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  • 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
• • 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
  • 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
  • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

• the invention relates to a new method for changing the genome of gram-positive bacteria, these bacteria and new vectors.
• the invention relates to a method for modifying Corynebacteria or Brevibacteria
• Corynebacterium glutamicum is a gram-positive, aerobic bacterium that (like other Corynebacteria, ie Corynebacterium and Brevibacterium species) is used in industry for the production of a number of fine chemicals and also for the degradation of hydrocarbons and the oxidation of terpenoids ( For an overview, see, for example, Liebl (1992) "The Genus Corynebacterium", in: The Procaryotes, Volume II, Balows, A. et al., Eds. Springer).
• DNA sequences can be introduced into the genome (newly introduced and / or existing sequences can be introduced in further copies), DNA sequence sections can also be removed from the genome (eg genes or parts of genes), but sequence exchanges can also be carried out (eg base exchanges) are carried out in the genome.
• sequence exchanges can also be carried out (eg base exchanges) are carried out in the genome.
• the change in the genome can be achieved by introducing DNA into the cell, which preferably does not replicate in the cell, and by recombining this introduced DNA with host genomic DNA and thus changing the genomic DNA. This procedure is described for example in van der Rest, ME et al. (1999) Appl. Microbiol. Biotechnol. 52, 541-545 and references therein.
• transformation marker used such as an antibiotic resistance gene
• This marker can then be used again in further transformation experiments.
• One way to do this is to use a conditionally negative dominant marker gene.
• a conditionally negative dominant marker gene is a gene that is disadvantageous (e.g. toxic) for the host under certain conditions, but has no negative effects on the host carrying the gene under other conditions.
• One example known from the literature is the URA3 gene from yeasts or fungi, an essential gene of pyrimidine biosynthesis, but which is disadvantageous for the host if the chemical 5-fluoro-0rotic acid is present in the medium (see for example DE19801120, Rothstein, R. (1991 ) Methods in Enzymology 194, 281-301).
• conditionally negatively dominant marker gene for removing DNA sequences (for example the transformation markers used and / or vector sequences and other sequence segments), also called “pop-out”, is described, for example, in Schwarzstein, R. (1991) Methods in Enzymology 194, 281-301.
• Galactose kinases (E.C.2.7.1.6, also called galactokinases) catalyze the phosphorilization of galactose to galactose phosphate.
• Numerous galactose kinases from different organisms are known, for example the galK gene from Escherichia coli (described in Debouck et al. (1985) Nucleic Acids Res. 13, 1841-1853), the galK gene from Bacillus subtilis (Glaser et al (1993) Mol. Microbiol. 10, 371-384) or the GALI gene from Saccharomyces cerevisiae (Citron & Donelson (1984) J. Bacteriol. 158, 269-278) each for a galactose kinase.
• genes for galactose kinases are good for use as conditionally dominant negative marker genes in gram-positive bacteria before Coryne bacteria are suitable.
• Genes for galactose kinases in Corynebacteria cause sensitivity to galactose in the nutrient medium (typically in a concentration range of 0.1 to 4% galactose in the medium).
• the invention relates to a plasmid vector which does not replicate in a target organism and contains the following components:
• Target organism is to be understood as the organism which is to be genetically modified by the methods and plasmid vectors according to the invention. These are preferably gram-positive bacteria, in particular bacterial strains from the genus Brevibacterium or Corynebacterium.
• the promoter d) is preferably heterologous to the galactose kinase gene used.
• Particularly suitable promoters are those from E. coli or C. gluta icum.
• the tac promoter is a particularly preferred promoter.
• the host organism in which the origin of replication a) is functionally active essentially serves to construct and multiply the plasmid vector according to the invention.
• All common microorganisms that can be genetically manipulated can be used as the host organism.
• Preferred host organisms are gram-negative bacteria such as Escherichia coli or yeasts, for example Saccharomyces cerevisiae.
• the host organism must be genetically different from the target organism, since replication of the plasmid vector should not take place in the target organism, while this is desired in the host organism through the use of the origin of replication a).
• Those sequences which are involved in increasing the production of fine chemicals are preferably exchanged in the target organism. Examples of such genes are given in WO 01/0842, 843 & 844, WO 01/0804 & 805, WO 01/2583.
