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
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
• • 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
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • 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
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • 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
  • C12N9/90—Isomerases (5.)
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/15—Corynebacterium

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 one or more desired molecules.
• a particularly suitable organism for this purpose is Corynebacterium glutamic ⁇ m, 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 Coryne acterium glutamicum or related types of bacteria.
• C. glutamicum is a gram-positive, aerobic bacterium which is commonly used in industry for the large-scale production of a number of fine chemicals, and also for the degradation of hydrocarbons (eg 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.
• glutamicum itself is not pathogenic, but it is related to other Corynebacterium species n, such as Corynebacterium diphtheriae (the causative agent of diphtheria), which are important pathogens in humans.
• 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 which serve as reference points for mapping the C. glutamicum genome or genomes of related organisms.
• MR proteins which are referred to here as metabolically regulatory (MR) proteins.
• MR proteins can, for example, perform a function which is involved in the transcription, translation or post-translational regulation of proteins which are important for the normal metabolic functioning of cells.
• cloning vectors for use 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 Brevibacteriu species (e.g. lactofermentum) Yoshiha a et al., J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol.
• nucleic acid molecules according to the invention can be used for the genetic manipulation of this organism in order to make it better and more efficient as a producer of one or more fine chemicals.
• the improved yield, production and / or efficiency of the production of a fine chemical can be based on a direct or indirect effect of the manipulation of a gene according to the invention.
• changes in C. glutamicum MR proteins that ordinarily regulate the yield, production, and / or efficiency of production of a fine chemical from a metabolic fine chemical pathway can have a direct impact on the overall production or production rate of one or more of these desired compounds have this organism.
• Changes in the proteins that are involved in this substance through its own way can also have an indirect effect on the yield, production and / or efficiency of the production of a desired fine chemical.
• the regulation of metabolism is necessarily complex, and the regulatory mechanisms which accomplish the different pathways can overlap in many places, so that more than one pathway can be set quickly according to a particular cell event.
• a regulatory protein for one pathway to have an effect on many other pathways, some of which may be involved in the biosynthesis or degradation of a desired fine chemical.
• modulating the effect of an MR protein can have an impact on the production of a fine chemical that is produced via a metabolic pathway that is different from that which is directly regulated by this MR protein.
• the nucleic acid and protein molecules according to the invention can be used to directly improve the yield, production and / or efficiency of the production of one or more desired fine chemicals from Corynebacterium glutamicum.
• one or more regulatory proteins according to the invention can be manipulated in such a way that their function is modulated.
• the mutation of an MR protein involved in the repression of the transcription of a gene encoding an enzyme required for the biosynthesis of an amino acid so that it is no longer capable of repression of the transcription causes an increase in the production of this amino acid.
• changing the activity of an MR protein which brings about increased translation, or activating the post-translational modification of a C. glutamicum protein which is involved in the biosynthesis of a desired fine chemical, can in turn increase the production of this chemical.
• the opposite situation can also be useful: by increasing the repression of transcription or translation or by post-translational negative modification of a C. glutamicum protein, which is involved in the regulation of the degradation pathway for a compound, the production of this chemical can be increased. In any case, the overall yield or the speed of production of the desired fine chemical can be increased.
• an MR protein according to the invention which usually suppresses the biosynthesis of nucleosides in response to a suboptimal extracellular nutrient supply (which prevents cell division), so that it has a lower repressive activity, one can do the biosynthesis of nucleosides and perhaps that Increase cell division.
• Changes in MR-Pro Tones that cause increased cell growth and division in culture, at least due to the increased number of cells that produce the chemical in culture, can increase the yield, production and / or efficiency of production of one or more desired fine chemicals evoke from culture.
• the invention provides new nucleic acid molecules that encode proteins, which are referred to here as metabolic regulatory (MR) proteins and can, for example, carry out an enzymatic step that is involved in the transcriptional, translational or post-translational regulation of metabolic pathways in C. glutamicum.
• MR metabolic regulatory
• Nucleic acid molecules that encode an MR protein are referred to here as MR nucleic acid molecules.
