EP1456232A2 - Genes codant pour des proteines de synthese membranaire et de transport membranaire - Google Patents

Genes codant pour des proteines de synthese membranaire et de transport membranaire

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Publication number
EP1456232A2
EP1456232A2 EP02781299A EP02781299A EP1456232A2 EP 1456232 A2 EP1456232 A2 EP 1456232A2 EP 02781299 A EP02781299 A EP 02781299A EP 02781299 A EP02781299 A EP 02781299A EP 1456232 A2 EP1456232 A2 EP 1456232A2
Authority
EP
European Patent Office
Prior art keywords
protein
mct
nucleic acid
cell
molecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP02781299A
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German (de)
English (en)
Inventor
Oskar Zelder
Markus Pompejus
Hartwig Schröder
Burkhard Kröger
Corinna Klopprogge
Gregor Haberhauer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Paik Kwang Industrial Co Ltd
Original Assignee
BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1456232A2 publication Critical patent/EP1456232A2/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • Certain products and by-products of naturally occurring metabolic processes in cells are used in many industries, including the food, feed, cosmetic and pharmaceutical industries.
  • These molecules collectively referred to as “fine chemicals", include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors and enzymes. They are best produced by growing large-scale bacteria that are designed to produce and secrete large quantities of one or more 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 CoryneJbacterium glutamicum or related types of bacteria.
  • C. glutamicum is a gram-positive, aerobic bacterium (eg. In the overflow of crude oil) in the industry for the large scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons and for the oxidation of terpenoids overall ⁇ meinhin used becomes.
  • 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, such as Corynebacterium diphtheriae (the causative agent of diphtheria), which are important pathogens in humans. The ability to identify the presence of Corynebacterium species can therefore also be of significant clinical importance, for example in diagnostic applications.
  • These nucleic acid molecules can also serve as reference points for mapping the C. glutamicum genome or organisms related to genomes.
  • These "new nucleic acid molecules encode proteins which are referred to here as membrane construction and membrane transport (MCT) proteins.
  • MCT proteins can, for example, perform a function which is involved in the metabolism (eg biosynthesis or degradation) of 5 compounds which are necessary for membrane biosynthesis, or support the membrane transport of one or more compounds into or out of the cell due to the availability of cloning vectors for use in Cor . y22ejbacfce.rium glutamicum, as disclosed, for example, in Sinskey et
  • the 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 production or efficiency of the production of a fine chemical can be attributed to a direct
  • MCT proteins which are involved in the export of the fine chemical molecules from the cell, can have a greater number or higher activity, so that larger
  • MCT proteins which are involved in the import of the nutrients necessary for the biosynthesis of one or more fine chemicals (e.g. phosphate, sulfate, nitrogen compounds, etc.),
  • fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins according to the invention which are involved in the biosynthesis of these compounds, or by impairing the activity of one or more MCT proteins which are involved in the degradation of these compounds, the yield, production and / or efficiency of the production of fatty acids and lipid molecules from C. glutamicum can be increased.
  • the mutagenesis of one or more MCT genes according to the invention can also produce MCT proteins with modified activities which indirectly impair the production of one or more desired fine chemicals from C. glutamicum. For example.
  • MCT proteins according to the invention which are involved in the export of waste products, have a greater number or higher activity, so that the normal metabolic waste of the cell (possibly in higher quantity due to the overproduction of the desired fine chemical) is efficiently exported before it contains the nucleotides and Can damage proteins in the cell (which would decrease cell viability) or interact with the fine chemical biosynthetic pathways (which would decrease the yield, production or efficiency of production of a desired fine chemical).
  • the relatively large intracellular amounts of the desired fine chemical can even be toxic to the cell. For example, increasing the number of transporters that can export these compounds from the cell can increase the viability of the cell in culture, which in turn causes that a larger number of cells in the culture produce the desired fine chemical.
  • the MCT proteins according to the invention can also be manipulated in such a way that the relative amounts of the different lipid and fatty acid molecules are produced. This can have a significant impact on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change the membrane fluidity. Changes in the membrane fluidity can influence the transport of the molecules across the membrane as well as the integrity of the cell, each of which has a considerable effect on the production of fine chemicals from C. glutamicum in large-scale fermenter cultures.
  • the invention provides new nucleic acid molecules which encode proteins which are referred to here as MCT proteins and are involved, for example, in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of molecules across these membranes .
