EP1444258A2 - Genes codant pour des proteines de stabilite genetique, d'expression genique et de repliement - Google Patents

Genes codant pour des proteines de stabilite genetique, d'expression genique et de repliement

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Publication number
EP1444258A2
EP1444258A2 EP02796537A EP02796537A EP1444258A2 EP 1444258 A2 EP1444258 A2 EP 1444258A2 EP 02796537 A EP02796537 A EP 02796537A EP 02796537 A EP02796537 A EP 02796537A EP 1444258 A2 EP1444258 A2 EP 1444258A2
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EP
European Patent Office
Prior art keywords
protein
proteins
ses
dna
nucleic acid
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.)
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EP02796537A
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German (de)
English (en)
Inventor
Oskar Zelder
Markus Pompejus
Hartwig Schröder
Burkhard Kröger
Corinna Klopprogge
Gregor Haberhauer
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BASF SE
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BASF SE
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Priority to EP05026934A priority Critical patent/EP1669369A2/fr
Priority to EP06110205A priority patent/EP1693380B1/fr
Publication of EP1444258A2 publication Critical patent/EP1444258A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • Certain products and by-products of naturally occurring metabolic processes in cells are used in many industries, including the food, feed, cosmetic and pharmaceutical industries. These molecules, collectively referred to as “fine chemicals", include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors and enzymes. Their production is best accomplished by growing large-scale bacteria that have been developed to produce and secrete large quantities of the desired molecule.
  • a particularly suitable organism for this purpose is Corynebacterium glutamicum, a gram-positive, non-pathogenic bacterium. Through strain selection, a number of mutant strains have been developed that produce a range of desirable compounds. However, selecting strains that are improved in the production of a particular molecule is a time consuming and difficult process.
  • This invention provides novel nucleic acid molecules that can be used to identify or classify Corynebacterium glutamicum or related types of bacteria.
  • C. glutamicum is a gram-positive, aerobic bacterium that is commonly used in industry for the large-scale production of a number of fine chemicals and also for the degradation of hydrocarbons (e.g. when crude oil overflows) and for the oxidation of terpenoids.
  • the nucleic acid molecules can therefore be used to identify microorganisms that can be used for the production of fine chemicals, for example by fermentation processes.
  • Corynebacterium diphtheriae the causative agent of diphtheria
  • the ability to identify the presence of Corynebacterium species can therefore also be of significant clinical importance, for example in diagnostic applications.
  • These nucleic acid molecules can also serve as reference points for mapping of the C. glutamicum genome or of genomes of related organisms.
  • SES proteins encode proteins, which are referred to here as gene stability, gene expression or protein secretion / folding (SES) proteins.
  • SES proteins can, for example, perform a function involved in the repair or recombination of DNA, transposition of genetic material, expression of genes (ie which are involved in transcription or translation), protein folding or protein secretion in C. glutamicum. Due to the availability of cloning vectors useful in Corynebacterium glutamicum, such as disclosed in Sinskey et al., U.S. Patent No. 4,649,119, and techniques for genetically manipulating C. glutamicum and the related Brevi acterium species (e.g. lactofermentum) (Yoshihama et al., J.
  • nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism so that it becomes a more efficient producer of one or more fine chemicals. This improved production or efficiency of the production of a fine chemical can take place due to a direct effect of the manipulation of a gene according to the invention or due to an indirect effect of such a manipulation.
  • Modifying proteins directly involved in transcription or translation e.g., polymerases or ribosomes
  • This increased cellular gene expression should include those proteins involved in fine chemical biosynthesis so that there can be an increase in the yield, production or efficiency in the production of one or more desired compounds.
  • Modifications of the transcription / translation protein machinery of C. glutamicum, so that the regulation of these proteins is changed, can also enable the increased expression of genes which are involved in the production of fine chemicals.
  • Modulating the activity of a number of proteins involved in peptide folding can increase the overall production of correctly folded molecules in the cell, thereby increasing the possibility will that desired proteins (eg fine chemical biosynthesis proteins) work properly. Furthermore, by mutating proteins involved in the secretion from C. glutamicum so that their number or activity is increased, it may be possible to increase the secretion of a fine chemical (for example an enzyme) from cells in the fermentation culture from which it is derived can be easily won.
  • a fine chemical for example an enzyme
  • the genetic modification of the SES molecules according to the invention can also lead to an indirect modulation of the production of one or more fine chemicals.
  • a DNA repair or recombination protein according to the invention by increasing the number or activity of a DNA repair or recombination protein according to the invention, the ability of the cell to detect and repair DNA damage can be increased. This should effectively increase the ability of the cell to hold an imitated gene in its genome and thereby increase the likelihood that a transgene introduced into C. glutamicum by genetic engineering (which, for example, encodes a protein which increases the biosynthesis of a fine chemical) will not occur during the Cultivation of the microorganism is lost.
  • genetic engineering which, for example, encodes a protein which increases the biosynthesis of a fine chemical
  • transposons proteins that are involved in the transposition or displacement of genetic elements in C. glutamicum (e.g. transposons). By mutagenesis of these proteins so that their number or activity is either increased or decreased, it is possible to simultaneously increase or decrease the genetic stability of the microorganism. This has an important effect on the fact that another mutation can be introduced into C. glutamicum and that the introduced mutation can be retained. Transposons also provide a suitable mechanism by which C. glutamicum mutagenesis can be carried out; the duplication of desired genes (e.g. of fine chemical biosynthesis genes) can easily be carried out by means of transposon mutagenesis, as well as the disruption of undesired genes (e.g. genes which are involved in the breakdown of desired fine chemicals).
  • desired genes e.g. of fine chemical biosynthesis genes
  • glutamicum cells that can produce fine chemicals during large scale cultivation.
  • the secretion apparatus for example the sec system
  • integral membrane proteins for example pores, channels or transporters
  • modulating the activity of proteins involved in C. glutamicum protein secretion can affect the ability of the cell to excrete waste products or to import necessary metabolites. If the activity of these secretory proteins is increased, the ability of the cell to produce fine chemicals can also be increased. If the activity of these secretory proteins is reduced, there may not be enough nutrients to support the overproduction of desired compounds, or waste products may interfere with this biosynthesis.
  • the invention provides new nucleic acid molecules which encode proteins, which are referred to here as SES proteins and, for example, in the repair or recombination of DNA, transposition of genetic material, expression of genes (ie the transcription or translation processes), protein folding or Protein secretion in Corynebacterium glutamicum may be involved.
  • Nucleic acid molecules that encode an SES protein are referred to here as SES nucleic acid molecules.
  • an SES protein is involved in improving or reducing the genetic stability in C. glutamicum, the expression of genes (e.g. during transcription or translation) or the protein folding in this organism or in the protein secretion from C. glutamicum. Examples of such proteins are those encoded by the genes shown in Table 1.
  • nucleic acid molecules for example cDNAs
  • isolated nucleic acid molecules comprising a nucleotide sequence which codes for an SES protein or biologically active sections thereof, and also nucleic acid fragments which code as primers or hybridization probes for the detection or amplification of SES codes -
  • the nucleic acid e.g. DNA or mRNA
  • the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region of one of these nucleotide sequences or a complement thereof.
  • the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B.
  • the preferred SES proteins according to the invention likewise preferably have at least one of the SES activities described here.
  • nucleic acid sequences of the sequence listing together with the sequence changes at the respective position described in Table 1 are defined as Appendix A.
  • polypeptide sequences of the sequence listing are defined as Appendix B together with the sequence changes at the respective position described in Table 1.
  • the isolated nucleic acid molecule is at least 15 nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence from Appendix A.
  • the isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule.
  • the isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum SES protein or a biologically active portion thereof.
  • a further aspect of the invention relates to vectors, for example recombinant expression vectors, which contain the nucleic acid molecules according to the invention, and host cells into which these vectors have been introduced.
  • this host cell is used to produce an SES protein by growing the host cell in a suitable medium. The SES protein can then be isolated from the medium or the host cell.
  • Another aspect of the invention relates to a genetically modified microorganism in which an SES gene has been introduced or modified.
  • the genome of the microorganism has been changed by introducing at least one nucleic acid molecule according to the invention which codes the mutated SES sequence as a transgene.
  • an endogenous SES gene in the genome of the microorganism has been changed, for example functionally disrupted, by homologous recombination with an altered SES gene.
  • the microorganism belongs to the genus Corynebacterium or Brevibacterium, Corynebacterium glutamicum being particularly preferred.
  • the microorganism is also used for the production of a desired compound, such as an amino acid, with lysine being particularly preferred.
  • an isolated SES protein or a section thereof for example a biologically active section thereof.
  • the isolated SES protein or its portion can participate in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e. transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum.
  • the isolated SES protein or a section thereof is sufficiently homologous to an amino acid sequence from Appendix B so that the protein or its section retains the ability, for example, to repair or recombine DNA, transposition of genetic material, Gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum.
  • host cells that have more than one of the nucleic acid molecules described in Appendix A.
  • Such host cells can be produced in various ways known to those skilled in the art. For example, they can be transfected by vectors which carry several of the nucleic acid molecules according to the invention. However, it is also possible to introduce one nucleic acid molecule according to the invention into the host cell with one vector and therefore to use several vectors either simultaneously or in a staggered manner. Host cells can thus be constructed which carry numerous up to several hundred of the nucleic acid sequences according to the invention. Such an accumulation can often achieve superadditive effects on the host cell with regard to fine chemical productivity.
  • the invention also provides an isolated SES protein preparation.
  • the SES protein comprises an amino acid sequence from Appendix B.
  • the invention relates to an isolated full length protein which essentially forms a complete amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is homologous.
  • the SES polypeptide or a biologically active portion thereof can be operably linked to a non-SES polypeptide to form a fusion protein.
  • this fusion protein has a different activity than that SES protein alone.
  • this fusion protein takes part in the repair or recombination of DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum. The integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.
  • Another aspect of the invention relates to a method for producing a fine chemical.
  • the method provides for the cultivation of a cell which contains a vector which brings about the expression of an SES nucleic acid molecule according to the invention, so that a fine chemical is produced.
  • this method also comprises the step of obtaining a cell which contains such a vector, the cell being transfected with a vector which brings about the expression of a SES nucleic acid.
  • this method also comprises the step in which the fine chemical is obtained from the culture.
  • the cell belongs to the genus Corynebacterium or Brevibactium.
  • Another aspect of the invention relates to methods for modulating the production of a molecule from a microorganism. These methods involve contacting the cell with a substance that modulates SES protein activity or SES nucleic acid expression so that a cell-associated activity is changed compared to the same activity in the absence of the substance.
  • the cell is modulated for one or more C. glufcamicum processes which are involved in genetic stability, gene expression, protein folding or protein secretion, so that the yield, production or efficiency in the production of a desired fine chemicals by this microorganism is improved .
  • the substance that modulates SES protein activity can be a substance that stimulates SES protein activity or SES nucleic acid expression.
  • Another aspect of the invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing into a cell a wild-type or mutant SES gene that either remains on a separate plasmid or is integrated into the genome of the host cell.
  • the integration into the genome can be random or by homologous recombination, so that the native gene is replaced by the inserted copy, which causes the production of the desired compound from the cell to be modulated.
  • these yields are increased.
  • the chemical is a fine chemical, which in an especially preferred embodiment is an amino acid. In a particularly preferred embodiment, this amino acid is L-lysine.