• the MR protein is involved in the transcription, translation or post-translational regulation of one or more metabolic pathways. 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 encodes an MR protein or biologically active sections thereof, and also nucleic acid fragments which act as primers or hybridization probes for the detection or amplification of MR coding nucleic acid (for example DNA or mRNA) are suitable.
• the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof of one of these nucleotide sequences.
• the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B.
• the preferred MR proteins according to the invention likewise preferably have at least one of the MR 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 mo lekül preferably corresponds to a naturally occurring nucleic acid olekül.
• the isolated nucleic acid more preferably encodes a naturally occurring C. glutamicum MR protein or a biologically active portion thereof.
• 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.
• a host cell that is grown in a suitable medium is used to produce an MR protein.
• the MR 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 MR 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 MR sequence as a transgene.
• an endogenous MR gene in the genome of the microorganism is changed by homologous recombination with an altered MR 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, particularly preferably lysine.
• 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 MR protein or a section, for example a biologically active section thereof.
• the isolated MR protein or its portion regulates one or more Metabolic pathways in C. glutamicum transcriptionally, translationally or post-translationally.
• the isolated MR protein or a portion thereof is sufficiently homologous to an amino acid sequence from Appendix B that the protein or its portion further has the ability to transcriptionally, translationally or post-translationally one or more metabolic pathways in C. glutamicum regulate.
• the invention also relates to an isolated MR protein preparation.
• the MR protein comprises an amino acid sequence from Appendix B.
• the invention relates to an isolated full-length protein which forms a complete amino acid sequence from Appendix B (which is encoded by an open reading frame in Appendix A) is essentially homologous.
• the MR polypeptide or a biologically active portion thereof can be operably linked to a non-MR polypeptide to form a fusion protein.
• this fusion protein has a different activity than the MR protein alone and, in other preferred embodiments, regulates one or more metabolic pathways in C. glutamicum transcriptionally, translationally or post-translationally.
• 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 MR 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 MR 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 Brevibacterium.
• 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 MR protein activity or MR nucleic acid expression so that cell-associated activity changes compared to the same activity in the absence of the substance. is changed.
• the cell is modulated in C. glutamicum with regard to one or more regulatory systems for metabolic pathways, so that the yields or the speed at which a desired fine chemical is produced by this microorganism is improved.
• the substance that modulates the MR protein activity stimulates, for example, the MR protein activity or the MR nucleic acid expression.
• Examples of substances that stimulate MR protein activity or MR nucleic acid expression include small molecules, active MR proteins and nucleic acids that encode MR proteins and have been introduced into the cell.
• Examples of substances that inhibit MR activity or expression include small molecules and antisense MR nucleic acid molecules.
• Another aspect of the invention relates to methods for modulating the yields of a desired compound from a cell, comprising introducing into a cell an MR 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 MR nucleic acid and protein molecules which are involved in the regulation of the Corynebacterium glutamicum metabolism, including the regulation of the fine chemical metabolism.
• the molecules according to the invention can either be used directly (for example where the modulation of the activity of a regulatory protein of the lysine pathway has a direct effect on the yield, production and / or efficiency of production of lysine from this organism) or indirectly, which still has an increase in the yield, production and / or efficiency of production of the desired compound (e.g.
• fine chemical is known in the art and includes molecules that are produced by an organism and have applications 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 dia inopimelic 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 citations 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", p 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 normal cell functions.
• 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 Ullmann's Encyclope - dia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim
• the amino acids can be in the D or L configuration, although L-amino acids are usually the only type which is found in naturally occurring proteins. Biosynthetic and degradation pathways of each of the 20 proteinogenic amino acids are well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)).
• the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), so called because they have to be included in the diet due to the complexity of their biosynthesis, are identified by simple biosynthetic pathways in the rest
• non-essential amino acids alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine
• 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 5-glutamate, MSG) and is 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 (euchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol 6, Chapter 14a, VCH: Weinheim). It has been discovered that these 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 Ullmann's Encyclopedia of Industrial Chemistry , Vol. A2, pp. 57-97, VCH, Weinheim, 1985 are suitable substances. 0
• 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 pentosephosphate pathway, erythrose-4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that 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 one
• 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 formed from oxa-acetate, an intermediate of the Ci cycle.
