EP1537223A2 - Procede de production par fermentation de produits chimiques fins contenant du soufre (metf) - Google Patents

Procede de production par fermentation de produits chimiques fins contenant du soufre (metf)

Info

Publication number
EP1537223A2
EP1537223A2 EP03794943A EP03794943A EP1537223A2 EP 1537223 A2 EP1537223 A2 EP 1537223A2 EP 03794943 A EP03794943 A EP 03794943A EP 03794943 A EP03794943 A EP 03794943A EP 1537223 A2 EP1537223 A2 EP 1537223A2
Authority
EP
European Patent Office
Prior art keywords
coding
gene
metf
sequence
dna
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.)
Withdrawn
Application number
EP03794943A
Other languages
German (de)
English (en)
Inventor
Burkhard Kröger
Oskar Zelder
Corinna Klopprogge
Hartwig Schröder
Stefan HÄFNER
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.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1537223A2 publication Critical patent/EP1537223A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P11/00—Preparation of sulfur-containing organic compounds
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • the invention relates to a process for the fermentative production of sulfur-containing fine chemicals, in particular L-methionine, using bacteria in which a nucleotide sequence coding for a methylene tetrahydrofolate reductase (metF) gene is expressed.
  • metalF methylene tetrahydrofolate reductase
  • Sulfur-containing fine chemicals such as methionine, homocysteine, S-adenosylmethionine, glutathione, cysteine, biotin, thiamine, lipoic acid are produced in cells via natural metabolic processes and are used in many branches of industry, including the food, feed, cosmetics and pharmaceutical industry.
  • These substances, collectively referred to as "sulfur-containing fine chemicals" include organic acids, both proteinogenic and non-proteinogenic amino acids, vitamins and cofactors. They are most conveniently produced on a large scale by growing bacteria that have been developed to produce and secrete large quantities of the desired substance. Organisms that are particularly suitable for this purpose are coryne-shaped bacteria, gram-positive non-pathogenic bacteria.
  • methionine analogues ⁇ -methyl-methionine, ethionine, norleucine, N-acetylnorleucine, S-trifluoromethyl homocysteine, 2-amino-5-heprenoic acid, selenomethionine, methionine sulfoximine, Methoxin, 1-aminocyclopentane carboxylic acid or auxotroph for regulatory metabolites and are sulfur-containing fine chemicals, such as. B. L-methionine.
  • the object of the invention was to provide a new process for the improved fermentative production of sulfur-containing fine chemicals, in particular L-methionine.
  • the above object is achieved by providing a method for the fermentative production of a sulfur-containing fine chemical, comprising the expression in a coryneform bacterium of a heterologous nucleotide sequence which codes for a protein with metF activity.
  • a first object of the invention is a process for the fermentative production of at least one sulfur-containing fine chemical, which comprises the following steps: a) fermentation of a coryneform bacterial culture producing the desired sulfur-containing fine chemical, wherein at least one heterologous nucleotide sequence is expressed in the coryneform bacteria which is for a protein encoded with methylene tetrahydrofolate reductase (metF) activity; b) accumulation of the sulfur-containing fine chemical in the medium or in the cells of the bacteria, and c) isolation of the sulfur-containing fine chemical, which preferably comprises L-methionine.
  • metalF methylene tetrahydrofolate reductase
  • the above heterologous metF-coding nucleotide sequence to the metF-coding sequence from Corynebacterium glutamicum ATCC 13032 preferably has a sequence homology of less than 100%, e.g. more than 70%, such as 75, 80, 85, 90 or 95%, or less than 70%, e.g. up to 60, 50, 40, 30, 20 or 10%.
  • the metF coding sequence is preferably derived from one of the following organisms from List I:
  • the metF-coding sequence used according to the invention preferably comprises a coding sequence according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 and 53 or a nucleotide sequence homologous thereto which encodes a protein with metF activity.
  • the metF-coding sequence used according to the invention also preferably codes for a protein with metF activity, the protein being an amino acid sequence according to SEQ ID NO: 1
  • the coding metF sequence is preferably a DNA or an RNA that is replicable in coryneform bacteria or stably integrated into the chromosome.
  • the method according to the invention is carried out by
  • a) uses a bacterial strain transformed with a plasmid vector which carries at least one copy of the coding metF sequence under the control of regulatory sequences, or b) uses a strain in which the coding metF sequence has been integrated into the chromosome of the bacterium.
  • coryneform bacteria are therefore fermented, in which at least one of the genes selected at the same time is selected
  • the lysC gene coding for an aspartate kinase b) the gene asd coding for an aspartate semialdehyde dehydrogenase c) the gene gap coding for glyceraldehyde-3-phosphate dehydrogenase, d) the gene pgk coding for 3-phosphoglycerate kinase , e) the pyc gene coding for the pyruvate carboxylase, f) the gene tpi coding for the triose phosphate isomerase, g) the gene metA coding for the homoserine O-acetyltransferase, h) the metB gene coding for the cystathionin gamma synthase, i) the metC gene coding for the cystathionin gamma lyase, j) the glyA gene coding for the serine hydroxymethyltransferase, k) the one for
  • coryneform bacteria are fermented in which at least one of the genes selected from genes of the above-mentioned groups a) to p) is mutated at the same time, so that the corresponding proteins are less compared to non-mutated proteins Measures or not influenced by metabolic metabolites in their activity and that in particular the production of the fine chemical is not impaired.
  • coryne-shaped bacteria are fermented, in which at least one of the genes selected from q) the gene coding for the homoserine kinase thrB, r) the gene coding for the threonine dehydratase ilvA, s) the at the same time for the threonine synthase coding gene thrC t) the gene coding for the meso-diaminopimelate D-dehydrogenase ddh u) the gene coding for the phosphoenolpyruvate carboxykinase pck, v) the gene coding for the glucose-6-phosphate-6-isomerase pgi , w) the poxB gene coding for the pyruvate oxidase, x) the gene dapA coding for the dihydrodipicolinate synthase, y) the gene dapB coding for the dihydrodip
  • coryne-shaped bacteria are fermented in which at least one of the genes of the above groups q) to z) is mutated at the same time, so that the enzymatic activity of the corresponding protein is partially or completely reduced.
  • Microorganisms of the type Corynebacterium glutamicum are preferably used in the process according to the invention.