  • MCT protein is involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of molecules across these membranes. Examples of these proteins include 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 MCT protein or biologically active sections thereof, and also nucleic acid fragments which can be used as primers or hybridization are suitable for the detection or amplification of MCT-coding nucleic acid (for example DNA or mRNA).
  • the isolated nucleic acid molecule comprises one of the nucleotide sequences listed in Appendix A or the coding region or a complement thereof from one of these nucleotide sequences.
  • the isolated nucleic acid molecule encodes one of the amino acid sequences listed in Appendix B.
  • the preferred MCT proteins according to the invention likewise preferably have at least one of the MCT activities described here.
  • nucleic acid sequences of the sequence listing together with the sequence changes at the respective position described in Table 1 are defined as Appendix A.
  • polypeptide sequences of the sequence listing are defined as Appendix B together with the sequence changes at the respective position described in Table 1.
  • the isolated nucleic acid molecule is at least 15 nucleotides long and hybridizes under stringent conditions to a nucleic acid molecule which comprises a nucleotide sequence from Appendix A.
  • the isolated nucleic acid molecule preferably corresponds to a naturally occurring nucleic acid molecule.
  • the isolated nucleic acid more preferably encodes a naturally occurring C. glufcarnicum MCT 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.
  • a host cell is used to produce an MCT protein, which is grown in a suitable medium.
  • the MCT 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 MCT 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 encodes the mutated MCT sequence as a transgene.
  • an endogenous MCT gene in the genome of the microorganism has been changed, for example functionally disrupted, by homologous recombination with an altered MCT gene.
  • the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum in particular is 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.
  • an isolated MCT protein or a section for example a biologically active section thereof.
  • the isolated MCT protein or its section can be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across the membranes.
  • the isolated MCT protein or a section thereof is sufficiently homologous to an amino acid sequence from Appendix B so that the protein or its section continues to participate in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or can be involved in the transport of molecules across the membranes.
  • the invention also relates to an isolated MCT protein preparation.
  • the MCT 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 MCT polypeptide or a biologically active portion thereof can be operably linked to a non-MCT polypeptide to form a fusion protein.
  • this fusion protein has a different activity than the MCT protein alone and, in other preferred embodiments, results in increased yields, increased production and / or Efficiency of producing a desired fine chemical from C. glutamicum.
  • the integration of this fusion protein into a host cell modulates the production of a desired compound from the cell in particularly preferred embodiments.
  • Another aspect of the invention relates to a method for producing a fine chemical.
  • the method provides for the cultivation of a cell which contains a vector which brings about the expression of an MCT 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 MCT 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 BreviJbacfcerium.
  • 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 MCT protein activity or MCT nucleic acid expression so that a cell-associated activity changes compared to the same activity in the absence of the substance.
  • the cell is modulated with respect to one or more C. glutamicum metabolic pathways for cell membrane components or with regard to the transport of compounds across the membranes, so that the yields or the production rate of a desired fine chemical are improved by this microorganism.
  • the substance that modulates the MCT protein activity stimulates, for example, the MCT protein activity or the MCT nucleic acid expression.
  • substances that stimulate MCT protein activity or MCT nucleic acid expression include small molecules, active MCT proteins, and nucleic acids that encode MCT proteins and have been introduced into the cell.
  • substances that inhibit MCT activity or expression include small molecules and antisense MCT 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 a MCT 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 take place randomly or by homologous recombination, so that the native gene can be recognized by the integrated copy. is set, 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 MCT nucleic acid and protein molecules which can be involved in the metabolism of compounds necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across the membranes.
  • the molecules of the invention can be used in the modulation of the production of fine chemicals from microorganisms, such as C. glutamicum, either directly (e.g. where the overexpression or optimization of a fatty acid biosynthetic protein has a direct effect on the yield, production and / or efficiency of production of the Fatty acid of modified C.
  • glutamicum or by an indirect effect which nevertheless causes an increase in the yield, production and / or efficiency in the production of the desired compound (for example where the modulation of the metabolism of the cell membrane components changes in the yield, production and / or efficiency of production or composition of the cell membrane, which in turn affects the production of one or more fine chemicals).
  • the modulation of the metabolism of the cell membrane components changes in the yield, production and / or efficiency of production or composition of the cell membrane, which in turn affects the production of one or more fine chemicals.
  • 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 diamino-pimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described, for example, in Kuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., ed. VCH: Weinheim and the 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 such 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 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 Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97 VCH: Weinheim (1985)).