  • the present invention provides SES nucleic acid and protein molecules that are involved in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e.
  • the molecules of the invention can be used to modulate the production of fine chemicals from microorganisms such as C. glutamicum, either directly (for example if the overexpression or optimization of the activity of a protein involved in the secretion of a fine chemical (for example an enzyme) has a direct effect on the yield, production and / or efficiency in the production of a fine chemical from the modified C. glutamicum) or by indirect effects which nevertheless lead to an increase in the yield, production and / or efficiency of the desired compound (for example if the modulation of the activity or number of copies C. glutamicum DNA repair protein changes in the ability of the microorganism to maintain the introduced mutation, which in turn can affect the production of one or more fine chemicals from this strain).
  • the aspects of the invention are further explained below.
  • fine chemical is known in the art and includes molecules that are produced by an organism and are used in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. These compounds include organic acids such as tartaric acid, itaconic acid and diaminopi melinic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6 , Rehm et al., Ed. VCH: Weinheim and the quotations contained therein, lipids, saturated and unsaturated fatty acids (e.g.
  • arachidonic acid arachidonic acid
  • diols e.g. propanediol and butanediol
  • carbohydrates e.g. hyaluronic acid and trehalose
  • aromatic compounds e.g. aromatic amines, vanillin and indigo
  • vitamins and cofactors as described in Ulimann's Encyclopedia of Industrial Cheistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and den citations therein; and Ong, AS, Niki, E. and Packer, L. (1995) "Nutrition, Lipids, Health and Disease” Proceedings of the UNESCO / Confederation of Scientific and Technological Associations in Malaysia and the Society for Free Radical Research - Asia, held on Sept.
  • amino acids comprise the basic structural units of all proteins and are therefore essential for the normal cell functions in all organisms.
  • amino acid is known in the art.
  • the proteinogenic amino acids of which there are 20 types, serve as structural units for proteins in which they are linked to one another via peptide bonds, whereas the non-proteinogenic amino acids (of which hundreds are known) are usually not found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)).
  • the amino acids can be in the optical D or L configuration, although L-amino acids are usually the only type found in naturally occurring proteins.
  • the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
  • the "essential" amino acids are routes of synthesis into the remaining 11 "non-essential" amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine).
  • higher Animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested with food for normal protein synthesis to take place.
  • Lysine is not only an important amino acid for human nutrition, but also for monogastric animals such as poultry and pigs.
  • Glutamate is most commonly used as a flavor additive (monosodium glutamate, MSG) and widely used in the food industry, as well as aspartate, phenylalanine, glycine and cysteine.
  • Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry.
  • Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are widespread feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol 6, Chapter 14a, VCH: Weinheim).
  • amino acids are also used as precursors for the synthesis of synthetic amino acids and proteins such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others in Ulmann's Encyclopedia of Industrial Chemistry , Vol. A2, pp. 57-97, VCH, Weinheim, 1985 are suitable substances.
  • Cysteine and glycine are each produced from serine, the former by condensation of homocysteine with serine, and the latter by transferring the side chain ⁇ -carbon atom to tetrahydrofolate in a reaction catalyzed by serine transhydroxymethylase.
  • Phenylalanine and tyrosine are prepared from the precursors of the glycolysis and pentose phosphate pathways, erythrosis-4-phosphate and phosphoenolpyruvate, in a 9-step bio synthesized synthetic route, which differs only in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in an 11-step process.
  • Tyrösin can also be prepared from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase.
  • Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis.
  • Aspartate is made from oxaloacetate, an intermediate of the citrate cycle.
  • Asparagine, methionine, threonine and lysine are each produced by converting aspartate.
  • Isoleucine is made from threonine.
  • histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
  • Amino acids the amount of which exceeds the protein biosynthesis requirement, cannot be stored and are instead broken down, so that intermediate products are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd edition, chapter 21 "Amino Acid Degradation and the Urea Cycle”; S 495-516 (1988)).
  • the cell is able to convert unwanted amino acids into useful metabolic intermediates, the production of amino acids is expensive in terms of energy, precursor molecules and the enzymes required for their synthesis.
  • amino acid biosynthesis is regulated by feedback inhibition, the presence of a particular amino acid slowing down or completely stopping its own production (for an overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L., Biochemistry , 3rd edition, chapter 24, "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988)).
  • the output of a certain amino acid is therefore restricted by the amount of this amino acid in the cell.
  • Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and must therefore absorb them, although they are easily synthesized by other organisms such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a number of metabolic pathways. In addition to their nutritional value, these compounds have a significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure, and for the industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, "Vitamins", Vol. A27, pp. 443-613, VCH: Weinheim, 1996).
  • vitamin is known in the art and encompasses nutrients which are required by an organism for normal function, but which cannot be synthesized by this organism itself.
  • the group of vitamins can include cofactors and nutraceutical compounds.
  • cofactor encompasses non-proteinaceous compounds which are necessary for the occurrence of normal enzyme activity. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferably organic.
  • nutraceutical encompasses food additives which are beneficial to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (eg polyunsaturated fatty acids).
  • vitamin Bi Thiamine
  • Riboflavin is synthesized from guanosine 5'-triphosphate (GTP) and ribose 5'-phosphate. Riboflavin in turn is used for the synthesis of flavinononucleotide (FMN) and flavin adenine dinucleotide (FAD).
  • the family of compounds that are collectively referred to as "vitamin B ⁇ " are all derivatives of the common structural unit 5-hydroxy-6-methylpyridine.
  • Panthothenate (pantothenic acid, R- (+) -N- (2,4-dihydroxy-3,3,3-dimethyl-1-oxobutyl) -ß-alanine) can be produced either by chemical synthesis or by fermentation.
  • the final steps in pantothenate biosynthesis consist of the ATP-driven condensation of ß-alanine and pantoic acid.
  • the enzymes responsible for the biosynthetic steps for the conversion into pantoic acid, into ⁇ -alanine and for the condensation into pantothenic acid are known.
  • the metabolically active form of pantothenate is coenzyme A, whose biosynthesis takes 5 enzyatic steps runs.
  • Pantothenate pyridoxal-5 '-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone, (R) - Panthenol (provitamin B 5 ), Pantethein (and its derivatives) and coenzyme A.
  • 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 that are all derived from folic acid, which in turn is derived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterine.
  • Corrinoids such as the cobalamins and especially vitamin B ⁇
  • the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system.
  • the biosynthesis of vitamin B ⁇ is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known.
  • Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called “niacin”.
  • Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
  • nucleotide includes the basic structural units of the nucleic acid molecules, which comprise a nitrogenous base, a pentose sugar (for RNA the sugar is ribose, for DNA the sugar is D-deoxyribose) and phosphoric acid.
  • nucleoside encompasses molecules which serve as precursors of nucleotides, but which, in contrast to the nucleotides, have no phosphoric acid unit.
  • 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; if this activity is specifically inhibited in cancer cells, the ability of tumor cells to divide and replicate can be inhibited.
  • nucleotides that do not form nucleic acid molecules but that serve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).
  • the purine nucleotides are synthesized from ribose 5-phosphate via a series of steps via the intermediate compound inosine 5 'phosphate (IMP), which leads to the production of guanosine 5' monophosphate (GMP) or adenosine 5 'monophosphate (AMP) leads from which the triphosphate forms used as nucleotides can be easily produced. These compounds are also used as energy stores, so that their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis takes place via the formation of uridine 5 'monophosphate (UMP) from ribose 5-phosphate. UMP in turn is converted to cytidine 5 'triphosphate (CTP).
  • IMP intermediate compound inosine 5 'phosphate
  • AMP adenosine 5 'monophosphate
  • the deoxy forms of all nucleotides are produced in a one-step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. After phosphorylation, these molecules can participate in DNA synthesis.
  • the production of a desired compound from a cell is the culmination of a large number of separate, yet interlinked processes, each of which is critical to the overall production and release of the compound from the cell.
  • each of these processes must be taken into account to ensure that the cell's biochemical machinery is compatible with this genetic manipulation.
  • Particularly important cellular mechanisms include the stability of the modified gene (s) when introduced into the cell, the ability of the mutant gene to be properly transcribed and translated (including codon usage) and the ability of the mutant protein product to be properly folded and / or to be secreted.
  • DNA repair and recombination are separate but interrelated ways for DNA repair and recombination.
  • the former is used to strictly correct errors in DNA molecules either by directly reversing the damage or by cutting out the damaged area and replacing it with the correct sequence.
  • the latter recombination system also repairs nucleic acid molecules, but only damage that results in damage in both strands of DNA, so that neither strand can be used as a template to correct the other.
  • Recombination repair and SOS response can easily lead to inversions, deletions, or other genetic rearrangements within or around the damaged area, which in turn promotes a certain level of genomic instability that increases the cell's ability to adapt to changing environments or stress, can contribute.
  • High-fidelity repair mechanisms include the direct reversal of DNA damage and the cutting out of the damage and the resynthesis using the information encoded in the opposite strand. Undoing the damage directly requires an enzyme with an activity that does the opposite of what originally damaged the DNA.
  • methylation of DNA can be corrected by the action of DNA repair methyltransferases, and nucleotide dimers generated by UV radiation can be repaired by the activity of deoxyribodipyrimidine photolyase, which cleaves the dimer back into the corresponding nucleotides in the presence of light (see Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York, and the references cited therein).
  • the repair system includes a double crossover event between the damaged area and another copy of the area on a homologous DNA molecule. This is possible because bacteria divide so quickly that a second copy of the genomic DNA is available before cell division actually takes place. This crossover event can easily lead to inversions, duplications, deletions, insertions and other genetic rearrangements and thus increases the overall genetic instability of the organism.
  • the SOS reaction is activated when there is sufficient damage in the DNA that the DNA polymerase III stops and cannot continue to replicate. Under these circumstances, single-stranded DNA is present.
  • the RecA protein is activated by binding to single-stranded DNA, and this activated form leads to activation of the LexA repressor, whereby the transcription block of more than 20 genes is released, including UvrA, UvrB, UvrC, Helicase II, DNA pol III, UmuC and UmuD.
  • the combined activities of these enzymes sufficiently fill the gap area so that DNA pol III can resume replication. However, these gaps are filled with bases that should not be present; this type of repair therefore leads to a fault-prone repair, which overall contributes to the genetic instability in the cell.
  • Transposons are genetic elements that can migrate from one site to another either within a chromosome or between a piece of extrachromosomal DNA (e.g. a plasmid) and a chromosome.
  • the transposition takes place in several ways; for example, the transposable element can be cut out of the donor site and integrated into the target site (non-replicative transposition), or the transposable element can alternatively be duplicated from the donor site to the target site, resulting in two copies of the element (replicative transposition). There is usually no sequence relationship between the donor and the target site.
  • This transposition event has a variety of possible outcomes.
  • the integration of a transposable element into a gene disrupts the gene, which usually completely eliminates its function.
  • An integration event that takes place in the DNA surrounding the gene cannot disrupt the coding sequence itself, but have a fundamental effect on the regulation of the gene and thus on its expression.
  • Recombination events between two copies of a transposable element located in different sections of the genome can lead to deletions, duplications, inversions, transpositions or amplifications of segments of the geno. It is also possible for different replicons to fuse.
  • IS elements The simplest transposon-like genetic elements are called insertion (IS) elements.
  • IS elements contain a nucleotide region of variable length (but usually less than 1500 bases) that contains no coding regions and is surrounded by inverted repeats at each end. Since the IS element does not encode proteins whose activity can be detected, the presence of an IS element is usually only observed due to a loss of function of one or more genes into which the IS element is inserted.