  • the invention further relates to a method for producing an L-methionine-containing animal feed additive from fermentation broths, which comprises the following steps: a) cultivation and fermentation of an L-methionine-producing microorganism in a fermentation medium; b) removal of water from the fermentation broth containing L-methionine; c) removal of the biomass formed during the fermentation in an amount of 0 to 100% by weight; and d) drying the fermentation broth obtained according to b) and / or c) in order to obtain the animal feed additive in the desired powder or granule form.
  • the invention likewise relates to the coding metF sequences isolated for the first time from the above microorganisms, the metF enzymes coded therefrom and the functional homologues of these polynucleotides or proteins.
  • Proteins with the activity of methylene tetrahydrofolate reductase are those proteins which are capable of 5,10-methylenetetrahydrofolate (CH 2 -H (4) folate) with oxidation of the cofactor NADH or NADPH to 5-methyltetrahydrofolate (CH 3 -H (4) folate).
  • metF protein Further details of the metF protein are known to the person skilled in the art: (Matthews RG. Sheppard C. Goulding C. European Journal of Pediatrics. 157 Suppl 2: S54-9, 1998, Trimmer EE. Ballou DP. Matthews RG. Biochemistry. 40 (21) : 6205-15, 2001).
  • the person skilled in the art can demonstrate the enzymatic activity of metF by means of enzyme tests, rules for this can be: Matthews, R.G., Methylenetetrahydrofolate reductasefrom pig liver. Methods in Enzymology. 122: 372-81, 1986.
  • sulfur-containing fine chemical encompasses any chemical compound which contains at least one sulfur atom covalently bonded and is accessible by a fermentation process according to the invention.
  • Non-limiting examples thereof are methionine, homocysteine, S-adenosylmethionine, in particular methionine, and S -Adenosyl-methionine.
  • the terms “L-methionine”, “methionine”, homocysteine and S-adenosylmethionine also include the corresponding salts, such as, for example, B. methionine hydrochloride or methionine sulfate.
  • Polynucleotides generally refers to polyribonucleotides (RNA) and polydeoxyribonucleotides (DNA), which can be unmodified RNA or DNA or modified RNA or DNA.
  • polypeptides are understood to mean peptides or proteins which contain two or more amino acids linked via peptide bonds.
  • metabolic metabolite denotes chemical compounds which occur in the metabolism of organisms as intermediates or end products and which, in addition to their property as chemical building blocks, can also have a modulating effect on enzymes and their catalytic activity. It is known from the literature that such Metabolism metabolites can have an inhibitory as well as a stimulating effect on the activity of enzymes (Biochemistry, Stryer, Lubert, 1995 WH Freeman & Company, New York, New York.) The literature also describes that it can be done by measures such as Mutation of genomic DNA by UV radiation, ionizing radiation or mutagenic substances and subsequent selection for certain phenotypes in organisms to produce those enzymes in which the influence of metabolic metabolites has been changed (Sahm H.
  • Enzymes can be influenced in their activity in such a way that the reaction rate is reduced or the affinity for the substrate changes or the reaction rates change.
  • the terms “express” or “amplification” or “overexpression” describe the production or increase in the intracellular activity of one or more enzymes in a microorganism that are encoded by the corresponding DNA introduce an organism, replace an existing gene with another gene, increase the number of copies of the gene or genes, Use tor or use a gene that codes for a corresponding enzyme with a high activity and you can combine these measures if necessary.
  • “Functional equivalents” or analogs of the specifically disclosed polypeptides are, within the scope of the present invention, different polypeptides which furthermore have the desired biological activity, such as substrate specificity.
  • “functional equivalents” are understood to mean, in particular, mutants which, in at least one of the sequence positions mentioned above, have a different amino acid than the one specifically mentioned but nevertheless have one of the above-mentioned biological activities.
  • “Functional equivalents” thus include the mutants obtainable by one or more amino acid additions, substitutions, deletions and / or inversions, the changes mentioned being able to occur in any sequence position as long as they lead to a mutant with the property profile according to the invention.
  • Functional equivalence is particularly given when the reactivity patterns between mutant and unchanged polypeptide match qualitatively, i.e. For example, the same substrates can be implemented at different speeds.
  • “Functional equivalents” naturally also include polypeptides that are accessible from other organisms, as well as naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.
  • “Functional equivalents” also include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.
  • Fusion equivalents are also fusion proteins which contain one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without mutual substantial functional impairment of the fusion protein parts
  • heterologous sequences are, for example, sig- nalpeptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.
  • “Functional equivalents” encompassed according to the invention are homologs to the specifically hard proteins. These have at least 30%, or about 40%, 50%, preferably at least about 60%, 65%, 70%, or 75%, in particular at least 85 %, such as 90%, 95% or 99%, homology to one of the specifically disclosed sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sei. (USA) 85 (8), 1988, 2444- 2448th
  • homologs of the proteins or polypeptides of the invention can be generated by mutagenesis, e.g. by point mutation or shortening of the protein.
  • the term "homolog” as used here refers to a variant form of the protein which acts as an agonist or antagonist of protein activity.
  • Homologs of the protein of the invention can be obtained by screening combinatorial libraries of mutants, e.g. Shortening mutants can be identified.
  • a varied library of protein variants can be generated by combinatorial mutagenesis at the nucleic acid level, e.g. by enzymatically ligating a mixture of synthetic oligonucleotides.
  • methods that can be used to generate banks of potential homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automated DNA synthesizer, and the synthetic gene can then be ligated into an appropriate expression vector.
  • degenerate gene set makes it possible to provide all sequences in a mixture which encode the desired set of potential protein sequences.
  • Methods for the synthesis of degenerate oligonucleotides are known to the person skilled in the art (eg Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Brachem. 53: 323; Itakura et al., (1984) Science 198: 1056 ; Ike et al. (1983) Nucleic Acids Res. 11: 477).
  • a bank of coding sequence fragments can be obtained by treating a double-stranded PCR fragment of a coding sequence with a nuclease under conditions under which nicking occurs only about once per molecule, denaturing the double-stranded DNA, renaturing the DNA to form double-stranded DNA , which may include sense / antisense pairs of various nodded products, removing single stranded sections from newly formed duplexes by treatment with S1 nuclease and ligating the resulting Fragment bank can be generated in an expression vector.
