DE10210962A1 - Preparation of amino acids, particularly threonine useful e.g. in animal nutrition, by growing Enterobacteriaceae having increased activity of e.g. lpd, aceE or aceF genes - Google Patents
Preparation of amino acids, particularly threonine useful e.g. in animal nutrition, by growing Enterobacteriaceae having increased activity of e.g. lpd, aceE or aceF genesInfo
- Publication number
- DE10210962A1 DE10210962A1 DE10210962A DE10210962A DE10210962A1 DE 10210962 A1 DE10210962 A1 DE 10210962A1 DE 10210962 A DE10210962 A DE 10210962A DE 10210962 A DE10210962 A DE 10210962A DE 10210962 A1 DE10210962 A1 DE 10210962A1
- Authority
- DE
- Germany
- Prior art keywords
- gene coding
- gene
- coding
- threonine
- genes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
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- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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Abstract
Description
Diese Erfindung betrifft ein Verfahren zur fermentativen Herstellung von L-Aminosäuren, insbesondere L-Threonin, unter Verwendung von Stämmen der Familie Enterobacteriaceae, in denen mindestens eines oder mehrere der Gene, ausgewählt aus der Gruppe lpd, aceE und aceF, verstärkt wird (werden). This invention relates to a method of fermentative Production of L-amino acids, especially L-threonine, using family tribes Enterobacteriaceae, in which at least one or more the genes selected from the group lpd, aceE and aceF, is strengthened.
Stand der TechnikState of the art
L-Aminosäuren, insbesondere L-Threonin, finden in der Humanmedizin und in der pharmazeutischen Industrie, in der Lebensmittelindustrie und ganz besonders in der Tierernährung Anwendung. L-amino acids, especially L-threonine, can be found in the Human medicine and in the pharmaceutical industry, in the Food industry and especially in the Animal nutrition application.
Es ist bekannt, dass L-Aminosäuren durch Fermentation von Stämmen der Enterobacteriaceae, insbesondere Escherichia coli (E. coli) und Serratia marcescens, hergestellt werden. Wegen der großen Bedeutung wird ständig an der Verbesserung der Herstellverfahren gearbeitet. Verfahrensverbesserungen können fermentationstechnische Maßnahmen, wie z. B. Rührung und Versorgung mit Sauerstoff, oder die. Zusammensetzung der Nährmedien wie z. B. die Zuckerkonzentration während der Fermentation, oder die Aufarbeitung zur Produktform, durch z. B. Ionenaustauschchromatographie, oder die intrinsischen Leistungseigenschaften des Mikroorganismus selbst betreffen. L-amino acids are known to be fermented by Strains of Enterobacteriaceae, especially Escherichia coli (E. coli) and Serratia marcescens. Because of the great importance it is constantly improving the manufacturing process worked. process improvements can fermentation-related measures such. B. Stirring and supply of oxygen, or the. Composition of the Culture media such as B. the sugar concentration during the Fermentation, or the processing to product form, by z. B. ion exchange chromatography, or the intrinsic Performance characteristics of the microorganism itself affect.
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 Threonin-Analogon α-Amino-β-Hydroxyvaleriansäure (AHV) oder auxotroph für regulatorisch bedeutsame Metabolite sind und L-Aminosäuren wie z. B. L-Threonin produzieren. To improve the performance characteristics of this Microorganisms become methods of mutagenesis, selection and mutant selection applied. This way you get Strains resistant to antimetabolites such as B. that Threonine analog α-amino-β-hydroxyvaleric acid (AHV) or are auxotrophic for regulatory metabolites and L-amino acids such as B. Produce L-threonine.
Seit einigen Jahren werden ebenfalls Methoden der rekombinanten DNA-Technik zur Stammverbesserung L- Aminosäuren produzierender Stämme der Familie Enterobacteriaceae eingesetzt, indem man einzelne Aminosäure-Biosynthesegene amplifiziert und die Auswirkung auf die Produktion untersucht. Methods of recombinant DNA technology for strain improvement L- Strains of the family producing amino acids Enterobacteriaceae used by individual Amino acid biosynthesis genes amplified and the impact examined for production.
Die Erfinder haben sich die Aufgabe gestellt, neue Maßnahmen zur verbesserten fermentativen Herstellung von L- Aminosäuren, insbesondere L-Threonin, bereitzustellen. The inventors set themselves the task of creating new ones Measures to improve the fermentative production of L- To provide amino acids, especially L-threonine.
Beschreibung der ErfindungDescription of the invention
Gegenstand der Erfindung ist ein Verfahren zur fermentativen Herstellung von L-Aminosäuren, insbesondere L-Threonin, unter Verwendung von Mikroorganismen der Familie Enterobacteriaceae, die insbesondere bereits L- Aminosäuren produzieren, und in denen mindestens eine oder mehrere der für die Gene kodierende(n) lpd, aceE und aceF Nukleotidsequenz(en) verstärkt wird (werden). The invention relates to a method for fermentative production of L-amino acids, in particular L-threonine, using microorganisms from the Family Enterobacteriaceae, which in particular already L- Produce amino acids, and in which at least one or several of the lpd, aceE and aceF encoding the genes Nucleotide sequence (s) is (are) amplified.