  • Transposons are mobile genetic elements that, in contrast to IS elements of repeats, contain nucleic acid sequences that can encode one or more proteins. It is not uncommon for these repeat areas to consist of IS elements.
  • the proteins encoded by the transposon are usually transpososes (proteins that catalyze the transposon's migration from one site to another) and antibiotic resistance genes.
  • the mechanisms and the regulation of the transposable elements are known in the art and were at least described, for example, in: Lengeier et al. (1999) Biology of Prokaryotes, Thieme Published by Stuttgart, pp. 375-361; Neidhardt et al.
  • RNA polymerase the operating DNA-transcribing enzyme
  • sigma factors which regulate gene transcription by converting the RNA polymerase into specific promoter-DNA. Direct sequences that recognize these factors).
  • the combination of RNA polymerase and sigma factors creates the RNA polymerase holoenzyme, an activated complex.
  • Gram-positive bacteria such as Coryne bacteria, contain only one type of RNA polymerase, but a number of different sigma factors, which are specific for different promoters, growth phases, environmental conditions, substrates, oxygen levels, transport processes and the like the organism can adapt to different environmental and metabolic conditions.
  • the promoter-transcription control is influenced by several repression or activation mechanisms.
  • Specific regulatory proteins that bind to promoters have the ability to block the binding of the RNA holoenzyme (repressors) or to support it (activators) and thus regulate the transcription.
  • the binding of these repressor and activator molecules is in turn regulated by their interactions with other molecules, such as proteins or other metabolic compounds.
  • the transcription can alternatively be regulated by factors which influence processes such as elongation or termination (see, for example, Sonenshein, AL, Hoch, JA, and Losick, R., ed. (1993) Bacillus subtilis, ASM Press: Washington , DC).
  • the ability to regulate the transcription of genes in response to a variety of environmental or metabolic signs allows the cells to control exactly when a gene can be expressed and how much of a gene product can be present in the cell at a time. This in turn prevents the unnecessary waste of energy or the unnecessary use of possibly rare interconnections or cofactors.
  • Translation is the process by which a polypeptide is synthesized from amino acids according to the information contained in an RNA molecule.
  • the main components of this process are ribosomes and specific initiation or elongation factors such as IF1-3, ERFINDUNGSGEM ⁇ SS-G and EFTu (eg Sonenshein, AL, Hoch, JA, and Losick, R., ed. (1993) Bacillus subtilis, ASM Press: Washington, DC).
  • tRNA transfer RNA
  • These molecules consist of an RNA single strand (between 60 and 100 bases), which is in an L-shaped three-dimensional structure with protruding areas or "arms". One of these arms forms base pairs with a specific codon sequence on the mRNA molecule. A second arm specifically interacts with a particular amino acid (encoded by the codon).
  • Other tRNA arms include the variable arm, the T ⁇ C arm (which carries thymidylate and pseudouridylate modifications) and the D arm (which carries a dihydrouridine modification). The function of these latter structures is still unknown, but their conservation between the tRNA molecules suggests a role in protein synthesis.
  • aminoacyl-tRNA synthetases For the nucleic acid-based tRNA molecule to pair with the correct amino acid, a family of enzymes called aminoacyl-tRNA synthetases must work. There are many different types of these enzymes, and each one is specific to a particular tRNA and amino acid. These enzymes bind the 3'-hydroxyl of the terminal tRNA adenosine ribose unit to the amino acid in a two-step reaction. First, the enzyme is activated by reaction with ATP and the amino acid, resulting in an aminoacyl-tRNA-synthetase-aminoacyl-adenylate complex. Second, the aminoacyl group is transferred from the enzyme to the target tRNA, where it remains in an energetic state.
  • amino acid-loaded tRNA occupies a binding site (the A site) next to a second site (the P site), which carries a tRNA molecule whose amino acid is bound to the nascent polypeptide chain ( Peptidyl tRNA).
  • the activated amino acid on the aminoacyl tRNA is sufficiently reactive that a peptide bond spontaneously forms between this amino acid and the next amino acid on the nascent polypeptide chain.
  • GTP hydrolysis provides the energy to transfer the tRNA now loaded with the polypeptide chain from the A site to the P site of the ribosome, and the process repeats until a stop codon is reached.
  • polypeptide chains which have to assume a three-dimensional shape before the protein can function normally.
  • the three-dimensional structure is achieved through a folding process.
  • isomerases e.g. trigger factor, cyclophilin and FKBP homologs
  • proteins from the heat shock protein group e.g. DnaK, DnaJ, GroEL, small heat shock proteins, HtpG and members of the Clp family (e.g. ClpA, ClpB, ClpW, ClpP and ClpX)
  • heat shock protein group e.g. DnaK, DnaJ, GroEL
  • small heat shock proteins e.g. DnaK, DnaJ, GroEL
  • HtpG e.g., small heat shock proteins, HtpG and members of the Clp family
  • ClpA, ClpB, ClpW, ClpP and ClpX members of the Clp family
  • the chaperones bind to the incorrectly folded protein and force it to return to an unfolded state. This cycle can be repeated until the protein is folded correctly.
  • the second group e.g. GroEL / ES
  • the GroEL / ES complex consists of two stacked 14-membered rings with a hydrophobic inner surface and a "lid" made of a 7-membered ring. The polypeptide is drawn into the channel in the center of this complex in an ATP-dependent reaction, where it can fold without interference from other polypeptides. Incorrectly folded proteins are not released from the complex.
  • Disulfide bonds are important for protein stability. Disulfide bonds are easily formed in aqueous solution, and it is difficult to reverse improper disulfide bridge formation without the help of a reducing environment.
  • thiol-containing molecules such as glutathione or thioredoxin and their corresponding oxidation / reduction systems can be found in the cytosol of most cells (Loferer, H., Hennecke, H. (1994) Trends in Biochemical Sciences 19 (4 ) .169-171).
  • the folding of nascent polypeptide chains is not desirable, for example when these proteins are to be secreted.
  • the folding process usually results in the hydrophobic regions of the protein being in the center of the protein, away from the aqueous solution, and the hydrophilic regions being presented on the outer surfaces of the protein.
  • This conformational arrangement produces one higher stability for the protein, but complicates the translocation of the protein across membranes, since the hydrophobic core of the membrane is inherently incompatible with the hydrophilic exterior of the protein.
  • the proteins synthesized by the cell, 5 which have to be secreted to the outside of the cell (eg cell surface enzymes and membrane receptors) or which have to be inserted into the membrane itself (eg transporter proteins and channel proteins), are usually secreted or inserted before folding ,
  • Polypeptide chains prevent, also prevent the folding of polypeptides until they are no longer needed.
  • these proteins can "escort" nascent polypeptide chains to a suitable location in the cell, where they are either removed so that folding is possible, or the protein can be transposed.
  • the machinery consists of a series of proteins, collectively referred to as the sec (type II secretion) system (for an overview, see Gilbert, M., et al. (1995) Critical Reviews in Biotechnology
  • the sec system consists of chaperones (e.g. SecA and SecB), integral membrane proteins, which are also referred to as translocases (e.g. SecY, SecE and SecG) and signal peptidases (e.g. LepB).
  • chaperones e.g. SecA and SecB
  • integral membrane proteins which are also referred to as translocases
  • signal peptidases e.g. LepB
  • SecB which it transfers to SecA on the inner surface of the cell membrane.
  • SecA binds to the prosequence and inserts into the membrane after ATP hydrolysis and also forces part of the polypeptide through the membrane.
  • the rest of the polypeptide is passed through the membrane through a complex of translocases such as SecY, 0 SecE and SecG.
  • the signal peptidase cleaves the prosequence, and the polypeptide is free on the extracellular side of the membrane, where it spontaneously folds.
  • the signal recognition particle dependent pathway involves binding a signal recognition particle (SRP) protein to the nascent polypeptide during its synthesis, causing the ribosom to stop.
  • a receptor for SRP on the inner surface of the membrane then binds the ribosome-polypeptide-SRP complex.
  • GTP hydrolysis provides the energy necessary to transfer the complex to the sec-translokase complex, where the polypeptide is passed through the ribosome across the membrane during its synthesis.
  • secretion mechanisms that are specific for only a few proteins.
  • the present invention is based at least in part on the discovery of new molecules, referred to here as SES nucleic acid and protein molecules, and on the repair or recombination of DNA in C. glutamicum, transposition or other rearrangement of C. glutaicur ⁇ DNA, Gene expression in C. glutamicum (ie transcription or translation processes), protein folding or protein secretion involve this microorganism.
  • the SES molecules participate in the repair or recombination of DNA, transposition of genetic material, gene expression (i.e., transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum.
  • the activity of the SES molecules according to the invention with regard to the repair or recombination of DNA, transposition of DNA, gene expression, protein folding or protein secretion has an effect on the production of a desired fine chemical by this microorganism.
  • the activity of the SES molecules according to the invention is modulated so that the activity of the C. glutamicum cell processes in which the SES proteins according to the invention are involved (eg repair or recombination of DNA, transposition of DNA, gene expression) , Protein folding or protein secretion) is changed, which leads directly or indirectly to a modulation of the yield, production and / or efficiency of the production of a desired fine chemical by C. glutamicum.
  • SES protein or "SES polypeptide” encompasses proteins which are involved in a number of cell processes which are related to the genetic stability, gene expression, protein folding or protein secretion of C. glutamicum.
  • an SES protein can be used for DNA repair or for recombination mechanisms in C. glutamicum, rearrangements of the genetic material of C. glutamicum (such as that mediated by transposons), the transcription or translation of genes in this microorganism, during the mediation protein folding in C. glutamicum (such as the activity of chaperones) or the secretion of protein from C. glutamicum cells (eg in the sec system).
  • SES proteins include those encoded by the SES genes listed in Table 1 and Appendix A.
  • SES gene or "SES nucleic acid sequence” encompass nucleic acid sequences which encode an SES protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of SES genes are those listed in Table 1.
  • production or “productivity” are known in the art and include the concentration of the fermentation product (for example the desired fine chemical) which is formed within a defined period of time and a defined fermentation volume (for example kg product per hour per 1 ).
  • production efficiency encompasses the time it takes to achieve a certain amount of production (for example how long it takes the cell to erect a certain rate of ejection of a fine chemical).
  • yield or "product / carbon yield” is known in the art and encompasses the efficiency of converting the carbon source into the product (ie, the fine chemical). For example, this is usually expressed as kg product per kg carbon source. Increasing the yield or production of the compound increases the amount of molecules or suitable molecules of this compound obtained in a given amount of culture over a predetermined period of time.
  • biosynthesis or “biosynthetic pathway” are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step or highly regulated process.
  • degradation or “degradation path” are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), e.g. in a multi-step or highly regulated Process.
  • metabolism is known in the art and encompasses the entirety of the biochemical reactions that take place in an organism. The metabolism of a particular compound (for example the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways in the cell which concern this compound.
  • DNA repair is known in the art and includes cellular mechanisms by which defects in the DNA (either due to damage such as, but not limited to, ultraviolet radiation, methylases, low-frequency replication or mutagens) are cut out and Getting corrected.
  • the term “recombination” or “DNA recombination” is known in the art and encompasses cellular mechanisms by which extensive DNA damage, which affects both strands of a DNA molecule, by homologous recombination with another undamaged corrected copy of the DNA molecule within the same cell can be corrected. These repairs are usually low-fidelity and can lead to gene rearrangements.
  • transposon is known in the art and encompasses a DNA element which can randomly insert into the genome of an organism and which can lead to the disruption of genes or their regulatory regions or to duplications, inversions, deletions and other gene rearrangements.
  • protein folding is known in the art and encompasses the migration of a polypeptide chain through several three-dimensional configurations until the stable, active, three-dimensional configuration is achieved. The formation of disulfide bonds and the sequestration of the hydrophobic region from the surrounding aqueous solution provide some of the driving forces for this protein folding process, and correct folding can be enhanced by the activity of chaperones.
  • secretion or "protein secretion” are known in the art and encompass the movement of proteins from the inside of the cell to the outside of the cell in a mechanism in which a system of secretion proteins allows them to pass across the cell membrane to the outside of the cell.
  • the SES molecules according to the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum.
  • a desired molecule such as a fine chemical
  • a microorganism such as C. glutamicum.
  • modulating proteins directly involved in transcription or translation eg polymerases or ribosomes
  • This increased cellular gene expression should include those proteins which are involved in fine chemical biosynthesis, so that an increase in the yield, production or efficiency in the production of one or more desired compounds can take place.
