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
  • 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
  • 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
• • 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
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

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, ipides 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 Corynehacterium 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.
• G & noms or organisms related to genomes These new nucleic acid molecules encode proteins, which are referred to here as DNA replication, riboson and pathogenesis (RRP) proteins. These RRP proteins can, for example, be directly or indirectly involved in the production of one or more fine chemicals in C. glutamicum.
• the RRP proteins according to the invention can also be involved in the degradation of hydrocarbons or in the oxidation of terpenoids. These proteins can be used to identify Corynejacterium glutamicum or organisms related to C. glutamicum; the presence of an RRP protein specific for C. glutamicum and related species in a protein mixture can indicate the presence of one of these bacteria in the sample. Furthermore, these RRP proteins can have homologs in plants or animals which are involved in a disease state or a condition; so these proteins can serve as useful pharmaceutical targets for drug screening and the development of therapeutic compounds.
• the nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to produce a or to modulate several fine chemicals.
• This modulation 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.
• a protein that regulates a fine chemical pathway one can directly influence whether the production of the desired compound is up or down regulated, both of which modulate the yield or efficiency of the production of the fine chemical from the cell.
• Indirect modulation of fine chemical production can also be done by modifying the activity of a protein according to the invention (ie by mutagenesis of the corresponding gene) so that the cell's ability to grow and divide or read. to remain viable and productive is increased overall.
• the production of fine chemicals from C. glutamicum is usually achieved by large-scale fermentation culture of these microorganisms, conditions that are often suboptimal for growth and cell division.
• a protein according to the invention for example a stress reaction protein, a cell wall protein or a protein which is involved in the metabolism of compounds which are necessary for the occurrence of cell division and growth, such as nucleotides and amino acids
• a protein according to the invention for example a stress reaction protein, a cell wall protein or a protein which is involved in the metabolism of compounds which are necessary for the occurrence of cell division and growth, such as nucleotides and amino acids
• glutamicum ie by changing the activity of one of the proteins according to the invention which is involved in such a pathway
• glutamicum it is possible to simultaneously change the activity or regulation of another metabolic pathway in this microorganism which may be directly involved in the synthesis or degradation of a fine chemical.
• Changes in the DNA replication proteins according to the invention can also bring about greater replication accuracy, and thereby increase the genetic stability and viability of the microorganisms and thereby reduce the risk that a further genetic modification which increases the production of the fine chemical is destroyed by faulty replication.
• This invention provides new nucleic acid molecules which encode proteins, which are referred to here as RRP proteins and, for example, modulate the production or efficiency of the production of one or more fine chemicals in C. glutamicum or can serve as identification markers for C. glutamicum or related organisms .
• Nucleic acid molecules that encode an RRP protein are referred to here as RRP nucleic acid molecules.
• the RRP protein can modulate the production or efficiency of the production of one or more fine chemicals in C. glutamicum or serve as an identification marker for C. glutamicum or related organisms. Examples of such proteins are those encoded by the genes shown in Table 1.
• isolated nucleic acid molecules for example cDNAs
• isolated nucleic acid molecules comprising a nucleotide sequence which encodes an RRP protein or biologically active sections thereof, and also nucleic acid fragments which act as primers or hybridization probes for the detection or amplification of RRP -code 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 RRP proteins according to the invention also preferably have at least one of the RRP 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.
• 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 encodes more strongly preferably a naturally occurring C. glutamicum RRP protein or a biologically active portion thereof.
• Another aspect of the invention relates to vectors, for example recombinant expression vectors, which contain the nucleic acid molecules according to the invention, and host cells, into which these vectors have been introduced.
• this host cell is used to produce an RRP protein by growing the host cell in a suitable medium. The RRP 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 RRP 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 RRP sequence as a transgene.
• an endogenous RRP gene in the genome of the microorganism is altered by homologous recombination with an altered RRP gene, e.g. functionally disrupted.
• the microorganism belongs to the genus Corynebacterium or Brevibacterium, Corynebacterium glutamicum being particularly preferred.
• the microorganism is also used to produce a desired compound, such as an amino acid, lysine being particularly preferred.
• host cells that have more than one of the nucleic acid molecules described in Appendix A.
• Such host cells can be produced in various ways known to those skilled in the art. For example, they can be transfected by vectors which carry several of the nucleic acid molecules according to the invention. However, it is also possible to introduce one nucleic acid molecule according to the invention into the host cell with one vector and therefore to use several vectors either simultaneously or in a staggered manner. Host cells can thus be constructed which carry numerous up to several hundred of the nucleic acid sequences according to the invention. Such an accumulation can often achieve superadditive effects on the host cell with regard to fine chemical productivity.
• Another aspect of the invention relates to an isolated KRP protein or a section thereof, for example a biologically active section thereof.
• the isolated RRP protein or its section can be used for production or ef- Modulate the efficiency of the production of one or more fine chemicals in C. glutamicum or serve as identification markers for C. glutamicum or related organisms.
