ -lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected.
  • the plasmids pIJlOl, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUBl 10, pC194, or pBD214 are suited for transformation of Bacillus species.
  • plasmids pUBl 10, pC194, or pBD214 are suited for transformation of Bacillus species.
  • plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBLl, pSA77, or pAJ667 (Pouwels et al, eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
  • One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
  • nucleic acid sequence of the nucleic acid is altered so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the SRT protein expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), 2 ⁇ , pAG-1, Yep6, Yepl3, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), and pYES2 (Invitrogen Co ⁇ oration, San Diego, CA).
  • Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi include those detailed in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy, et al, eds., p. 1-28, Cambridge
  • the SRT proteins of the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al.
  • the SRT proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants (e.g., the spermatophytes, such as crop plants).
  • plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W.
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ.
  • the invention further provides a recombinant expression vector comprising a
  • DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to SRT mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an SRT protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • suitable host cells are known to those of ordinary skill in the art.
  • Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA)) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • foreign nucleic acid e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector)
  • nucleic acid in the form of a vector e.g., a plasmid, phage, phas
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker e.g., resistance to antibiotics
  • Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SRT protein or can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • drug selection e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die.
  • a vector is prepared which contains at least a portion of an SRT gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the SRT gene.
  • this SRT gene is a Corynebacterium glutamicum SRT gene, but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source.
  • the vector is designed such that, upon homologous recombination, the endogenous SRT gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous SRT gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous SRT protein).
  • the altered portion of the SRT gene is flanked at its 5' and 3' ends by additional nucleic acid of the SRT gene to allow for homologous recombination to occur between the exogenous SRT gene carried by the vector and an endogenous SRT gene in a microorganism.
  • the additional flanking SRT nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • are included in the vector see e.g., Thomas, K.R., and Capecchi, M.R.
  • the vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced SRT gene has homologously recombined with the endogenous SRT gene are selected, using art-known techniques.
  • recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of an SRT gene on a vector placing it under control of the lac operon permits expression of the SRT gene only in the presence of IPTG.
  • Such regulatory systems are well known in the art.
  • an endogenous SRT gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur.
  • an endogenous or introduced SRT gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional SRT protein.
  • one or more of the regulatory regions (e.g. , a promoter, repressor, or inducer) of an SRT gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the SRT gene is modulated.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an SRT protein.
  • the invention further provides methods for producing SRT proteins using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an SRT protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered SRT protein) in a suitable medium until SRT protein is produced.
  • the method further comprises isolating SRT proteins from the medium or the host cell.
  • Another aspect of the invention pertains to isolated SRT proteins, and biologically active portions thereof.
  • An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of SRT protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of SRT protein having less than about 30% (by dry weight) of non-SRT protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-SRT protein, still more preferably less than about 10% of non-SRT protein, and most preferably less than about 5% non-SRT protein.
  • non-SRT protein also referred to herein as a "contaminating protein”
  • the SRT protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of SRT protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of SRT protein having less than about 30% (by dry weight) of chemical precursors or non-SRT chemicals, more preferably less than about 20% chemical precursors or non-SRT chemicals, still more preferably less than about 10% chemical precursors or non-SRT chemicals, and most preferably less than about 5% chemical precursors or non-SRT chemicals.
  • isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the SRT protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum SRT protein in a microorganism such as C. glutamicum.
  • an isolated SRT protein or a portion thereof of the invention can contribute to the resistance or tolerance of C. glutamicum to one or more chemical or environmental stresses or hazards, or has one or more of the activities set forth in Table 1.
  • the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability to mediate the resistance or tolerance of C. glutamicum to one or more chemical or environmental stresses or hazards.
  • the portion of the protein is preferably a biologically active portion as described herein.
  • an SRT protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing.
  • the SRT protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing).
  • the SRT protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to one of the nucleic acid sequences of the invention , or a portion thereof.
  • Ranges and identity values intermediate to the above-recited values, are also intended to be encompassed by the present invention.
  • ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included.
  • the preferred SRT proteins of the present invention also preferably possess at least one of the SRT activities described herein.
  • a preferred SRT protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can increase the resistance or tolerance of C. glutamicum to one or more environmental or chemical stresses, or which has one or more of the activities set forth in Table 1.
  • the SRT protein is substantially homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above.