  • the amino acids can be in the D or L configuration, although L-amino acids are usually the only type 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)).
  • essential amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
  • amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
  • amino acids are identified by simple biosynthetic pathways converted into the remaining 11 "non-essential” amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine and tyrosine).
  • Higher animals have the ability to synthesize some of these amino acids, but the essential amino acids must be ingested in order for normal protein synthesis to take place.
  • Lysine is not an important amino acid only 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 Ullmann'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 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 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 formed from oxa acetate, an intermediate of the citrate cycle.
  • Asparagine, methionine, threonine and lysine are each converted 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 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
  • 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 necessary for normal enzyme activity to occur are. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferably organic.
  • nutraceutical encompasses food additives which are harmful to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (eg 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).
  • the family of compounds commonly referred to as "Vitamin B6" e.g. pyridoxine, pyridoxamine, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride
  • Vitamin B6 e.g. pyridoxine, pyridoxamine, pyridoxal 5 'phosphate and the commercially used pyridoxine hydrochloride
  • Panthothenate (antothenic acid, R- (+) -N- (2,4-dihydroxy-3,3,3-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 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.
  • 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 known as "nia ⁇ in”.
  • 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, do not contain a phosphoric acid unit exhibit.
  • RNA and DNA synthesis 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 carcinogenic cells, the ability of tumor cells to divide and replicate can be inhibited.
  • AMP energy stores
  • FAD and NAD coenzymes
  • the purine and pyrimidine bases, nucleosides and nucleotides also have other possible 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 commonly used as flavor enhancers (e.g. IMP or GMP) or for many medical applications (see e.g. Kuninaka, A., (1996) "Nucleotides and Related Compounds in Biotechnology Vol. 6, Rehm et al., ed VCH: Weinheim, pp. 561-612)
  • Enzymes which 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 being developed.
  • the purine nucleotides are removed via a series of steps via the intermediate compound inosine 5 'phosphate (IMP) from Ri- synthesized bose-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 prepared.
  • IMP inosine 5 'phosphate
  • GMP guanosine 5 'monophosphate
  • 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 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., Nishimoto et al., (1998) US Patent No. 5,759,610; Singer, MA and Lindguist, 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.
  • the cell membranes serve a number of functions in a cell. First of all, a membrane differentiates the cell content from the environment, so that the cell maintains integrity. The membranes also serve as barriers so that dangerous or undesired connections cannot flow in and desired connections cannot flow out.
  • cell membranes are inherently impermeable to the not facilitated diffusion of hydrophilic compounds such as proteins, water molecules and ions: a double layer of lipid molecules in which the polar head groups protrude outwards (out of the cell or into the cell interior) and the non-polar tails protrude to the middle of the double layer and form a hydrophobic core (for a general overview of the structure and function of the membrane see Gennis, RB (1989) Biomembranes, Molecular Structure and Function, Springer: Heidelberg).
  • This barrier allows that the cells contain a relatively higher concentration of desired compounds and a relatively lower concentration of undesired compounds than the surrounding medium, since the diffusion of these compounds through the membrane is efficiently blocked.
  • the membrane also provides an effective barrier against the import of desired molecules and the export of waste molecules.
  • the cell membranes contain many types of transporter proteins that can facilitate the transmembrane transport of various types of compounds: pores or channels and transporters.
  • the former are integral membrane proteins, sometimes protein complexes, that form a regulated opening through the membrane.
  • This regulation or "gating" is usually specific to those to be transported through the pore or channel, so that these transmembrane constructs are specific to a specific class of substrates; For example, a potassium channel is constructed in such a way that only ions with a charge and size similar to potassium can pass through.
  • Channel and pore proteins have certain hydrophobic and hydrophilic domains so that the hydrophobic portion of the protein can attach to the interior of the membrane, whereas the hydrophilic portion defines the interior of the channel, providing a protected hydrophilic environment through which the selected hydrophilic Molecule can arrive.
  • Many such pores / channels are known in the art, including those for potassium, calcium, sodium and chloride ions.
  • This pore and channel mediated system is limited to very small molecules, such as ions, since pores or channels that are large enough to allow complete proteins to pass through by facilitating diffusion would not be able to pass through smaller molecules prevent.
  • the transport of molecules through this process is sometimes referred to as "facilitated diffusion” because the driving force of a concentration gradient is required for the transport to take place.