• the isolated RRP protein or a portion thereof is sufficiently homologous to an amino acid sequence of Appendix B so that the protein or its portion retains the ability, e.g., the production or efficiency of the production of one or more fine chemicals in C. modulate glutamicum or serve as an identification marker for C. glutamicum or related organisms.
• the invention also relates to an isolated RRP protein preparation.
• the RRP 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 RRP polypeptide or a biologically active portion thereof can be operably linked to a non-RRP polypeptide to form a fusion protein.
• this fusion protein has a different activity than the RRP protein alone.
• this fusion protein can modulate the yield, production and / or efficiency of production of one or more fine chemicals in C. glutamicum or serve as an identification marker for C. glutamicum or related organisms.
• the integration of this fusion protein in a host cell modulates the production of a desired compound from the cell.
• 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 RRP 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 an RRP 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 Brevijbacterium.
• 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 RRP protein activity or RRP 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 with respect to one or more C. g utamici-jn RRP protein activities, so that the yield, production and / or efficiency of the production of a desired fine chemical by this microorganism is improved.
• the substance that modulates RRP protein activity can be a substance that stimulates RRP protein activity or RRP nucleic acid expression. Examples of substances that stimulate RRP protein activity or RRP nucleic acid expression include small molecules, active RRP proteins and nucleic acids that encode RRP proteins and have been introduced into the cell. Examples of substances that inhibit RRP activity or expression include small molecules and RRP antisense nucleic acid molecules.
• Another aspect of the invention relates to methods for modulating the yields, the production and / or the efficiency of producing a desired compound from a cell, comprising introducing into a cell a RRP wild-type or mutant gene which either remains on a separate plasmid or is integrated into the genome of the host cell.
• the integration into the genome can be random or by homologous recombination, so that the native gene is replaced by the integrated 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 RRP nucleic acid and protein molecules that are used to identify Corynebacterium glutamicum or related organisms, to map the C. glutamumcum genome (or the genome of a closely related organism), or to identify microorganisms that can be used for the production of fine chemicals, for example, through fermentations.
• the proteins encoded by these nucleic acids can be used for direct or indirect modulation of the production or efficiency of the production of a or several fine chemicals in C. glutamicum, as identification markers for C. glutamicum or related organisms, for the oxidation of terpenoids or for the degradation of hydrocarbons or as targets for the development of therapeutic pharmaceutical compounds.
• 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 diaminopimelic 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 re - lated compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim and the quotes 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 Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", Pp. 443-613 (1996) VCH: Weinheim and the citations contained therein; and Ong, AS, Niki, E. and Packer, L.
• 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 Ul- mann'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.
• 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 can also be used as precursors for the synthesis of synthetic amino acids and proteins such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others in Ulann'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 synthesized from the precursors of the glycolysis and pentose phosphate pathways, erythrosis-4-phosphate and phosphoenolpyruvate, in a 9-step biosynthetic pathway that only differs in the last two steps after the synthesis of prephenate. Tryptophan is also produced from these two starting molecules, but its synthesis takes place in an 11-step process.
• Tyrosine can also be produced from phenylalanine in a reaction catalyzed by phenylalanine hydroxylase.
• Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis.
• Aspartate is 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 emission of a The correct 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, activity and 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 includes non-proteinaceous compounds that are necessary for normal enzyme activity to occur. 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 (e.g. polyunsaturated fatty acids).
• Thiamine (vitamin Bi) is formed by chemical coupling of pyrimidine and thiazole units.
• Riboflavin (vitamin B) is synthesized from guanosine 5 'triphosphate (GTP) and ribose 5'-phosphate. Riboflavin in turn is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
• vitamin Bg for example pyridoxine, pyridoxa in, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride
• Panthothenate pantothenic acid, R - (+) -N- (2, 4-dihydroxy-3, 3-dimethyl-l-oxobutyl) -ß-alanine
• the final steps in pantothenate biosynthesis consist of the ATP-driven condensation of ß-alanine and pantoic acid.
• pantothenate The enzymes responsible for the biosynthesis 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 place over 5 enzymatic steps.
• Pantothenate, pyridoxal-5'-phosphate, cysteine and ATP are the precursors of coenzyme A.
• These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone, (R) - Panthenol (provitamin B 5 ), Pantethein (and its derivatives) and coenzyme A.
• the biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively investigated and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in the Fe cluster synthesis and belong to the class of the nifS proteins.
• the lipoic acid is derived from octanoic acid and serves as a coenzyme in energy metabolism, where it is a component 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 ⁇ 2
• 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 known as "Niacin” can be called.
• 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 and pyrimidine bases, nucleosides and nucleotides also have other possible uses: as intermediates in the biosynthesis of various fine chemicals (eg thiamine, S-adenosyl methionine, folate or riboflavin), as an energy source for the cell (e.g. ATP or GTP) ) and for chemicals themselves, which are usually used as flavor enhancers (for example IMP or GMP) or for many medical applications (see for example Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al VCH: Weinheim, pp. 561-612)
• Enzymes that are involved in the purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly used as targets against chemicals for crop protection, including Fungicides, herbicides and insecticides are developed.
• 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' onophosphate (GMP) or adenosine 5 'monophosphate (AMP), from which the triphosphate forms used as nucleotides can be easily produced.