  • the SRT protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to an entire amino acid sequence of the invention and which has at least one of the SRT activities described herein.
  • Ranges and identity values intermediate to the above-recited values are also intended to be encompassed by the present invention.
  • ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included.
  • the invention pertains to a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention .
  • Biologically active portions of an SRT protein include peptides comprising amino acid sequences derived from the amino acid sequence of an SRT protein, e.g., an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an SRT protein, which include fewer amino acids than a full length SRT protein or the full length protein which is homologous to an SRT protein, and exhibit at least one activity of an SRT protein.
  • biologically active portions peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length
  • biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.
  • the biologically active portions of an SRT protein include one or more selected domains/motifs or portions thereof having biological activity.
  • SRT proteins are preferably produced by recombinant DNA techniques.
  • a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the SRT protein is expressed in the host cell.
  • the SRT protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • an SRT protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques.
  • native SRT protein can be isolated from cells (e.g. , endothelial cells), for example using an anti-SRT antibody, which can be produced by standard techniques utilizing an SRT protein or fragment thereof of this invention.
  • an SRT "chimeric protein” or “fusion protein” comprises an SRT polypeptide operatively linked to a non-SRT polypeptide.
  • An "SRT polypeptide” refers to a polypeptide having an amino acid sequence corresponding to SRT
  • a non-SRT polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the SRT protein, e.g., a protein which is different from the SRT protein and which is derived from the same or a different organism.
  • the term "operatively linked" is intended to indicate that the SRT polypeptide and the non-SRT polypeptide are fused in-frame to each other.
  • the non- SRT polypeptide can be fused to the N-terminus or C-terminus of the SRT polypeptide.
  • the fusion protein is a GST-SRT fusion protein in which the SRT sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant SRT proteins.
  • the fusion protein is an SRT protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an SRT protein can be increased through use of a heterologous signal sequence.
  • an SRT chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An SRT- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the SRT protein.
  • Homologues of the SRT protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the SRT protein.
  • the term "homologue” refers to a variant form of the SRT protein which acts as an agonist or antagonist of the activity of the SRT protein.
  • An agonist of the SRT protein can retain substantially the same, or a subset, of the biological activities of the SRT protein.
  • An antagonist of the SRT protein can inhibit one or more of the activities of the naturally occurring form of the SRT protein, by, for example, competitively binding to a downstream or upstream member of the SRT system which includes the SRT protein.
  • the C. glutamicum SRT protein and homologues thereof of the present invention may increase the tolerance or resistance of C. glutamicum to one or more chemical or environmental stresses.
  • homologues of the SRT protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the SRT protein for SRT protein agonist or antagonist activity.
  • a variegated library of SRT variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of SRT variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential SRT sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g.
  • libraries of fragments of the SRT protein coding can be used to generate a variegated population of SRT fragments for screening and subsequent selection of homologues of an SRT protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an SRT coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the SRT protein.
  • REM Recursive ensemble mutagenesis
  • cell based assays can be exploited to analyze a variegated SRT library, using methods well known in the art.
  • nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of genomes of organisms related to C. glutamicum; identification and localization of C. glutamicum sequences of interest; evolutionary studies; determination of SRT protein regions required for function; modulation of an SRT protein activity; modulation of the activity of an SRT pathway; and modulation of cellular production of a desired compound, such as a fine chemical.
  • the SRT nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms.
  • the invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present.
  • Corynebacterium glutamicum itself is nonpathogenic, it is related to pathogenic species, such as Corynebacterium diphtheriae.
  • Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology.
  • a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells; the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body.
  • Degenerative changes brought about by the inhibition of protein synthesis in these tissues which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease.
  • Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.
  • the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject.
  • This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
  • C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C. glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.
  • the nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of C.
  • nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum.
  • the SRT nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The resistance processes in which the molecules of the invention participate are utilized by a wide variety of cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed.
  • genes of the invention e.g., the gene encoding LMRB (SEQ ID NO: l) or other gene of the invention encoding a chemical or environmental resistance or tolerance protein (e.g., resistance against one or more antibiotics), may be used as genetic markers for the genetic transformation of (e.g., the transfer of additional genes into or disruption of preexisting genes of) organisms such as C. glutamicum or other bacterial species.
