DE102004009453A1 - Process for the preparation of L-amino acids using coryneform bacteria - Google Patents
Process for the preparation of L-amino acids using coryneform bacteria Download PDFInfo
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- DE102004009453A1 DE102004009453A1 DE200410009453 DE102004009453A DE102004009453A1 DE 102004009453 A1 DE102004009453 A1 DE 102004009453A1 DE 200410009453 DE200410009453 DE 200410009453 DE 102004009453 A DE102004009453 A DE 102004009453A DE 102004009453 A1 DE102004009453 A1 DE 102004009453A1
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- methionine
- asur
- amino acids
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Classifications
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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
Die Erfindung betrifft ein Verfahren zur fermentativen Herstellung von L-Aminosäuren, insbesondere L-Methionin, bei dem man die die gewünschte L-Aminosäure produzierenden coryneformen Bakterien fermentiert, bei denen man den Regulator AsuR abschwächt, insbesondere ausschaltet oder auf niedrigem Niveau exprimiert. Die Erfindung betrifft weiterhin diese rekombinanten Mikroorganismen.The invention relates to a process for the fermentative production of L-amino acids, in particular L-methionine, in which fermenting the desired L-amino acid producing coryneform bacteria, in which one attenuates the regulator AsuR, in particular switches off or expressed at a low level. The invention further relates to these recombinant microorganisms.
Description
Gegenstand der Erfindung ist ein Verfahren zur fermentativen Herstellung von L-Aminosäuren, insbesondere L-Methionin, unter Verwendung von coryneformen Bakterien, in denen das asuR-Gen abgeschwächt wird. Das asuR-Gen kodiert für den Regulator AsuR.object The invention relates to a process for the fermentative production of L-amino acids, especially L-methionine, using coryneform bacteria, in which the asuR gene is attenuated. The asuR gene codes for the regulator AsuR.
Chemische Verbindungen, mit denen insbesondere L-Aminosäuren, Vitamine, Nukleoside und Nukleotide und D-Aminosäuren gemeint sind, finden in der Humanmedizin, in der pharmazeutischen Industrie, in der Kosmetik, in der Lebensmittelindustrie und in der Tierernährung Anwendung.Dry Compounds with which in particular L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids are found in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal nutrition application.
Zahlreiche dieser Verbindungen werden durch Fermentation von Stämmen coryneformer Bakterien, insbesondere Corynebacterium glutamicum, hergestellt. Wegen der großen Bedeutung wird ständig an der Verbesserung der Herstellverfahren gearbeitet. Verfahrensverbesserungen können fermentationstechnische Maßnahmen wie zum Beispiel Rührung und Versorgung mit Sauerstoff, oder die Zusammensetzung der Nährmedien wie zum Beispiel die Zuckerkonzentration während der Fermentation, oder die Aufarbeitung zur Produktform durch zum Beispiel Ionenaustauschchromatographie oder die intrinsischen Leistungseigenschaften des Mikroorganismus selbst betreffen.numerous These compounds are coryneformer by fermentation of strains Bacteria, in particular Corynebacterium glutamicum, produced. Because of the big one Meaning becomes constant worked on the improvement of the manufacturing process. process improvements can fermentation measures such as emotion and oxygen supply, or the composition of the nutrient media such as the sugar concentration during fermentation, or the work-up to the product form by, for example, ion exchange chromatography or the intrinsic performance characteristics of the microorganism concern yourself.
Zur Verbesserung der Leistungseigenschaften dieser Mikroorganismen werden Methoden der Mutagenese, Selektion und Mutantenauswahl angewendet. Auf diese Weise erhält man Stämme, die resistent gegen Antimetabolite wie z.B. das Lysin-Analogon S-(2-Aminoethyl)-Cystein oder die Methionin-Analoga α-Methyl-Methionin, Ethionin, Norleucin, N-acetylnorleucin, S-Trifluoromethylhomocystein, 2-amino-5- heprenoitsäure, Seleno-Methionin, Methioninsulfoximin, Methoxin, 1-Aminocyclopentan-Carboxylsäure sind oder auxotroph für regulatorisch bedeutsame Metabolite sind und L-Aminosäuren produzieren.to Improve the performance characteristics of these microorganisms Methods of mutagenesis, selection and mutant selection applied. In this way receives man tribes, resistant to antimetabolites, e.g. the lysine analog S- (2-aminoethyl) cysteine or the methionine analogues α-methyl-methionine, Ethionine, norleucine, N-acetylnorleucine, S-trifluoromethyl homocysteine, 2-amino-5-heprenoic acid, seleno-methionine, methionine sulfoximine, Methoxine, 1-aminocyclopentane-carboxylic acid or are auxotrophic for regulatory are significant metabolites and produce L-amino acids.
Seit einigen Jahren werden ebenfalls Methoden der rekombinanten DNA-Technik zur Stammverbesserung L-Aminosäure produzierender Stämme von Corynebacterium glutamicum eingesetzt, indem man einzelne Aminosäure-Biosynthesegene amplifiziert und die Auswirkung auf die L-Aminosäure-Produktion untersucht.since Several years ago, methods of recombinant DNA technology were also used for strain improvement producing L-amino acid strains of Corynebacterium glutamicum by amplifying single amino acid biosynthesis genes and the effect on L-amino acid production examined.
Aufgabe der ErfindungObject of the invention
Die Erfinder haben sich die Aufgabe gestellt, neue Grundlagen für verbesserte Verfahren zur fermentativen Herstellung von L-Aminosäuren, insbesondere L-Methionin, mit coryneformen Bakterien bereitzustellen.The Inventors have set themselves the task of new foundations for improved Process for the fermentative production of L-amino acids, in particular L-methionine to provide with coryneform bacteria.
Beschreibung der Erfindungdescription the invention
Werden im folgenden L-Aminosäuren oder Aminosäuren erwähnt, sind damit eine oder mehrere der proteinen Aminosäuren einschließlich ihrer Salze, ausgewählt aus der Gruppe L-Asparaginsäure, L-Asparagin, L-Threonin, L-Serin, L-Glutaminsäure, L-Glutamin, L-Glycin, L-Alanin, L-Cystein, L-Valin, L-Methionin, L-Isoleucin, L-Leucin, L-Tyrosin, L-Phenylalanin, L-Histidin, L-Lysin, L-Tryptophan, L-Arginin und L-Prolin gemeint. Besonders bevorzugt ist L-Methionin.Become in the following L-amino acids or amino acids mentioned, are thus one or more of the protein amino acids including theirs Salts, selected from the group L-aspartic acid, L-asparagine, L-threonine, L-serine, L-glutamic acid, L-glutamine, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and L-proline. Especially preferred is L-methionine.