• IMP inosine 5 'phosphate
• GMP guanosine 5' onophosphate
• AMP adenosine 5 'monophosphate
• 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).
• 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.
• Trehalose consists of two glucose molecules that are linked by an ⁇ , ⁇ -l, 1 bond. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen foods, and in beverages. applies. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, e.g., Nishi oto et al., (1998) U.S. Patent No. 5,759,610; Singer, MA and Lindquist, S. (1998 ) Trends Biotech. 16: 460-467; Paiva, CLA and Panek, AD (1996) Biotech Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium from which it can be obtained by methods known in the art.
• the cell In order for a certain type of bacteria to survive in an environment, at least three activities are necessary. First, the cell must be able to divide efficiently so that the cell population is at least maintained or increased. Second, the cell must efficiently express those genes that encode proteins that are necessary for normal cell functions. Finally, the cell must be able to interact with its environment, either by adapting to the prevailing environmental conditions or by physically moving into preferred environments or by acting directly on the environment in a way that improves its survivability. The critical steps involved in each of these activities include replication of the bacterial genome, ribosomal activity in protein biosynthesis, and anti-cellular or cell lytic activities (as are involved in the pathogenesis of an organism).
• the present invention is based at least in part on the discovery of new molecules, which are referred to here as RRP nucleic acid molecules.
• RRP nucleic acid molecules are not only suitable for the identification of C. glutamicum or related types of bacteria, but also as markers for mapping the C. glutamicum genome and for the identification of bacteria which are suitable for the production of fine chemicals by, for example, fermentative methods.
• the present invention is also based, at least in part, on the RRP protein molecules which are encoded by these RRP nucleic acid molecules. These RRP molecules can modulate the yield, production and / or efficiency of the production of one or more fine chemicals in C. glutamicum, serve as identification markers for C.
• the RRP molecules according to the invention are direct or indirectly involved in the metabolic pathway of one or more fine chemicals in C. glutamicum.
• the activity of the RRP molecules according to the invention to participate indirectly or directly in such metabolic pathways has an effect on the production of a desired fine chemical by this microorganism.
• the activity of the RRP molecules according to the invention is modulated, so that the C. glutamicum metabolic pathways in which the RRP proteins according to the invention are involved are modulated in terms of efficiency or output, which directly or indirectly affects the Production or efficiency of production of a desired fine chemical modulated by C. glutamicum.
• RRP protein or "RRP polypeptide” includes proteins that modulate the yield, production, and / or efficiency of production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, as a target protein for drug screening or design or can serve as identification markers for C. glutamicum or related organisms.
• RRP proteins include those encoded by the RRP genes listed in Table 1 and Appendix A.
• RRP gene or "RRP nucleic acid sequence” encompass nucleic acid sequences which encode an RRP protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of RRP genes are those listed in Table 1.
• production or “productivity” are known in the art and include the concentration of the fermentation product (e.g. the desired fine chemical), which is formed within a specified period and a specified fermentation volume (e.g. kg product per hour per 1).
• concentration of the fermentation product e.g. the desired fine chemical
• fermentation efficiency encompasses the time it takes to achieve a certain amount of production (for example, how long it takes the cell to establish a certain rate of ejection of a fine chemical).
• yield or “product / carbon yield” is known in the art and encompasses the efficiency of the conversion of the carbon source into the product (ie the fine chemical). For example, this is usually expressed as kg product per kg carbon source.
• 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 involve 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 all 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
• the metabolism of an amino acid, such as glycine then encompasses all biosynthesis, modification and degradation pathways in the cell which concern this compound.
• the RRP molecules according to the invention are capable of directly or indirectly 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.
• one or more RRP proteins of the invention can be manipulated so that their function is modulated. This modulation of the function can lead to modulation of the yield, production and / or efficiency of the production of one or more fine chemicals from C. glutamicum.
• the ability of the cell to synthesize or degrade this compound can be directly modulated, thereby reducing the yield and / or modulate the efficiency of fine chemical production.
• modulating the activity of a protein that regulates a fine chemical pathway one can directly influence whether the production of the desired compound is up or down, both of which modulate the yield or efficiency of the production of the fine chemical from the cell.
• Indirect modulation of fine chemical production can also be done by modifying the activity of a protein of the invention (ie, mutagenizing the corresponding gene) so that the overall ability of the cell to grow and divide, or to remain viable and productive, is increased.
• the production of fine chemicals from C. glutamicum is usually achieved by fermentation culture on a large scale of these microorganisms, conditions that are often suboptimal for growth and cell division.
• a protein according to the invention for example a stress reaction protein, a cell wall protein or proteins which are involved in the metabolism of compounds which are necessary for the occurrence of cell growth and division, such as nucleotides and amino acids), so that better survival, It is possible to grow and multiply in these conditions is, it may be possible to increase the number and productivity of these modified C.
• glutamicum cells in cultures on a large scale, which in turn should lead to increased yields and / or increased efficiency in the production of one or more desired fine chemicals.
• the metabolic pathways of a cell are necessarily interdependent and co-regulated.