  • Permeases also facilitate the easier diffusion of larger molecules, such as glucose or other sugars, into the cell if the concentration of these molecules is greater on one side of the membrane than on the other (also referred to as "uniport").
  • these integral proteins (which often have 6 to 14 membrane-spanning ⁇ -helices) do not form open channels through the membrane, but they do bind to the target molecule on the membrane surface and then undergo a conformational change, so that the target molecule is released on the opposite side of the membrane.
  • lipid molecules The synthesis of membranes is a well characterized process involving many components, the most important of which are the lipid molecules. Lipid synthesis can be divided into two parts: the synthesis of fatty acids and their binding to sn-glycerol-3-phosphate and the addition or modification of a polar head group. Usual lipids found in bacterial membranes used include phospholipids, glycolipids, sphingolipids and phosphoglycerides. Fatty acid synthesis begins with the conversion of acetyl-CoA into malonyl-CoA by acetyl-CoA-carboxylase or in acetyl-ACP by acetyl transacylase.
  • CFA cyclopropane fatty acids
  • the present invention is based, at least in part, on the discovery of new molecules, which are referred to here as MCT nucleic acid and protein molecules and control the production of cell membranes in C. glutamicum and effect the movement of molecules across these membranes.
  • the MCT molecules are involved in the metabolism of compounds which are involved in the construction of the cell membranes in C. glutamicum or in the transport of the molecules across these membranes.
  • the activity of the MCT molecules according to the invention for regulating the production of membrane components has an effect on the production of a desired fine chemical by this organism.
  • the activity of the modulated MCT molecules is modulated such that the C.
  • glutamicum Metabolic pathways that are regulated by the MCT proteins according to the invention, are modulated with regard to yield, production and / or production efficiency and are modified with regard to the efficiency of the transport of the compounds through the membranes, which increases the yield, production and / or efficiency of the Production of a desired fine chemical modulated either directly or indirectly by C. glutamicum.
  • MCT protein or “MCT polypeptide” encompasses proteins that are involved in the metabolism of compounds that are essential for the construction of
  • MCT proteins include those encoded by the MCT genes listed in Table 1 and Appendix A.
  • MCT gene or “MCT nucleic acid sequence” encompass nucleic acid sequences which encode an MCT protein which consists of a coding region and corresponding untranslated 5 'and 3' sequence regions. Examples of MCT 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 production quantity (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 known in the art and encompasses the efficiency of converting the carbon source to the product (ie, the fine chemical), for example, usually expressed as kg product per kg carbon source.
  • Increasing the yield or production of the compound will increase the amount of molecules or molecules recovered suitable obtained molecules of this compound in a certain Culture volume increased over a fixed period.
  • biosynthesis or “biosynthetic pathway” are known in the art and encompass the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds, for example in a multi-step or highly regulated process.
  • degradation or “degradation path” are known in the art and include the cleavage of a compound, preferably an organic compound, by a cell into degradation products (more generally, smaller or less complex molecules), e.g. in a multi-step or highly regulated Process.
  • metabolism is known in the art and encompasses all of the biochemical reactions that take place in an organism. The metabolism of a particular compound (e.g. the metabolism of an amino acid, such as Gly- a) then includes all biosynthesis, modification and degradation pathways of this compound in the cell.
  • the MCT molecules according to the invention are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism, such as C. glutamicum.
  • a desired molecule such as a fine chemical
  • C. glutamicum stai ⁇ x which contains such a modified protein.
  • MCT proteins that are involved in the import of the nutrients that are necessary for the biosynthesis of one or more fine chemicals can accordingly be present in greater numbers or with higher activity, so that these precursor, cofactor or intermediate compounds are present in the cell in higher concentrations.
  • fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or increasing the number of one or more MCT proteins according to the invention, which are involved in the biosynthesis of these compounds, or by influencing the activity of one or more MCT proteins, which are involved in the degradation of these compounds to increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from C. glutamicum.
  • the mutagenesis of one or more genes according to the invention can also produce MCT proteins with modified activities which influence the production of one or more fine chemicals from C. glutamicum.
  • MCT proteins according to the invention which are involved in the export of waste products, can be present in greater number or higher activity, so that the normal metabolic waste of the cell (possibly in higher quantity due to overproduction of the desired fine chemical) can be exported efficiently before being nucleotides and Damage proteins within the cell (which reduces cell viability) or interact with other fine chemical pathways (which reduces the yield, production or efficiency of production of the desired fine chemical).