Unter proteinogenen Aminosäuren versteht man die Aminosäuren, die in natürlichen Proteinen, das heißt in Proteinen von Mikroorganismen, Pflanzen, Tieren und Menschen vorkommen. Sie dienen als Struktureinheiten für Proteine, in denen sie über Peptidbindungen miteinander verknüpft sind.Under proteinogenic amino acids one understands the amino acids, in natural Proteins, that is in proteins of microorganisms, plants, animals and humans occurrence. They serve as structural units for proteins in which they have peptide bonds linked together are.
Werden im folgenden L-Methionin oder Methionin erwähnt, sind damit auch die Salze wie z.B. Methionin-Hydrochlorid oder Methionin-Sulfat gemeint.Become in the following L-methionine or methionine are mentioned, so are the salts such as. Methionine hydrochloride or methionine sulfate meant.
Der Regulator AsuR ist ein Aktivator von Genen, die in die Aufnahme und Verwertung von Schwefel-haltigen Verbindungen, insbesondere von Sulfonaten, involviert sind. Er wird durch Sulfat reprimiert. Weiterhin reprimiert AsuR Gene der Cystein-Biosynthese. Die Cystein-Biosynthese ist von grosser Bedeutung für die Methionin-Biosynthese, da der für die Biosynthese von Methionin benötigte Schwefel aus Sulfit und Cystein aus der Cystein-Biosynthese stammt. Die Bezeichnung „asuR" leitet sich ab von „alternate sulfur source utilization regulator".The regulator AsuR is an activator of genes involved in the uptake and utilization of sulfur-containing compounds, in particular sulfonates. He is repressed by sulfate. Furthermore, AsuR represses genes of cysteine biosynthesis. Cysteine biosynthesis is of great importance for methionine biosynthesis, as the sulfur required for the biosynthesis of methionine comes from sulfite and cysteine from cysteine biosynthesis. The term "asuR" is derived from "alternate sulfur source utili zation regulator ".
Regulatorische Proteine, welche die Expression anderer Gene regulieren sind solche Proteine, die beispielsweise durch eine spezifische Proteinstruktur, genannt Helix-Turn-Helix-Motiv, an DNA binden können und so die Transkription anderer Gene entweder verstärken oder abschwächen können.regulatory Proteins that regulate the expression of other genes are those Proteins, for example, by a specific protein structure, called helix turn-helix motif, can bind to DNA and so that transcription of other genes can either increase or decrease.
Es wurde gefunden, dass die Funktion des Regulators AsuR aus Corynebacterium glutamicum die Expression von Genen, die in die Aufnahme und Verwertung von Schwefel-haltigen Verbindungen, insbesondere Sulfonaten, involviert sind, aktiviert und Gene der Cystein-Biosynthese reprimiert.It It was found that the function of the regulator AsuR from Corynebacterium glutamicum the expression of genes involved in the uptake and recovery sulfur-containing compounds, in particular sulfonates involved are activated and repressed genes of cysteine biosynthesis.
Das Abschwächen, insbesondere Ausschalten des für den Regulator AsuR kodierenden asuR-Gens verbessert die Produktion von L-Methionin in den entsprechenden coryneformen Bakterien im Vergleich zu den Ausgangsorganismen ohne Abschwächung oder Ausschaltung dieses Gens.The weaken, in particular turning off the for the regulator AsuR coding asuR gene improves the production of L-methionine in the corresponding coryneform bacteria in the Comparison to the parent organisms without weakening or elimination of this Gene.
Gegenstand der Erfindung ist ein Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung von coryneformen Bakterien, die insbesondere bereits L-Aminosäuren produzieren und in denen das für das regulatorische Protein AsuR kodierende asuR-Gen abgeschwächt, insbesondere ausgeschaltet ist oder auf niedrigem Niveau exprimiert wird.object The invention relates to a process for the fermentative production of L-amino acids using coryneform bacteria, in particular already L-amino acids produce and in which for attenuated the regulatory protein AsuR coding asuR gene, in particular is off or is expressed at a low level.
Gegenstand dieser Erfindung ist weiterhin ein Verfahren zur fermentativen Herstellung von L-Aminosäuren, in dem folgende Schritte durchgeführt werden:
- a) Fermentation der L-Aminosäure produzierenden rekombinanten coryneformen Bakterien in einem Medium, wobei in diesen das für den Regulator AsuR kodierende Gen asuR abgeschwächt, insbesondere ausgeschaltet ist oder auf niedrigem Niveau exprimiert wird,
- b) Anreicherung der L-Aminosäuren im Medium oder in den Zellen der Bakterien, und
- c) Isolierung der gewünschten L-Aminosäuren, wobei gegebenenfalls Bestandteile der Fermentationsbrühe und/oder der Biomasse in Anteilen (> 0 bis 100 %) oder in ihren Gesamtmengen im Endprodukt verbleiben.
- a) fermentation of the L-amino acid-producing recombinant coryneform bacteria in a medium, in which the AsuR gene coding for the regulator AsuR is attenuated, in particular switched off or is expressed at a low level,
- b) accumulation of L-amino acids in the medium or in the cells of the bacteria, and
- c) isolation of the desired L-amino acids, optionally with components of the fermentation broth and / or the biomass remaining in proportions (> 0 to 100%) or in their total amounts in the end product.
Die eingesetzten coryneformen Bakterien produzieren bevorzugt bereits vor der Abschwächung oder dem Ausschalten des asuR-Gens L-Aminosäuren, insbesondere L-Methionin.The used coryneform bacteria produce preferred already before the weakening or switching off the asuR gene L-amino acids, especially L-methionine.