  • nucleic acid molecules permits efficient selection of organisms which have inco ⁇ orated a given transgene cassette (e.g., a plasmid, phage, phasmid, phagemid, transposon, or other nucleic acid element), based on a trait which permits the survival of the organism in an otherwise hostile or toxic environment (e.g., in the presence of an antimicrobial compound).
  • a given transgene cassette e.g., a plasmid, phage, phasmid, phagemid, transposon, or other nucleic acid element
  • glutamicum and related bacteria it is possible, as described herein, to use homologs (e.g., homologs from other organisms), allelic variants or fragments of the gene retaining desired activity.
  • homologs e.g., homologs from other organisms
  • allelic variants or fragments of the gene retaining desired activity e.g., allelic variants or fragments of the gene retaining desired activity.
  • 5' and 3' regulatory elements of the genes of the invention may be modified as described herein (e.g., by nucleotide substitution, insertion, deletion, or replacement with a more desirable genetic element) to modulate the transcription of the gene.
  • an LMRB variant in which the nucleotide sequence in the region from -1 to -200 5' to the start codon has been altered to modulate (preferably increase) the transcription and/or translation of LMRB may be employed, as can constructs in which a gene of the invention (e.g., the LMRB gene (SEQ ID NO:l)) is functionally coupled to one or more regulatory signals (e.g., inducer or repressor binding sequences) which can be used for modulating gene expression.
  • regulatory signals e.g., inducer or repressor binding sequences
  • more than one copy of a gene (functional or inactivated) of the invention may be employed.
  • genes of the invention are in the discovery of new antibiotics which are active against Corynebacteria and/or other bacteria.
  • a gene of the invention may be expressed (or overexpressed) in a suitable host to generate an organism with increased resistance to one or more drugs or antibiotics (in the case of LMRB, lincosamides in particular, especially lincomycin).
  • This type of resistant host can subsequently be used to screen for chemicals with bacteriostatic and/or bacteriocidal activity, such as novel antibiotic compounds.
  • the genes of the invention e.g., the LMRB gene
  • the invention provides methods for screening molecules which modulate the activity of an SRT protein, either by interacting with the protein itself or a substrate or binding partner of the SRT protein, or by modulating the transcription or translation of ⁇ SRT nucleic acid molecule of the invention.
  • a microorganism expressing one or more SRT proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the SRT protein is assessed.
  • Manipulation of the SRT nucleic acid molecules of the invention may result in the production of SRT proteins having functional differences from the wild-type SRT proteins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
  • the goal of such manipulations is to increase the viability and activity of the cell when the cell is exposed to the environmental and chemical stresses and hazards which frequently accompany large-scale fermentative culture.
  • a heat-shock-regulated protease one may increase the ability of the cell to destroy incorrectly folded proteins, which may otherwise interfere with normal cellular functioning (for example, by continuing to bind substrates or cofactors although the protein lacks the activity to act on these molecules appropriately).
  • the ove ⁇ roduction or increase in activity by mutagenesis of proteins involved in the development of cellular resistance to chemical stresses of various kinds should increase the fitness of the organism in the environment containing the hazardous substance (i.e., large-scale fermentative culture), and thereby may permit relatively larger numbers of cells to survive in such a culture.
  • the net effect of all of these mutagenesis strategies is to increase the quantity of fine-chemical-producing compounds in the culture, thereby increasing the yield, production, and/or efficiency of production of one or more desired fine chemicals from the culture.
  • the nucleic acid and protein molecules of the invention may be utilized to generate C. glutamicum or related strains of bacteria expressing mutated SRT nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved.
  • This desired compound may be any natural product of C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C glutamicum strain of the invention.