• Asparagine, methionine, threonine and lysine are each produced by converting aspartate.
• Isoleucine is made from threonine.
• histidine is formed from 5-phospho ibosyl-1-pyrophosphate, an activated sugar.
• Amino acids the amount of which exceeds the cell's protein biosynthesis requirements, cannot be stored and are instead broken down, so that intermediates are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd ed. 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.
• 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 also 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, Ullmann'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.
• nutrazeutiku 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 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 mononu Kleotide (FMN) and flavin adenine dinucleotide (FAD) used.
• GTP guanosine 5 'triphosphate
• FMN flavin mononu Kleotide
• FAD flavin adenine dinucleotide
• the family of compounds which are collectively referred to as "vitamin B6" are all derivatives of the common structural unit 5-hydroxy-6-methylpyridine.
• Panthothenate (pantothenic acid, R - (+) - N- (2, 4-dihydroxy-3, 3-dimethyl-l-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 for. the biosynthetic steps for the conversion into pantoic acid, into ß-alanine and for the condensation into pantothenic acid are known enzymes.
• the metabolically active form of pantothenate is Coenzy A, whose biosynthesis takes place in 5 enzymatic steps.
• Pantothenate pyridoxal-5 '-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of 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 oc-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.
• 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 uses: as intermediates in the biosynthesis of various Fine chemicals (e.g. thiamine, S-adenosyl-methionine, folate or riboflavin), as energy sources for the cell (e.g. ATP or GTP) and for chemicals themselves, are usually used as flavor enhancers (e.g. IMP or GMP) or for many medical see applications (see, for example, Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim, pp. 561-612).
• Enzymes which are based on purine, Pyrimidine, nucleoside or nucleotide metabolism are also increasingly becoming targets against which crop protection chemicals, including fungicides, herbicides and insecticides, are being developed.
• the purine nucleotides are synthesized in a series of steps via the intermediate compound inosine 5'-phosphate (IMP) from ribose 5-phosphate, which leads to the production of guanosine 5 'monophosphate (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 'monophosphate
• AMP adenosine 5' -monophosphate
• Pyridin biosynthesis takes place via the formation of uridine 5 'monophosphate (UMP) from ribose 5-phosphate. UMP in turn is converted into 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 via an ⁇ , ⁇ -l, 1 bond. It is commonly used in the food industry as a sweetener, as an additive for dried or frozen food and in beverages. However, it is also used in the pharmaceutical, cosmetics and biotechnology industries (see, e.g., Nishi oto et al., (1998) US Patent No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech. 16 (1998) 460-467; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 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.
• All living cells have complex catabolic and anabolic abilities with many interconnected metabolic pathways.
• the cell uses a fine-tuned regulatory network. By regulating enzyme synthesis and enzyme activity, either independently or simultaneously, the cell can regulate the activity of completely different metabolic pathways, so that the changing needs of the cell are satisfied.
• proteins that bind to the DNA which either increases the expression of a gene (positive regulation, as in the case of the ara operon from E. coli) or decreases (negative regulation, as in the case of the lac operon from E) coli).
• These expression-modulating transcription factors can themselves be subject to regulation. Their activity can be regulated, for example, by binding small-molecule compounds to the DNA-binding protein, which stimulates the binding of these proteins to the appropriate binding site on the DNA (as in the case of arabinose for the ara operon) or inhibits it ( as in the case of lactose for the lac operon) (see, for example, Helmann, JD and Chamberlin, MJ (1988) "Structure and function of bacterial sigma factors" Ann.
• Protein synthesis is regulated not only at the level of transcription, but often also at the level of translation. This regulation can be accomplished through many mechanisms, including altering the ability of the ribosome to bind to one or more mRNAs, binding the ribosome to mRNA, maintaining or removing the secondary mRNA structure, using common or less common codons for a particular gene , the degree of abundance of one or more tRNAs and special regulatory mechanisms such as attenuation (see Vellanoweth, RI (1993) Translation and its regulation in Bacillus subtilis and other gram-positive bacteria, Sonenshein, AL et al. Ed. ASM: Washington, DC , Pp. 699-711 and references cited therein.