  • Modifications of the transcription / translation protein machinery of C. glutamicum so that the regulation of these proteins is changed can also enable the increased expression of genes which are involved in the production of fine chemicals. Modulating the activity of a number of proteins involved in peptide folding can increase the overall production of correctly folded molecules in the cell, increasing the possibility that desired proteins (eg, fine chemical biosynthesis proteins) can function properly.
  • mutating proteins involved in C. glutamicum secretion so that their number or activity is increased, it may be possible to increase the secretion of a fine chemical (eg an enzyme) from cells in the fermentation culture from which it can be easily obtained .
  • the genetic modification of the SES molecules according to the invention can also lead to an indirect modulation of the production of one or more fine chemicals.
  • a DNA repair or recombination protein of the invention by increasing the number or activity of a DNA repair or recombination protein of the invention, the ability of the cell to detect and repair DNA damage can be increased. This should effectively increase the ability of the cell to hold a mutated gene in its genome and thereby increase the likelihood that a transgene introduced into C. glutamicum by genetic engineering (which, for example, encodes a protein that increases the biosynthesis of a fine chemical) will not is lost during the growth of the microorganism.
  • genetic engineering which, for example, encodes a protein that increases the biosynthesis of a fine chemical
  • transposons proteins that are involved in the transposition or rearrangement of genetic elements in C. glutamicum (e.g. transposons). By mutagenesis of these proteins so that their number or activity is either increased or decreased, it is possible to simultaneously increase or decrease the genetic stability of the microorganism. This has an important effect on the fact that another mutation can be introduced into C. glutamicum and that the introduced mutation can be retained. Transposons also provide a suitable mechanism by which C. glutamicum mutagenesis can be carried out; the duplication of desired genes (e.g. of fine chemical biosynthesis genes) can easily be carried out by means of transposon mutagenesis, as well as the disruption of undesired genes (e.g. genes which are involved in the breakdown of desired fine chemicals).
  • desired genes e.g. of fine chemical biosynthesis genes
  • certain bacterial protein secretion pathways for example the sec system
  • integral membrane proteins for example receptors, channels, pores or transporters
  • the activity of proteins involved in protein secretion can be modulated from C. glutamicum are involved, influence the ability of the cell to excrete waste products or to import necessary metabolites. If the activity of these secretory proteins is increased, the cell's ability to produce fine chemicals can also be increased (due to an increased presence of transporters / channels in the membrane which can import nutrients or excrete waste products). If the activity of these secretory proteins is reduced, there may not be enough nutrients to support the overproduction of desired compounds, or waste products may interfere with this biosynthesis.
  • the genome of a Corynebacterium glutamicum strain which is available from the American Type Culture Collection under the name ATCC 13032, is suitable as a starting point for producing the nucleic acid sequences according to the invention.
  • nucleic acid sequences according to the invention can be produced from these nucleic acid sequences by conventional methods using the changes described in Table 1.
  • the SES protein according to the invention or a biologically active section or fragment thereof can participate in the repair or recombination of DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum or one or more of the activities described in Table 1.
  • nucleic acid molecule which encode SES polypeptides or biologically active sections thereof, and to nucleic acid fragments which are used as hybridization probes or primers for identifying or amplifying SES-coding nucleic acids (for example SES-DNA).
  • SES-DNA for example SES-DNA
  • nucleic acid molecule as used here is intended to encompass DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g. mRNA) as well as DNA or RNA analogs which are generated by means of nucleotide analogs.
  • This term also includes the untranslated sequence located at the 3 'and 5' end of the coding gene region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3 'end of the coding region of the gene.
  • the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example, sequences that are located at the 5 'or 3' end of the nucleic acid ).
  • the isolated SES nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotide sequences that naturally comprise the nucleic acid molecule in the genomic Flank the DNA of the cell from which the nucleic acid originates (for example a C. glutamicum cell).
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can also be substantially free of other cellular material or culture medium if it is produced by recombinant techniques, or of chemical precursors or other chemicals if it is chemically synthesized.
  • a nucleic acid molecule according to the invention for example a nucleic acid molecule with a nucleotide sequence from Appendix A or a section thereof, can be produced using standard molecular biological techniques and the sequence information provided here.
  • a C. utamicum SES cDNA can be isolated from a C. glutamicum library by using a complete sequence from Appendix A or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch , EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • a isolate a small acid molecule comprising a complete sequence from Annex A or a section thereof by polymerase chain reaction, using oligonucleotide primers which have been prepared on the basis of this sequence can be isolated by polymerase chain reaction using oligonucleotide primers which have been prepared based on this same sequence from Appendix A).
  • oligonucleotide primers which have been prepared based on this same sequence from Appendix A can be isolated from normal endothelial cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al.
  • cDNA can be converted using reverse transcriptase (for example Moloney-MLV reverse -Transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase for example 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 the amplification via polymerase chain reaction can be created on the basis of one of the nucleotide sequences shown in Appendix A.
  • a nucleic acid according to the invention can be amplified using cDNA or alternatively genomic DNA as a template and suitable oligonucleotide primers according to standard PCR amplification techniques.
  • nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis.
  • Oligonucleotides which correspond to an SES nucleotide sequence can also be produced by standard synthesis methods, for example using an automatic DNA synthesizer.
  • an isolated nucleic acid molecule according to the invention comprises one of the nucleotide sequences listed in Appendix A.
  • an isolated nucleic acid molecule according to the invention comprises a nucleic acid molecule which is complementary to one of the nucleotide sequences shown in Appendix A or a portion thereof, which is a nucleic acid molecule which is sufficiently complementary to one of the nucleotide sequences shown in Appendix A that it can hybridize to one of the sequences given in Appendix A, creating a stable duplex.
  • the nucleic acid molecule of the invention encodes a protein or a portion thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion thereof retains the ability to repair or recombine DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding tion or protein secretion in Corynebacterium glutamicum.
  • the term "sufficiently homologous" refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent (e.g., an amino acid residue with a side chain similar to an amino acid residue in one of the sequences of Appendix B) amino acid residues to an amino acid sequence from Appendix B so that the protein or a portion thereof can participate in the repair or recombination of DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum. Proteins involved in the genetic stability, gene expression, protein folding or protein secretion of C. glutamicum, as described here, can play a role in the production and secretion of one or more fine chemicals. Examples of these activities are also described here. Thus, the "function of an SES protein” directly or indirectly contributes to the yield, production and / or efficiency of the production of one or more fine chemicals. Examples of SES proteins are shown in Table 1.
  • Sections of proteins which are encoded by the SES nucleic acid molecules according to the invention are preferably biologically active sections of one of the SES proteins.
  • the term “biologically active section of an SES protein” as used here is intended to encompass a section, for example a domain / motif of an SES protein, which is involved in the repair or recombination of DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum or has one of the activities shown in Table 1.
  • SES protein or a biologically active section thereof can participate in the repair or recombination of DNA, transposition of genetic material, gene expression (ie transcription or translation processes), protein folding or protein secretion in Corynebacterium glutamicum, a test of the enzymatic Activity.
  • nucleotide sequence of Appendix A which leads to a change in the amino acid sequence of the encoded SES protein without affecting the functionality of the SES protein.
  • nucleotide substitutions attached to "non- essential "amino acid residues lead to amino acid substitutions, in a sequence of Appendix A.
  • A" non-essential "amino acid residue is a residue that can be changed in the wild-type sequence by one of the SES proteins (Appendix B) without the activity of the SES -Proteins is changed, whereas an "essential" amino acid residue is required for SES protein activity.
  • other amino acid residues for example non-conserved or only semi-preserved amino acid residues in the domain with SES activity
  • An isolated nucleic acid molecule that encodes an SES protein that is homologous to a protein sequence from Appendix B can be prepared by introducing one or more nucleotide substitutions,
  • additions or deletions are generated in a nucleotide sequence from Appendix A, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • the mutations can be introduced into one of the sequences from Appendix A using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are preferably introduced at one or more of the predicted non-essential amino acid residues.
  • the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families 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
  • non-polar side chains e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g. threonine, valine, Isoleucine
  • aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine.
  • a predicted non-essential amino acid residue in an SES protein is thus preferably replaced by another amino acid residue of the same side chain family.
  • the mutations can alternatively be introduced randomly over all or part of the SES coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for SES activity described here in order to identify mutants, that maintain SES activity.
  • the encoded protein can be expressed recombinantly and the activity of the pro Teins can be determined, for example, using the tests described here (see example 8 of the example section).
  • vectors preferably expression vectors, which contain a nucleic acid which encode an SES protein (or a portion thereof).
  • vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is attached.
  • plasmid which is a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, whereby additional DNA segments can be ligated into the viral genome.
  • Certain vectors can replicate autonomously in a host cell into which they have been introduced (for example bacterial vectors with a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell when introduced into the host cell and thereby replicated together with the host genome.
  • certain vectors can control the expression of genes to which they are operably linked. These vectors are referred to here as "expression vectors".
  • the expression vectors that can be used in recombinant DNA techniques are usually in the form of plasmids.
  • plasmid and “vector” can be used interchangeably because the plasmid is the most commonly used vector form.
  • the invention is intended to encompass other expression vector forms, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.
  • the recombinant expression vectors according to the invention comprise a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, that is to say that the recombinant expression vectors comprise one or more regulatory sequences, selected on the basis of the host cells to be used for expression, with the are operably linked to the nucleic acid sequence to be expressed.
  • “operably linked” means that the nucleotide sequence of interest is bound to the regulatory sequence (s) in such a way that expression of the nucleotide sequence is possible (for example in an in vitro transcription) - / translation system or in a host cell if the vector is introduced into the host cell).
  • regulatory sequence is intended to encompass promoters, enhancers and other expression control elements (for example polyadenylation signals). This regulatory sequences are described, for example, in Goeddel - Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that control the constitutive expression of a nucleotide sequence in many host cell types and those that control the expression of the nucleotide sequence only in certain host cells. The person skilled in the art is aware that the design of an expression vector can depend on factors such as the choice of the host cell to be transformed, the desired level of protein expression etc.
  • the expression vectors according to the invention can be introduced into the host cells, so that proteins or peptides are thereby , including the fusion proteins or peptides encoded by the nucleic acids as described herein (e.g., SES proteins, mutated forms of SES proteins, fusion proteins, etc.).
  • the recombinant expression vectors according to the invention can be designed for the expression of SES proteins in prokaryotic or eukaryotic cells.
  • SES genes in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA et al. (1992) "Foreign gene expression in yeast: a review ", Yeast 8: 423-488; van den Hondel, CAMJJ et al. (1991)” Heterologous gene expression in filamentous fungi "in: More Gene Manipulations in Fungi, JW Bennet & LL Lasure,
  • Suitable host cells are further discussed in Goeddel , Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can alternatively, for example with regulatory sequences of the T7 promoter and T7 polymerase, be transcribed and translated in vitro.
  • Proteins are usually expressed in prokaryotes using vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins.