Es wurde gefunden, dass Mikroorganismen, bevorzugt coryneforme Bakterien nach Abschwächung, insbesondere Ausschalten des Regulators AsuR, in verbesserter Weise L-Aminosäuren, insbesondere L-Methionin, produzieren.It was found to be microorganisms, preferably coryneform bacteria after weakening, in particular Turn off the regulator AsuR, in an improved way L-amino acids, in particular L-methionine, produce.
Die Nukleotidsequenzen des genannten Gens von Corynebacterium glutamicum gehören zum Stand der Technik und können verschiedenen Patentanmeldungen sowie der Datenbank des National Center for Biotechnology Information (NCBI) der National Library of Medicin (Bethesda, MD, USA) entnommen werden.The Nucleotide sequences of said gene of Corynebacterium glutamicum belong to the state of the art and can various patent applications and the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicin (Bethesda, MD, USA).
Die Nukleotidsequenz des für den Regulator AsuR von Corynebacterium glutamicum kodierenden Gens kann der Patentanmeldung EP1108790 als Sequenz Nr. 12 sowie als Sequenz Nr. 1 entnommen werden.The Nucleotide sequence of for the regulator AsuR of Corynebacterium glutamicum coding gene can the patent application EP1108790 as Sequence No. 12 and as Sequence no. 1 are removed.
Die Nukleotidsequenz ist ebenfalls in der Datenbank des National Center for Biotechnology Information (NCBI) der National Library of Medicin (Bethesda, MD, USA) unter der Accession Number AX120096 und unter der Accession Number AX120085 hinterlegt. Weiterhin ist sie unter der Accession Number BX927148 von Nukleotid 11177 bis 10104 der angegebenen Sequenz zu finden, die Aminosäuresequenz des zugehörigen Proteins ist unter der Accession Number CAF18575 hinterlegt.The Nucleotide sequence is also in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under the Accession Number AX120096 and under the Accession Number AX120085 is stored. Furthermore she is under Accession Number BX927148 from nucleotide 11177 to 10104 of to find the sequence indicated, the amino acid sequence of the associated protein is stored under the Accession Number CAF18575.
Die in der angegebenen Textstelle beschriebene Sequenz kodierend für das Gen asuR kann erfindungsgemäß verwendet werden. Weiterhin können Allele des genannten Gens verwendet werden, die sich aus der Degeneriertheit des genetischen Kodes oder durch funktionsneutrale Sinnmutationen („sense mutations") ergeben.The in the given passage described sequence coding for the gene AsuR can be used according to the invention become. Furthermore you can Alleles of the mentioned gene are used, resulting from the degeneration of the genetic code or by functionally neutral sense mutations ( "Sense mutations ").
Bevorzugte Ausführungsformen finden sich in den Ansprüchen.preferred embodiments can be found in the claims.
Der Begriff „Abschwächung" bzw. „Abschwächen" beschreibt in diesem Zusammenhang die Verringerung oder Ausschaltung der intrazellulären Aktivität eines oder mehrerer Enzyme bzw. Proteine in einem Mikroorganismus, die durch die entsprechende DNA kodiert werden, indem man beispielsweise einen schwachen Promotor verwendet oder ein Gen bzw. Allel verwendet, das für ein entsprechendes Enzym mit einer niedrigen Aktivität kodiert bzw. das entsprechende Gen oder Enzym bzw. Protein inaktiviert und gegebenenfalls diese Maßnahmen kombiniert.The term "attenuation" or "attenuation" in this context describes the reduction or elimination of the intracellular activity of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or a gene or allele that codes for a corresponding enzyme with a low activity or the corresponding gene or enzyme or protein inactivated and given if these measures combined.
Durch die Maßnahmen der Abschwächung wird die Aktivität oder Konzentration des entsprechenden Proteins im allgemeinen auf 0 bis 75%, 0 bis 50%, 0 bis 25%, 0 bis 10% oder 0 bis 5% der Aktivität oder Konzentration des Wildtyp-Proteins, beziehungsweise der Aktivität oder Konzentration des Proteins im Ausgangs-Mikroorganismus, abgesenkt.By the measures the weakening becomes the activity or concentration of the corresponding protein in general 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration the wild-type protein, or the activity or concentration of the protein in the starting microorganism, lowered.
Die Herabsetzung der Proteinkonzentration ist über 1- und 2-dimensionale Proteingelauftrennung und anschließende optische Identifizierung der Proteinkonzentration mit enstprechender Auswertesoftware im Gel nachweisbar. Eine gebräuchliche Methode zur Präparation der Proteingele bei coryneformen Bakterien und zur Identifizierung der Proteine ist die von Hermann et al. (Electrophoresis, 22: 1712–23 (2001)) beschriebene Vorgehensweise. Die Proteinkonzentration kann ebenfalls durch Western-Blot-Hybridisierung mit einem für das nachzuweisende Protein spezifischen Antikörper (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) und anschließender optischer Auswertung mit ensprechender Software zur Konzentrationsbestimmung (Lohaus und Meyer (1998) Biospektrum 5: 32–39; Lottspeich, Angewandte Chemie 111: 2630–2647 (1999)) analysiert werden. Die Aktivität von DNA-bindenden Proteinen kann mittels DNA Band-Shift-Assays (auch als Gelretardation bezeichnet) gemessen werden wie beispielsweise im Lehrbuch „Bioanalytik" (Lottspeich/Zorbas, Spektrum Akademischer Verlag Gmbh, Heidelberg, Deutschland, 1998) beschrieben und bei Wilson et al. (J. Bacteriol. 183: 2151–2155 (2001)) angewendet. Die Wirkung von DNA-bindenden Proteinen auf die Expression anderer Gene kann durch verschiedene gut beschriebene Methoden des Reportergen-Assays nachgewiesen werden (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The reduction of the protein concentration is detectable by 1 and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration with the corresponding evaluation software in the gel. A common method for preparing the protein gels in coryneform bacteria and for identifying the proteins is that described by Hermann et al. (Electrophoresis, 22: 1712-23 (2001)). The protein concentration can also be determined by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and subsequent optical evaluation with corresponding software for concentration determination (Lohaus and Meyer (1998) Biospektrum 5: 32-39; Lottspeich, Angewandte Chemie 111: 2630-2647 (1999)). The activity of DNA-binding proteins can be measured by DNA band-shift assays (also called gel retardation) as described, for example, in the textbook "Bioanalytics" (Lottspeich / Zorbas, Spektrum Akademischer Verlag Gmbh, Heidelberg, Germany, 1998) and Wilson The effect of DNA-binding proteins on the expression of other genes can be demonstrated by various well-described methods of the reporter gene assay (Sambrook et al., Molecular cloning. a laboratory manual 2 nd Ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)..