  • An expression bank can be derived by this method, which encodes N-terminal, C-terminal and internal fragments with different sizes of the protein according to the invention.
  • REM Recursive ensemble mutagenesis
  • the invention also relates to nucleic acid sequences (single and double-stranded DNA and RNA sequences, such as cDNA and mRNA), coding for one of the above metF enzymes and their functional equivalents, which e.g. are also accessible using artificial nucleotide analogs.
  • the invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active sections thereof, and to nucleic acid fragments which e.g. can be used for use as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.
  • nucleic acid molecules according to the invention can also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region
  • nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and, moreover, can be essentially free of other cellular material or culture medium if used by recombinant technology. ken is manufactured, or be free of chemical precursors or other chemicals when it is chemically synthesized.
  • the invention further includes the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.
  • the nucleotide sequences according to the invention enable the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms.
  • probes or primers usually comprise a nucleotide sequence region which, under stringent conditions, can be attached to at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand are hybridized.
  • nucleic acid sequences according to the invention are derived from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 or 53 and differ from them by addition, substitution, insertion or deletion of one or more nucleotides, but continue to code for polypeptides with the desired property profile.
  • These can be polynucleotides that correspond to the above sequences in at least about 50%, 55%, 60%, 65%, 70%, 80% or 90%, preferably in at least about 95%, 96%, 97%, 98% or 99 % of the sequence positions are identical.
  • nucleic acid sequences which comprise so-called silent mutations or which have been changed in accordance with the codon usage of a specific source or host organism, in comparison to a specifically named sequence, as well as naturally occurring variants, such as e.g. Splice variants or allele variants, thereof. Sequences obtainable also by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility).
  • the invention also relates to the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population due to natural variation. These natural variations usually cause a variance of 1 to 5% in the nucleotide sequence of a gene.
  • the invention also encompasses nucleic acid sequences which hybridize with the above-mentioned coding sequences or are complementary thereto.
  • These polynucleotides read can be found by screening genomic or cDNA banks and, if necessary, using PCR to prime them with suitable primers and then isolate them, for example, with suitable probes.
  • Another possibility offers the transformation of suitable microorganisms with polynucleotides or vectors according to the invention, the multiplication of the microorganisms and thus the polynucleotides and their subsequent isolation.
  • polynucleotides according to the invention can also be synthesized chemically.
  • the property of being able to “hybridize” to polynucleotides means the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions non-specific bindings between non-complementary partners are avoided.
  • the sequences should be closed 70-100%, preferably 90-100%, of complementary nature
  • the property of complementary sequences to be able to specifically bind to one another is made for example in the Northern or Southern blot technique or in primer binding in PCR or RT -PCR. Usually, oligonucleotides with a length of 30 base pairs or longer are used.
  • metF genes coding for the enzyme methylene tetrahydrofolate reductase from the organisms in list I above can be isolated in a manner known per se.
  • E. coli Escherichia coli
  • the creation of gene banks is described in detail in well-known textbooks and manuals. Examples include the textbook by Winnacker: Genes and Clones, An Introduction to Genetic Technology (Verlag Chemie, Weinheim, Germany, 1990), or the handbook by Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989).
  • a very well-known gene bank is that of the E. coli K-12 strain W3110, which was developed by Kohara et al. (Cell ⁇ O, 495-508 (198)) in ⁇ vectors.
  • cosmids such as the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings ofthe National Academyof Sciences USA, 84: 2160-2164), but also plasmids such as pBR322 (BoliVal ; Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268) can be used.
  • E. coli strains that are defective in terms of restriction and recombination are particularly suitable as hosts. An example of this is the DH ⁇ mcr strain, which was described by Grant et al.
  • DNA sequences of organisms coding for the metF genes according to list I above were found.
  • DNA sequences according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51 and 53 found.
  • amino acid sequences of the corresponding proteins were derived from these DNA sequences using the methods described above.
  • SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 , 48, 50, 52 and 54 the resulting amino acid sequences of the metF gene products are shown.
  • Coding DNA sequences resulting from the sequences according to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 , 37, 39, 41, 43, 45, 47, 49, 51 and 53 resulting from the degeneration of the genetic code are also the subject of the invention.
  • DNA sequences which hybridize with these sequences or sequence parts derived therefrom are the subject of the invention.
  • Amino acid sequences which are correspondingly derived from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54 result are also part of the invention.
  • microorganisms serving as host cells in particular coryneform bacteria, which contain a vector, in particular a pendulum vector or plasmid vector, which carries at least one metF gene according to the invention, or in which a metF gene according to the invention is expressed or amplified.
  • microorganisms can produce sulfur-containing fine chemicals, in particular L-methionine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol.
  • These are preferably coryneform bacteria, in particular of the genus Corynebacterium. From the genus Corynebacterium, the species Corynebacterium glutamicum should be mentioned in particular, which is known in the art for its ability to produce L-amino acids.
  • suitable strains of coryneform bacteria are those of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum (C. glutamicum), such as
  • the abbreviation KFCC means the Korean Federation of Culture Collection, the abbreviation ATCC the American type strain culture collection, and the abbreviation FERM BP the collection of the National institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan ,