  • Fusion vectors contribute a number of amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein. These fusion vectors usually have three functions: 1) to increase the expression of reco binant protein; 2) increasing the solubility of the recombinant protein; and 3) supporting the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is often introduced at the junction of the fusion unit and the recombinant protein, so that the recombinant protein can be separated from the fusion unit after the fusion protein has been purified.
  • These enzymes and their corresponding recognition sequences include factor Xa, thrombin and entokinase.
  • Common fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ), in which Glutathione-S-transferase (GST), maltose E-binding protein or protein A is fused to the recombinant target protein.
  • GST Glutathione-S-transferase
  • the coding sequence of the SES protein is cloned into a pGEX expression vector so that a vector is generated which encodes a fusion protein comprising from the N-terminus to the C-terminus: GST - thrombin cleavage site - X protein.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin.
  • the recombinant SES protein, which is not fused with GST can be obtained by
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET lld (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990 ) 60-89).
  • Target gene expression from the pTrc vector is based on transcription by host RNA polymerase from a hybrid trp-lac fusion promoter.
  • the target gene expression from the pETIld vector is based on the transcription from a T7-gnl0-lac fusion promoter, which is mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by the BL 21 (DE3) or HMS174 (DE3) host strains from a resident ⁇ prophage which harbors a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize the expression of the recombinant protein is to express the protein in a host bacterium whose ability to proteolytically cleave the recombinant protein is impaired (Gottesman, S. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 119-128).
  • Another strategy is to change the Nucleic acid sequence of the nucleic acid to be inserted into an expression vector, so that the individual codons for each amino acid are those which are preferably used in a bacterium selected for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20: from 2111 to 2118). This change in the nucleic acid sequences according to the invention can be carried out using standard DNA synthesis techniques.
  • the SES protein expression vector is a yeast expression vector.
  • yeast expression vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943 ), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).
  • Vectors and methods of constructing vectors suitable for use in other fungi, such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J.
  • the SES proteins according to the invention can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell Biol .. 3: 2156-2165) and pVL series (Lucklow and Summers (1989) Virology 170: 31-39).
  • the SES proteins according to the invention can be expressed in cells of single-cell plants (such as algae) or in plant cells of higher plants (for example spermatophytes, such as crops).
  • plant expression vectors include those which are described in detail in: Bekker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximally to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721.
  • a nucleic acid according to the invention is expressed in mammalian cells with a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195).
  • pCDM8 Seed, B. (1987) Nature 329: 840
  • pMT2PC Kaufman et al. (1987) EMBO J. 6: 187-195.
  • the control functions of the expression vector often provided by viral regulatory elements. Promoters commonly used are derived, for example, from Polyoma, Adenovirus 2, Cytomegalievirus and Simian Virus 40.
  • the recombinant mammalian expression vector can preferably bring about the expression of the nucleic acid in a specific cell type (for example, tissue-specific regulatory elements are used 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) EMBO J.
  • mice hox promoters Kessel and Gruss (1990) Science 249: 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537 -546).
  • the invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction.
  • the DNA molecule is operatively linked to a regulatory sequence in such a way that expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to the SES mRNA becomes possible.
  • Regulatory sequences can be selected which are operably linked to a nucleic acid cloned in the antisense direction and which control the continuous expression of the antisense RNA molecule in a multiplicity of cell types, for example viral promoters and / or enhancers or regulatory sequences can be selected that control the 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 are present in which antisense nucleic acids are produced under the control of a highly effective regulatory region, the activity of which is determined by the cell type into which the vector is introduced.
  • Another aspect of the invention relates to host cells into which a recombinant expression vector according to the invention has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably here. It goes without saying that these terms refer not only to a specific target cell, but also to the descendants or potential descendants of this cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, these offspring are not necessarily identical to the parental cell, but are still included in the scope of the term as used here.
  • a host cell can be a prokaryotic or eukaryotic cell.
  • an SES 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 the person skilled in the art.
  • Microorganisms which are related to Corynebacterium glutamicum and which can be suitably used as host cells for the nucleic acid and protein molecules according to the invention are listed in Table 3.
  • vector DNA can be introduced into prokaryotic or eukaryotic cells.
  • transformation and “transfection”, “conjugation” and “transduction” as used herein are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (e.g. DNA) into a host cell, including calcium phosphate - or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemically mediated transmission or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sabbrook et al. (Molecular Cloning: A Laboratory Manual.
  • a gene encoding a selectable marker (eg resistance to antibiotics) is usually introduced into the host cells together with the gene of interest.
  • selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate.
  • a nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an SES protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified, for example, by drug selection (for example, cells that have integrated the selectable marker survive, whereas the other cells die).
  • a vector which contains at least a section of an SES gene, into which a deletion, addition or substitution has been introduced in order to change the SES gene, for example to functionally disrupt it.
  • This SES gene is preferably a Corynebacterium glutamicum SES gene, but a homologue from a related bacterium or even from a mammalian, yeast, or insect source can be used.
  • the vector is designed in such a way that the endogenous SES gene is functionally disrupted when homologous recombination occurs (ie no longer encodes a functional protein; also referred to as a "knockout" vector).
  • the vector can be designed in such a way that the endogenous SES gene is mutated or otherwise altered in the case of homologous recombination, but still encodes the functional protein (for example the upstream regulatory region can be altered in such a way that the expression of the endogenous SES protein thereby is changed.).
  • the modified portion of the SES gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the SES gene, which is a homologous recombination between the exogenous SES gene carried by the vector and one endogenous SES gene in a microorganism.
  • the additional flanking SES nucleic acid is long enough for successful homologous recombination with the endogenous gene.
  • the vector usually contains several kilobases flanking DNA (both at the 5 'and 3' ends) (see, for example, Thomas, KR and Capecchi, MR (1987) Cell 51: 503 for a description of homologous recombination vectors).
  • the vector is introduced into a microorganism (eg by electroporation), and cells, in where the introduced SES gene is homologously recombined with the endogenous SES gene are selected using methods known in the art.
  • recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene. Inclusion of an SES gene in a vector, thereby placing it under the control of the Lac operon, enables e.g. expression of the SES gene only in the presence of IPTG. These regulatory systems are known in the art.
  • a host cell according to the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e. express) an SES protein.
  • the invention also provides methods for producing SES proteins using the host cells of the invention.
  • the method comprises culturing the host cell according to the invention (into which a recombinant expression vector encoding an SES protein has been introduced, or into whose genome a gene encoding a wild-type or modified SES protein has been introduced) in a suitable medium until the SES protein has been produced.
  • the method comprises isolating the SES proteins from the medium or the host cell.
  • the nucleic acid molecules, proteins, protein homologs, fusion proteins, primers, vectors and host cells described here can be used in one or more of the following methods: identification of C. glutamicum and related organisms, mapping of genomes of organisms related to C. glutamicum , Identification and localization of C. glutamicum sequences of interest, evolution studies, determination of SES protein areas that are necessary for their function, modulation of the activity of an SES protein, - modulation of the metabolism of one or more cell membrane components; Modulation of the transmembrane transport of one or more compounds and modulation of the cellular production of a desired compound, such as a fine chemical.
  • the SES nucleic acid molecules according to the invention have a multitude of uses.
  • Corynebacterium glutamicum can initially be used to identify an organism as Corynebacterium glutamicum or a close relative of it. They can also 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 a number of C. glutamicum genes. By probing the extracted genomic DNA from a culture of a uniform or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene that is unique to this organism, one can determine whether this organism is present is.
  • Corynebacterium glutamicum itself is not pathogenic, but it is related to pathogenic species such as Corynebacterium diptheriae. The detection of such an organism is of significant clinical importance.
  • the nucleic acid and protein molecules according to the invention can also serve as markers for certain regions of the genome. This is not only suitable for mapping the genome, but also for functional studies of C. glutamicum proteins.
  • the C. glutamicum genome can be cleaved, for example, and the fragments incubated with the DNA-binding protein.
  • those who bind the protein can additionally be probed with the nucleic acid molecules according to the invention, preferably with easily detectable labels, - the binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genomic map of C.
  • nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can serve as markers for the construction of a genomic map in related bacteria (e.g. Brevibacterium lactofermentum).
  • the SES nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies.
  • the metabolic and transport processes in which the molecules according to the invention are involved are exploited by a large number of procaryotic and eukaryotic cells;
  • the degree of evolutionary kinship of the organisms can be determined. Accordingly, such a comparison enables the determination of which sequence regions are conserved and which are not, which can be helpful in determining those regions of the protein which are essential for the enzyme function. This type of determination is valuable for protein technology studies and can give an indication of how much mutagenesis the protein can tolerate without losing its function.
  • SES nucleic acid molecules according to the invention can bring about the production of SES proteins with functional differences from the wild-type SES proteins. These proteins can be improved in their efficiency or activity, can be present in the cell in larger numbers than usual or can be weakened in their efficiency or activity.
  • This modulation of the activity of proteins involved in DNA repair, recombination or transposition in C. glutamicum should affect the genetic stability of the cell. For example, by reducing the number or activity of proteins involved in DNA repair mechanisms, the cell's ability to correct genetic errors can be reduced, which makes it easier to introduce desired mutations into the genome (such as those involved in fine chemical production) Encoding proteins). Increasing the activity or number of transposons should also lead to an increased mutation rate in the genome and can easily double the desired genes (e.g. those that encode proteins involved in fine chemical production) or disruption of undesired genes (e.g. those that Encoding breakdown proteins).
  • the modulation of proteins involved in transcription and translation in C. glutamicum can have direct and indirect effects on the production of fine chemicals from these microorganisms. For example, by manipulating a protein that directly translates a gene (eg a polymerase) or directly regulates transcription (a repressor or activator protein), it is possible to directly influence the expression of the target gene. For genes that encode a protein that is on the Biosynthesis or degradation of a fine chemical, this type of genetic manipulation should have a direct effect on the production of this fine chemical. Mutagenesis of a repressor protein so that it can no longer repress its target gene, or mutagenesis of an activator protein so that its activity is optimized, should lead to increased transcription of the target gene.
  • a protein that directly translates a gene eg a polymerase
  • transcription a repressor or activator protein
  • the target gene is a fine chemical biosynthesis gene
  • an increased production of this chemical can result due to the overall larger number of available transcripts of this gene, which should also lead to an increased number of the protein.
  • transcription factors e.g sigma factors
  • translation repressors / activators that regulate transcription in C. glutamicum globally in response to environmental or metabolic factors
  • unfavorable conditions e.g. high temperature, low oxygen, high levels of waste products