  • NRRL ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA
  • NCIMB National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK
  • NT (NT) (GAP) Deposit rxa00062 1521 GB_HTG2 AC007366 185001 AC007366 Homo sapiens clone NH0501G22, * " SEQUENCING IN PROGRESS " * , 3 Homo sapiens 39,080 5-Jun-99 unordered pieces
  • gspG general secretory pathway protein H
  • gspH general secretory pathway protein I
  • gspl general secretory pathway protein I
  • gspl general secretory pathway protein J
  • gspK general secretory pathway protein K
  • gspL general secretory pathway protein L
  • gspM general secretory pathway protein M
  • GB STS G37084 384 G37084 SHGC-56832 Human Homo sapiens STS genomic, sequence tagged site Homo sapiens 40,420 30-MAR-1 rxa00404 2439 GB_BA1 MTCY22D7 31859 Z83866 Mycobacterium tuberculosis H37Rv complete genome, segment 133/162 Mycobacterium 60,271 17-Jun-9 tuberculosis
  • GB_BA1 ECU82598 136742 U82598 Escherichia co genomic sequence of minutes 9 to 12 Escherichia coh 54,256 15-Jan-9
  • GB_BA2 AE000165 12003 AE000165 Escherichia coh K-12 MG1655 section 55 of 400 of the complete genome Escherichia coh 54,256 12-Nov-9 rxa00479 2313
  • GB GSS2 CNS015U4 1036 AL105910 Drosophila melanogaster genome survey sequence SP6 end of BAC BACN14G08 Drosophila melanogaster 37,573 26-Jul-99 of DrosBAC library from Drosophila melanogaster (fruit fly), genomic survey sequence
  • GB_PR3 HSA494016 50502 AL1 17328 Human DNA sequence from clone 494016 on chromosome 22, complete Homo sapiens 36,475 23-Nov-9 sequence rxa00497 420 GB_BA1 MTCY78 33818 Z77165 Mycobacterium tuberculosis H37Rv complete genome, segment 145/162 Mycobacterium 40,250 17-Jun-98 tuberculosis
  • GB_BA1 MSGY223 42061 AD000019 Mycobacterium tuberculosis sequence from clone y223 Mycobacterium 37,217 10-DEC-1 t l ⁇ U ihU ⁇ CrI iU ilInUcil
  • GB_BA1 BSUB0014 213420 Z99117 Bacillus subtilis complete genome (section 14 of 21 ) from 2599451 to 2812870 Bacillus subtilis 36,553 26-N0V-9 rxa00605 1603
  • GB_BA2 AF069070 2776 AF069070 Endosymbiont of Onchoeerea volvulus catalase gene, complete eds endosymbiont of 55,396 25-Nov-9 Onchoeerea volvulus
  • GB_BA2 PAU93274 8008 U93274 Pseudomonas aeruginosa YafE (yafE), LeuB (leuB), Asd (asd), FimV (fimV), and Pseudomonas 35,895 23-Jun-98
  • HisT hisT genes, complete eds, TrpF (trpF) gene, partial eds, and unknown aeruginosa
  • GB_BA2 PAU93274 8008 U93274 Pseudomonas aeruginosa YafE (yafE), LeuB (leuB), Asd (asd), FimV (fimV), and Pseudomonas 41 ,417 23-Jun-9
  • HisT (hisT) genes complete eds, TrpF (trpF) gene, partial eds, and unknown aeruginosa rxa00803 1353 GBJN2 CELH34C03 27748 AF100662 Caenorhabditis elega ⁇ s cosmid H34C03 Caenorhabditis elegans 34,152 28-OCT-1
  • GBJHTG3 AC011283 87295 AC011283 Homo sapiens clone MS2016A09, *** SEQUENCING IN PROGRESS *** , 1 Homo sapiens 35,448 07-OCT-1 unordered pieces rxa00981 753 GB_OV GGA245664 512 AJ245664 Gallus gallus partial mRNA for ATP-citrate lyase (ACL gene) Gallus gallus 37,538 28-Sep-9 GB_PL2 AC007887 159434 AC007887 Genomic sequence for Arabidopsis thaliana BAC F1504 from chromosome I, Arabidopsis thaliana 37,600 04-OCT-1 complete sequence
  • PROGRESS *** in unordered pieces rxa01345 1575 GB_PR3 AC005224 166687 AC005224 Homo sapiens chromosome 17, clone hRPK 214_0_1 , complete sequence Homo sapiens 38,195 14-Aug-9 GB_PR3 AC005224 166687 AC005224 Homo sapiens chromosome 17, clone hRPK 214_0_1 , complete sequence Homo sapiens 36,61 1 14-Aug-9 GB HTG3 AC011500 1300851 AC011500 Homo sapiens chromosome 19 clone CIT978SKB_60E11 , '" SEQUENCING IN Homo sapiens 36,446 AC01150