  • coryneform bacteria advantageously produce sulfur-containing fine chemicals, in particular L-methionine, after overexpression of a metF gene from List I organisms.
  • the person skilled in the art can take different measures individually or in combination to achieve overexpression.
  • the number of copies of the corresponding genes can be increased, or the promoter and regulatory region or the ribosome binding site located upstream of the structural gene can be mutated.
  • Expression cassettes which are installed upstream of the structural gene, act in the same way. Inducible promoters also make it possible to increase expression in the course of fermentative L-methionine production. Expression is also improved by measures to extend the life of the mRNA. Furthermore, preventing the breakdown of the enzyme protein also increases the enzyme activity.
  • the genes or gene constructs can either be present in plasmids with different copy numbers or be integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can also be achieved by changing the media composition and culture management.
  • the invention therefore also relates to expression constructs containing, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for an inventive polypeptide; and vectors comprising at least one of these expression constructs.
  • Such constructs according to the invention preferably comprise a promoter 5 'upstream of the respective coding sequence and a terminator sequence 3' downstream and optionally further customary regulatory elements, in each case operatively linked to the coding sequence.
  • An “operative linkage” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence. Examples of sequences which can be linked operatively are activation sequences and Enhancers, etc.
  • Other regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San
  • the natural regulatory sequence can still be present before the actual structural gene. This natural regulation can possibly be switched off by genetic modification and the expression of the genes increased or decreased.
  • the gene construct can also have a simpler structure, ie no additional regulation signals are inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased or decreased.
  • the nucleic acid sequences can be contained in one or more copies in the gene construct.
  • promoters examples include: the promoters, ddh, amy, lysC, dapA, lysA from Corynebacterium glutamicum, but also gram-positive promoters SPO2 such as those found in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press, District of Columbia, Washington and Patek M. Eikmanns BJ. Patek J. Sahm H. Microbiology.
  • inducible promoters such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P r P r promoter.
  • inducible promoters such as, for example, light-inducible and in particular temperature-inducible promoters, such as the P r P r promoter.
  • all natural promoters with their regulatory sequences can be used.
  • synthetic promoters can also be used advantageously.
  • the regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it.
  • the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers".
  • an increase in translation is also possible, for example, by improving the stability of the mRNA.
  • An expression cassette is produced by fusing a suitable promoter, a suitable Shine-Dalgarnow sequence with a metF nucleotide sequence and a suitable termination signal.
  • Common recombination and cloning techniques are used for this, such as those used in Current Protocols in Molecular Biology, 1993, John Wiley & Sons, Incorporated, New York New York, PCR Methods, Gelfand, David H., Innis, Michael A., Sninsky, John J. 1999, Academic Press, Incorporated, California, San Diego,., PCR Cloning Protocols, Methods in Molecular Biology Ser., Vol. 192, 2nd ed., Humana Press, New Jersey, Totowa. T. Maniatis, E.F.
  • the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables optimal expression of the genes in the host.
  • Vectors are well known to the person skilled in the art and can be found, for example, from “Cloning Vectors" (Pouwels PH et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985).
  • vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.
  • plasmids for example.
  • Suitable plasmids are those which are replicated in coryneform bacteria.
  • Numerous known plasmid vectors such as. B. pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKExl (Eikmanns et al., Gene 102: 93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • Other plasmid vectors such as. B.
  • pCLiK5MCS or those based on pCG4 (US-A 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)) or pAG1 (US-A 5,158,891) can in can be used in the same way.
  • plasmid vectors are also suitable with the aid of which the method of gene amplification by integration into the chromosome can be used, as described, for example, by Remscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for the duplication or amplification of the hom-thrB operon.
  • the complete gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum.
  • vectors examples include pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al. 1991, Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342) in question.
  • the plasmid vector containing the gene to be amplified is then transformed into the desired strain of C. glutamicum by transformation.
  • Enzymes can be influenced in their activity by mutations in the corresponding genes in such a way that there is a partial or complete reduction in the reaction rate of the enzymatic reaction. Examples of such mutations are known to the person skilled in the art (Motoyama H. Yano H. Terasaki Y. Anazawa H. Applied & Environmental Microbiology. 67: 3064-70, 2001, Eikmanns BJ. Eggeling L. Sahm H. Antonie van Leeuwenhoek. 64: 145 -63, 1993-94.)
  • sulfur-containing fine chemicals in particular L-methionine
  • one or more enzymes of the respective biosynthetic pathway, the cysteine pathway, aspartate semialdehyde synthesis, glycolysis, anaplerotic, pen to strengthen tose-phosphate metabolism, the citric acid cycle or the export of amino acids in addition to expression or amplification of a metF gene according to the invention, one or more enzymes of the respective biosynthetic pathway, the cysteine pathway, aspartate semialdehyde synthesis, glycolysis, anaplerotic, pen to strengthen tose-phosphate metabolism, the citric acid cycle or the export of amino acids.
  • one or more of the following genes can be amplified for the production of sulfur-containing fine chemicals, in particular L-methionine:
  • the microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch process (batch cultivation) or in the fed batch (feed process) or repeated fed batch process (repetitive feed process) for the production of sulfur-containing fine chemicals, in particular L-methionine.
  • batch cultivation batch cultivation
  • feed process fed batch
  • repetitive feed process repeated fed batch process
  • sulfur-containing fine chemicals in particular L-methionine.