  • Modulating the activity or number of proteins involved in polypeptide folding can overall increase the production of correctly folded molecules in the cell. This has two effects: first, an overall increase in the number of proteins in the cell due to the fact that fewer proteins are incorrectly folded and broken down, and secondly, an increase in the number of counter protein that is folded correctly and is therefore active (see, for example, Thomas, JG, Baneyx, F. (1997) Protein Expression and Purification 11 (3): 289-296; Luo, ZH, and Hua, ZC (1998) Biochemistry and Molecular Biology International 46 (3): 471-477; Dale, GE, et al. (1994) Protein Engineering 7 (7): 925-931; Amrein, KE et al. (1995) Proc.
  • proteins involved in polypeptide folding eg chaperones
  • the manipulation of proteins involved in the secretion of C. glutamicum polypeptides so that their activity or number is improved can directly improve the secretion of a proteinaceous fine chemical (e.g. an enzyme) from this microorganism. It is much easier to harvest and clean fine chemicals if they are secreted into the medium of a culture on a large scale than if they are retained in the cell, so the yield and production of a fine chemical should be increased by this change in the secretion system.
  • the genetic manipulation of these secretion proteins can also lead to direct improvements in the production of one or more fine chemicals. First, the increased or decreased activity of one or more C.
  • glutamicum secretion systems (as achieved by mutagenesis of one or more SES proteins involved in these pathways) can result in overall increased or decreased secretion rates from the cell.
  • Many of these secreted proteins have functions that are important for cell viability (e.g. cell surface proteases or receptors). Changing the secretion pathway so that these proteins are more easily transported to their extracellular location can affect the overall viability of the
  • the nucleic acid and protein molecules of the invention can be used to generate C. glutamicum or related bacterial strains expressing mutant SES nucleic acid and protein molecules so that yield, production and / or efficiency of production of a desired connection is improved.
  • the desired compound can be any product produced by C. glutamicum, including the end products of biosynthetic pathways and intermediates of naturally occurring metabolic pathways, as well as molecules that are not naturally occurring in the C. glutamicum metabolism, but which are produced by a C. glutamicum strain according to the invention.
  • Example 1 Preparation of the entire genomic DNA from Corynebacterium glutamicum ATCC13032
  • a culture of Corynebacterium glutamicum 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 stated volumes are calculated for 100 ml culture volume).
  • composition of buffer I 140.34 g / 1 sucrose, 2.46 g / 1 MgS0 4 ⁇ 7 H 2 0, 10 ml / 1 KH 2 P0 4 solution (100 g / l, with KOH to pH 6, 7 adjusted), 50 ml / 1 M12 concentrate (10 g / 1 (NH 4 ) 2 S0 4 , 1 g / 1 NaCl, 2 g / 1 MgS0 4 • 7 H 2 0, 0.2 g / 1 CaCl 2 , 0.5 g / 1 yeast extract (Difco), 10 ml / 1 trace element mixture (200 mg / 1 FeS0 • H0, 10 mg / 1 ZnS0 • 7 H 2 0, 3 mg / 1 MhCl 2 • 4 H 2 0, 30 mg / 1 H 3 B0 3 , 20 mg / 1 CoCl 2 • 6 H 2 0, 1 mg / 1 NiCl 2 • 6 H 2 0, 3 mg / 1 Na 2 Mo0 4 • 2 H 2 0, 500
  • Lysozyme was added to the suspension in a final concentration of 2.5 mg / ml. After about 4 hours of incubation at 37 ° C., the cell wall was broken down and the protoplasts obtained were harvested by centrifugation. The pellet was washed once with 5 ml of buffer I and once with 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added. After adding proteinase K at a final concentration of 200 ⁇ g / ml, the suspension was incubated at 37 ° C. for about 18 hours.
  • the DNA was purified by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol using standard procedures. Then the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by incubation for 30 min at -20 ° C and 30 min centrifugation at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) , The DNA was dissolved in 1 ml of TE buffer containing 20 ⁇ g / ml RNase A and dialyzed against 1000 ml of TE buffer at 4 ° C. for at least 3 hours. During this time the buffer was exchanged 3 times.
  • Plasmids pBR322 (Sutcliffe, JG (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741) found particular use; pACYC177 (Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); Plasmids of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or cosmids, such as SuperCosl (Stratagene, LaJolla, USA) or Lorist6 (Gibson, TJ Rosenthal, A., and Waterson, RH (1987) Gene 53: 283-286.
  • Genomic banks as described in Example 2, were used for DNA sequencing according to standard methods, in particular the chain termination method with ABI377 sequencing machines (see, for example, Fleischman, RD et al. (1995) "Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science 269: 496-512)
  • the sequencing primers with the following nucleotide sequences were used: 5 '-GGAAACAGTATGACCATG-3' or 5 '-GTA ⁇ AACGACGGCCAGT-3'.
  • In vivo mutagenesis of Corynebacterium glutamicum can be carried out by passing a plasmid (or other vector) DNA through E. coli or other microorganisms (eg Bacillus spp. Or yeasts such as Saccharomyces cerevisiae), which maintain the integrity of their cannot maintain genetic information.
  • E. coli or other microorganisms eg Bacillus spp. Or yeasts such as Saccharomyces cerevisiae
  • Common mutator strains have mutations in the genes for the DNA repair system (eg, mutHLS, mutD, mutT, etc., for comparison see Rupp, WD (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277 -2294, ASM: Washington). These strains are known to the person skilled in the art. The use of these strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.
  • Example 5 DNA transfer between Escherichia coli and Corynebacterium glutamicum
  • Corynebacterium and BreviJacteri um species contain endogenous plasmids (such as, for example, pHMl519 or pBLl) which replicate autonomously (for an overview, see, for example, Martin, JF et al. (1987) Biotechnology 5: 137-146).
  • Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel , FM et al.
  • origins of replication are preferably taken from endogenous plasmids which have been isolated from Corynebacterium and Brevi.foac'tertiuz ⁇ species.
  • transformation markers for these species are genes for kanamycin resistance (such as those derived from Tn5 or Tn-903 transposon) or for Chloramphenicol (Winnacker, EL (1987) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim).
  • C. glutamicum can be carried out by protoplast transformation (Kastsumata, R. et al., (1984) J. Bacteriol. 159: 306-311), electroporation (Liebl, E. et al., (1989) FEMS Microbiol. Letters , 53: 399-303) and, in cases where special vectors are used, can also be achieved by conjugation (as described, for example, in Schwarzfer, A., et (1990) J. Bacteriol. 172: 1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C.
  • a suitable method for determining the amount of transcription of the mutant gene is to carry out a Northern blot (see, for example, Ausubel et al., (1988) Current Protocols) in Molecular Bio-logy, Wiley, New York), wherein a primer designed to bind to the gene of interest is provided with a detectable (usually radioactive or chemiluminescent) label so that when the total RNA of a culture of the organism is extracted, separated on a gel, transferred to a stable matrix and incubated with this probe - the binding and the quantity of binding of the probe indicate the presence and also the amount of mRNA for this gene.
  • Total cell RNA can be isolated from Corynebacterium glutamicum by various methods known in the art, such as in Bormann, ER et al., (1992) Mol. Microbiol. 6: 317-326.
  • Standard techniques such as Western blot, can be used to determine the presence or the relative amount of protein that is translated from this mRNA (see, for example, Ausubel et al. (1988) "Current Protocols in Molecular Biology", Wiley, New York).
  • total cell proteins are extracted, separated by gel electrophoresis, transferred to a matrix, such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein.
  • This probe is usually provided with a chemiluminescent or colorimetric label that is easy to detect. The presence and amount of label observed indicates the presence and amount of the mutant protein sought in the cell.
  • Example 7 Growth of genetically modified Corynebacterium glutamicum media and growing conditions
  • Corynebacteria are grown in synthetic or natural growth media.
  • a number of different growth media for Corynebakterian are known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4,120,867; Liebl (1992) "The Genus Corynebacterium", in: The Procaryotes, Vol. II, Balows, A., et al., Ed. Springer-Verlag).
  • These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • Very good carbon sources are, for example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose.
  • Sugar can also be added to the media through complex compounds such as molasses or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources.
  • Other possible carbon sources are alcohols and organic acids such as methanol, ethanol, acetic acid or lactic acid.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds.
  • Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH 4 CI or (NH 4 ) 2 S0 4 , NH 4 OH, nitrates, urea, amino acids or complex nitrogen sources, such as maize spring water, soy flour, soy protein, yeast extract, meat extract and others.
  • ammonia gas or ammonium salts such as NH 4 CI or (NH 4 ) 2 S0 4 , NH 4 OH, nitrates, urea
  • amino acids or complex nitrogen sources such as maize spring water, soy flour, soy protein, yeast extract, meat extract and others.
  • Inorganic salt compounds that may be contained in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
  • Chelating agents can be added to the medium to keep the metal ions in solution.
  • Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
  • the media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
  • Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like.
  • the exact composition of the media connections strongly depends on the respective experiment and is decided individually for each specific case. Information on media optimization is available from the textbook "Applied Microbiological Physiology, A Practical Approach” (ed. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).
  • Growth media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) or others.
  • All media components are sterilized either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration.
  • the components can be sterilized either together or, if necessary, separately. All media components can be present at the beginning of the cultivation or can be added continuously or in batches.
  • the growing conditions are defined separately for each experiment.
  • the temperature should be between 15 ° C and 45 ° C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by adding buffers to the media.
  • An exemplary buffer for this purpose is a potassium phosphate buffer.
  • Synthetic buffers such as MOPS, HEPES; ACES etc. can be used alternatively or simultaneously.
  • the cultivation pH can also be kept constant during the cultivation by adding NaOH or NH 4 OH. If complex media components, such as yeast extract, are used, the need for additional buffers is reduced, since many complex compounds have a high buffer capacity.
  • When using a Fermenters for the cultivation of microorganisms can also regulate the pH value with gaseous ammonia.
  • the incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth.
  • the disclosed growth experiments can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes.
  • the microorganisms should be grown in microtiter plates, glass tubes or shake flasks, either with or without baffles.
  • 100 ml shake flasks are used, which are filled with 10% (by volume) of the required growth medium.
  • the flasks should be shaken on a rotary shaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, a mathematical correction should be carried out for the evaporation losses.
  • the medium is inoculated onto an ODeoo v ° n 0.5-1.5, using cells which are placed on agar plates, such as CM plates (10 g / 1 glucose, 2.5 g / 1 NaCl, 2 g / 1 Urea, 10 g / 1 polypeptone, 5 g / 1 yeast extract, 5 g / 1 meat extract, 22 g / 1 agar pH 6.8 with 2 M NaOH), which had been incubated at 30 ° C., were grown.
  • the inoculation of the media is carried out either by introducing a saline solution of C. glutaznicum cells from CM plates or by adding a liquid preculture of this bacterium.
  • DNA band shift assays also referred to as gel retardation assays
  • reporter gene assays as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904 and the references cited therein. Reporter gene test systems are well known and established for use in pro- and eukaryotic cells using enzymes such as beta-galactosidase, green fluorescent protein and several others.
  • membrane transport proteins The activity of membrane transport proteins can be determined according to techniques as described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, pp. 85-137; 199-234; and 270-322 are described.
  • Example 9 Analysis of the influence of mutated protein on the production of the desired product
  • the effect of the genetic modification in C. glutamicum on the production of a desired compound can be determined by growing the modified microorganisms under suitable conditions (such as those described above) and the medium and / or the cellular components with respect to the increased production of the desired product (ie an amino acid) is investigated.
  • suitable conditions such as those described above
  • Such analysis techniques are well known to the person skilled in the art and include spectroscopy, thin-layer chromatography, staining methods of various types, enzymatic and microbiological methods and analytical chromatography, such as high-performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. A2) 89-90 and pp.
  • the analytical methods include measurements of the amount of nutrients in the medium (e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways and measurements of gases that are generated during fermentation. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, P.M. Rhodes and P.F. Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein.
  • nutrients in the medium e.g. sugar, hydrocarbons, nitrogen sources, phosphate and other ions
  • Example 10 Purification of the desired product from C. glutamicum culture
  • the supernatant fraction from both purification processes is subjected to chromatography with a suitable resin, the desired molecule either being retained on the chromatography resin, but not many impurities in the sample, or the impurities remaining on the resin, but the sample not.
  • chromatography steps can if necessary can be repeated using the same or different chromatography resins.
  • the person skilled in the art is skilled in the selection of the suitable chromatography resins and their most effective application for a particular molecule to be purified.
  • the purified product can be 5 concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.
  • the identity and purity of the isolated compounds can be determined by prior art techniques. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme test or microbiological tests. This analysis method