  • the relatively large intracellular amounts of the desired fine chemical per se can be toxic to the cell.
  • the MCT proteins according to the invention can be manipulated in such a way that the relative amounts of different lipid and fatty acid molecules are produced. This can have a significant impact on the lipid composition of the cell membrane. Since each type of lipid has different physical properties, changing the lipid composition of a membrane can significantly change the membrane fluidity. Changes in membrane fluidity can affect the transport of molecules across the membrane as well as cell integrity, each of which has a significant impact on the production of fine chemicals from C. glutamicum in large-scale fermenter cultures.
  • 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 MCT protein according to the invention or a biologically active section or fragments thereof can be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum or in the transport of molecules across these membranes, or one or more of the molecules in Activities listed in Table 1.
  • nucleic acid molecule which encode MCT polypeptides or biologically active sections thereof, and to nucleic acid fragments which are sufficient for use as hybridization probes or primers for identifying or amplifying MCT-coding nucleic acids (for example MCT-DNA).
  • nucleic acid molecule is intended to encompass DNA molecules (eg cDNA or genomic DNA) and RNA molecules (eg mRNA) as well as DNA or RNA analogs that are generated by means of nucleotide analogs.
  • This term also includes the untranslated sequence located at the 3 'and 5' end of the coding gene region: at least about 100 nucleotides of the sequence upstream of the 5 'end of the coding region and at least about 20 nucleotides of the sequence downstream of the 3' end of the coding gene region.
  • the nucleic acid molecule can be single-stranded or double-stranded, but is preferably a double-stranded DNA.
  • An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid.
  • An "isolated" nucleic acid preferably has no sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid originates (for example, sequences that are located on the 5 'or
  • the isolated MCT 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 DNA of the Flank 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 any other cellular material or culture medium if it is produced by recombinant techniques, or of chemical precursors or other chemicals if it is chemically synthesized.
  • a nucleic acid molecule according to the invention for example a nucleic acid molecule with a nucleotide sequence from Appendix A or a section thereof, can be produced by means of standard molecular biological techniques and the sequence information provided here.
  • a C. glutamicur ⁇ -MCT cDNA can be isolated from a C. glufcamicum bank by using a complete sequence from Appendix A or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch , EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd to 1st 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 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 converted using reverse transcriptase (for example Moloney-MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase for example Moloney-MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD, or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for 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 an MCT 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 can hybridize to one of the sequences given in Appendix A, creating a stable duplex.
  • the nucleic acid molecule according to the invention encodes a protein or a portion thereof which comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B that the protein or a portion thereof further participates in the metabolism of compounds which are essential for the construction of the cell membranes in C. glutamicum are necessary, or may be involved in the transport of the molecules across these membranes.
  • the term "sufficiently homologous" 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 ami - Show nos acid sequence from Appendix B, so that the protein or a portion thereof can also be involved in the metabolism of compounds which are necessary for the construction of the cell membranes in C. glutamicum, or in the transport of the molecules across these membranes. Protein components of these pathways for membrane components or membrane transport systems, as described here, can play a role in the production and secretion of one or more fine chemicals. Examples of these Activities are also described here.
  • the "function of an MCT protein” relates either directly or indirectly to the yield, production and / or efficiency of the production of one or more fine chemicals. Table 1 shows examples of MCT protein activities.
  • Sections of proteins which are encoded by the MCT nucleic acid according to the invention are preferably biologically active sections of one of the MCT proteins.
  • the term “biologically active section of an MCT protein”, as used here, is intended to include a section, for example a domain or a motif, of an MCT protein which is involved in the metabolism of compounds which are essential for the structure of the cell membranes in C. glutamicum are necessary, or may be involved in the transport of molecules across these membranes, or has an activity given in Table 1.
  • a test of the enzymatic activity can be carried out be performed. These test methods, as described in detail in Example 8 of the example part, are familiar to the person skilled in the art.
  • nucleotide sequence of Appendix A which leads to a change in the amino acid sequence of the encoded MCT protein without affecting the functionality of the MCT 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 modified in a wild-type sequence from one of the MCT proteins (Appendix B) without changing the activity of the MCT protein, whereas an "essential" amino acid residue is required for the MCT protein activity.
  • other amino acid residues for example non-conserved or only semi-preserved amino acid residues in the domain with MCT activity
  • An isolated nucleic acid molecule that encodes an MCT 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 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.