Die Mikroorganismen, die Gegenstand der vorliegenden Erfindung sind, können Aminosäuren aus Glucose, Saccharose, Lactose, Fructose, Maltose, Melasse, Stärke, Cellulose oder aus Glycerin und Ethanol herstellen. Es kann sich um Vertreter coryneformer Bakterien insbesondere der Gattung Corynebacterium handeln. Bei der Gattung Corynebacterium ist insbesondere die Art Corynebacterium glutamicum zu nennen, die in der Fachwelt für ihre Fähigkeit bekannt ist, L-Aminosäuren zu produzieren.The Microorganisms which are the subject of the present invention can amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative Coryneformer bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum, which in the professional world for their ability is known, L-amino acids to produce.
Geeignete
Stämme
der Gattung Corynebacterium, insbesondere der Art Corynebacterium
glutamicum, sind besonders die bekannten Wildtypstämme
Corynebacterium
glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium
acetoacidophilum ATCC13870
Corynebacterium melassecola ATCC17965
Corynebacterium
thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium
lactofermentum ATCC13869 und
Brevibacterium divaricatum ATCC14020
oder
wie beispielsweise der L-Methionin produzierende Stamm
Corynebacterium
glutamicum ATCC21608.Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium molassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
or such as the L-methionine producing strain
Corynebacterium glutamicum ATCC21608.
Stämme mit der Bezeichnung „ATCC" können von der American Type Culture Collection (Manassas, VA, USA) bezogen werden. Stämme mit der Bezeichnung „FERM" können vom National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) bezogen werden. Der genannte Stamm von Corynebacterium thermoaminogenes (FERM BP-1539) ist in der US-A-5.250.434 beschrieben.With tribes the designation "ATCC" can of of the American Type Culture Collection (Manassas, Va, USA) become. strains labeled "FERM" can be dated from National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) be obtained. The mentioned strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A-5,250,434.
Zur Erzielung einer Abschwächung können entweder die Expression der Gene oder die katalytischen oder regulatorischen Eigenschaften der Enzymproteine herabgesetzt oder ausgeschaltet werden. Gegebenenfalls können beide Maßnahmen kombiniert werden.to Achieving a slowdown can either the expression of the genes or the catalytic or regulatory Properties of the enzyme proteins reduced or eliminated become. If necessary, you can both measures be combined.
Die Genexpression kann durch geeignete Kulturführung oder durch genetische Veränderung (Mutation) der Signalstrukturen der Genexpression verringert werden. Signalstrukturen der Genexpression sind beispielsweise Repressorgene, Aktivatorgene, Operatoren, Promotoren, Attenuatoren, Ribosomenbindungsstellen, das Startkodon und Terminatoren. Angaben hierzu findet der Fachmann z.B. in der Patentanmeldung WO 96/15246, bei Boyd und Murphy (Journal of Bacteriology 170: 5949–5952 (1988)), bei Voskuil und Chambliss (Nucleic Acids Research 26: 3584–3590 (1998), bei Pátek et al. (Microbiology 142: 1297–309 (1996) und Journal of Biotechnology 104: 311–323 (2003)) und in bekannten Lehrbüchern der Genetik und Molekularbiologie wie z.B. dem Lehrbuch von Knippers („Molekulare Genetik", 6. Auflage, Georg Thieme Verlag, Stuttgart, Deutschland, 1995) oder dem von Winnacker („Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Deutschland, 1990).Gene expression can be reduced by appropriate culture or by genetic modification (mutation) of the signal structures of gene expression. Signaling structures of gene expression include repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon, and terminators. The skilled artisan, for example, finds this in the patent application WO 96/15246, Boyd and Murphy (Journal of Bacteriology 170: 5949-5952 (1988)), Voskuil and Chambliss (Nucleic Acids Research 26: 3584-3590 (1998)) Pátek et al (Microbiology 142: 1297-309 (1996) and Journal of Biotechnology 104: 311-323 (2003)) and in known textbooks of genetics and molecular biology such as the textbook by Knippers ("Molecular Genetics", 6th Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or Winnacker's ("Gene and Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990).
Ein Beispiel für die gezielte Regulation der Genexpression ist die Klonierung des abzuschwächenden Gens unter die Kontrolle eines durch Zugabe dosierter Mengen von IPTG (Isopropyl-β-D-thiogalactopyranosid) induzierbaren Promotors wie zum Beispiel des trc-Promotors oder des tac-Promotors. Hierzu eignen sich Vektoren wie beispielsweise der Escherichia coli Expressionsvektor pXK99E (WO0226787; hinterlegt gemäß Budapester Vertrag am 31. Juli 2001 in DH5alpha/pXK99E als DSM14440 bei der Deutschen Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Deutschland)) oder pVWEx2 (Wendisch, Ph. D. thesis, Berichte des Forschungszentrums Jülich, Jül-3397, ISSN 0994-2952, Jülich, Deutschland (1997)), die eine IPTGabhängige Expression des klonierten Gens in Corynebacterium glutamicum ermöglichen.One example for the targeted regulation of gene expression is the cloning of the to be attenuated gene under the control of an addition of metered amounts of IPTG (Isopropyl-β-D-thiogalactopyranoside) inducible Promoter such as the trc promoter or the tac promoter. For this vectors such as the Escherichia coli expression vector are suitable pXK99E (WO0226787, filed under the Budapest Treaty on 31. July 2001 in DH5alpha / pXK99E as DSM14440 at the Deutsche Sammlung for microorganisms and cell cultures (DSMZ, Braunschweig, Germany)) or pVWEx2 (Wendisch, Ph. D. thesis, Reports of Forschungszentrum Jülich, Jül-3397, ISSN 0994-2952, Jülich, Germany (1997)), which cloned an IPTG-dependent expression of the Gens in Corynebacterium glutamicum.