  • GBJHTG3 AC010831 70233 AC010831 Homo sapiens clone 6_L_24, LOW-PASS SEQUENCE SAMPLING Homo sapiens 35,764 23-Sep-99 GB_PR3 AC004058 38400 AC004058 Homo sapiens chromosome 4 clone B241 P19 map 4q25, complete sequence Homo sapiens 40,778 30-Sep-98 rxa01408 324 GB_PR4 AF152365 246546 AF152365 Homo sapiens constitutive fragile region FRA3B sequence Homo sapiens 41 ,234 1-Aug-99 GB HTG3 AC007890 121256 AC007890 Drosophila melanogaster chromosome 3 clone BACR02G21 (D722) RPCI-98 Drosophila melanogaster 39,432 3-Sep-99
  • PROGRESS "*, 20 unordered pieces GB_HTG3 AC009919 134724 AC009919 Homo sapiens clone 115_l_23, LOW-PASS SEQUENCE SAMPLING Homo sapiens 35,409 8-Sep-99 rxa01922 1275 GB_BA1 ECONEUC 1676 M84026 E coh protein p7 (neu C) gene, complete eds Escherichia coh 35,189 26-Apr-93
  • GB_PR4 AC005552 167228 AC005552 Homo sapiens chromosome 17, clone hRPK 212_E_8, complete sequence Homo sapiens 36,249 26-NOV-9 rxa01984 420 GB_PR1 HS169C8F 245 Z57239 H sapiens CpG island DNA genomic Mse1 fragment, clone 169c8, forward read Homo sapiens 45,679 18-OCT-1 cpg169c8 ft1a
  • PROGRESS * ", 54 unordered pieces rxa02159 636 GB BA2 AF049897 9196 AF049897 Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC), Corynebacterium 99,843 1-Jul-98 ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine glutamicum transaminase (argD), ornithine carbamoyltransferase (argF), arginine repressor
  • argR argininosuccinate synthase
  • argH argininosuccinate lyase
  • GB_BA2 AF031518 2045 AF031518 Corynebacterium glutamicum ornithine carbamolytra ⁇ sferase (argF) gene, Corynebacterium 88,679 5-Jan-99 complete eds glutamicum
  • GB_BA2 AF041436 516 AF041436 Corynebacterium glutamicum arginine repressor (argR) gene complete eds Corynebacterium 100,000 5-Jan-99 glutamicum rxa02184 504
  • GB_GSS5 AQ766877 545 AQ766877 HS_2017_B2_B08_MR CIT Approved Human Genomic Sperm Library
  • PROGRESS *** in unordered pieces rxa02446 558 GB_BA2 AF036166 895 AF036166 Xanthomonas campest ⁇ s organic hydroperoxide resistance protein (ohr) gene, Xanthomonas campestns 49,369 19-MAY-1 complete eds
  • GB_BA1 MTY13E10 35019 Z95324 Mycobacterium tuberculosis H37Rv complete genome, segment 18/162 Mycobacterium 72,028 17-Jun-98 tuberculosis rxa02586 393 GBJN2 AC006472 156362 AC006472 Drosophila melanogaster, chromosome 2R, region 45E1-46A2, BAC clone Drosophila melanogaster 37,958 30-Jan-99
  • Example 1 Preparation of total genomic DNA of Corynebacterium glutamicum ATCC 13032 A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30°C with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture — all indicated volumes have been calculated for 100 ml of culture volume).
  • composition of buffer-I 140.34 g/1 sucrose, 2.46 g/1 MgSO 4 x 7H 2 O, 10 ml/1 KH 2 PO 4 solution ( 100 g/1, adjusted to pH 6.7 with KOH), 50 ml/1 Ml 2 concentrate (10 g/1 (NH 4 ) 2 SO 4 , 1 g/1 NaCl, 2 g/1 MgSO 4 x 7H 2 O, 0.2 g/1 CaCl 2 , 0.5 g/1 yeast extract (Difco), 10 ml/1 trace-elements-mix (200 mg/1 FeSO 4 x H 2 O, 10 mg/1 ZnSO 4 x 7 H 2 O, 3 mg/1 MnCl 2 x 4 H 2 O, 30 mg/1 H 3 BO 3 20 mg/1 CoCl 2 x 6 H 2 O, 1 mg/1 NiCl 2 x 6 H 2 O, 3 mg/1 Na 2 MoO 4 x 2 H 2 O, 500 mg/1 complexing agent (EDTA or critic acid), 100 ml/1 vitamins-mix (0.2 mg/1
  • Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37°C, the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) are added. After adding of proteinase K to a final concentration of 200 ⁇ g/ml, the suspension is incubated for ca.18 h at 37°C.
