EP1537224A2 - Procedes pour la production, par fermentation, de produits chimiques fins (meta) contenant du soufre - Google Patents

Procedes pour la production, par fermentation, de produits chimiques fins (meta) contenant du soufre

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Publication number
EP1537224A2
EP1537224A2 EP03794944A EP03794944A EP1537224A2 EP 1537224 A2 EP1537224 A2 EP 1537224A2 EP 03794944 A EP03794944 A EP 03794944A EP 03794944 A EP03794944 A EP 03794944A EP 1537224 A2 EP1537224 A2 EP 1537224A2
Authority
EP
European Patent Office
Prior art keywords
coding
gene
meta
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.)
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Application number
EP03794944A
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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.)
Evonik Operations GmbH
Original Assignee
BASF SE
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Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1537224A2 publication Critical patent/EP1537224A2/fr
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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 especially L-methionine, using bacteria in which a nucleotide sequence coding for a homoserine-O-acetyl transferase (metA) gene is expressed.
  • metalA homoserine-O-acetyl transferase
  • 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.
  • Process improvements can include fermentation-related measures, such as stirring and supply of oxygen, or the composition of the nutrient media, such as, for example, the sugar concentration during fermentation, or working up to the product, for example by ion exchange chromatography, or the intrinsic performance properties of the microorganism itself affect.
  • 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 metA 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 homoserine O-acetyl transferase (metA) 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.
  • the above heterologous metA-coding nucleotide sequence for the metA-coding sequence from Corynebacterium glutamicum ATCC 13032 preferably has a sequence homology of less than 100% and preferably of more than 70%.
  • the metA coding sequence is preferably derived from one of the following organisms from List I: list
  • the metA 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 and 45 or a nucleotide sequence homologous thereto, which codes for a protein with metA activity.
  • the metA coding sequence used according to the invention also preferably codes for a protein with metA activity, the protein being an amino acid sequence according to 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 and 46 or an amino acid sequence homologous thereto, which stands for a protein with metA activity.
  • the coding metA sequence is preferably a DNA or an RNA which can be replicated in coryneform bacteria or which is 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 metA sequence under the control of regulatory sequences, or b) uses a strain in which the coding metA 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
  • 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 pyruvate carboxylase, f) the gene tpi coding for triosephosphate isomerase, g) the gene metH coding for methionine synthase, h) the gene metB coding for cystathionine gamma synthase, i) the metC gene coding for the cystathionin gamma-lyase, j) the glyA gene coding for the serine hydroxymethyltransferase, k) the metY gene coding for the O-acetylhom
  • coryneform bacteria are fermented, in which at least one of the genes selected from genes of the above-mentioned group a) to p) is mutated at the same time, so that the corresponding proteins or, to a lesser extent, compared to non-mutated proteins their activity is not influenced by metabolic metabolites and that in particular the production of the fine chemical according to the invention is not impaired.
  • coryneform 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) that for the ThrC gene encoding threonine synthase thr) t) the ddh gene coding for meso-diaminopimelate D-dehydrogenase u) the pck gene coding for phosphoenolpyruvate carboxykinase, v) the pgi gene coding for glucose-6-phosphate-6-isomerase, 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 dihydrodipicolinate reduct
  • coryneform 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 also relates to the coding metA sequences isolated for the first time from the above microorganisms, the homoserine-O-acetyl transferase coded therefrom and the functional homologues of these polynucleotides or proteins.
  • Proteins with the activity of homoserine-O-acetyl transferase are proteins that are able to convert homoserine and acetyl-co-enzyme A to O-acetyl-homoserine.
  • the person skilled in the art distinguishes the activity of the homoserine-O-acetyl-transferase from the homoserine-O-succinyl-transferase, which is also called metA in the literature. In the latter enzyme, succinyl-coenzyme A and not acetyl-coenzyme A serves as the substrate for the reaction.
  • 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 of this are methionine, homocysteine, S-adenosylmethionine, in particular methionine. and S-adenosyl methionine.