Eingesetzt wurde diese Methode beispielsweise in der Patentschrift WO0226787 zur regulierten Expression des deaD-Gens durch Integration des Vektors pxK99EdeaD in das Genom von Corynebacterium glutamicum und von Simic et al. (Applied and Environmental Microbiology 68: 3321–3327 (2002)) zur regulierten Expression des glyA-Gens durch Integration des Vektors pK18mobglyA' in Corynebacterium glutamicum.used For example, this method has been described in WO0226787 for the regulated expression of the deaD gene by integration of the vector pxK99EdeaD into the genome of Corynebacterium glutamicum and Simic et al. (Applied and Environmental Microbiology 68: 3321-3327 (2002)) for regulated expression of the glyA gene by integration of the vector pK18mobglyA 'in Corynebacterium glutamicum.
Eine weitere Methode zur spezifischen Verringerung der Genexpression ist die Antisense-Technik, wobei kurze Oligodesoxyukleotide oder Vektoren zur Synthese längerer Antisense-RNA in die Zielzellen gebracht werden. Die Antisense-RNA kann dort an komplementäre Abschnitte spezifischer mRNAs binden und deren Stabilität verringern oder die Translatierbarkeit blocken. Ein Beispiel hierzu findet der Fachmann bei Srivastava et al. (Applied Environmental Microbiology 2000 Oct; 66 (10): 4366–4371).A another method for the specific reduction of gene expression is the antisense technique, wherein short oligodeoxy nucleotides or Vectors for the synthesis of longer Antisense RNA can be brought into the target cells. The antisense RNA can be complementary there Bind sections of specific mRNAs and reduce their stability or block the translatability. An example of this can be found the expert in Srivastava et al. (Applied Environmental Microbiology 2000 Oct; 66 (10): 4366-4371).
Mutationen, die zu einer Veränderung bzw. Herabsetzung der katalytischen Eigenschaften von Enzymproteinen führen, sind aus dem Stand der Technik bekannt; als Beispiele seien die Arbeiten von Qiu und Goodman (Journal of Biological Chemistry 272: 8611–8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760–1762 (1997)) und Möckel (Ph. D. thesis, Berichte des Forschungszentrums Jülich, Jül-2906, ISSN09442952, Jülich, Deutschland (1994)) genannt. Zusammenfassende Darstellungen können bekannten Lehrbüchern der Genetik und Molekularbiologie wie z.B. dem von Hagemann („Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) entnommen, werden.mutations the change or reduction of the catalytic properties of enzyme proteins to lead, are known in the art; as examples are the Works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (Ph. D. thesis, Reports of Forschungszentrum Jülich, Jül-2906, ISSN09442952, Jülich, Germany (1994)). Summary presentations may be known textbooks genetics and molecular biology e.g. that of Hagemann ("Allgemeine Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).
Als Mutationen kommen Transitionen, Transversionen, Insertionen und Deletionen in Betracht. In Abhängigkeit von der Wirkung des Aminosäureaustausches auf die Enzymaktivität wird von Fehlsinnmutationen („missense mutations") oder Nichtsinnmutationen („nonsense mutations") gesprochen. Insertionen oder Deletionen von mindestens einem Basenpaar in einem Gen führen zu Rasterverschiebungsmutationen („frame shift mutations"), in deren Folge falsche Aminosäuren eingebaut werden oder die Translation vorzeitig abbricht. Deletionen von mehreren Kodonen führen typischerweise zu einem vollständigen Ausfall der Enzymaktivität. Anleitungen zur Erzeugung derartiger Mutationen gehören zum Stand der Technik und können bekannten Lehrbüchern der Genetik und Molekularbiologie wie z.B. dem Lehrbuch von Knippers („Molekulare Genetik", 6. Auflage, Georg Thieme Verlag, Stuttgart, Deutschland, 1995), dem von Winnacker („Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Deutschland, 1990) oder dem von Hagemann („Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) entnommen werden.When Mutations include transitions, transversions, insertions and Deletions into consideration. Dependent on from the effect of amino acid exchange on the enzyme activity is caused by missense mutations ("missense mutations ") or Nonsense mutations ("nonsense mutations "). Insertions or deletions of at least one base pair in one Lead gene to frame shift mutations, as a result wrong amino acids be installed or the translation aborts prematurely. deletions lead by several codons typically to a complete one Failure of enzyme activity. Instructions for producing such mutations belong to State of the art and can known textbooks genetics and molecular biology e.g. the textbook by Knippers ( "Molecular Genetics ", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ( "Gene and Clones ", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann's ( "General Genetics ", Gustav Fischer Verlag, Stuttgart, 1986).
Eine gebräuchliche Methode, Gene von C. glutamicum zu mutieren, ist die von Schwarzer und Pühler (Bio/Technology 9, 84–87 (1991)) beschriebene Methode der Gen-Unterbrechung (gene disruption") und des Gen-Austauschs („gene replacement").A common The method of mutating genes of C. glutamicum is that of Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)) method of gene disruption and gene replacement ( "Gene replacement ").