• the transcription and translation regulation can be directed to a single protein (sequential regulation) or simultaneously to several proteins in different metabolic pathways (coordinated regulation). Genes whose expression is regulated in a coordinated manner are often close together in an operon or regulon in the genome.
• This up- or down-regulation of gene transcription and translation is controlled by the cellular or extracellular amounts of various factors, such as substrates (precursors and intermediate molecules that are used in one or more metabolic pathways), catabolites (molecules that are caused by biochemical metabolic pathways) which are related to the production of energy from the breakdown of complex organic molecules such as sugar) and end products (the molecules obtained at the end of a metabolic pathway).
• genes that encode enzymes that are necessary for the activity of a particular metabolic pathway is induced by high amounts of substrate molecules for this metabolic pathway. Accordingly, this gene expression is repressed when there are high intracellular amounts of the end product of the route (Snyder, L. and Champness, W. (1977) The Molecular Biology of Bacteria ASM: Washington). Gene expression can also be regulated by other external and internal factors, such as environmental conditions (e.g. heat, oxidative stress, or hunger). These global environmental changes cause changes in the expression of specialized modulating genes that trigger gene expression directly or indirectly (via additional genes or proteins) by binding to DNA and thereby induce or repress transcription (see, for example, Lin, ECC and Lynch, AS Ed. (1995) Regulation of Gene Expression in Escherichia coli, Chapman & Hall: New York).
• Another mechanism by which cellular metabolism can be regulated is at the level of the protein. This regulation takes place either via the activities of other enzymes or by binding low-molecular components that prevent or enable the normal function of the protein. Examples of the compounds of low molecular weight binding protein regulation by comparison include the binding of 'GTP or NAD. The binding of low molecular weight chemicals is usually reversible as with the GTP-binding proteins. These proteins occur in two states (with bound GTP or GDP), one state being the active form of the protein and the other the inactive form.
• Regulation of protein activity by the action of other enzymes is usually done by covalent modification of the protein (i.e., phosphorylation of amino acid residues such as histidine or aspartate, or methylation).
• This covalent modification is usually reversible, which is accomplished by an enzyme with the opposite activity.
• An example of this is the opposite activity of kinases and phosphorylases in protein phosphorylation: protein kinases phosphorylate specific residues on a target protein (e.g. serine or threonine), whereas protein phosphorylases remove the phosphate groups from these proteins.
• Enzymes that modulate the activity of other proteins are usually modulated by external stimuli themselves. These stimuli are mediated by proteins that act as sensors.
• Control systems for downward regulation of the metabolic pathways can be removed or reduced in order to improve the synthesis of desired chemicals, and accordingly those for the upward regulation of the metabolic pathway for a desired product can be constitutively activated or optimized with regard to activity (as shown in Hirose, Y. and Okada, H. (1979) "Microbial Production of Amino Acids", in: Peppler, HJ and Perlman, D. (ed.) Microbial Technology 2nd ed., vol. 1, chap. 7, Academic Press, New York).
• the present invention is based at least in part on the discovery of new molecules, which are referred to here as MR nucleic acid and protein molecules and which regulate one or more metabolic pathways in C. glutamicum through transcriptional, translational or post-translational measures.
• the MR molecules regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally.
• the activity of the MR molecules according to the invention for regulating one or more metabolic pathways in C. glutamicum has an effect on the production of a desired fine chemical by this organism.
• the MR molecules according to the invention have modulated activity, so that the metabolic pathways of C. glutamicum which regulate the MR proteins according to the invention are modulated in terms of their efficiency or their throughput, which either directly or indirectly affects the Yield, production and / or efficiency of production of a desired fine chemical modulated by C. glutamicum.
• MR protein or "MR polypeptide” encompasses proteins which regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally.
• MR proteins include those encoded by the MR genes listed in Table 1 and Appendix A.
• MR gene or "MR nucleic acid sequence” include 'Nukleinklareseguenzen encoding an MR protein, which comprises a coding region and corresponding untranslated 5' and 3 'consists -Sequenz Symposium Programen. Examples of MR genes are 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)
• 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 set up a certain throughput rate of a fine chemical).