  • the culture medium to be used has to meet the requirements of the respective strains in a suitable manner. Descriptions of culture media of various microorganisms are contained in the manual "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington DC, USA, 1981).
  • These media which can be used according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.
  • Preferred carbon sources are sugars, such as mono-, di- or polysaccharides.
  • sugars such as mono-, di- or polysaccharides.
  • very good carbon sources are 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 of sugar refining. It may also be advantageous to add mixtures of different carbon sources.
  • Other possible carbon sources are oils and fats such as e.g. B. Soybean oil. Sunflower oil.
  • Peanut oil and coconut fat fatty acids such as As palmitic acid, stearic acid or linoleic acid, alcohols such as. As glycerol, methanol or ethanol and organic acids such as. B. 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 ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract and others.
  • the nitrogen sources can be used individually or as a mixture.
  • Inorganic salt compounds that may be present in the media include the chlorine rid, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron
  • Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source for the production of sulfur-containing fine chemicals, in particular methionine.
  • Phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.
  • 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 fermentation media used according to the invention 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 originate from complex media components such as yeast extract, brass, corn steep liquor and the like. Suitable precursors can also be added to the culture medium.
  • the exact composition of the media connections strongly depends on the respective experiment and is decided individually for each specific case. Information on media optimization is available from the textbook "Applied Microbiol. Physiology, A Practical Approach” (ed. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 199635773).
  • 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 optionally added continuously or in batches.
  • the temperature of the culture is normally between 15 ° C and 45 ° C, preferably 25 ° C to 40 ° C and can be kept constant or changed during the experiment.
  • the pH of the medium should be in the range from 5 to 8.5, preferably around 7.0.
  • the pH for cultivation can be checked during the cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid.
  • Antschaummitte.l such.
  • B. fatty acid polyglycol esters can be used.
  • suitable selectively acting substances such as, for. B. antibiotics.
  • oxygen or oxygen-containing gas mixtures such as e.g. B. ambient air
  • the temperature of the culture is usually 20 ° C to 45 ° C.
  • the culture is continued until a maximum of the desired product has formed. This goal is usually achieved within 10 hours to 160 hours.
  • the fermentation broths obtained in this way in particular those containing L-methionine, usually have a dry matter of 7.5 to 25% by weight.
  • the fermentation is carried out with limited sugar at least at the end, but in particular for at least 30% of the fermentation period. This means that during this time the concentration of usable sugar in the fermentation medium is kept at> 0 to 3 g / l, or is lowered.
  • the fermentation broth is then processed further.
  • the biomass can be wholly or partially separated by methods such as. B. centrifugation, filtration, decanting or a combination of these methods from the fermentation broth or completely left in it.
  • the fermentation broth can then be prepared using known methods, such as, for. B. with the help of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, or by nanofiltration, thickened or concentrated.
  • This concentrated fermentation broth can then be worked up by freeze drying, spray drying, spray granulation or by other methods.
  • the product-containing broth is subjected to chromatography with a suitable resin after the biomass has been separated off, the desired product or the impurities being wholly or partly retained on the chromatography resin.
  • chromatography steps can be repeated if necessary using the same or different chromatography resins.
  • the person skilled in the art is skilled in the selection of the suitable chromatography resins and their most effective application.
  • the cleaned product can be concentrated by filtration or ultrafiltration and kept at a temperature at which the stability of the product is at a maximum.
  • the identity and purity of the isolated compound (s) can be determined by techniques of the prior art Technology can be determined. 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, p.
  • FIG. 1 shows the plasmid map for plasmid pClysC
  • FIG. 2 shows the plasmid map for plasmid pCISIysCthr311 ile
  • Figure 3 shows the plasmid map for plasmid pC_metF_Cd.
  • KanR stands for Kanamycin resistance gene
  • ask stands for aspartate kinase gene
  • ampicillin resistance and origin of replication of the vector pBR322 with the oligonucleotides p1.3 (SEQ ID NO: 55) and p2.3 (SEQ ID NO: 56) were amplified using the polymerase chain reaction (PCR).
  • the oligonucleotide p1.3 (SEQ ID NO: 55) in 5'-3 'direction contains the interfaces for the restriction endonucleases Smal, BamHI, Nhel and Ascl and the oligonucleotide p2.3 (SEQ ID NO: 56) in 5'-3 'direction the interfaces for the restriction endonucleases Xbal, Xhol, Notl and Dral.
  • the PCR reaction was carried out according to standard method such as Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
  • the DNA fragment with a size of approximately 2.1 kb obtained was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the blunt ends of the DNA fragment were ligated together using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation approach was carried out using standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing ampicillin (50 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is called pCLiKL
  • a kanamycin resistance cassette was amplified with the oligonucleotides neol (SEQ ID NO: 57) and neo2 (SEQ ID NO: 58).
  • neo2 (SEQ ID NO: 58): 5'-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3 '
  • the oligonucleotide neol in the 5'-3 'direction contains the interfaces for the restriction endonucleases Xbal, Smal, BamHI, Nhel and the oligonucleotide neo2 (SEQ ID NO: 58) in the 5'-3' direction for the restriction endonucleases Ascl and Nhel.
  • the PCR reaction was carried out according to standard methods such as Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
  • the DNA fragment with a size of approximately 1.3 kb was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the DNA fragment was cut with the restriction endonucleases Xbal and Ascl (New England Biolabs, Beverly, USA) and then purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the vector pCLiKI was also cut with the restriction endonucleases Xbal and Ascl and with alkaline phosphatase (Röche Diagnostics, Mannheim) according to the manufacturer dephosphorylated.
  • the linearized vector (approx. 2.1 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA).