Bei der Methode der Gen-Unterbrechung wird zum Beispiel ein zentraler Teil der Kodierregion des interessierenden Gens in einen Plasmidvektor kloniert, der in einem Wirt (typischerweise E. coli), nicht aber in C. glutamicum replizieren kann. Als Vektoren kommen beispielsweise pSUP301 (Simon et al., Bio/Technology 1, 784–791 (1983)), pK18mob, pK19mob, pK18mobsacB oder pK19mobsacB (Schäfer et al., Gene 145, 69–73 (1994)), pGEM-T (Promega corporation, Madison, WI, USA), pCR2.1-TOPO (Firma Invitrogen, Groningen, Niederlande; Shaman (1994). Journal of Biological Chemistry 269: 32678–84; US-Patent 5,487,993), pCR®Blunt (Firma Invitrogen, Groningen, Niederlande; Bernard et al., Journal of Molecular Biology, 234: 534–541 (1993)) oder pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510–4516) in Frage. Der Plasmidvektor, der den zentralen Bereich der Kodierregion des Gens enthält, wird anschließend durch Konjugation oder Transformation in den gewünschten Stamm von C. glutamicum überführt. Die Methode der Konjugation ist beispielsweise bei Schäfer et al. (Journal of Bacteriology 172: 1663–1666 (1990) und Applied and Environmental Microbiology 60: 756–759 (1994)) beschrieben. Methoden zur Transformation sind beispielsweise bei Thierbach et al. (Applied Microbiology and Biotechnology 29, 356–362 (1988)), Dunican und Shivnan (Bio/Technology 7, 1067–1070 (1989)) und Tauch et al. (FEMS Microbiological Letters 123, 343–347 (1994)) beschrieben. Nach homologer Rekombination mittels eines "cross-over"-Ereignisses wird die Kodierregion des betreffenden Gens durch die Vektorsequenz unterbrochen und man erhält zwei unvollständige Allele, denen jeweils das 3'- bzw. das 5'-Ende fehlt. Diese Methode wurde beispielsweise von Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575–580 (1994)) zur Ausschaltung des recA-Gens von C. glutamicum verwendet.For example, in the method of gene disruption, a central part of the coding region of the gene of interest is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum. Examples of vectors which are used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob, pK19mob, pK18mobsacB or pK19mobsacB (Schäfer et al., Gene 145, 69-73 (1994)), pGEM- T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Invitrogen, Groningen, The Netherlands; Shaman (1994) Journal of Biological Chemistry. 269: 32678-84; US Patent 5,487,993), pCR ® Blunt ( Invitrogen, Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510-4516). The plasmid vector containing the central region of the coding region of the gene is then converted by conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation is described by Schäfer et al. (Journal of Bacteriology 172: 1663-1666 (1990) and Applied and Environmental Microbiology 60: 756-759 (1994)). Methods for transformation are example as in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a "cross-over" event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, each of which lacks the 3 'or the 5' end. This method has been described, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.
Gegenstand der Erfindung sind ebenfalls Vektoren, die mindestens 15, bevorzugt 25 aufeinanderfolgende Nukleotide des zentralen Teils der Kodierregion des Gens asuR enthalten.object The invention also includes vectors which are at least 15 25 consecutive nucleotides of the central part of the coding region of the gene included.
Bei der Methode des Genaustausches („gene replacement") wird eine Mutation wie z.B. eine Deletion, Insertion oder Basenaustausch in dem interessierenden Gen in-vitro hergestellt. Das hergestellte Allel wird wiederum in einen für C. glutamicum nicht replikativen Vektor kloniert und dieser anschließend durch Transformation oder Konjugation in den gewünschten Wirt von C. glutamicum überführt. Nach homologer Rekombination mittels eines ersten, Integration bewirkenden "cross-over"-Ereignisses und eines geeigneten zweiten, eine Exzision bewirkenden "cross-over"-Ereignisses im Zielgen bzw. in der Zielsequenz erreicht man den Einbau der Mutation bzw. des Allels. Diese Methode ist bei Scharzer und Pühler (Bio/Technology 9: 84–87 (1991) beschrieben und wurde beispielsweise von Peters-Wendisch et al. (Microbiology 144, 915–927 (1998)) verwendet, um das pyc-Gen von C. glutamicum durch eine Deletion auszuschalten oder von Wehmeier et al. (Microbiology 144: 1853–1862 (1998)) zum Einfügen einer Deletion in das rel-Gen von C. glutamicum.at the method of gene replacement becomes a mutation such as. a deletion, insertion or base exchange in the one of interest Gene produced in vitro. The produced allele will turn into one for C. glutamicum is not cloned replicative vector and this subsequently by Transformation or conjugation into the desired host of C. glutamicum. To homologous recombination by means of a first, cross-over event causing integration and a suitable second excision-causing cross-over event in the target gene or in the target sequence to achieve the incorporation of the mutation or of the allele. This method is described by Scharzer and Pühler (Bio / Technology 9: 84-87 (1991) and has been described, for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) was used to delete the pyc gene of C. glutamicum by a deletion turn off or by Wehmeier et al. (Microbiology 144: 1853-1862 (1998)) to paste a deletion in the rel gene of C. glutamicum.
Einen Überblick über verschiedene gentechnische Methoden bei C. glutamicum geben Kirchner und Tauch (Journal of Biotechnology 104: 287–299 (2003).An overview of different Genetic engineering methods for C. glutamicum are given by Kirchner and Tauch (Journal of Biotechnology 104: 287-299 (2003).
In eines oder mehrere der Gene, ausgewählt aus der Gruppe yaeC, abc unc yaeE kann auf diese weise eine Deletion, Insertion oder ein Basenaustausch eingebaut werden.In one or more of the genes selected from the group yaeC, abc Uncut can in this way be a deletion, insertion or a Base exchange to be installed.
Weiterhin kann es für die Produktion von L-Aminosäuren vorteilhaft sein, zusätzlich zur Abschwächung des Regulators AsuR eines oder mehrere Enzyme des jeweiligen Biosyntheseweges, der Glykolyse, der Anaplerotik, des Zitronensäure-Zyklus, des Pentosephosphat-Zyklus, des Aminosäure-Exports und gegebenenfalls regulatorische Proteine entweder zu verstärken, insbesondere überzuexprimieren, oder abzuschwächen, insbesondere auszuschalten oder die Expression zu verringern.Farther can it for the production of L-amino acids be beneficial, in addition to mitigate the Regulators AsuR one or more enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, of amino acid export and, where appropriate, either to enhance, in particular to overexpress, regulatory proteins, or mitigate, in particular, turn off or reduce expression.
Der Begriff „Verstärkung" bzw. „Verstärken" beschreibt in diesem Zusammenhang die Erhöhung der intrazellulären Aktivität oder Konzentration eines oder mehrerer Enzyme bzw. Proteine in einem Mikroorganismus, die durch die entsprechende DNA kodiert werden, indem man beispielsweise die Kopienzahl des Gens bzw. der Gene erhöht, einen starken Promotor oder ein Gen bzw. Allel verwendet, das für ein entsprechendes Enzym bzw. Protein mit einer hohen Aktivität kodiert und gegebenenfalls diese Maßnahmen kombiniert.Of the The term "reinforcement" or "reinforcing" describes in this Related the increase the intracellular activity or concentration of one or more enzymes or proteins in one Microorganism encoded by the corresponding DNA, for example, by increasing the copy number of the gene (s), one strong promoter or a gene or allele used for a corresponding enzyme or protein with a high activity codes and optionally these measures combined.