  • L-methionine In the context of the present invention, the terms “L-methionine”, “methionine”, homocysteine and S-adenosylmethionine also include the corresponding salts, such as, for. 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 own properties as chemical building blocks, can also have a modulating effect on enzymes and their catalytic activity. It is known from the literature that that such metabolic 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). It is also described in the literature that it is possible through 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 by metabolic metabolites has been changed (Sahm H.
  • 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 a strong promoter or use a gene which codes for a corresponding enzyme with a high activity and these measures can be combined 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.
  • “Functional 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, signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.
  • “Functional equivalents” encompassed according to the invention are homologs to the specifically disclosed proteins. These have at least 20%, or about 30%, 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 by Pearson and Lipman, Proc. Natl. Acad, Be. (USA) 85 (8), 1988, 2444-2448.
  • 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 homologs 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. Biochem. 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 library into 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.
  • Several techniques are known in the art for screening combinatorial library gene products produced by point mutations or truncation and for screening DNA banks for gene products with a selected property. These techniques can be adapted to the rapid screening of the gene banks which have been generated by combinatorial mutagenesis of homologs according to the invention.
  • 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 metA 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 or 45 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. Furthermore, the invention also encompasses nucleic acid sequences which hybridize with the above-mentioned coding sequences or are complementary thereto. These polynucleotides can be found when screening genomic or cDNA libraries and, if appropriate, can be amplified therefrom using suitable primers by means of PCR and then isolated, for example, using suitable probes.
  • 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 bind specifically to one another is demonstrated, for example, in the Northern or Southern blot technique or in primer binding in PCR or RT-PCR, usually using oligonucleotides from a length of 30 base pairs.
  • metA genes coding for the enzyme homoserine-O-acetyl transferase 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 Winnacker's textbook: Genes and Clones, An Introduction to Genetic Technology (Verlag Chemie, Weinheim, Germany, 1990), or the manual 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 of the National Academy of 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 metA 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 and 45 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 and 46 the resulting amino acid sequences of the metA 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 and 45 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.
  • the person skilled in the art can find instructions for identifying DNA sequences by means of hybridization in the manual "The DIG System Users Guide for Filter Hybridization” by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260).
  • PCR polymerase chain reaction
  • 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 and 46 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 metA gene according to the invention, or in which a metA 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 Corynebacterium glutamicum ATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes FERM BP-1539, Corynebacterium melassecola ATCC 17965
  • C. glutamicum Corynebacterium glutamicum ATCC 13032
  • Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium acetoacidophilum ATCC 13870
  • Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium melassecola ATCC 17965
  • Brevibacterium flavum ATCC 14067 Brevibacterium lactofermentum ATCC 13869 and Brevibacterium divaricatum ATCC 14020 to name; or strains derived therefrom, such as Corynebacterium glutamicum KFCC10065 Corynebacterium glutamicum ATCC21608
  • 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 metA 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 are not present in plasmids with different copy numbers or are integrated and amplified in the chromosome. Alternatively, overexpression of the genes in question can be achieved by changing the media composition and culture management.
  • the invention therefore also relates to expression constructs which contain, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide according to the invention; 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.
  • 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 Diego, CA (1990).
  • 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 regulatory signals are inserted in front of the structural gene and the natural before the 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 SP02 as described in Bacillus Subtilis and Its Closest Relatives, Sonenshein, Abraham L., Hoch, James A., Losick, Richard; ASM Press, Districtof 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 amplified 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 metA nucleotide sequence and a suitable termination signal.
  • a suitable promoter for this purpose, common recombination and cloning techniques are used, as described, for example, 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.
  • 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 those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. 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.
  • metA genes according to the invention were overexpressed, for example, using episomal plasmids.
  • 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, the anaplerotic, the phosphate metabolism, the citric acid cycle or the export of amino acids in addition to expression or amplification of a metA gene according to the invention, one or more enzymes of the respective biosynthetic pathway, the cysteine pathway, aspartate semialdehyde synthesis, glycolysis, the anaplerotic, the 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 metB gene coding for the cystahionin gamma synthase (EP 1 108790 A2; DNA SEQ NO. 3491), the metC gene coding for the cystahionin gamma lyase (EP 1 108790 A2; DNA SEQ NO. 3061) ) the glyA gene coding for the serine hydroxymethyltransferase (EP 1 108790 A2; DNA-SEQ NO. 1110),
  • 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 American Society for Bacteriology's “Manual of Methods for General 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. B. glycerol, methanol or ethanol and organic acids such as. B. acetic acid or lactic acid.