• the isolated nucleic acid sequences according to the invention are located in the genome of a Corynebacterium glutamicum strain which is available from the American Type Culture Collection under the name ATCC 13032.
• the nucleotide sequence of the isolated C. glutamicum RRP nucleic acid molecules and the predicted amino acid sequences of the C. glutamicum RRP proteins are shown in Appendix A and B, respectively. Computer analyzes were carried out which classified and / or identified many of these nucleotide sequences as sequences with homology to E. coli or Bacillus subtil ⁇ s genes.
• the present invention also relates to proteins whose amino acid sequence is essentially homologous to an amino acid sequence in Appendix B.
• a protein whose amino acid sequence is essentially homologous to a selected amino acid sequence is at least about 50% homologous to the selected amino acid sequence, for example the entire selected amino acid sequence.
• a protein whose amino acid sequence is essentially homologous to a selected amino acid sequence can also be at least about 50-60%, preferably at least about 60-70%, more preferably at least about 70-80%, 80-90% or 90- 95%, and most preferably at least about 96%, 97%, 98%, 99% or even more homologous to the selected amino acid sequence.
• An RRP protein according to the invention or a biologically active section or fragment thereof can modulate the yield, production and / or efficiency of the production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or as an identification marker serve for C. glutamicum or related organisms.
• Various aspects of the invention are described in more detail in the subsections below:
• nucleic acid molecules which encode RRP molecules or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for the identification or amplification of RRP-coding nucleic acids (for example RRP-DNA).
• RRP-coding nucleic acids for example RRP-DNA
• These nucleic acid molecules can be used to identify C. glutamicum or related organisms, to map the genome of C. glutamicum or related organisms or to identify microorganisms that are used to produce fine chemicals, e.g. by fermentation processes are suitable.
• the term "nucleic acid molecule" as used herein is intended to encompass DNA molecules (e.g. cDNA or genomic DNA) and RNA molecules (e.g.
• nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
• 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 which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example sequences which are located at the 5 'or 3' end of the nucleic acid).
• the isolated RRP 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 isolated using standard molecular biological techniques and the sequence information provided here become.
• a C. glutamicum RRP cDNA can be isolated from a C. glutamicum bank by using a complete sequence from Appendix A or a portion 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 nucleic acid molecule comprising a complete sequence from Appendix A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been created on the basis of this sequence (for example a nucleic acid molecule comprising a complete sequence can be used Appendix A, or a portion thereof, can be isolated by polymerase chain reaction using oligonucleotide primers made from this same sequence from Appendix A).
• mRNA can be isolated from normal endothelial cells (for example by the guanidinium thiocyanate extraction method of Chirgwin et al.
• cDNA can be obtained by means of reverse transcriptase (for example Moloney-MLV reverse transcriptase) at Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL) and using random polynucleotide primers or oligonucleotide primers based on one of the nuances shown in Appendix A - Kleotide sequences are produced.
• 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.
• the nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis.
• Oligonucleotides which correspond to a KRP 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.
• the sequences of Appendix A correspond to the RRP cD ⁇ As according to the invention from Corynebacterium glutamicum. These cD ⁇ As include sequences, the RRP proteins (ie the "coding region” shown in each sequence in Appendix A), and the 5 'and 3' untranslated sequences, which are also given in Appendix A.
• the nucleic acid molecule can alternatively include only the coding region of one of the sequences in Appendix A.
• the nucleic acid molecule according to the invention can moreover comprise only a section of the coding region of one of the sequences in Appendix A, for example a fragment which can be used as a probe or primer or fragment which encodes a biologically active section of an RRP protein.
• the nucleotide sequences determined from the cloning of the RRP genes from C. glutamicum enable the generation of probes and primers which are used to identify and / or clone RRP homologs in other cell types and organisms and RRP homologs from other Corynebak. teries or related species are designed.
• the probe or primer usually comprises essentially purified oligonucleotide.
• the oligonucleotide usually comprises a nucleotide sequence region which, under stringent conditions, comprises at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand from one of the sequences given in Appendix A, an antisense strand of hybridizes one of the sequences given in Appendix A or naturally occurring mutants thereof.
• Primers based on a nucleotide sequence from Appendix A can be used in PCR reactions for cloning RRP homologs. Probes based on the RRP nucleotide sequences can be used to detect transcripts or genomic sequences that encode the same or homologous proteins.
• the probe also comprises a label group attached to it, for example a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.
• a label group attached to it for example a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.
• the nucleic acid molecule according to the invention encodes a protein or a section thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a section thereof retains the ability to increase the yield, production and / or To modulate the efficiency of the production of one or more fine chemicals in C. glutamicum, to degrade hydrocarbons, to oxidize terpenoids, to serve as a target for drug development or to serve as an identification marker for C. glutamicum or related organisms.
• the term "sufficiently homologous" refers to proteins or portions thereof, their Amino acid sequences have a minimal number of identical or equivalent (for example 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 form an amino acid sequence from Appendix B, so that the protein or a portion thereof the yield, production and / or modulate the efficiency of the production of one or more fine chemicals in C. glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or as an identification marker for C. glutamicum or related organisms. Examples of these activities are also described here.