Durch die Maßnahmen der Verstärkung, insbesondere Überexpression, wird die Aktivität oder Konzentration des entsprechenden Proteins im allgemeinen um mindestens 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% oder 500%, maximal bis 1000% oder 2000% bezogen auf die des Wildtyp- Proteins beziehungsweise der Aktivität oder Konzentration des Proteins im Ausgangs-Mikroorganismus erhöht.By the measures the reinforcement, in particular overexpression, becomes the activity or concentration of the corresponding protein in general at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000% based on the wild-type protein or the activity or increased concentration of the protein in the starting microorganism.
Die Verwendung endogener Gene wird im allgemeinen bevorzugt. Unter „endogenen Genen" oder „endogenen Nukleotidsequenzen" versteht man die in der Population einer Art vorhandenen Gene beziehungsweise Nukleotidsequenzen.The Use of endogenous genes is generally preferred. Under "endogenous Genes "or" endogenous Nucleotide sequences " the genes present in the population of a species Nucleotide sequences.
So kann beispielsweise für die Herstellung von L-Methionin neben der Abschwächung des Regulators AsuR eines oder mehrere der Gene ausgewählt aus der Gruppe der Gene oder Allele der Methioninproduktion verstärkt, insbesondere überexprimiert werden. Unter „Gene oder Allele der Methioninproduktion" sind sämtliche, bevorzugt endogene, offene Leserahmen, Gene oder Allele zu verstehen, deren Verstärkung/Überexpression eine Verbesserung der Methioninproduktion bewirken kann.So can be for example the production of L-methionine in addition to the attenuation of the regulator AsuR one or more of the genes selected amplified from the group of genes or alleles of methionine production, in particular overexpressed become. Under "Genes or alleles of methionine production "are all, preferably endogenous, open reading frames, genes or alleles to understand their amplification / overexpression can improve methionine production.
Hierzu
gehören
unter anderem folgende offene Leserahmen, Gene oder Allele: accBC,
accDA, aecD, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dps, eno,
fda, gap, gap2, gdh, gnd, glyA, hom, homFBR,
lysC, lysCFBR, metA, metB, metE, metH, metY,
msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA,
pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC,
sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf und zwf A213T.
Diese sind in Tabelle 1 zusammengefasst und erläutert. Tabelle
1 Gene
und Allele der Methioninproduktion
Weiterhin kann es für die Produktion L-Methionin vorteilhaft sein, zusätzlich zur Abschwächung des Regulators AsuR gleichzeitig eines oder mehrere der Gene, ausgewählt aus der Gruppe der Gene oder Allele, die für das Wachstum oder die Methioninproduktion nicht essentiell sind, abzuschwächen, insbesondere auszuschalten oder die Expression zu verringern.Farther can it for the production of L-methionine may be beneficial, in addition to the attenuation of the regulator AsuR simultaneously one or more of the genes selected from the group of genes or alleles responsible for growth or methionine production are not essential to mitigate in particular, turn off or reduce expression.
Hierzu
gehören
unter anderem folgende offene Leserahmen, Gene oder Allele: brnQ,
ccpA1, ccpA2, citA, citB, citE, ddh, gluA, gluB, gluC, gluD, luxR,
luxS, lysR1, lysR2, lysR3, menE, metD, metK, pck, pgi, poxB und
zwa2. Diese sind in Tabelle 2 zusammengefasst und erläutert. Tabelle
2 Gene
und Allele, die für
die Methioninproduktion nicht essentiell sind
Schließlich kann es für die Produktion von Aminosäuren, insbesondere L-Methionin, vorteilhaft sein, neben der Abschwächung Regulators AsuR unerwünschte Nebenreaktionen auszuschalten (Nakayama: „Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).Finally, can it for the production of amino acids, especially L-methionine, be beneficial, in addition to the attenuation regulator AsuR unwanted To eliminate side reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms " in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).
Die erfindungsgemäß hergestellten Mikroorganismen sind ebenfalls Gegenstand der Erfindung und können kontinuierlich oder diskontinuierlich im batch – Verfahren (Satzkultivierung) oder im fed batch (Zulaufverfahren) oder repeated fed batch Verfahren (repetitives Zulaufverfahren) zum Zwecke der Produktion von L-Aminosäuren kultiviert werden. Eine Zusammenfassung über bekannte Kultivierungsmethoden ist im Lehrbuch von Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) oder im Lehrbuch von Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)) beschrieben.The produced according to the invention Microorganisms are also the subject of the invention and can be continuous or batchwise in a batch process (batch culturing) or in the fed batch or repeated fed batch process (repetitive feed method) for the purpose of producing L-amino acids become. A summary about known cultivation methods is in the textbook by Chmiel (Bioprozesstechnik 1. Introduction in bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook of Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).
Das zu verwendende Kulturmedium muß in geeigneter Weise den Ansprüchen der jeweiligen Stämme genügen. Beschreibungen von Kulturmedien verschiedener Mikroorganismen sind im Handbuch „Manual of Methods for General Bacteriology„ der American Society for Bacteriology (Washington D.C., USA, 1981) enthalten.The to be used culture medium must be in Appropriate to the claims satisfy the respective strains. descriptions of culture media of various microorganisms are described in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981).
Als Kohlenstoffquelle können Zucker und Kohlehydrate wie z.B. Glucose, Saccharose, Lactose, Fructose, Maltose, Melasse, Stärke und Cellulose, Öle und Fette wie z.B. Sojaöl, Sonnenblumenöl, Erdnußöl und Kokosfett, Fettsäuren wie z.B. Palmitinsäure, Stearinsäure und Linolsäure, Alkohole wie z.B. Glycerin und Ethanol und organische Säuren wie z.B. Essigsäure verwendet werden. Diese Stoffe können einzeln oder als Mischung verwendet werden.When Carbon source can Sugars and carbohydrates, e.g. Glucose, sucrose, lactose, fructose, Maltose, molasses, starch and cellulose, oils and fats such as e.g. Soybean oil, Sunflower oil, Peanut oil and coconut oil, fatty acids such as. palmitic acid, stearic acid and linoleic acid, Alcohols such as e.g. Glycerine and ethanol and organic acids like e.g. acetic acid be used. These substances can used singly or as a mixture.