  • fatty acids such as As palmitic acid, stearic acid or linoleic acid
  • alcohols such as. B. glycerol, methanol or ethanol
  • 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 can be contained in the media include the chloride, 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 come from complex media components such as yeast extract, molasses, 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. Physiologe A Practical Approach" (ed. P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 9635773).
  • 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.
  • 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 containing L-methionine, usually have a dry matter of 7.5 to 25% by weight. It is also advantageous if 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 the concentration of usable sugar in the fermentation medium is kept to> 0 to 3 g / l or reduced during this time.
  • 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 prior art techniques. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin-layer chromatography, NIRS, enzyme tests or microbiological tests. These analysis methods are summarized in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.
  • FIG. 1 shows the plasmid map for plasmid pClysC
  • FIG. 2 shows the plasmid map for plasmid pCISIysCthr311ile
  • Figure 3 shows the plasmid map for plasmid pC_metA_Cd.
  • KanR Kanamycin resistance gene
  • ask aspartate kinase gene
  • ampicillin resistance and origin of replication of the vector pBR322 with the oligonucleotides p1.3 (SEQ ID NO: 47) and p2.3 (SEQ ID NO: 48) were amplified using the polymerase chain reaction (PCR).
  • the oligonucleotide p1.3 contains in 5'-3 'direction the interfaces for the restriction endonucleases Smal, BamHI, Nhel and Ascl and the oligonucleotide p2.3 (SEQ ID NO: 48) in 5'-3 'direction the interfaces for the restriction endonucleases Xbal, Xhol, Notl and Dral.
  • the PCR reaction was carried out according to the standard method 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 2.1 kb 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 set 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-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 named pCLiKI.
  • a kanamycin resistance cassette was amplified with the oligonucleotides neol (SEQ ID NO: 49) and neo2 (SEQ ID NO: 50).
  • neol (SEQ ID NO: 49): 5'-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGGA-3 *
  • neo2 (SEQ ID NO: 50): 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: 50) 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 dephosphorylated with alkaline phosphatase (Röche Diagnostics, Mannheim) according to the manufacturer.
  • the linearized vector (approx. 2.1 kb) was isolated using the GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharma, Freiburg) according to the manufacturer's instructions.
  • This vector fragment was prepared using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer ligated 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).
  • 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-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 named pCLiK3.
  • the replication origin pHM1519 was amplified with the oligonucleotides cg1 ((SEQ ID NO: 51) and cg2 (SEQ ID NO: 52).
  • the oligonucleotides contain cg1 (SEQ ID NO: 51) and cg2 (SEQ ID NO: 52) interfaces for the restriction endonuclease Notl.
  • the PCR reaction was carried out according to the standard method 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. After electrophoresis in a 0.8% agarose gel, 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 using the Rapid DNA Ligation Kit (Röche Diagnostics, Mannheim) according to the manufacturer's instructions with the cut PCR fragment and the ligation approach was performed 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 given the name pCLiK5.
  • HS445 (SEQ ID NO: 53) and HS446 (SEQ ID NO: 54)
  • SEQ ID NO: 53 the two synthetic, largely complementary oligonucleotides
  • HS446 SEQ ID NO: 54
  • 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.
  • the vector pCLiK5 was cut with the restriction endonuclease 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 pCLiK5MCS.
  • 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 pCLiK5MCS is listed as SEQ ID NO: 57.
  • Bacillus subtilis sacB gene (coding for Levan sucrase) was amplified with the oligonucleotides BK1732 and BK1733.
  • 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 with a size of approximately 1.9 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 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 pCLiK5MCS (produced according to Example 1) was also cut with the restriction endonuclease Not1 and dephosphorylated with alkaline phosphatase (I (Röche Diagnostics, Mannheim)) according to the manufacturer. After electrophoresis in a 0.8% agarose gel, an approximately 2.4 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 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 pCLiK5MCS integrative sacB.
  • 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 pCLiK5MCS integrative sacB is listed as SEQ ID NO: 58.