• the "function of an RRP protein” thus contributes to the overall regulation of the metabolic pathway of one or more fine chemicals or to the degradation of a hydrocarbon or to the oxidation of a terpenoid.
• Sections of proteins which are encoded by the RRP nucleic acid molecules according to the invention are preferably biologically active sections of one of the RRP proteins.
• the term “biologically active section of an RRP protein”, as used here, is intended to include a section, for example a domain / a motif of an RRP protein, which / which the yield, production and / or efficiency of the Production of one or more fine chemicals modulated in C. glutamicum, degrades hydrocarbons, oxidizes terpenoids, serves as a target for drug development or as an identification ark for C. glutamicum or related organisms.
• a test of the enzymatic activity can be carried out become.
• Additional nucleic acid fragments that encode biologically active sections of an RRP protein can be determined by isolating a section from one of the sequences in Appendix B, expressing the encoded section of the RRP protein or peptide (for example by recombinant expression in vitro) and produce the activity of the encoded portion of the RRP protein or peptide.
• the invention also encompasses nucleic acid molecules that differ from one (and portions thereof) of one of the nucleotide sequences shown in Appendix A because of the degenerate genetic code and thus encode the same RRP protein as that encoded by the nucleotide sequences shown in Appendix A.
• an isolated one according to the invention Nucleic acid molecule is a nucleotide sequence that encodes a protein with an amino acid sequence shown in Appendix B.
• the nucleic acid molecule according to the invention encodes a full-length C. glutamicum protein which is essentially homologous to an amino acid sequence from Appendix B (encoded by an open reading frame shown in Appendix A).
• nucleotide sequence of Appendix A which leads to a change in the amino acid sequence of the encoded RRP protein without affecting the functionality of the RRP protein.
• nucleotide substitutions which lead to amino acid substitutions at "non-essential" amino acid residues can be prepared in a sequence from Appendix A.
• a "non-essential" amino acid residue is a residue that can be changed in the wild-type sequence by one of the RRP proteins (Appendix B) without changing the activity of the RRP protein, whereas an "essential" amino acid residue for the RRP- Protein activity is required.
• other amino acid residues for example non-conserved or only semi-preserved amino acid residues in the domain with RRP activity
• a further aspect of the invention consequently relates to nucleic acid molecules which encode RRP proteins which contain modified amino acid residues which are not essential for RRP activity. These RRP proteins differ in amino acid sequence from a sequence in Appendix B, but still retain at least one of the RRP activities described here.
• the isolated nucleic acid molecule in one embodiment, comprises a nucleotide sequence encoding a protein that comprises an amino acid sequence that has at least about 50% homology to an amino acid sequence from Appendix B and the yield, production, and / or efficiency of production of one or more fine chemicals in C. modulate glutamicum, degrade hydrocarbons, oxidize terpenoids, serve as a target for drug development or can serve as an identification marker for C. glutamicum or related organisms.
• An isolated nucleic acid molecule that encodes an RRP protein that is homologous to a protein sequence from Appendix B can be prepared by introducing one or more nucleotide substitutions,
• additions or deletions are generated in a nucleotide sequence from Appendix A, so that one or more amino acid substances ttions, additions or deletions are introduced into the encoded protein.
• the mutations can be introduced into one of the sequences from Appendix A using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.
• Conservative amino acid substitutions are preferably introduced on one or more of the predicted non-essential amino acid residues.
• 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.
• amino acids with basic side chains eg lysine, arginine, histidine
• acidic side chains eg aspartic acid, glutamic acid
• uncharged polar side chains eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
• polar side chains e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
• beta-branched side chains e.g. threonine, valine, isoleucine
• aromatic side chains e.g. tyrosine, phenylalanine, tryptophan, histidine
• a predicted non-essential amino acid residue in an RRP 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 RRP coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for RRP activity described here in order to identify mutants, who maintain RRP activity.
• the encoded protein can be expressed recombinantly, and the activity of the protein can be determined, for example, using the tests described here (see Example 8 of the example part).
• vectors preferably expression vectors, containing a nucleic acid encoding an RRP 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). Other vectors (e.g.
• non-episomal mammalian vectors are integrated into the genome of a host cell when introduced into the host cell and thereby replicated together with the host genome.
• certain vectors can control the expression of genes with which they are operably linked. These vectors are referred to here as "expression vectors".
• expression vectors usually the expression vectors that can be used in recombinant DNA techniques are 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 include promoters, repressor binding sites, activator binding sites, enhancer areas and other expression control elements (for example terminators, other elements of the m-RNA secondary structure or polyadenylation signals). These regulatory sequences are described, for example, in Goeddel: Gene Expression Technology: Methods in Enzyology 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, including of the fusion proteins or peptides encoded by the nucleic acids as described herein (e.g., RRP proteins, mutated forms of RRP proteins, fusion proteins, etc.).
• the recombinant expression vectors according to the invention can be designed for the expression of RRP proteins in prokaryotic or eukaryotic cells.