• yield or “product / carbon Yield” is well known in the art and includes the efficiency of converting the carbon source to 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 include 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.
• the term "metabolism” is known in the art and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a certain compound (eg the metabolism of an amino acid, such as glycine) then encompasses all biosynthesis, modification and degradation pathways of this compound in the cell.
• the term “regulation” is known in the art and encompasses the activity of one protein to control the activity of another protein.
• transcription regulation is known in the art and includes the activity of a protein to inhibit or activate the conversion of a DNA encoding a target protein into mRNA.
• transcription regulation is known in the art and includes the activity of a protein to inhibit or activate the conversion of an mRNA encoding a target protein into a protein molecule.
• post-translational regulation is known in the art "and includes the activity of a protein to inhibit or improve the activity of a target protein by covalently modifying the target protein (for example by methylation, glycosylation or phosphorylation).
• the MR 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.
• Genre combination techniques can be used to manipulate one or more regulatory proteins according to the invention for metabolic pathways in such a way that their function is modulated.
• a biosynthesis enzyme can be improved in efficiency or its allosteric control region can be destroyed so that the inhibition of the production of the compound is prevented.
• a degradation enzyme can be deleted or modified by substitution, deletion or addition in such a way that its degradation activity for the desired compound is reduced without the viability of the cell being impaired. In any case, the total yield or the production rate of one of these desired fine chemicals can be increased.
• these changes in the protein and nucleotide molecules according to the invention can improve the production of fine chemicals in an indirect manner.
• the regulatory mechanisms of the metabolic pathways in the cell are necessarily linked, and the activation of one metabolic pathway can cause the repression or activation of another in an accompanying manner.
• By modulating the activity of one or more proteins of the invention the production or efficiency of the activity of other fine chemical biosynthesis or degradation pathways can be affected.
• By lowering the ability of an MR protein to repress the transcription of a gene encoding a particular amino acid biosynthetic protein other amino acid biosynthetic pathways can be depressed because these pathways are linked.
• the growth and division of cells from their extracellular surroundings can be decoupled to a certain degree; by affecting an MR protein that usually represents the biosynthesis of a nucleotide when the extracellular conditions for growth and cell division are suboptimal so that it now lacks this function, growth can be allowed to occur even if the extracellular Conditions are bad. This is particularly important in large-scale fermentation farming, where the conditions in the culture are often suboptimal in terms of temperature, nutrient supply or aeration, but which still promote growth and cell division when the cellular regulatory systems for these factors are eliminated.
• 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 the production of 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 MR protein according to the invention or a biologically active section or fragments thereof can regulate a metabolic pathway in C. srlutamicum transcriptionally, translationally or post-translationally, or can have one or more of the activities listed in Table 1.
• a metabolic pathway in C. srlutamicum transcriptionally, translationally or post-translationally or can have one or more of the activities listed in Table 1.
• One aspect of the invention relates to isolated nucleic acid molecules which encode MR polypeptides 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 nucleic acids coding for MR (e.g. MR-DNA).
• nucleic acid molecule as used here is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg 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' ends of the coding 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 gene region.
• the nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA.
• 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 MR 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 contain the nucleic acid molecule in the genomic Flank the DNA of the cell from which the nucleic acid originates (for example a C. srlutamicum cell).
• An "isolated" nucleic acid molecule, such as a cDNA molecule can also be essentially free of another cellular material or culture medium if it is produced by recombinant techniques, or free of chemical precursors or other chemicals if it is chemically synthesized becomes.
• 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. glutaznicum MR cDNA can be isolated from a C. glutaznicum library 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. 2. On1. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
• a nucleic acid molecule comprising a complete sequence from Annex A or a section thereof can be isolated by polymerase chain reaction, using the oligonucleotide primers which have been prepared 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 OH gonucleotide 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 by 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. Russia, 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.
• the nucleic acid so amplified can be cloned into a suitable vector and characterized by DNA sequence analysis.
• Oligonucleotides which correspond to an MR nucleotide sequence can 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 is compatible with one of the sequences given in Appendix A can hybridize, resulting in a stable duplex.