  • plasmid-bearing cells were achieved by plating on LB agar (Lennox, 1955, Virology, 1: 190) containing ampicillin (50 ⁇ g / ml) and kanamycin (20 ⁇ g / ml).
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is named pCLiK2.
  • the vector pCLiK2 was cut with the restriction endonuclease Dral (New England Biolabs, Beverly, USA). After electrophoresis in a 0.8% agarose gel, an approximately 2.3 kb vector fragment was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. This vector fragment was religated using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation approach was carried out according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB Agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is named pCLiK3.
  • the replication origin pHM1519 was amplified with the oligonucleotides cg1 ((SEQ ID NO: 59) and cg2 (SEQ ID NO: 60).
  • cg1 (SEQ ID NO: 59): 5'-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3 '
  • cg2 (SEQ ID NO: 60): 5'-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3 '
  • the oligonucleotides cg1 (SEQ ID NO: 59) and cg2 (SEQ ID NO: 60) contain interfaces for the restriction endonuclease Notl.
  • the PCR reaction was carried out according to standard methods such as Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
  • the resulting DNA fragment with a size of approximately 2.7 kb was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the DNA fragment was cut with the restriction endonuclease Notl (New England Biolabs, Beverly, USA) and then purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the vector pCLiK3 was also cut with the restriction endonuclease Not1 and dephosphorylated with alkaline phosphatase (Röche Diagnostics, Mannheim)) according to the manufacturer.
  • the linearized vector (approx. 2.3 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation approach according to standard methods as in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is given the name pCLiK5.
  • HS445 (SEQ ID NO: 61) and HS446 (SEQ ID NO: 62)
  • the interfaces for the restriction endonucleases Swal, Xhol , Aatl, Apal, Asp718, Mlul, Ndel, Spei, EcoRV, Sall, Clal, BamHI, Xbal and Smal combined by heating to 95 ° C. and slowly cooling to a double-stranded DNA fragment.
  • HS445 (SEQ ID NO: 61): 5'-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAG TTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGATGGGGGTATA HS446 (SEQ ID NO: 62):
  • the vector pCLiK ⁇ was cut with the restriction endonucleases Xhol and BamHI (New England Biolabs, Beverly, USA) and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% agarose gel, the linearized vector (approx. 5.0 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • This vector fragment was ligated using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions with the synthetic double-stranded DNA fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190).
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is called pCLiK ⁇ MCS.
  • Sequencing reactions were carried out according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
  • the resulting plasmid pCLiK ⁇ MCS is listed as SEQ ID NO: 65.
  • Bacillus subtilis sacB gene (coding for Levan sucrase) was amplified with the oligonucleotides BK1732 and BK1733.
  • BK1732 (SEQ ID NO.63): ⁇ '-GAGAGCGGCCGCCGATCCTTTTTAACCCATCAC-3 '
  • BK1733 (SEQ ID NO: 64): 5'-AGGAGCGGCCGCCATCGGCATTTTCTTTTGCG-3 '
  • the oligonucleotides BK1732 and BK1733 contain interfaces for the restriction endonuclease Notl.
  • the PCR reaction was carried out according to standard methods such as Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo Polymerase (Stratagene, La Jolla, USA).
  • the DNA fragment obtained with a size of approximately 1.9 kb was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the DNA fragment was cut with the restriction endonuclease Notl (New England Biolabs, Beverly, USA) and then purified again with the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions.
  • the vector pCLiK ⁇ MCS (produced according to Example 1) was also cut with the restriction enduclease Notl and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% 'strength by weight agarose an approximately 2.4 kb vector fragment using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) was isolated according to the manufacturer. This vector fragment was ligated with the aid of the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer with the cut PCR fragment and the ligation approach according to standard methods as described in Sambrook et al. (Molecular Cloning.
  • the plasmid DNA of an individual clone was isolated using the Qiaprep Spin Miniprep Kit (Qiagen, Hilden) according to the manufacturer's instructions and checked by restriction digestion.
  • the plasmid obtained in this way is given the name pCLiK ⁇ MCS integrative sacB.
  • Sequencing reactions were carried out according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: ⁇ 463- ⁇ 467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
  • the resulting plasmid pCLiK ⁇ MCS integrative sacB is listed as SEQ ID NO: 66. Further vectors which are suitable for the expression or overproduction of metF genes according to the invention can be produced in an analogous manner.
  • Example 3 Isolation of the lysC gene from the C. glutamicum strain LU1479
  • an allelic exchange of the lysC wild-type gene coding for the enzyme aspartate kinase in C. glutamicum ATCC13032, hereinafter referred to as LU1479 is to be carried out.
  • a nucleotide exchange is to be carried out in the LysC gene so that the amino acid Thr at position 311 in the resulting protein is replaced by the amino acid III.
  • the oligonucleotide primers SEQ ID NO: 67 and SEQ ID NO: 68 were used to amplify lysC using the Pfu-Turbo PCR system (Stratagene USA) according to the manufacturer.
  • Chromosomal DNA from C. glutamicum ATCC 13032 was developed according to Tauch et al. (199 ⁇ ) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828.
  • the amplified fragment is flanked at its 5 'end by a Sall restriction cut and at its 3' end by a Mlul restriction cut. Before cloning, the amplified fragment was digested by these two restriction enzymes and purified using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
  • the polynucleotide obtained was cloned integrally via the Sall and Mlul restriction sections into pCLIK5 MCS SacB (hereinafter called pCIS; SEQ ID NO: 66 from Example 2) and transformed into E.coli XL-1 blue.
  • Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 19 ⁇ , Virology, 1: 190).
  • the plasmid was isolated and the expected nucleotide sequence was confirmed by sequencing.
  • the plasmid DNA was prepared using methods and materials from Quiagen. Sequencing reactions were carried out according to Sanger et al.
  • sequence SEQ ID NO: 69 comprises the following essential sub-areas: LOCUS pCIS ysC 6860 bp DNA circular FEATURES Location / Qualifiers CDS 1) 1 ⁇ 6..1420
  • the directed mutagenesis of the lysC gene from C. glutamicum was carried out using the QuickChange Kit (from Stratagene / USA) according to the manufacturer's instructions.
  • the mutagenesis was carried out in the plasmid pCIS lysC, SEQ ID NO: 69.