• RRP genes in bacterial cells such as C. glutamicum, insect cells (with Baculovirus expression vectors), yeast and other fungal cells (see Romanos, MA et al.
• the recombinant expression vector can be transcribed and translated in vitro, for example using regulatory sequences of the T7 promoter and T7 polymerase.
• Proteins are usually expressed in prokaryotes using vectors which contain constitutive or inducible promoters which control the expression of fusion or non-fusion proteins.
• Fusion vectors contribute a number of amino acids to a protein encoded therein, usually at the amino terminus of the recombinant protein. These fusion vectors usually have three functions: 1) to increase the expression of recombinant protein; 2) increasing the solubility of the recombinant protein; and 3) supporting the purification of the recombinant protein by acting as a ligand in affinity purification.
• a proteolytic cleavage site is often introduced at the junction of the fusion unit and the recombinant protein, so that the recombinant protein can be separated from the fusion unit after the fusion protein has been purified.
• These enzymes and their corresponding recognition sequences include factor Xa, thrombin and enterokinase.
• Common fusion expression vectors include pGEX (Pharmacia Biotech Ine; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ), in which Glutathione-S-Transferase (GST), maltose E-binding protein or protein A to the recombinant target protein is fused.
• GST Glutathione-S-Transferase
• the coding sequence of the RRP 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 RRP protein that is not fused to GST can be obtained by cleaving the fusion protein with thrombin.
• 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: 2111-2118). This change in the nucleic acid sequences according to the invention can be carried out using standard DNA synthesis techniques.
• the RRP 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 NEN include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, JF Peberdy et al., ed. , Pp. 1-28, Cambridge University Press: Cambridge.
• the RRP 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 RRP 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., Keper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left border ", Plant Mol. Biol. 20: 1195-1197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 8711-8721.
• 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).
• the control functions of the expression vector are often provided by viral regulatory elements. Commonly used promoters come, for example, from polyoma, adenovirus 2, cytomegalovirus and si ian 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 for the expression of 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, and in particular promoters of T (Ca lame and Eaton (1988) Adv Im- munol 43235-275%) -Zellrezeptoren
• neuron-specific promoters e.g. the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477
• pancreatic-specific promoters Edlund et al.
• mammary specific promoters e.g., milk serum promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166.
• Development-regulated promoters are also included, for example the mouse hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the ⁇ -fetoprotein promoter (Ca pes 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. That that the DNA molecule is operably linked to a regulatory sequence in such a way that expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the RRP 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 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.
• 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 the host cells into which a recombinant expression vector according to the invention has been introduced.
• the terms "host cell” and “recombinant host cell” are used interchangeably here. It goes without saying that these terms refer not only to a specific target cell, but also to the descendants or potential descendants of this cell. Because determined in successive generations due to mutation or environmental influences Modifications can occur, these offspring are not necessarily identical to the parental cell, but are still included in the scope of the term as used here.
• a host cell can be a prokaryotic or eukaryotic cell.
• an RRP 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 natural ones Competence, chemically mediated transmission, calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection or electroporation.
• Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.
• 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 RRP 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. (eg cells that have integrated the selectable marker survive, whereas the other cells die).
• a vector which contains at least a section of an RRP gene into which a deletion, addition or substitution has been introduced in order to change the RRP gene, for example to functionally disrupt it.
• This RRP gene is preferably a Corynebacterium glutamicun.-RRP gene, however a ho ologon from a related bacterium or even from a mammalian, yeast or insect source can be used.
• the vector is designed such that the endogenous RRP gene is functionally disrupted in homologous recombination (i.e. 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 RRP gene is mutated or otherwise altered when homologous recombination occurs, 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 RRP protein is changed.).
• the altered portion of the RRP gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the RRP gene, which is a homologous recombination between the exogenous RRP gene carried by the vector and one endogenous RRP gene in a microorganism.
• the additional flanking RRP nucleic acid is long enough for successful homologous recombination with the endogenous gene.
• the vector contains less than one kilobase of flanking DNA (at both the 5 'and 3' ends) (see, e.g., Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503 for a description of homologous recombination vectors).
• the vector is introduced into a microorganism (e.g., by electroporation), and cells in which the introduced RRP gene is homologously recombined with the endogenous RRP 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 RRP gene in a vector, thereby placing it under the control of the Lac operon, enables e.g. expression of the RRP gene only in the presence of IPTG.
• These regulatory systems are known in the art.
• a host cell according to the invention such as a prokaryotic or eukaryotic host cell in culture, can be used for the production (ie expression) of an RRP protein.
• the invention also provides methods for the production of RRP 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 which encodes an RRP protein has been introduced, or into whose genome a gene has been introduced which is a wild-type or modified RRP protein encoded) in a suitable medium until the RRP protein has been produced.
• the method comprises isolating the RRP 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 RRP protein areas that are necessary for the function, modulation of the activity of an RRP protein; Modulating the activity of one or more metabolic pathways and modulating the cellular production of a desired compound, such as a fine chemical.
• the RRP nucleic acid molecules according to the invention have a multitude of uses. They can initially be used to identify an organism as Corynebacterium glutamicum or a close relative of it.