• nucleic acid molecules for example complete genes
• disruptions for example deletions or integrative disruptions
• sequence changes for example single or multiple point mutations, complete gene exchangers.
• Preferred disruptions are those which lead to a reduction of by-products of the desired fermentation product
• preferred integrations are those which increase a desired metabolism to a fermentation product and / or reduce or eliminate "bottlenecks" (de-bottlenecking).
• Appropriate etabolic adjustments are preferred for sequence changes.
• the fermentation product is preferably a fine chemical.
• the transfer of DNA into the target organism can be carried out by methods customary to the person skilled in the art, preferably by conjugation or electroporation.
• DNA that is to be transferred to the target organism by conjugation contains special sequence sections (hereinafter referred to as mob sequences) that make this possible.
• mob sequences and their use for conjugation are described, for example, in Schaefer, A. et al. (1991) J. Bacteriol. 172, 1663-1666.
• a genetic marker is a selectable property that is mediated by a gene. These are preferably genes, the expression of which brings about resistance to antibiotics, in particular resistance to canycin, chloramphenicol, tetrahydroclinic or anti-picillin.
• a medium containing galactose is understood to mean in particular a medium with at least 0.1% and at most 10% (by weight) of galactose.
• Coryne bacteria in the sense of the invention are understood to mean all Coryne bacterium species, Brevibacterium species and Mycobacterium species. Corynebacterium species and Brevibacterium species are preferred.
• Corynebacterium species and Brevibacterium species are: Brevibacterium brevis, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium glutamicum, Corynebacterium diphtheriae, Corynebacterium lacto-fermentum.
• Mycobacterium species are: Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, Mycobacterium smeg atis.
• strains given in the following table are particularly preferred as target organisms:
• Another object of the invention is a method for producing a marker-free mutant target organism comprising the following steps:
• Clones by culturing in a galactose-containing medium for the presence of galactose sensitivity Clones by culturing in a galactose-containing medium for the presence of galactose sensitivity.
• the invention further relates to the mutagenized gram-positive bacteria (mutants) produced using this method, in particular the mutagenized Corynebacteria.
• mutants generated in this way can then be used for the production of fine chemicals or, for example in the case of C. diphtheriae, for the production e.g. of vaccines with weakened or non-pathogenic agents.
• Fine chemicals are understood to mean: organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors as well as enzymes.
• fine chemical is known in the art and includes molecules produced by an organism and used in various industries, such as, but not limited to, the pharmaceutical, agricultural, and cosmetic industries. These compounds include organic acids such as tartaric acid, itaconic acid and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides and nucleotides (as described for example in Kuninaka, A. (1996) Nucleotides and related compounds, S 561-612, in Biotechnology Vol. 6, Rehm et al., Ed.
• VCH Weinheim and the citations contained therein
• lipids saturated and unsaturated fatty acids (for example arachidonic acid), diols (for example propanediol and butanediol), carbohydrates (for example Hyaluronic acid and trehalose), aromatic compounds (e.g. aromatic amines, vanillin and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A27, "Vitamins", pp. 443-613 (1996) VCH: Weinheim and the contained therein quotes; 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, though
• 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)).
• the "essential" amino acids histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine
• amino acids so designated because they have to be included in the diet due to the complexity of their biosynthesis, 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 with food for normal protein synthesis to take place.
• Lysine is not only an important amino acid for human nutrition, but also for monogastric animals such as poultry and pigs.
• Glutamate is most commonly used as a flavor additive (monosodium glutamate, MSG) and widely used in the food industry, as well as aspartate, phenylalanine, glycine and cysteine.
• Glycine, L-methionine and tryptophan are all used in the pharmaceutical industry.
• Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are used in the pharmaceutical and cosmetic industries. Threonine, tryptophan and D- / L-methionine are widespread feed additives (Leuchtenberger, W. (1996) Amino acids - technical production and use, pp. 466-502 in Rehm et al., (Ed.) Biotechnology Vol. 6, chapter 14a, VCH: Weinheim).
• amino acids are also used as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S) -5-hydroxytryptophan and others, in Ulimann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97, VCH, Weinheim, 1985 are suitable substances.
• Phenylalanine and tyrosine are synthesized from the precursors of the glycolysis and pentose phosphate pathways, 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 be catalyzed in a phenylalanine hydroxylase also produce the reaction from phenylalanine. Alanine, valine and leucine are each biosynthetic products from pyruvate, the end product of glycolysis.