• the nucleic acid molecule according to the invention encodes a protein or a section thereof, the one
• amino acid sequence that is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion of which still has the ability to regulate a metabolic pathway in C. glutamicum transcriptionally, translationally or post-translationally refers to proteins or portions thereof whose amino acid sequences have a minimal number of identical or equivalent amino acid residues (e.g. an amino acid residue with a side chain similar to an amino acid residue in one of the sequences of Appendix B) to an amino acid sequence from Appendix B, so that the protein or a portion thereof can regulate a metabolic pathway in C. grutamicum transcriptionally, translationally or post-translationally.
• Protein components of these pathways can regulate the biosynthesis or degradation of one or more fine chemicals. Examples of these activities are also described here.
• the "function of an MR protein” relates to the overall regulation of one or more metabolic fine chemical pathways. Table 1 shows examples of MR protein activities.
• Sections of proteins which are encoded by the MR nucleic acid molecules according to the invention are preferably biologically active sections of one of the MR proteins.
• biologically active section of an MR protein is intended to include a section, for example a domain or a motif, of an MR protein that transcriptionally, translationally or post-translationally a metabolic pathway in C. glutamicum can regulate, or has an activity given in Table 1.
• a test of the enzymatic activity can be carried out.
• nucleotide sequence of Appendix A which leads to a change in the amino acid sequence of the encoded MR protein without affecting the functionality of the MR 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 can be changed in a wild-type sequence from one of the MR proteins (Appendix B) without changing the activity of the MR protein, whereas an "essential" amino acid residue is required for the MR protein activity.
• other amino acid residues e.g. non-preserved or single Semiconserved amino acid residues in the domain with MR activity cannot be essential for the activity and can therefore probably be changed without changing the MR activity.
• An isolated nucleic acid molecule encoding an MR protein that is homologous to a protein sequence from Appendix B can be generated by introducing one or more nucleotide substitutions, additions or deletions into 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 by 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. In a "conservative amino acid substitution", 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 do not include 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) and aromatic side chains (e.g.
• 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,
• a predicted non-essential amino acid residue in an MR protein is therefore 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 MR coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the MR activity described here in order to identify mutants that maintain MR activity. After mutagenesis of one of the sequences from Appendix A, 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 section).
• vectors preferably expression vectors, which contain a nucleic acid which encode an MR protein (or a section thereof).
• vector refers to a nucleic acid molecule that can transport another nucleic acid to which it is bound.
• plasmid which stands for 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 (e.g. bacterial vectors with bacterial origin of replication and episomal mammalian vectors).
• vectors eg non-episomal mammalian vectors
• Other vectors are integrated into the genome of a host cell when it is 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 called "expression vectors".
• expression vectors usually the expression vectors 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 these other expression vector forms, such as viral vectors (for example replication-deficient retroviruses, adenoviruses and adeno-related viruses), which perform similar functions.
• the recombinant expression vector according to the invention comprises a nucleic acid according to the invention in a form which is suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors one or more regulatory sequences, selected on the basis of the host cells to be used for expression, the is 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). These 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 direct expression of the nucleotide sequence only in certain host cells. Those skilled in the art are aware that the design of an expression vector can depend on factors such as the choice of the transforming host cell, the level of expression of the desired protein, etc.
• the expression vectors of the invention can be introduced into the host cells so that proteins or peptides, including fusion proteins or peptides, which are encoded by the nucleic acids as described herein, are thereby produced (e.g. MR proteins, mutated forms of MR proteins, fusion proteins, etc.).
• MR genes can be found 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, ed., Pp.
• the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
• Proteins are usually expressed in prokaryotes using vectors that contain constitutive or inducible promoters that 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 Detection 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 is fused to the recombinant target protein.
• GST glutathione-S-transferase
• the coding sequence of the MR 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 MR protein that is not fused to GST can be obtained by cleaving the fusion protein with thrombin.
• Suitable inducible non-fusion expression vectors from E. coli include pTrc (Amann et al., (1988) Gene 69: 301-315) and pETIld (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 pETlld 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).