• the invention provides the nucleic acid sequences of a number of C. glutamicum genes. By probing the extracted genomic DNA of a culture of a uniform or mixed population of microorganisms under stringent conditions with a probe 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 cells in the sample can first be grown in a suitable liquid or on a suitable solid culture medium in order to increase the number of cells in the culture. These cells are lysed, and all of the DNA contained is extracted and, if necessary, cleaned to remove cell debris and protein material that could interfere with the subsequent analysis.
• Polymerase chain reaction or a similar technique known in the art is performed (see a general overview of methodologies commonly used for nucleic acid sequence amplification in Mullis et al., U.S. Patent No. 4,683,195, Mullis et al., U.S. Patent No. 4965188 and Innis, MA, and Gelfand, DH (1989) PCR-Protocols, A guide to Methods and Applications, Academic Press, pp. 3-12, and (1988) Bio-technology 6: 1197, and International patent application no.
• primers which are specific for an RRP nucleic acid molecule according to the invention are incubated with the nucleic acid sample so that this particular RRP nucleic acid sequence, if present in the sample, is amplified.
• the particular nucleic acid sequence to be amplified is selected based on its exclusive presence in the genome of C. glutamicum and only a few closely related bacteria. The presence of the desired amplification product indicates the presence of C. glutamicum or an organism closely related to C. glutamicum.
• the nucleic acid and protein molecules according to the invention can also serve as markers for specific regions of the genome. Using techniques known in the art, it is possible to prove the physical localization of the RRP nucleic acid molecules according to the invention on the C. glutamicum genome, which in turn can be used for easier localization of other nucleic acid molecules and genes on the map.
• the nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can also enable the construction of a genomic map in such bacteria (e.g. Brevibacterium lactofermentum).
• the nucleic acid and protein molecules according to the invention are not only suitable for mapping the genome, but also for functional studies of C. glutamicum proteins.
• the C. gIutamicu_n genome can be split, for example, and the fragments incubated with the DNA-binding protein.
• Those that 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. glutamicum, and if so several times with different ones Enzymes is carried out, it facilitates a rapid determination of the nucleic acid sequence to which the protein binds.
• the RRP nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies.
• the metabolic processes in which the molecules according to the invention are involved are exploited by a large number of prokaryotic 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.
• the RRP proteins according to the invention can be used as markers for classifying an unknown bacterium as C. glutamicum or for identifying C. glutamicum or closely related bacteria in a sample.
• cells in a sample may be amplified (eg, by culturing in a suitable medium) to increase the sample size and then lysed so that the proteins contained therein are released. If necessary, this sample can be cleaned to remove cell debris and nucleic acid molecules that could interfere with the subsequent analysis.
• Antibodies that are specific for a selected RRP protein according to the invention can be incubated with the protein sample in a typical Western test format (see, for example, Ausubel et al., (1988) Current Protocols in Molecular Biology, Wiley: New York), wherein the antibody binds to its target protein when this protein is present in the sample.
• An RRP protein is selected for this type of test if it is unique or almost unique to C. glutamicum or C. glutamicum and very closely related bacteria.
• the proteins in the sample are then separated by gel electrophoresis and transferred to a suitable matrix, such as nitrocellulose.
• a suitable second antibody with a detectable label eg chemiluminescent or colorimetric
• the presence or absence of the marker indicates the presence or absence of the target protein in the sample. If the protein is present, this indicates the presence of C. glutamicum.
• a similar procedure allows an unknown bacterium to be classified as C. glutamicum; if a series of proteins specific to C. glutamicum are not detected in the protein samples prepared by the unknown bacterium, this bacterium is probably not C. glutamicum.
• RRP proteins with functional differences from the wild-type RRP proteins. These proteins can be improved in terms of their efficiency or activity, can be present in the cell in larger numbers than usual or can be weakened in terms of their efficiency or activity.
• Indirect modulation of fine chemical production can also be done by modifying the activity of a protein of the invention (ie, mutagenizing the corresponding gene) so that the overall ability of the cell to grow and divide, or to remain viable and productive, is increased.
• the production of fine chemicals from C. glutamicum is usually achieved by large-scale fermentation culture of these microorganisms, conditions that are often suboptimal for growth and cell division.
• a protein according to the invention for example a stress reaction protein, a cell wall protein or proteins which are involved in the metabolism of compounds which are necessary for the occurrence of cell growth and division, such as nucleotides and amino acids), so that better survival, Growing and multiplying in these conditions is possible, it may be possible to increase the number and productivity of these altered C.
• the nucleic acid and protein molecules of the invention can be used to generate C. glutamicum or related bacterial strains expressing mutant RRP nucleic acid and protein molecules so that the yield, production and / or efficiency of production of a desired connection is improved.
• the desired compound can be any product made 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 Corynejacteriuz ⁇ glutamicum (ATCC 13032) was grown overnight at 30 ° C. with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded, and the cells were resuspended in 5 ml buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml culture volume).