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  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

L'invention concerne de nouvelles molécules d'acide nucléique, leur utilisation dans la construction de micro-organismes génétiquement améliorés et un procédé de fabrication de produits chimiques fins, en particulier d'acides aminés, à l'aide desdits micro-organismes génétiquement améliorés.
EP02796537A 2001-11-05 2002-10-31 Genes codant pour des proteines de stabilite genetique, d'expression genique et de repliement Withdrawn EP1444258A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP05026934A EP1669369A2 (fr) 2001-11-05 2002-10-31 Séquence d'acides nucléiques codante pour le gène OPCA
EP06110205A EP1693380B1 (fr) 2001-11-05 2002-10-31 Séquence d'acide nucléique codante pour le gène OPCA

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10154180A DE10154180A1 (de) 2001-11-05 2001-11-05 gene die für genetische Stabilitäts-, genexpressions-und Faltungsproteine codieren
DE10154180 2001-11-05
PCT/EP2002/012138 WO2003040180A2 (fr) 2001-11-05 2002-10-31 Genes codant pour des proteines de stabilite genetique, d'expression genique et de repliement

Related Child Applications (2)

Application Number Title Priority Date Filing Date
EP05026934A Division EP1669369A2 (fr) 2001-11-05 2002-10-31 Séquence d'acides nucléiques codante pour le gène OPCA
EP06110205A Division EP1693380B1 (fr) 2001-11-05 2002-10-31 Séquence d'acide nucléique codante pour le gène OPCA