  • 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 MCT 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 MCT coding sequence, for example by saturation mutagenesis, and the resulting mutants can be examined for the MCT activity described here in order to identify mutants, that maintain MCT 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 that contain a nucleic acid that encode an MCT 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 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. 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 extent of expression of the desired protein, etc.
  • the expression vectors according to the invention can be introduced into the host cells, so that proteins or peptides are thereby obtained , including fusion proteins or peptides that are encoded by the nucleic acids as described herein (e.g., MCT proteins, mutant forms of MCT proteins, fusion proteins, etc.).
  • the recombinant expression vectors according to the invention can be designed for the expression of MCT proteins in prokaryotic or eukaryotic cells.
  • MCT 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.
  • 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.
  • 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 MCT 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 MCT protein that is not fused to GST can be obtained by cle
  • 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 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 is carried out by standard DNA synthesis techniques.
  • the MCT 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, suitable 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 MCT 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 MCT 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 (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, Adenovirus2, Cytomegalievirus and Simian Virus 40.
  • suitable expression systems for prokaryotic and eukaryotic cells see chapters 16 and 17 from Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • 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 (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors
  • pancreatic-specific promoters e.g. neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477
  • pancreatic-specific promoters e.g. milk serum promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166
  • mammary-specific promoters 10 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. 3:15 537-546).
  • the invention also provides a recombinant expression vector comprising a DNA molecule according to the invention which is cloned into the expression vector in the antisense direction.
  • the DNA molecule is operatively linked to a regulatory sequence such that expression (by transcription of the DNA-.Molecule) of an RNA molecule, which is antisense to the MCT mRNA, is possible.
  • Regulatory sequences can be selected that are functional to an antisense rich
  • cloned nucleic acid are bound and which control the continuous expression of the antisense RNA molecule in a variety of cell types, for example.
  • Viral promoters and / or enhancers or regulatory sequences can be selected which are constitutive, tissue-specific or cell-type-specific expression
  • 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 whose activity is determined by the cell type, the vector is introduced into the •. 5
  • a highly effective regulatory region whose activity is determined by the cell type, the vector is introduced into the •. 5
  • a further 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 MCT protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • suitable host cells are known to the person skilled in the art.
  • Microorganisms which are related to Corynebacterium glutamicum and which can be suitably used as host cells for the nucleic acid and protein molecules according to the invention are listed in Table 3.
  • vector DNA can be introduced into prokaryotic or eukaryotic cells.
  • transformation and “transfection”, “conjugation” and “transduction” as used herein are intended to encompass a variety of methods known in the art for introducing foreign nucleic acid (e.g. DNA) into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection 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 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 MCT protein, or can be introduced on a separate vector.
  • a vector which contains at least a section of an MCT gene into which a deletion, addition or substitution has been introduced in order to change the MCT gene, for example to functionally disrupt it.
  • This MCT gene is preferably a Corynebacterium glutamicum MCT gene, however a homologue from a related bacterium or even from a mammalian, yeast or insect source can be used.
  • the vector is designed in such a way that the endogenous MCT 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 MCT 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 MCT protein is thereby effected is changed.).
  • the modified portion of the MCT gene is flanked in the homologous recombination vector at its 5 'and 3' ends by additional nucleic acid of the MCT gene, which is a homologous recombination between the exogenous MCT gene carried by the vector and one endogenous MCT gene in a microorganism.
  • the additional flanking MCT nucleic acid is long enough for successful homologous recombination with the endogenous gene.
  • the vector usually contains several kilobase 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 MCT gene is homologously recombined with the endogenous MCT 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.
  • the inclusion of an MCT gene in a vector under the control of the lac operon enables e.g. expression of the MCT 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 for the production (ie expression) of an MCT protein.
  • the invention also provides methods of producing MCT proteins using the host cells of the invention.
  • the method comprises growing the invention host cell (into which a recombinant expression vector encoding an MCT protein has been introduced, or into whose genome a gene encoding a wild-type or altered MCT protein has been introduced) in a suitable medium until the MCT Protein has been produced.
  • the method comprises isolating the MCT 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 are of interest, identification and localization of C. glufcamicum sequences, evolution studies, determination of MCT protein areas which are necessary for the function, modulation of the activity of an MCT protein; Modulating the activity of an MCT path; and modulating the cellular production of a desired compound, such as a fine chemical.