Als Stickstoffquelle können organische Stickstoff-haltige Verbindungen wie Peptone, Hefeextrakt, Fleischextrakt, Malzextrakt, Maisquellwasser, Sojabohnenmehl und Harnstoff oder anorganische Verbindungen wie Ammoniumsulfat, Ammoniumchlorid, Ammoniumphosphat, Ammoniumcarbonat und Ammoniumnitrat verwendet werden. Die Stickstoffquellen können einzeln oder als Mischung verwendet werden.When Nitrogen source can organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, Malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can used singly or as a mixture.
Als Phosphorquelle können Phosphorsäure, Kaliumdihydrogenphosphat oder Dikaliumhydrogenphosphat oder die entsprechenden Natrium haltigen Salze verwendet werden. Das Kulturmedium muß weiterhin Salze von Metallen enthalten wie z.B. Magnesiumsulfat oder Eisensulfat, die für das Wachstum notwendig sind. Schließlich können essentielle Wuchsstoffe wie Aminosäuren und Vitamine zusätzlich zu den oben genannten Stoffen eingesetzt werden. Dem Kulturmedium können überdies geeignete Vorstufen zugesetzt werden. Die genannten Einsatzstoffe können zur Kultur in Form eines einmaligen Ansatzes hinzugegeben oder in geeigneter Weise während der Kultivierung zugefüttert werden.Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. The culture medium must further contain salts of metals such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth factors such as amino acids and vitamins can be added to those mentioned above substances are used. In addition, suitable precursors can be added to the culture medium. The said feedstocks may be added to the culture in the form of a one-time batch or fed in a suitable manner during the cultivation.
Zur pH-Kontrolle der Kultur werden basische Verbindungen wie Natriumhydroxid, Kaliumhydroxid, Ammoniak bzw. Ammoniakwasser oder saure Verbindungen wie Phosphorsäure oder Schwefelsäure in geeigneter Weise eingesetzt. Zur Kontrolle der Schaumentwicklung können Antischaummittel wie z.B. Fettsäurepolyglykolester eingesetzt werden. Zur Aufrechterhaltung der Stabilität von Plasmiden können dem Medium geeignete selektiv wirkende Stoffe wie z.B. Antibiotika hinzugefügt werden. Um aerobe Bedingungen aufrechtzuerhalten, werden Sauerstoff oder Sauerstoffhaltige Gasmischungen wie z.B. Luft in die Kultur eingetragen. Die Temperatur der Kultur liegt normalerweise bei 20°C bis 45°C und vorzugsweise bei 25°C bis 40°C. Die Kultur wird solange fortgesetzt, bis sich ein Maximum des gewünschten Produktes gebildet hat. Dieses Ziel wird normalerweise innerhalb von 10 Stunden bis 160 Stunden erreicht.to pH control of the culture are basic compounds such as sodium hydroxide, Potassium hydroxide, ammonia or ammonia water or acidic compounds like phosphoric acid or sulfuric acid used in a suitable manner. To control foaming can Antifoam agents such as e.g. fatty acid be used. To maintain the stability of plasmids can the medium suitable selective substances such. antibiotics added become. To maintain aerobic conditions, oxygen is used or oxygen-containing gas mixtures such as e.g. Air in the culture entered. The temperature of the culture is usually 20 ° C to 45 ° C, and preferably at 25 ° C up to 40 ° C. The culture is continued until a maximum of the desired Product has formed. This goal is usually within from 10 hours to 160 hours.
Mit den Methoden der Erfindung kann die Leistung der Bakterien oder des Fermentationsprozesses bezüglich der Produkt-Konzentration ((Produkt pro Volumen), der Produkt-Ausbeute (gebildetes Produkt pro verbrauchter Kohlenstoff-Quelle), der Produkt-Bildung (gebildetes Produkt pro Volumen und zeit) oder anderer Prozess-Parameter und Kombinationen davon um mindestens 0,5%, mindestens 1% oder mindestens 2% verbessert werden.With The methods of the invention may be the performance of the bacteria or of the fermentation process with respect to the product concentration ((product per volume), the product yield (formed Product Per Used Carbon Source), Product Formation (Formed Product per volume and time) or other process parameters and Combinations of at least 0.5%, at least 1% or at least 2% be improved.
Methoden zur Bestimmung von L-Aminosäuren sind aus dem Stand der Technik bekannt. Die Analyse kann so wie bei Spackman et al. (Analytical Chemistry, 30, (1958), 1185–1190) beschrieben durch Anionenaustauschchromatographie mit anschließender Ninhydrin Derivatisierung erfolgen, oder sie kann durch reversed phase HPLC erfolgen, so wie bei Lindroth et al. (Analytical Chemistry (1979) 51: 1167–1174) beschrieben. Informationen dazu findet der Fachmann auch bei Ashman et al. (in: Tschesche (Hrsg), Modern Methods in Protein Chemistry. 155–172, de Gruyter, Berlin 1985).methods for the determination of L-amino acids are known from the prior art. The analysis can be as well as Spackman et al. (Analytical Chemistry, 30, (1958), 1185-1190) by anion exchange chromatography followed by ninhydrin Derivatization can take place, or it can be done by reversed phase HPLC carried out, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174) described. The expert also finds information on this at Ashman et al. (in: Tschesche (eds), Modern Methods in Protein Chemistry. 155-172, de Gruyter, Berlin 1985).
Das erfindungsgemäße Verfahren dient zur fermentativen Herstellung von L-Methionin.The inventive method is used for the fermentative production of L-methionine.
Die Konzentration von L-Methionin im Endprodukt kann gegebenenfalls durch den Zusatz von L-Methionin auf den gewünschten Wert eingestellt werden.The Concentration of L-methionine in the final product may optionally be adjusted by the addition of L-methionine to the desired value.
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| EP05700861A EP1756279A1 (en) | 2004-02-27 | 2005-01-13 | Method for producing l-amino acids by means of recombinant coryneform bacteria with reduced activity asur regulators |
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- 2005-01-13 EP EP05700861A patent/EP1756279A1/en not_active Withdrawn
- 2005-01-13 CN CNA2005800057916A patent/CN1922322A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN1922322A (en) | 2007-02-28 |
| EP1756279A1 (en) | 2007-02-28 |
| WO2005083082A1 (en) | 2005-09-09 |
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