  • the DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform- isoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at -20°C and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing 20 ⁇ g/ml RNaseA and dialysed at 4°C against 1000 ml TE-buffer for at least 3 hours. During this time, the buffer was exchanged 3 times.
  • Example 2 Construction of genomic libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032.
  • plasmids pBR322 Suddens, J.G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741
  • pACYC177 Choange & Cohen (1978) J. Bacteriol 134:1 141-1156
  • plasmids of the pBS series pBSSKX, pBSSK- and others; Stratagene, LaJolla, USA
  • cosmids as SuperCosl (Stratagene, LaJolla, USA) or Lorist ⁇ (Gibson, T.J., Rosenthal A. and Waterson, R.H. (1987) Gene 53:283-286.
  • Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J Microbiol. Biotechnol. 4: 256-263).
  • Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R.D. et al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496- 512). Sequencing primers with the following nucleotide sequences were used: 5'- GGAAACAGTATGACCATG-3' or 5'-GTAAAACGACGGCCAGT-3'.
  • Example 4 In vivo Mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information.
  • E. coli or other microorganisms e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae
  • Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W.D. (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.)
  • Such strains are well known to those of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Calla
  • Corynebacterium and Brevibacterium species contain endogenous plasmids (as e.g., pHM1519 or pBLl) which replicate autonomously (for review see, e.g., Martin, J.F. et al. (1987) Biotechnology, 5:137-146).
  • Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al.
  • origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species.
  • transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E.L. (1987) "From Genes to Clones —
  • Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCGl (U.S. Patent No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N.D. and Joyce, CM. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180).
  • genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
  • Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or through the use of transposons.
  • REMI restriction endonuclease mediated integration
  • a gene of interest by modifying the regulatory regions (e.g., a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI).
  • Site-directed methods such as homologous recombination
  • random events such as transposon mutagenesis or REMI.
  • Nucleic acid sequences which function as transcriptional terminators may also be inserted 3' to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E.L. (1987) From Genes to Clones - Introduction to Gene Technology. VCH: Weinheim.
  • Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein.
  • a useful method to ascertain the level of transcription of the mutant gene is to perform a Northern blot (for reference see, for example, Ausubel et al.
  • RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene.
  • a detectable tag usually radioactive or chemiluminescent
  • Example 7 Growth of Genetically Modified Corynebacterium glutamicum — Media and Culture Conditions
  • Corynebacteria are cultured in synthetic or natural growth media.
  • a number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-210; von der Osten et tf/. (1998) Biotechnology Letters, 11 :11-16; Patent DE 4,120,867; Liebl (1992) "The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al, eds. Springer- Verlag).
  • These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements.
  • Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides.
  • sugars such as mono-, di-, or polysaccharides.
  • glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources.
  • sugar to the media via complex compounds such as molasses or other by-products from sugar refinement.
  • Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds.
  • Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH 4 C1 or (NH 4 ) 2 SO 4 , NH 4 OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
  • ammonia gas or ammonia salts such as NH 4 C1 or (NH 4 ) 2 SO 4 , NH 4 OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
  • Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate- salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Chelating compounds can be added to the medium to keep the metal ions in solution.
  • Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin.
  • the exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook "Applied Microbiol. Physiology, A Practical Approach (eds. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.
  • All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121°C) or by sterile filtration.
  • the components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.
  • the temperature should be in a range between 15°C and 45°C.
  • the temperature can be kept constant or can be altered during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media.
  • An exemplary buffer for this purpose is a potassium phosphate buffer.
  • Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH 4 OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the micro- organisms, the pH can also be controlled using gaseous ammonia.
  • the incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth.
  • the disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes.
  • the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles.
  • 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium.
  • the flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100 - 300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.
  • the medium is inoculated to an OD 600 of O.5 - 1.5 using cells grown on agar plates, such as CM plates (10 g/1 glucose, 2,5 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 agar, pH 6.8 with 2M NaOH) that had been incubated at 30 ° C. Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.