  • the MCT 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. glutamicum genes.
  • the invention provides the nucleic acid sequences of a number of C. glutamicum genes.
  • Corynebacterium glutamicum itself is not pathogenic, but it is related to pathogenic species such as Corynebacterium diptheriae. The detection of such an organism is of significant clinical importance.
  • the nucleic acid and protein molecules according to the invention can 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. glufca icum proteins.
  • the C. glutamicum genome can be cleaved, for example, and the fragments incubated with the DNA-binding protein.
  • Those that bind the protein can additionally with the nucleic acid molecules according to the invention, pre- preferably probed with easily detectable markings; the binding of such a nucleic acid molecule to the genome fragment enables the fragment to be located on the genomic map of C.
  • nucleic acid molecules according to the invention can also be sufficiently homologous to the sequences of related species so that these nucleic acid molecules can serve as markers for the construction of a genomic map in related bacteria, such as Brevijbacfcerium lactofermentum.
  • the MCT nucleic acid molecules according to the invention are also suitable for evolution and protein structure studies.
  • the metabolic and transport processes in which the molecules according to the invention are involved are used in 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 give an indication of which protein can tolerate mutagenesis without losing its function.
  • MCT nucleic acid molecules can bring about the production of MCT proteins with functional differences from the wild-type MCT proteins. These proteins can be improved in their efficiency or activity, can be present in the cell in larger numbers than usual, or can be weakened in their efficiency or activity.
  • the efficient overproduction of one or more fine chemicals requires increased amounts of cofactors, precursor molecules and intermediates for the appropriate biosynthetic pathways.
  • transporter proteins involved in the import of nutrients such as carbon sources (ie sugars), nitrogen sources (ie amino acids, ammonium salts), phosphates and sulfur, the production of a fine chemical can be reduced improve any nutritional constraints in the biosynthesis process.
  • fatty acids and lipids are themselves desirable fine chemicals; by optimizing the activity or by increasing the number of one or more MCT proteins according to the invention which are involved in the biosynthesis of these compounds, or by influencing the activity of one or more MCT proteins which are involved in the degradation of these compounds, can increase the yield, production and / or efficiency of the production of fatty acid and lipid molecules from C. glutamicum.
  • the genetic manipulation of one or more MCT genes according to the invention can likewise produce MCT proteins with modified activities which indirectly influence the production of one or more desired fine chemicals from C. glutamicum.
  • the normal biochemical metabolic processes for example, produce a large number of waste products (e.g. hydrogen peroxide and other reactive oxygen species) that can actively interact with the same metabolic processes (e.g., peroxynitrite nitrates, as is known, tyrosine side chains, whereby some enzymes are inactivated with tyrosine in the active center (Groves, JT (1999) Curr. Opin. Chem. Biol. 3 (2); 226-235). Although these waste products are usually excreted, the C.
  • glutamicum strains used for large-scale fermentative production are used for the overproduction of one or However, several fine chemicals have been optimized and can thus produce more waste products than is usual for a C. glutamicum wild type.
  • MCT proteins according to the invention By optimizing the activity of one or more MCT proteins according to the invention, which are involved in the export of waste molecules, the cell's viability can be improved improve and be an efficient metabolic activity
  • the presence of high intracellular amounts of the desired fine chemical can be toxic to the cell, so increasing the cell's ability to secrete these compounds can improve cell viability.
  • the MCT proteins according to the invention can be manipulated so that the relative amounts of different lipid and fatty acid molecules are changed. This can have a significant impact on the lipid composition of the cell membrane.
  • membrane fluidity can significantly change membrane fluidity. Changes in membrane fluidity can affect the transport of molecules across the membrane, which, as explained above, can modify the export of waste products or the fine chemicals produced or the import of necessary nutrients. These membrane fluidity changes can also significantly affect cell integrity; Cells with relatively weaker membranes are more susceptible to mechanical stress in a large fermenter environment, which can damage or kill the cells.
  • MCT proteins which are involved in the production of fatty acids and lipids for membrane construction, so that the membrane composition of the resulting membrane should be more sensitive to the environmental conditions in the cultures used to produce fine chemicals a larger proportion of C. glutamicum cells survive and multiply. Larger amounts of C. glutamicum cells in a culture should result in greater yields, production or efficiency in the production of the fine chemical from the culture.