  • Further vectors which are suitable for the expression or overproduction of metA genes according to the invention can be prepared 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: 59 and SEQ ID NO: 60 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 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 referred to as pCIS; SEQ ID NO: 58 from Example 2) and transformed into E.coli XL-1 blue.
  • Selection for plasmid-carrying cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 195 ⁇ , 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: 61 comprises the following essential sub-areas:
  • CDS complement 2 (393 ⁇ .. ⁇ 3 ⁇ 6)
  • 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: 61.
  • the following oligonucleotide primers were synthesized for the exchange of thr311 after 311ile using the Quickchange method (Stratagene)
  • oligonucleotide primers in the Quickchange reaction leads to an exchange of the nucleotide in position 932 (from C to T) in the lysC gene (see SEQ ID NO: 64) and in the corresponding enzyme to an amino acid substitution in position 311 (Thr-> lle) (see SEQ ID NO: 6 ⁇ ).
  • the resulting amino acid exchange Thr311Ile in the lysC gene was confirmed by sequencing after transformation into E.coli XL1-blue and plasmid preparation.
  • the plasmid was named pCIS lysC thr311 ile and is listed as SEQ ID NO: 66.
  • the corresponding plasmid map is shown in Figure 2.
  • sequence SEQ ID NO: 66 comprises the following essential sub-areas:
  • CDS 1 156..1420
  • CDS complement 2 (393 ⁇ ..63 ⁇ 6)
  • the sacB gene contained in the vector pCIS lysC thr311ile converts sucrose into a toxic product, only those colonies can grow which 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 the 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 give an ethionine resistance (cheese, H. Nakayama K.Agr. Biol. Chem. 39 153-106 1975 L-methionine production by methionine analogously -resistant mutants of Corynebacterium glutamicum): An overnight culture in BHI medium (Difco) was washed in citrate buffer (50mM pH ⁇ , ⁇ ) and at 30 ° C for 20 min with N-methyl-nitrosoguanidine (10mg / ml in 50mM Citrate pH5, ⁇ ) treated.
  • the cells were washed nitrosoguanidine (citrate buffer ⁇ OmM pH 5.5) and plated on a medium which was composed of the following components, calculated on 500 ml: 10 g (NH 4 ) 2 SO 4 , 0.5 g KH 2 PO 4 , 0. ⁇ g K 2 HP0 4 , 0.125 g MgS0 4 .7H 2 0, 21 g MOPS, 50 mg CaCl 2 , 15 mg proteocatechuate, 0.5 mg biotin, 1 mg thiamine, 5 g / l D, L-ethionine (Sigma Chemicals Germany), pH 7.0 ,
  • the medium contained O. ⁇ ml of a trace salt solution of: 10g / l FeS0 4 * 7H 2 0, 1g / l MnS0 4 ⁇ 2 0, 0.1 g / l ZnS0 4 * 7H 2 0, 0.02g / l CuSO 4, 0.002g / l
  • Mutagenized cells were applied to plates with the medium described and incubated at 30 ° C. for 3-7 days. 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 311ile ET-16 strain.
  • Example 5 The strains prepared in Example 5 were grown on an agar plate with CM medium for 2 days at 30 ° C.
  • the cells were then scraped off the plate and resuspended in saline.
  • 10 ml of Medium II and 0.5 g of autoclaved CaC0 3 (Riedel de Haen) in a 100 ml Erlenmeyer flask were inoculated with the cell suspension up to an OD ⁇ OOnm of 1.5 and for 72 hours on an orbital shaker at 200 rpm at 30 ° C incubated.
  • 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: 67 and SEQ ID NO: 68 the chromosomal DNA from C. diphtheriae as 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. 1.4 kb which contains the metA gene including a 5 'non-coding region (promoter region). The amplified fragment is flanked at its ⁇ '-end by an Xhol restriction site and at the 3'-end by a Nel restriction site which was 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 Ndel (Röche Diagnostics, Mannheim) and separated by gel electrophoresis. The approximately 1.4 kb DNA fragment was then purified from the agarose using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
  • the vector pClik ⁇ MCS SEQ ID NO: 67 hereinafter called pC, was cut with the restriction enzymes Xhol and Ndel (Röche Diagnostics, Mannheim) and an approximately 5 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, 1955, 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 metA_Cd (Corynebacterium diphtheriae) is listed as SEQ ID NO: 69.