  • CM plates 10 g/1 glucose, 2,5 g/1 NaCl, 2 g/1 urea, 10 g/1 polypeptone, 5 g/1 yeast extract, 5 g/1 meat extract, 22 g/1 agar, pH 6.8 with
  • DNA band-shift assays also called gel retardation assays
  • reporter gene assays such as that described in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and references cited therein. Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
  • membrane-transport proteins The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
  • Example 9 Analysis of Impact of Mutant Protein on the Production of the Desired Product
  • a desired compound such as an amino acid
  • suitable conditions such as those described above
  • analyzing the medium and/or the cellular component for increased production of the desired product i.e., an amino acid.
  • Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p.
  • Example 10 Purification of the Desired Product from C. glutamicum Culture Recovery of the desired product from the C. glutamicum cells or supernatant of the above-described culture can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C. glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supernate fraction is retained for further purification.
  • the supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not.
  • chromatography steps may be repeated as necessary, using the same or different chromatography resins.
  • One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified.
  • the purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
  • the identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1 1 : 27- 32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p.
  • HPLC high-performance liquid chromatography
  • NIRS thin layer chromatography
  • enzymatic assay or microbiologically.
  • Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60:
  • EXAMPLE 11 Cloning of a Corynebacterium glutamicum Gene Involved in
  • Plasmid pSL130 was constructed by ligation of the aceB promoter region (paceB) of C. glutamicum (Kim, H.J. et al. (1997) J. Microbiol. Biotechnol. 7: 287-292) into the poly linker of the lac operon fusion vector pRS415, which lacks a promoter (Simon, R.W. et al. (1987) Gene 53: 85-96). Plasmid pSL145 was constructed by ligating the resulting paceB-lac region into the E. coli cloning vector pACYC184. E. coli DH5 ⁇ F' was transformed with pSL145 and the resulting strain was used as a host for screening of a genomic C. glutamicum library (in pSL109).
  • Transformants were screened by growth on agar medium containing 5-bromo-4- chloro-3-indolyl-beta-D-glalactopyranoside (X-Gal).
  • X-Gal 5-bromo-4- chloro-3-indolyl-beta-D-glalactopyranoside
  • a white colony containing DNA influencing lacZ expression, was selected for further analysis.
  • This clone was found to contain a 4 kB fragment from the gene library.
  • Subclones were constructed in pSL109 and a subclone which retained the white phenotype on X-Gal plates was identified. This subclone was found to contain a 2.6 kB BamHl-XhoI fragment (plasmid pSL149-5). The fragment was sequenced and identified as a membrane protein-encoding gene (LMRB gene).
  • LMRB gene membrane protein-encoding gene
  • the 1442 nucleotides of the coding sequence of the LMRB gene encode a polypeptide of 481 amino acid residues with a high percentage of hydrophobic amino acids.
  • a Genbank search determined that the LMRB protein is 40% identical to the protein product of the ImrB gene from Bacillus subtilis (Genbank Accession AL009126, TREMBL Accession P94422), as determined using a CLUSTAL W analysis (using standard parameters).
  • the LMRN protein contains a sequence pattern: 158-A-P-A-L-G-P-T-L-S-G-167 (SEQ ID NO:301), which resembles the known multi-drug-resistance-protein consensus motif G-X-X-X-G-P-X-X-G-G (SEQ ID NO:302) (Paulsen, I.T., and Skurray, R.A. (1993) Gene 124: 1-11). Therefore, the LMRB protein was classified as a drug resistance protein.
  • the ImrB gene was overexpressed in C. glutamicum ASO19E12 (Kim, H.J. et al. (1997) J Microbiol. Biotechnol. 1: 287-292) using the plasmid pSL149-5, described above. Disruption of the LMRB gene was accomplished by use of the vector pSL18- lmrB.
  • This vector was constructed as follows: an internal fragment of the LMRB gene was amplified by PCR under standard conditions using primers 5'- CTCCAGGATTGCTCCGAAGG-3' (SEQ ID NO:303) and 5'- CACAGTGGTTGACCACTGGC-3' (SEQ ID NO:304).
  • the resulting PCR product was treated with T7 DNA polymerase and T7 polynucleotide kinase, and was cloned into the Smal site of plasmid pSL18 (Kim, Y.H. and H.-S. Lee (1996) J. Microbiol. Biotechnol. 6: 315-320).
  • the disruption of the LMRB gene in C. glutamicum ASO19E12 was performed by conjugation, as previously described (Schwarzer and Puhler (1991) Bio/Technology 9:84-87).