• the MR 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 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 MR 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 MR proteins according to the invention can be expressed in single-cell plant cells (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 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 (Kauf an 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, Adenovirus2, 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 regulatory elements are known in the art.
• 10 gnete tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Cala e and Eaton (1988) Adv. Immunol. 43: 235-275 ), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulin
• milk serum promoter 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 (Campes and Tilghman (1989) Genes Dev. 3:
• 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 such that expression (by transcription of the DNA. Molecule) of an RNA molecule that is antisense to MR mRNA is possible. Regulatory sequences can be selected that are functional to an antisense rich
• the antisense expression vector can be in the form of a recombinant plas ids, 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.
• 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. 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 MR 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” as used here are intended to encompass a large number of methods known in the prior art for introducing foreign nucleic acid (for example DNA) into a host cell, including 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 that encodes 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 are resistant to medicinal products. elements 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 MR protein, or can be introduced on a separate vector. Cells that have been stably transfected with the introduced nucleic acid can be identified 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 MR gene into which a deletion, addition or substitution has been introduced in order to change the MR gene, for example to functionally disrupt it.
• This MR gene is preferably a Corynebacterium glutamicum MR gene, but a homologue from a related bacterium or even from a source of mammals, yeasts or insects can be used.
• the vector is designed in such a way that the endogenous MR 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 alternatively be designed in such a way that the endogenous MR gene is mutated or otherwise altered in the case of homologous recombination, but still codes the functional protein (for example the upstream regulatory region can be altered in such a way that the expression of the endogenous MR protein is thereby effected is changed.).
• the modified portion of the MR gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the MR gene, which is a homologous recombination between the exogenous MR gene carried by the vector. and an endogenous MR gene in a microorganism.
• the additional flanking MR 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 which the introduced gene MR-MR with the endogenous gene has homologously recombined are selected using art-known procedures.
• recombinant microorganisms can be produced which contain selected systems which allow regulated expression of the introduced gene.
• the inclusion of an MR gene in a vector under control of the Lac operon, for example, enables expression of the MR gene only in the presence of IPTG.
• 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 MR protein.
• the invention also provides methods for the production of MR proteins using the host cells according to the invention.
• the method comprises culturing the host cell according to the invention (into which a recombinant expression vector which encodes an MR protein has been introduced, or into whose genome a gene has been introduced which is a wild-type or modified MR protein encoded) in a suitable medium until the MR protein has been produced.
• the method comprises isolating the MR proteins from the medium or the host cell.
• the nucleic acid molecules, proteins, protein hologa, 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 that are related to C. glutamicum related, identification and localization of C. grlufcamicum sequences of interest, evolution studies, determination of MR protein areas that are necessary for the function, modulation of the activity of an MR protein; Modulating the activity of an MR path; and modulating the cellular production of a desired compound, such as a fine chemical.
• the MR 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 close relatives thereof.
• the invention provides the nucleic acid sequences of a number of C. gluta icum genes. By probing the extracted genomic DNA from a culture of a unitary or mixed population of microorganisms under stringent conditions with a probe that spans a region of a C. ermutamicum gene that is unique to that organism, one can determine whether this organism is present is.
• Corynebacterium glutamicum itself is not pathogenic, but it is associated with pathogenic species such as Corynebacterium diptheriae Wundt. The detection of such an organism is of significant clinical importance.
• the nucleic acid and protein molecules according to the invention can serve as markers for specific regions of the genome. This is useful not only when mapping the genome, but also for functional studies of C. grlutamicum proteins.
• the C. grlutamicum genome can be cleaved, 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.
• nucleic acid molecules according to the invention can moreover 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, such as Brevibacterium lactofermentum.
• the MR 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 used by many 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 provide an indication of which protein can tolerate mutagenesis without losing function.
• the manipulation of the MR nucleic acid molecules according to the invention can bring about the production of MR proteins with functional differences from the wild-type MR proteins. These proteins can be improved in terms of their efficiency or activity, and can be present in the cell in greater numbers than usual. against, or may be weakened in their efficiency or activity.
• By increasing the yield, production and / or efficiency of production by activating the expression of one or more lysine biosynthesis enzymes one can simultaneously increase the expression of other compounds, such as other amino acids, which the cell usually needs in larger amounts if lysine is needed in larger quantities.