  • the corresponding plasmid map is shown in Figure 3.
  • Example 8 Transformation of the LU1479 lysC 311 ile ET-16 strain with the plasmid pC metA_Cd
  • the LU1479 lysC 311 ile ET-16 strain was transformed with the plasmid pC metA_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 Kan-resistant clones were picked and isolated. The methionine productivity of the clones was examined in a shake flask test (see Example 6).
  • the strain LU1479 lysC 311ile ET-16 pC metA_Cd produced significantly more methionine compared to LU1479 lysC 311ile ET-16.

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Abstract

L'invention concerne des procédés pour la production, par fermentation, de produits chimiques fins contenant du soufre, notamment de L-méthionine, au moyen de bactéries dans lesquelles est exprimée une séquence nucléotidique codant pour un gène de la méthionine synthase (methA).
EP03794944A 2002-08-26 2003-08-26 Procedes pour la production, par fermentation, de produits chimiques fins (meta) contenant du soufre Withdrawn EP1537224A2 (fr)

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DE10239073 2002-08-26
DE10239073A DE10239073A1 (de) 2002-08-26 2002-08-26 Verfahren zur fermentativen Herstellung schwefelhaltiger Feinchemikalien
PCT/EP2003/009452 WO2004024932A2 (fr) 2002-08-26 2003-08-26 Procedes pour la production, par fermentation, de produits chimiques fins (meta) contenant du soufre

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EP1537224A2 true EP1537224A2 (fr) 2005-06-08

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KR (1) KR20050034745A (fr)
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AU (1) AU2003264108A1 (fr)
BR (1) BR0313759A (fr)
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DE10239308A1 (de) 2002-08-27 2004-03-11 Basf Ag Verfahren zur fermentativen Herstellung von schwefelhaltigen Feinchemikalien
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
KR100905381B1 (ko) 2006-07-28 2009-06-30 씨제이제일제당 (주) Ｌ-메치오닌 전구체 생산 균주 및 상기 ｌ-메치오닌전구체로부터의 ｌ-메치오닌 및 유기산의 생산방법
KR20080061801A (ko) * 2006-12-28 2008-07-03 씨제이제일제당 (주) L-메티오닌 생산능을 향상시키는 폴리펩타이드, 상기 폴리펩타이드를 과발현하는 미생물 및 상기 미생물을 이용한 l- 메티오닌 생산방법
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US9005952B2 (en) * 2008-04-04 2015-04-14 Cj Cheiljedang Corporation Microorganism producing L-methionine precursor and the method of producing L-methionine precursor using the microorganism
UA112050C2 (uk) * 2008-08-04 2016-07-25 БАЄР ХЕЛСКЕР ЛЛСі Терапевтична композиція, що містить моноклональне антитіло проти інгібітора шляху тканинного фактора (tfpi)
WO2011073738A1 (fr) 2009-12-14 2011-06-23 Metabolic Explorer Utilisation de promoteurs inductibles dans la production de méthionine
SG10201903166PA (en) 2010-03-01 2019-05-30 Bayer Healthcare Llc Optimized monoclonal antibodies against tissue factor pathway inhibitor (tfpi)
DK2657250T3 (en) * 2010-12-21 2017-12-11 Cj Cheiljedang Corp Polypeptide variant having homoserine acetyl transferase activity and microorganism expressing this
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
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US7238502B2 (en) 2007-07-03
WO2004024932A3 (fr) 2004-04-22
US20070218526A1 (en) 2007-09-20
WO2004024932A2 (fr) 2004-03-25
CN1788090A (zh) 2006-06-14
JP2005537024A (ja) 2005-12-08
KR20050034745A (ko) 2005-04-14
DE10239073A1 (de) 2004-03-11
US20060003425A1 (en) 2006-01-05
BR0313759A (pt) 2005-06-21
AU2003264108A1 (en) 2004-04-30
CN100462440C (zh) 2009-02-18

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