• composition of buffer I 140.34 g / 1 sucrose, 2.46 g / 1 MgSO 4 • 7 H 2 0, 10 ml / 1 KH 2 PO 4 solution (100 g / 1, 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 4 • H 2 0, 10 mg / 1 ZnS0 4 • 7 H 2 0, 3 mg / 1 MnCl 2 • 4 H 2 0, 30 mg / 1 H 3 B0 3 , 20 mg / 1 CoCl 2 • 6 H 2 0, 1 mg / 1 NiCl 2 • 6 H 2 0, 3 mg / 1 Na 2 Mo0 4 • 2 H 2 0, 500 mg /
• 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 washed resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added.
• 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, pheno1-chloroform isoamyl alcohol and chlorofoirai isoamyl alcohol using standard methods. 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 of the pBS series (pBSSK +, pBSSK- and others; Stratagene, LaJolla, USA) or Cosmide, 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 '-GTAAAACGACGGCCAGT-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 (e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae), which maintain the integrity of their genetic information cannot maintain.
• E. coli or other microorganisms e.g. 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, S 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 CoryneJba ⁇ terium glutamicum
• Corynebacterium and Brevi acterium species contain endogenous plasmids (such as pHM1519 or pBLl) that replicate autonomously (for an overview see, for example, Martin, J.F. et al. (1987) Biotechnology 5: 137-146).
• Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can easily be constructed using standard vectors for E. coli (Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold Spring
• Origin of replication and a suitable marker from Corynebacterium glutamicum is added.
• origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibactertium species.
• transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn-903 transposon) or for chloramphenicol (Winnacker, EL (1987) "Fro Genes to Clones - Introduction to Gene Technology, VCH, Weinheim)
• kanamycin resistance such as those derived from the Tn5 or Tn-903 transposon
• chloramphenicol Winnacker, EL (1987) "Fro Genes to Clones - Introduction to Gene Technology, VCH, Weinheim)
• gene overexpression see, for example, Yoshihama, M. et al. (1985) J. Bacteriol. 162: 591-597, Martin, JF et al., (1987) Biotechnology, 5: 137-146 and Eikmanns, BJ et al. (1992) Gene 102 : 93-98).
• C. glutamicum can be carried out by protoplast transformation (Kastsumata, R. et al., (1984) J. Bacteriol. 159: 306-311), electroporation (Liebl, E. et al., (1989) FEMS Microbiol. Letters , 53: 399-303) and, in cases where special vectors are used, can also be achieved by conjugation (as described, for example, in Schaefer, A., et (1990) J. Bacteriol. 172: 1663-1666).
• 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 Biology, Wiley: New York), wherein a primer which is designed in such a way that it binds to the gene of interest, with a detectable
• the label usually radioactive or chemiluminescent is labeled 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 indicates the presence and also the amount of RNA 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 the observed amount of labeling indicate the presence and the 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 obtained via complex Compounds such as molasses or other by-products of sugar refining to the media.
• Sources of different carbon sources are alcohols and organic see acids such as methanol, ethanol, acetic acid or lactic acid.
• Sources of nitrogen 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 C1 or (NH 4 ) S0 4 , NH 4 OH, nitrates,
• Urea amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract and others.
• Inorganic salt compounds that may be included in the media include the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.
• Chelating agents can be added to the medium to keep the metal ions in solution.
• Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.
• the media usually also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.
• Growth factors and salts often come from complex media components such as yeast extract, molasses, corn steep liquor and the like.
• the exact composition of the media connections strongly depends on the respective experiment and is decided individually for each specific case. Information on media optimization is available from the textbook "Applied Microbiology. 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) and the like.
• All media components are sterilized either by heat (20 min at 1.5 bar and 121 ° C) or by sterile filtration.
• the components can either be sterilized 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 value can be keep constant 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.
• the pH value can also be regulated with gaseous ammonia.
• the incubation period is usually in the range of several hours to several days. This time is selected so that the maximum amount of product accumulates in the broth.
• the disclosed growth experiments can be carried out in a variety of containers, such as microtiter plates, glass tubes, glass flasks or glass or metal fermenters of different sizes.
• 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 made for the evaporation losses.
• the medium is inoculated to an ODgoo of 0.5-1.5 using cells that 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. glutamicum 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.
• 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. 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 desired product can be obtained from C. glutamicum cells or from the supernatant of the culture described above by various methods known in the art. If the desired product is not secreted by the cells, the cells can be harvested from the culture by slow centrifugation, the cells can be lysed by standard techniques such as mechanical force or ultrasound treatment. The cell debris is removed by centrifugation and the supernatant fraction containing the soluble proteins is obtained for further purification of the desired compound. If the product is secreted by the C. glutamicum cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is kept for further purification.
• the supernatant fraction from both purification procedures is subjected to chromatography with a suitable resin, the desired molecule either being retained on the chromatography resin, but not many impurities in the sample, 5 or the impurities remaining on the resin, but the sample is not.
• chromatography steps can be repeated if necessary 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 10 for a particular molecule to be purified.
• the purified product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is maximum.
• 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,
• routine 40 determines many equivalents of the specific embodiments of the invention. These equivalents are intended to be encompassed by the claims below.

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