• the regulation of the metabolism can also be changed in the entire cell in such a way that the cell under the environmental conditions of a fermentative culture (where the nutrient and oxygen supply can be poor and toxic waste products can be present in large quantities in the environment). and can grow or replicate better. For example.
• the nucleic acid and protein molecules according to the invention can be used to generate C. glutamicum or related bacterial strains which express mutated MR nucleic acid and protein molecules, so that the yield, production and / or Production efficiency of a desired connection is improved.
• the desired compound can be a natural product of C. glutamicum, which comprises the end products of the biosynthetic pathways and intermediates of naturally occurring metabolic routes as well as molecules which do not occur naturally in the metabolism of C. glutamicum, but which are derived from a C. glutamicum according to the invention. micum stems are produced.
• 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 of buffer I (5% of the original volume of the culture - all stated volumes are calculated for 100 ml of culture volume).
• 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, adjusted to pH with KOH 6.7), 50 ml / 1 M12 concentrate (10 g / 1 (NH 4 ) 2 S ⁇ 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
• 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 M 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.
• 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 the standard method. 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, J.G. (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
• Cos ide like 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 (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, utHLS, utD, mutT, etc., for comparison see Rupp, WD (1996) DNA repair mechanisms in Escher i-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, for example, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34 illustrates.
• Example 5 DNA transfer between Escherichia coli and Corynebacterium glutamicum
• Corynebacterium and Brevibacterium species contain endogenous plasmids (such as 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.
• 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 in which special vectors are used, can also be achieved by conjugation (as described, for example, in Schaefer, 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.
• Example 6 Determination of the expression of the mutated protein
• a suitable method for determining the amount of transcription of the mutated 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 which is designed in such a way that it binds to the gene of interest is provided with a detectable (usually radioactive or chemiluminescent) label so that - if 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, as described 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 via complex compounds such as molasses or other by-products from 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.
• 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 CI or (NH 4 ) SO 4 , NH 4 OH, nitrates, urea, amino acids or complex nitrogen sources, such as maize spring water, soy flour, soy protein, yeast extracts, meat extracts and others.
• ammonia gas or ammonium salts such as NH 4 CI or (NH 4 ) SO 4 , NH 4 OH, nitrates, urea
• amino acids or complex nitrogen sources such as maize spring water, soy flour, soy protein, yeast extracts, meat extracts 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 depends heavily on the respective experiment and is decided individually for each case. Information about media optimization is available from the textbook "Applied Microbiol. 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 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.
• 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 growth experiments disclosed 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 with or without baffles. Preferably 100 ml shake flasks are used, which contain 10%
• 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 OD ß oo of 0.5-1.5 using cells grown on agar plates, such as CM plates (10 g / 1 glucose, 2.5 g / 1 NaCl, 2 g / 1 urea, 10 g / 1 polypeptone, 5 g / 1 yeast extract, 5 g / 1 meat extract, 22 g / 1 agar pH 6.8 with 2 M NaOH), which have been incubated at 30 ° C.
• the inoculation of the media is carried out either by introducing a saline solution of C. srlutamicuzn 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 Kol ar, 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 the techniques described in Gennis, R.B. (1989) "Pores, Channels and Transporters", in Biome branes, Molecular
• 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 are examined for the increased production of the desired product (ie an amino acid).
• 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 from 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 a 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, and the cells can be lysed using standard techniques such as mechanical force or ultrasound. 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. ⁇ rlutam ⁇ cum cells, the cells are removed from the culture by slow centrifugation and the supernatant fraction is retained for further purification.
• the supernatant fraction from both purification procedures is subjected to chromatography with an appropriate resin, either with the desired molecule retained on the chromatography resin but not with many contaminants in the sample, or with the contaminants remaining on the resin but not the sample. If necessary, these chromatography steps can be repeated using the same or different chromatography resins.
• the specialist is in training choose the appropriate chromatography resins and the most effective application 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 at a maximum.
• the identity and purity of the isolated compounds can be determined by standard techniques in the art. These include

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