  • the following oligonucleotide primers were synthesized for the exchange of thr311 for 311ile using the QuickChange method (Stratagene)
  • sequence SEQ ID NO: 74 comprises the following essential sub-areas:
  • the plasmid pCIS lysC thr311 He was in C. glutamicum LU1479 by means of electroporation as in Liebl, et al. (1989) FEMS Microbiology Letters 63: 299-303. Modifications to the protocol are described in DE-A-10046870.
  • the chromosomal arrangement of the lysC locus of individual transformants was determined using standard methods by Southemblot and hybridization, as described in Sambrook et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. This ensured that the transformants are those which have integrated the transformed plasmid by homologous recombination at the lysC locus. After overnight growth of such colonies in media containing no antibiotic, the cells were placed on a sucrose CM agar medium (10% Sucrose) and incubated at 30 ° C for 24 hours.
  • the sacB gene contained in the vector pCIS lysC thr311 ile converts sucrose into a toxic product, only those colonies can grow that have deleted the sacB gene by a second homologous recombination step between the wild-type lysC gene and the mutated lysC thr311 ile gene. During homologous recombination, either the wild-type gene or the mutated gene can be deleted together with the sacB gene. If the sacB gene is removed together with the wild-type gene, a mutant transformant results.
  • the obtained strain LU1479 lysC 311 ile (Example 4) was treated to become ethionine-resistant (cheese, H. Nakayama K.Agr. Biol. Chem. 395 ⁇ 3-106 1975 L-methionine production by methionine analogue resistant mutants of Corynebacterium glutamicum): An overnight culture in BHI medium (Difco) was washed in citrate buffer (50mM pH 5.5) and at 30 ° C for 20 min with N-methyl-nitrosoguanidine (10mg / ml in 50mM citrate pH5.5) treated.
  • the cells were washed (citrate buffer 50 mM pH ⁇ , 5) and plated on a medium which was composed of the following components, calculated on 500 ml: 10 g (NH 4 ) 2 SO 4 , 0. ⁇ g KH 2 PO 4 , 0. ⁇ g K 2 HPO 4 , 0.12 ⁇ g MgSO 4 .7H 2 O, 21g MOPS, 60mg CaCI 2 , 1 ⁇ mg Proteocatechuate, O. ⁇ mg Biotin, 1 mg Thiamine, ⁇ g / l D, L-ethionine (Sigma Chemicals Germany), pH 7.0.
  • the medium also contained O. ⁇ ml of a trace salt solution consisting of: 10g / l FeSO .7H 2 O, 1g / l MnSO 4 * H 2 0, 0.1 g / l ZnSO 4 * 7H 2 0, 0.02g / l CuSO 4, 0.002g / l NiCI 2 * 6H 2 O, all salts were dissolved in 0.1 M HCl.
  • the completely assembled medium was sterile filtered and, after adding 40 ml of sterile 50% glucose solution, mixed with liquid sterile agar in a final concentration of 1. ⁇ % agar and poured out into culture dishes.
  • Mutagenized cells were applied to plates with the medium described and 3-7 Incubated for days at 30 ° C. Clones obtained were isolated, isolated at least once on the selection medium and then examined for their methionine productivity in a shake flask in medium II (see Example 6
  • Example 6 Preparation of methionine with the LU1479 lysC 311 ile ET-16 strain.
  • Example ⁇ The strains produced in Example ⁇ were grown on an agar plate with CM medium for 2 days at 30 ° C.
  • CM agar 10.0 g / l D-glucose, 2, ⁇ g / l NaCl, 2.0 g / l urea, 10.0 g / l Bacto Peptone (Difco), ⁇ , 0 g / l yeast extract (Difco), ⁇ , 0 g / l beef extract (Difco), 22.0 g / l agar (Difco), autoclaved (20 min., 121 ° C)
  • the cells were then scraped off the plate and resuspended in saline.
  • 10 ml of Medium II and 0.1 g of autoclaved CaCO 3 (Riedel de Haen) were inoculated with the cell suspension in a 100 ml Erlenmeyer flask to an OD600 nm of 1.5 and for 72 hours on an orbital shaker at 200 rpm at 30 ° C incubated.
  • Methionine as well as other amino acids formed in the culture broth were analyzed using the Agilent amino acid determination method on an Agilent 1100 Series LC System HPLC.
  • a derivatization with ortho-phthalaldehyde before the column separation allowed the quantification of the amino acids formed.
  • the separation of the amino acid mixture took place on one Hypersil AA column (Agilent) instead.
  • Chromosomal DNA from Corynebacterium diphtheriae was obtained from the American Type Strain Culture Collection (ATCC, Atlanta-USA) with the order number 700971 D from the ATCC 700971 strain.
  • oligonucleotide primers SEQ ID NO: 7 ⁇ and SEQ ID NO: 76 the chromosomal DNA from C. diphtheriae as a template and Pfu Turbo Polymerase (from Stratagene), the polymerase chain reaction (PCR) was carried out using standard methods such as Innis et al , (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press amplified a DNA fragment of approx. 1.2 kb which contains the metF gene including a non-coding ⁇ 'region (promoter region). The amplified fragment is flanked at its ⁇ '-end by an Xhol restriction site and at the 3'-end by an Xbal restriction site which were introduced via the oligonucleotide primers.
  • the DNA fragment obtained was purified using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) according to the manufacturer's instructions. Subsequently, it was cleaved with the restriction enzymes Xhol and Xbal (Röche Diagnostics, Mannheim) and separated by gel electrophoresis. The approximately 1.2 kb DNA fragment was then purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
  • the vector pCIik ⁇ MCS SEQ ID NO: 6 ⁇ hereinafter referred to as pC, was cut with the restriction enzymes Xhol and Xbal (Röche Diagnostics, Mannheim) and an approximately ⁇ kb fragment after electrophoretic separation with GFX TM PCR, DNA and Gel Band Purification Kit isolated.
  • the vector fragment was ligated together with the PCR fragment using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions and the ligation approach was carried out according to standard methods as described in Sambrook et al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, described (1989)), transformed into competent E.coli XL-1Blue (Stratagene, La Jolla, USA). Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 19 ⁇ , Virology, 1: 190).
  • the plasmid DNA was prepared using methods and materials from Quiagen. Sequencing reactions were carried out according to Sanger et al. (1977) Proceedings of the National Academy of Sciences USA 74: 5463-5467. The sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
  • the resulting plasmid pC metF_Cd (Corynebacterium diphtheriae) is listed as SEQ ID NO: 77.
  • the corresponding plasmid map is shown in Figure 3.
  • the LU1479 lysC 311ile ET-16 strain was transformed with the plasmid pC metF_Cd according to the method described (Liebl, et al. (1989) FEMS Microbiology Letters 53: 299-303).
  • the transformation mixture was plated on CM plates which additionally contained 20 mg / l kanamycin in order to achieve a selection for plasmid-containing cells. Obtained can-resistant clones were picked and isolated. The methionine productivity of the clones was shown in a shake flask test (see Example 6).
  • the LU1479 lysC 311 ile ET-16 pC metF_Cd strain produced significantly more methionine compared to LU1479 lysC 311 ile ET-16.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Fodder In General (AREA)