Publications (1)

Publication Number Publication Date
EP1444258A2 true EP1444258A2 (fr) 2004-08-11

Family

ID=7704614

Family Applications (3)

Application Number Title Priority Date Filing Date
EP02796537A Withdrawn EP1444258A2 (fr) 2001-11-05 2002-10-31 Genes codant pour des proteines de stabilite genetique, d'expression genique et de repliement
EP06110205A Expired - Lifetime EP1693380B1 (fr) 2001-11-05 2002-10-31 Séquence d'acide nucléique codante pour le gène OPCA
EP05026934A Ceased EP1669369A2 (fr) 2001-11-05 2002-10-31 Séquence d'acides nucléiques codante pour le gène OPCA

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP06110205A Expired - Lifetime EP1693380B1 (fr) 2001-11-05 2002-10-31 Séquence d'acide nucléique codante pour le gène OPCA
EP05026934A Ceased EP1669369A2 (fr) 2001-11-05 2002-10-31 Séquence d'acides nucléiques codante pour le gène OPCA

Country Status (10)

Country Link
US (5) US7138513B2 (fr)
EP (3) EP1444258A2 (fr)
KR (1) KR100861746B1 (fr)
CN (1) CN1323087C (fr)
AT (1) ATE429443T1 (fr)
AU (1) AU2002361951A1 (fr)
BR (1) BR0213771A (fr)
DE (2) DE10154180A1 (fr)
WO (1) WO2003040180A2 (fr)
ZA (1) ZA200404424B (fr)

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Publication number Priority date Publication date Assignee Title
JP2006340603A (ja) * 2003-06-23 2006-12-21 Ajinomoto Co Inc Ｌ−グルタミン酸の製造法
DE10359594A1 (de) * 2003-12-18 2005-07-28 Basf Ag PEF-TU-Expressionseinheiten
DE10359661A1 (de) * 2003-12-18 2005-07-28 Basf Ag Genvarianten die für Proteine aus dem Stoffwechselweg von Feinchemikalien codieren
DE102004035065A1 (de) 2004-07-20 2006-02-16 Basf Ag P-ET-TS-Expressionseinheiten
DE102004061846A1 (de) * 2004-12-22 2006-07-13 Basf Ag Mehrfachpromotoren
DE102005023829A1 (de) 2005-05-24 2006-11-30 Degussa Ag Allele des opcA-Gens aus coryneformen Bakterien
US20070072194A1 (en) * 2005-09-28 2007-03-29 Alper Hal S Global transcription machinery engineering
DE102007044134A1 (de) * 2007-09-15 2009-03-19 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
JP7081832B2 (ja) * 2016-06-13 2022-06-07 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア α（Ｖ）β（６）インテグリン結合ペプチドおよびその使用方法
AU2018395054A1 (en) * 2017-12-29 2020-07-16 The Scripps Research Institute Unnatural base pair compositions and methods of use

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DE122007000007I2 (de) 1986-04-09 2010-12-30 Genzyme Corp Genetisch transformierte Tiere, die ein gewünschtes Protein in Milch absondern
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
DE4120867A1 (de) 1991-06-25 1993-01-07 Agfa Gevaert Ag Fotografisches verarbeitungsverfahren und vorrichtung dafuer
DE69529026T2 (de) 1994-07-19 2003-07-17 Hayashibara Biochem Lab Trehalose, ihre Herstellung und ihre Verwendung
DE19929365A1 (de) 1999-06-25 2000-12-28 Basf Lynx Bioscience Ag Teilsequenzen der Gene des Primär- und Sekundärmetabolismus aus Corynebacterium glutamicum und ihr Einsatz zur mikrobiellen Herstellung von Primär- und Sekundärmetaboliten
MXPA01012844A (es) * 1999-06-25 2002-07-09 Basf Ag Genes de corynebacterium glutamicum que codifican proteinas de tolerancia y resistencia al estres.
SK3012001A3 (en) * 1999-07-09 2002-07-02 Degussa Nucleotide sequences which code for the opca gene and a process for the fermentative preparation of l-amino acids
JP4623825B2 (ja) 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド

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Also Published As

Publication number Publication date
US7339048B2 (en) 2008-03-04
US20050009152A1 (en) 2005-01-13
WO2003040180A2 (fr) 2003-05-15
US20070037262A1 (en) 2007-02-15
US20070077631A1 (en) 2007-04-05
KR100861746B1 (ko) 2008-10-29
ATE429443T1 (de) 2009-05-15
CN1582299A (zh) 2005-02-16
EP1693380A2 (fr) 2006-08-23
ZA200404424B (en) 2005-06-06
CN1323087C (zh) 2007-06-27
US7355028B2 (en) 2008-04-08
KR20050042246A (ko) 2005-05-06
WO2003040180A3 (fr) 2004-04-01
DE50213492D1 (de) 2009-06-04
BR0213771A (pt) 2004-10-19
EP1669369A2 (fr) 2006-06-14
DE10154180A1 (de) 2003-05-15
EP1693380B1 (fr) 2009-04-22
EP1693380A3 (fr) 2007-01-03
US20070054381A1 (en) 2007-03-08
US7138513B2 (en) 2006-11-21
US7323559B2 (en) 2008-01-29
US20070072273A1 (en) 2007-03-29
AU2002361951A1 (en) 2003-05-19

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