  • C. glutamicum cells transformed with pSL 149-5 displayed similar resistances as untransformed cells against erythromycin, penicillin G, tetracycline, chloramphenicol, spectinomycin, nalidixic acid, gentamycin, streptomycin, ethidium bromide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and sodium dodecyl sulfate. Significant differences were observed, however, in the resistance of transformed and untransformed cells to lincomycin.
  • LMRB-overexpressing C. glutamicum cells were found to be able to grow in the presence of 20 ⁇ g/ml lincomycin. In contrast, cells which do not overexpress LMRB (or cells carrying a LMRB disruption) were not able to grow on agar media containing 5 ⁇ g/ml lincomycin. This effect was clearly visible in liquid culture. LMRB overexpression led to a 9-fold increased resistance (compared to wild-type) against lincomycin and LMRB disruption resulted in a decreased resistance (28% of wild-type) to this antibiotic.
  • Gapped BLAST can be utilized as described in Altschul et al. , (1997) Nucleic Acids Res. 25(17):3389-3402.
  • BLAST and Gapped BLAST programs When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence being analyzed.
  • Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci. 4: 11- 17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.
  • a comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York).
  • the gene sequences of the invention were compared to genes present in Genbank in a three-step process.
  • a BLASTN analysis e.g., a local alignment analysis
  • a subsequent FASTA search (e.g., a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits.
  • Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP program in the GCG software package (using standard parameters).
  • the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4.
  • Example 13 Construction and Operation of DNA Microarrays
  • the sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA arrays are well known in the art, and are described, for example, in Schena, M. et al. (1995) Science 270: 467-470; Wodicka, L. et al (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J.L. et al. (1997) Science 278: 680-686).
  • DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).
  • sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction.
  • a nucleic acid amplification reaction such as the polymerase chain reaction.
  • the choice and design of the 5' or 3' oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470).
  • Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359- 1367.
  • Photolithographic methods precisely defined regions of the matrix are exposed to light.
  • Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification.
  • Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions.
  • Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.
  • nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays.
  • These nucleic acid molecules can be labeled according to standard methods.
  • nucleic acid molecules e.g., mRNA molecules or DNA molecules
  • Hybridization of labeled nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra).
  • Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
  • the application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C. glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.
  • Protein populations of interest include, but are not limited to, the total protein population of C. glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.
  • Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis.
  • Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety of electrophoretic techniques.
  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE
  • Isoelectric focusing polyacrylamide gel electrophoresis Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein).
  • Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451-1463).
  • Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art. Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling.
  • Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes).
  • fluorescent dyes such as Sypro Ruby (Molecular Probes).
  • the inclusion of radioactively labeled amino acids or other protein precursors e.g., 35 S-methionine, S-cysteine, 14 C-labelled amino acids, 15 N-amino acids, 15 NO or 15 NH 4 + or ' C-labelled amino acids
  • fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.
  • Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used.
  • the amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such techniques are well-known in the art. To determine the identity of any given protein, direct sequencing or other standard techniques may be employed.
  • N- and/or C-terminal amino acid sequencing may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1 192)).
  • mass spectrometry in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1 192)).
  • MALDI mass spectrometry
  • ESI electrophoresis 18: 1184-1 192
  • the information obtained by these methods can be used to compare patterns of protein presence, activity, or modification between different samples from various biological conditions (e.g., different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.

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ES2184658T1 (es) 2003-04-16
SK18882001A3 (sk) 2002-09-10
TR200103709T2 (tr) 2002-08-21
JP2003525593A (ja) 2003-09-02
AU5836900A (en) 2001-01-31
JP2007244391A (ja) 2007-09-27
HUP0203340A2 (hu) 2003-01-28
PL359863A1 (en) 2004-09-06
WO2001000804A3 (en) 2001-08-02
KR20070087095A (ko) 2007-08-27
JP2007244392A (ja) 2007-09-27
KR100878335B1 (ko) 2009-01-14
MXPA01012844A (es) 2002-07-09
AU783703B2 (en) 2005-11-24
CA2380870A1 (en) 2001-01-04
WO2001000804A2 (en) 2001-01-04
KR20070087094A (ko) 2007-08-27
KR100834986B1 (ko) 2008-06-03
KR20060118631A (ko) 2006-11-23

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