Abstract

L'invention concerne un procédé de production par fermentation de produits chimiques fins contenant du soufre, en particulier de L-méthionine, à l'aide de bactéries dans lesquelles est exprimée une séquence nucléotidique codant pour un gène de la méthionine synthase (metF).
EP03794943A 2002-08-27 2003-08-26 Procede de production par fermentation de produits chimiques fins contenant du soufre (metf) Withdrawn EP1537223A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10239308 2002-08-27
DE10239308A DE10239308A1 (de) 2002-08-27 2002-08-27 Verfahren zur fermentativen Herstellung von schwefelhaltigen Feinchemikalien
PCT/EP2003/009451 WO2004024931A2 (fr) 2002-08-27 2003-08-26 Procede de production par fermentation de produits chimiques fins contenant du soufre (metf)

Publications (1)

Publication Number Publication Date
EP1537223A2 true EP1537223A2 (fr) 2005-06-08

Family

ID=31501993

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03794943A Withdrawn EP1537223A2 (fr) 2002-08-27 2003-08-26 Procede de production par fermentation de produits chimiques fins contenant du soufre (metf)

Country Status (9)

Country Link
US (1) US7282357B2 (fr)
EP (1) EP1537223A2 (fr)
JP (1) JP2005537023A (fr)
KR (1) KR20050034746A (fr)
CN (1) CN100588714C (fr)
AU (1) AU2003258667A1 (fr)
BR (1) BR0313760A (fr)
DE (1) DE10239308A1 (fr)
WO (1) WO2004024931A2 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10239073A1 (de) 2002-08-26 2004-03-11 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
DE10239082A1 (de) 2002-08-26 2004-03-04 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
GB0326192D0 (en) * 2003-11-10 2003-12-17 Univ Kent Canterbury Proteins involved in signal transduction
DE10359668A1 (de) 2003-12-18 2005-07-14 Basf Ag Verfahren zur Herstellung von Methionin
US8399214B2 (en) * 2005-07-18 2013-03-19 Evonik Degussa Gmbh Use of dimethyl disulfide for methionine production in microoraganisms
EP1907559A1 (fr) * 2005-07-18 2008-04-09 Basf Se Micro-organismes recombinés producteurs de méthionine
BRPI0620880B1 (pt) * 2006-01-04 2018-10-09 Evonik Degussa Gmbh método para a produção de metionina, pelo cultivo de um microorganismo, e, microorganismo
RU2010101001A (ru) * 2007-06-15 2011-07-20 Эвоник Дегусса ГмБх (DE) Микроорганизмы с дерегулированной системой витамина в12
WO2011073738A1 (fr) * 2009-12-14 2011-06-23 Metabolic Explorer Utilisation de promoteurs inductibles dans la production de méthionine
CN103289928B (zh) * 2013-05-31 2015-03-04 浙江工业大学 荧光假单胞菌及其在生物合成蛋氨酸中的应用
WO2017009009A1 (fr) * 2015-07-10 2017-01-19 Evonik Degussa Gmbh Production d'acide aminé
US11680279B2 (en) * 2017-11-29 2023-06-20 Ajinomoto Co., Inc. Method for producing objective substance

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5835197A (ja) * 1981-08-26 1983-03-01 Kyowa Hakko Kogyo Co Ltd プラスミドｐｃｇ２
US4601893A (en) * 1984-02-08 1986-07-22 Pfizer Inc. Laminate device for controlled and prolonged release of substances to an ambient environment and method of use
GB2165546B (en) * 1984-08-21 1989-05-17 Asahi Chemical Ind A plasmid containing a gene for tetracycline resistance and dna fragments derived therefrom
DE4027453A1 (de) 1990-08-30 1992-03-05 Degussa Neue plasmide aus corynebacterium glutamicum und davon abgeleitete plasmidvektoren
WO1993017112A1 (fr) 1992-02-20 1993-09-02 Genencor International, Inc. Biosynthese de methionine au moyen d'une source reduite de soufre
DE4440118C1 (de) * 1994-11-11 1995-11-09 Forschungszentrum Juelich Gmbh Die Genexpression in coryneformen Bakterien regulierende DNA
JPH10229891A (ja) 1997-02-20 1998-09-02 Mitsubishi Rayon Co Ltd マロン酸誘導体の製造法
JP4623825B2 (ja) 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド
EP1307477A2 (fr) * 2000-08-02 2003-05-07 Degussa AG Sequences nucleotidiques codant pour le gene metf
DE10046870A1 (de) * 2000-09-20 2002-03-28 Basf Ag Verfahren zur Veränderung des Genoms von Corynebakterien
DE10217058A1 (de) 2002-04-17 2003-11-27 Basf Ag Verfahren zur Herstellung von schwefelhaltigen Feinchemikalien
DE10222858A1 (de) 2002-05-23 2003-12-04 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
DE10239082A1 (de) 2002-08-26 2004-03-04 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
DE10239073A1 (de) 2002-08-26 2004-03-11 Basf Ag Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BLANCO J. ET AL: "The folate branch of the methionine biosynthesis pathway in Streptomyces lividans: disruption of the 5,10-methylenetetrahydrofolate reductase gene leads to methionine auxotrophy", JOURNAL OF BACTERIOLOGY, vol. 180, no. 6, March 1998 (1998-03-01), pages 1586 - 1591, XP002191540 *

Also Published As

Publication number Publication date
US20060068476A1 (en) 2006-03-30
AU2003258667A1 (en) 2004-04-30
CN100588714C (zh) 2010-02-10
KR20050034746A (ko) 2005-04-14
WO2004024931A2 (fr) 2004-03-25
US7282357B2 (en) 2007-10-16
WO2004024931A3 (fr) 2004-04-22
JP2005537023A (ja) 2005-12-08
CN1688706A (zh) 2005-10-26
BR0313760A (pt) 2005-07-12
DE10239308A1 (de) 2004-03-11

Similar Documents

Publication Publication Date Title
EP1769080B1 (fr) Micro-organismes pour la production de composes sulfures
EP2235194B1 (fr) Procédé de production fermentative de 1,5-diaminopentane
US20070218526A1 (en) Method for zymotic production of fine chemicals containing sulphur (metA)
CN101230351B (zh) Psod表达单元
EP1908842A2 (fr) Unités d'expression PEF-TU
EP1516059B1 (fr) Procede pour produire par fermentation des produits de chimie fine contenant du soufre
EP1497443A2 (fr) Procede de production de produits chimiques fins sulfureux aux moyen de bacteries coryneformes codant pour le gene meth
WO2004024931A2 (fr) Procede de production par fermentation de produits chimiques fins contenant du soufre (metf)
WO2004024933A2 (fr) Procedes pour la production, par fermentation, de produits chimiques fins (mety) contenant du soufre
DE10109690A1 (de) Neue für das metY-Gen kodierende Nukleotidsequenzen
DE10136986A1 (de) Für Gene cysD, cysN, cysK, cysE und cysH kodierende Nukleotidsequenzen
DE10109689A1 (de) Neue für das metE-Gen kodierende Nukleotidsequenzen
DE10109687A1 (de) Neue für das metH-Gen kodierende Nukleotidsequenzen
DE10109688A1 (de) Neue für die Gene metR und metZ kodierende Nukleotidsequenzen
DE10109686A1 (de) Neue für das metf-Gen kodierende Nukleotidsequenzen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20050329

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20061130

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: BASF SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20080903