  • the nucleic acid and protein molecules according to the invention can be used to generate C. glutamicum or related bacterial strains which express mutant MCT 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 pathways as well as molecules which do not occur naturally in the metabolism of C. glutamicum, but which are derived from a C. gluta- micum strain can be 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 Ml2 concentrate (10 g / 1 (NH 4 ) 2 S0, 1 g / 1 NaCl, 2 g / 1 MgS0 • 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 • H0, 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 mM Tris-HCl, 1 mM EDTA, pH 8).
  • the pellet was resuspended in 4 ml TE buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) were added.
  • proteinase K at a final concentration of 200 ⁇ g / l, the suspension was incubated at 37 ° C. for about 18 hours.
  • the DNA was purified by extraction with phenol, phenol-chloroform-isoamyl alcohol and chloroform-isoamyl alcohol using standard procedures. Then the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by incubation for 30 min at -20 ° C and 30 min centrifugation at 12000 rpm in a high-speed centrifuge with an SS34 rotor (Sorvall) , The DNA was dissolved in 1 ml of TE buffer containing 20 ⁇ g / ml RNase A and dialyzed against 1000 ml of TE buffer at 4 ° C. for at least 3 hours. During this time the buffer was exchanged 3 times.
  • Plasmids pBR322 (Sutcliffe, 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
  • Cosmids such as SuperCosl (Stratagene, LaJolla, USA) or Lorist ⁇ (Gibson, T.J. Rosenthal, A., and Waterson, R.H. (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 performed by passing a plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae) that maintain the integrity of their genetic information cannot maintain.
  • E. coli or other microorganisms e.g. Bacillus spp. Or yeasts such as Saccharomyces cerevisiae
  • Usual mutator strains show 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, pp. 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 Breviipa ⁇ fcerium species contain endogenous plasmids (such as pHMl519 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 Harbor Laboratory Press or Ausubel , FM et al.
  • origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevijbacfcertium 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) "From 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) "From Genes to Clones - Introduction to Gene Technology, VCH, Weinheim
  • glutamicum and which can be used for various purposes, including gene overexpression (see, e.g., 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).
  • the observations of the activity of a mutant protein in a transformed host cell are based on the fact that the mutant protein is expressed in a similar manner and in a similar amount as the wild-type protein.
  • a suitable method for determining the amount of transcription of the mutant gene is to carry out a Northern blot (see e.g.
  • RNA can be isolated from CoryneJbacterium glutamicum by various methods known in the art, as described in Bormann, E.R. 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 easy 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.
  • Nitrogen sources are usually organic or inorganic nitrogen compounds or materials containing these compounds. Exemplary nitrogen sources include ammonia gas or ammonium salts, such as NH 4 CI or (NH 4 ) 2 SO 4 , NH 4 OH, nitrates,
  • Urea amino acids or complex nitrogen sources such as corn steep liquor, 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. PM Rhodes, PF 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 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 with or without baffles.
  • 100 ml shake flasks are used, which are filled with 10% (by volume) of the required growth medium.
  • the flasks should be shaken on a rotary shaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Evaporation losses can be reduced by maintaining a humid atmosphere; alternatively, a mathematical correction should be carried out for the evaporation losses.
  • the medium is inoculated 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. 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 the techniques as described in Gennis, RB (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 for the increased production of the desired product (ie an amino acid) is examined.
  • 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 analysis methods include measurements of the amounts of nutrients in the medium (for example sugar, hydrocarbons, nitrogen sources, phosphate and other ions), measurements of the biomass composition and growth, analysis of the production of ordinary metabolites from biosynthetic pathways and measurements of gases generated during fermentation become. Standard methods for these measurements are in Applied Microbial Physiology; A Practical Approach, PM Rhodes and PF Stanbury, ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN: 0199635773) and the literature references specified therein.
  • 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. 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 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.
  • Those skilled in the art are skilled in the selection of 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 maximum.
  • the identity and purity of the isolated compounds can be determined by standard techniques in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.

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Abstract

L'invention concerne de nouvelles molécules d'acide nucléique, leur utilisation dans la construction de micro-organismes génétiquement améliorés et un procédé de fabrication de produits chimiques fins, en particulier d'acides aminés, à l'aide desdits micro-organismes génétiquement améliorés.
EP02781299A 2001-11-05 2002-10-31 Genes codant pour des proteines de synthese membranaire et de transport membranaire Ceased EP1456232A2 (fr)

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