• Aspartate is made from oxaloacetate, an intermediate of the citrate cycle. Asparagine, methionine, threonine and lysine are each produced by converting aspartate. Isoleucine is made from threonine. In a complex 9-step process, histidine is formed from 5-phosphoribosyl-1-pyrophosphate, an activated sugar.
• Amino acids the amount of which exceeds the protein biosynthesis requirements of the cell, cannot be stored and are instead broken down, so that intermediate products are provided for the main metabolic pathways of the cell (for an overview see Stryer, L., Biochemistry, 3rd ed. Chap. 21 "Amino Acid Degradation and the Urea Cycle”; S 495-516 (1988)).
• the cell is able to convert unwanted amino acids into useful metabolic intermediates, the production of amino acids is expensive in terms of energy, precursor molecules and the enzymes required for their synthesis.
• amino acid biosynthesis is regulated by feedback inhibition, where the presence of a particular amino acid slows or stops its own production (for an overview of the feedback mechanism in amino acid biosynthetic pathways, see Stryer, L ., Biochemistry, 3rd ed., Chapter 24, "Biosynthesis of Amino Acids and Heme", pp. 575-600 (1988)).
• the output of a certain amino acid is therefore restricted by the amount of this amino acid in the cell.
• Vitamins, cofactors and nutraceuticals comprise another group of molecules. Higher animals have lost the ability to synthesize them and must therefore absorb them, although they are easily synthesized by other organisms such as bacteria. These molecules are either biologically active molecules per se or precursors of biologically active substances that serve as electron carriers or intermediates in a number of metabolic pathways. In addition to their nutritional value, these compounds also have a significant industrial value as dyes, antioxidants and catalysts or other processing aids. (For an overview of the structure,
• vitamin is known in the art and encompasses nutrients which are required by an organism for normal function, but which cannot be synthesized by this organism itself.
• the group of vitamins can include cofactors and nutraceutical compounds.
• cofactor includes non-proteinaceous compounds that are necessary for normal enzyme activity to occur. These compounds can be organic or inorganic; the inventive
• Cofactor molecules are preferably organic.
• the term “nutraceutical” encompasses food additives which are beneficial to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and also certain lipids (e.g. polyunsaturated fatty acids).
• Thiamine (vitamin Bi) is formed by chemical coupling of pyrimidine and thiazole units.
• Riboflavin (vitamin B) is synthesized from guanosine 5 'triphosphate (GTP) and ribose 5' phosphate. Riboflavin in turn is used to synthesize flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
• 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 (pantothenic acid, R- (+) -N- (2, 4-di-hydroxy-3, 3-dimethyl-l-oxobutyl) -? - alanine) can be produced either by chemical synthesis or by fermentation.
• the final steps in pantothenate biosynthesis consist of the ATP-driven condensation of ⁇ -alanine and pantoic acid. Those for the biosynthesis steps for the conversion into pantoic acid, into? -Alanine and for the condensation Enzymes responsible for pantothenic acid are known.
• the metabolically active form of pantothenate is coenzyme A, whose biosynthesis takes place over 5 enzymatic steps.
• Pantothenate pyridoxal-5 '-phosphate, cysteine and ATP are the precursors of coenzyme A. These enzymes not only catalyze the formation of pantothenate, but also the production of (R) -pantoic acid, (R) -pantolactone, (R) - Panthenol (provitamin B 5 ), Pantethein (and its derivatives) and coenzyme A.
• the biosynthesis of biotin from the precursor molecule pimeloyl-CoA in microorganisms has been extensively investigated and several of the genes involved have been identified. It has been found that many of the corresponding proteins are involved in the Fe cluster synthesis and belong to the class of the nifS proteins.
• the lipoic acid is from the
• Octanoic acid is derived and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the? -Ketoglutarate dehydrogenase complex.
• 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.
• GTP guanosine 5'-triphosphate
• Corrinoids such as the cobalamins and especially vitamin B ⁇ 2
• the porphyrins belong to a group of chemicals that are characterized by a tetrapyrrole ring system.
• the biosynthesis of vitamin B ⁇ 2 is sufficiently complex that it has not been fully characterized, but a large part of the enzymes and substrates involved is now known.
• Nicotinic acid (nicotinate) and nicotinamide are pyridine derivatives, which are also called “niacin”.
• Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
• purine and pyrimidine metabolism and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections.
• purine or pyrimidine encompasses nitrogenous bases which are part of the nucleic acids, coenzymes and nucleotides.
• nucleotide includes the basic structural units of the nucleic acid molecules, which are a nitrogenous base, a pentose sugar (RNA is the ribose sugar, DNA is the D-deoxyribose sugar) and
• nucleoside encompasses molecules which serve as precursors of nucleotides, but which, in contrast to the nucleotides, have no phosphoric acid unit.
• nucleotides that do not form nucleic acid molecules, but that serve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).
• S-adenosyl-methionine, folate or riboflavin as an energy source for the cell
• ATP for example ATP or GTP
• 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 that are involved in the purine, pyrimidine, nucleoside or nucleotide metabolism are also increasingly used as targets against chemicals for crop protection, including fungicides, herbicides and insecticides be developed.
• the purine nucleotides are synthesized via a series of steps via the intermediate compound inosine 5 'phosphate (IMP) from ribose 5 phosphate, which leads to the production of guanosine 5' monophosphate (GMP) or adenosine 5 'monophosphate (AMP) leads from which the triphosphate forms used as nucleotides can be easily produced.
• 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 to cytidine 5 'triphosphate (CTP).
• CTP cytidine 5 'triphosphate
• 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 pharmaceutical pharmaceutical industry, the cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) US Pat. No. 5,759,610; Singer, MA and Lindquist, S. Trends Biotech. 16 (1998) 460-467 ; Paiva, CLA and Panek, AD Biotech Ann. Rev. 2 (1996) 293-314; and Shiosaka, MJ Japan 172 (1997) 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium from which it can be obtained by methods known in the art.
• oligonucleotides can be used as primers, which can be defined on the basis of published sequences for galactose kinases (for example Genbank entry X02306).
• the preparation of the template for the PCR (the genomic DNA from E. coli) and the PCR can be carried out according to methods which are well known to the person skilled in the art and are described, for example, in Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) "Current Protocols in Molecular Biology", John Wiley & Sons.
• the gene for galactose kinase (galK gene), consisting of the sequence coding for the protein and 30 bp5 'of the coding sequence (ribosome binding site), can be provided with terminal interfaces for restriction endonucleases (for example EcoRI) in the course of the PCR and then this can be done PCR product can be cloned into suitable vectors (such as the plasmids pUCl ⁇ or pWST4B (Liebl et al. (1989) FEMS Microbiol. Lett. 65, 299-304)) which have the suitable interfaces for restriction endonucleases.
• suitable vectors such as the plasmids pUCl ⁇ or pWST4B (Liebl et al. (1989) FEMS Microbiol. Lett. 65, 299-304)
• Corynebacterium glutamicum R163 is described, for example, in Liebl et al. (1992) J. Bacteriol. 174, 1854-1861.
• the galK gene from E. coli was initially brought under the control of a heterologous promoter.
• the E. coli tac promoter was cloned by PCR methods.
• the tac promoter and the galK gene were then cloned into the plasmid pWST4B (Liebl et al. (1989) FEMS Microbiol. Lett. 65, 299-304), a shuttle vector which was found both in E. coli and in C. glutamicum is replicable and mediates resistance to chloramphenicol. After DNA transfer to C. glutamicum (see for example WO 01/02583) and selection of chloramphenicol-resistant colonies, these were examined for galactose sensitivity.
• Example 3 Inactivation of the Corynebacterium glutamicum ddh gene
• the two PCR products can be fused using known methods in such a way that the resulting product does not result in a functional ddh gene.
• This inactive form of the ddh gene and the galK gene from E. coli can be cloned into pSLl ⁇ (Kim, YH & H.-S. Lee (1996) J. Microbiol. Biotechnol. 6, 315-320) and thus the Get vector pSL18galK? Ddh.
• the procedure is familiar to the person skilled in the art.
• the transfer of this vector into Corynebacterium is known to the person skilled in the art and is possible, for example, by conjugation or electroporation.
• the integrants can be selected with kanamycin, the "pop-out" selection as described in Example 2.
• the inactivation of the ddh gene can be shown, for example, by a lack of Ddh activity. Ddh activity can be measured by known methods (see e.g. Misono et al. (1986) Agric. Biol. Che. 50, 1329-1330).

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