Werden im folgenden L-Aminosäuren oder Aminosäuren erwähnt, sind damit eine oder mehrere Aminosäuren einschließlich ihrer Salze, ausgewählt aus der Gruppe L-Asparagin, L- Threonin, L-Serin, L-Glutamat, L-Glycin, L-Alanin, L- Cystein, L-Valin, L-Methionin, L-Isoleucin, L-Leucin, L- Tyrosin, L-Phenylalanin, L-Histidin, L-Lysin, L-Tryptophan und L-Arginin gemeint. Besonders bevorzugt ist L-Threonin. If L-amino acids or amino acids are mentioned below, are one or more amino acids included their salts, selected from the group L-asparagine, L- Threonine, L-serine, L-glutamate, L-glycine, L-alanine, L- Cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L- Tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine meant. L-threonine is particularly preferred.
Der Begriff "Verstärkung" beschreibt in diesem Zusammenhang die Erhöhung der intrazellulären Aktivität 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 oder Allel verwendet, das für ein entsprechendes Enzym bzw. Protein mit einer hohen Aktivität kodiert und gegebenenfalls diese Maßnahmen kombiniert. The term "reinforcement" describes in this context the increase in the intracellular activity of one or several enzymes or proteins in a microorganism that can be encoded by the appropriate DNA by using for example the copy number of the gene or genes increased, a strong promoter or a. Gene or allele used that for a corresponding enzyme or protein encoded with a high activity and possibly this Combined measures.
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. Through the measures of reinforcement, in particular Overexpression, the activity or concentration of the corresponding protein in general by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to 1000% or 2000% based on that of the wild type Protein or the activity or concentration of the protein in the starting microorganism increased.
Das Verfahren ist dadurch gekennzeichnet, dass man folgende
Schritte durchführt:
- a) Fermentation der die gewünschte L-Aminosäure produzierenden Mikroorganismen der Familie Enterobacteriaceae, in denen man eines oder mehrere der Gene, ausgewählt aus der Gruppe lpd, aceE und aceF, oder dafür kodierende Nukleotidsequenzen verstärkt, insbesondere überexprimiert,
- b) Anreicherung der gewünschten L-Aminosäure im Medium oder in den Zellen der Mikroorganismen, und
- c) Isolierung der gewünschten L-Aminosäure wobei gegebenenfalls Bestandteile der Fermentationsbrühe und/oder die Biomasse in ihrer Gesamtheit oder Anteilen (≥ 0 bis 100) davon im Produkt verbleiben.
- a) fermentation of the desired L-amino acid-producing microorganisms of the Enterobacteriaceae family, in which one or more of the genes selected from the group lpd, aceE and aceF, or nucleotide sequences coding therefor are amplified, in particular overexpressed,
- b) enrichment of the desired L-amino acid in the medium or in the cells of the microorganisms, and
- c) isolation of the desired L-amino acid, components of the fermentation broth and / or the biomass in their entirety or parts (≥ 0 to 100) thereof remaining in the product, if appropriate.
Die Mikroorganismen, die Gegenstand der vorliegenden Erfindung sind, können L-Aminosäuren aus Glucose, Saccharose, Lactose, Fructose, Maltose, Melasse, gegebenenfalls Stärke, gegebenenfalls Cellulose oder aus Glycerin und Ethanol herstellen. Es handelt sich um Vertreter der Familie Enterobacteriaceae, ausgewählt aus den Gattungen Escherichia, Erwinia, Providencia und Serratia. Die Gattungen Escherichia und Serratia werden bevorzugt. Bei der Gattung Escherichia ist insbesondere die Art Escherichia coli und bei der Gattung Serratia insbesondere die Art Serratia marcescens zu nennen. The microorganisms that are the subject of the present L-amino acids from glucose, Sucrose, lactose, fructose, maltose, molasses, optionally starch, optionally cellulose or Make glycerin and ethanol. It is a matter of Representative of the Enterobacteriaceae family, selected from the genera Escherichia, Erwinia, Providencia and Serratia. The genera Escherichia and Serratia are prefers. In the genus Escherichia is particularly Kind Escherichia coli and in the genus Serratia especially the species Serratia marcescens.
Geeignete, insbesondere L-Threonin produzierende Stämme der
Gattung Escherichia, insbesondere der Art Escherichia coli
sind beispielsweise
Escherichia coli TF427
Escherichia coli H4578
Escherichia coli KY10935
Escherichia coli VNIIgenetika MG442
Escherichia coli VNIIgenetika M1
Escherichia coli VNIIgenetika 472T23
Escherichia coli BKIIM B-3996
Escherichia coli kat 13
Escherichia coli KCCM-10132.
Suitable, in particular L-threonine-producing strains of the genus Escherichia, in particular of the type Escherichia coli, are for example
Escherichia coli TF427
Escherichia coli H4578
Escherichia coli KY10935
Escherichia coli VNIIgenetic MG442
Escherichia coli VNIIgenetics M1
Escherichia coli VNIIgenetics 472T23
Escherichia coli BKIIM B-3996
Escherichia coli kat 13
Escherichia coli KCCM-10132.
Geeignete L-Threonin produzierende Stämme der Gattung
Serratia, insbesondere der Art Serratia marcescens sind
beispielsweise
Serratia marcescens HNr21
Serratia marcescens TLr156
Serratia marcescens T2000.
Suitable strains of the genus Serratia, in particular of the species Serratia marcescens, which produce L-threonine are, for example
Serratia marcescens HNr21
Serratia marcescens TLr156
Serratia marcescens T2000.
L-Threonin produzierende Stämme aus der Familie der Enterobacteriaceae besitzen bevorzugt, unter anderen, ein oder mehrere der genetischen bzw. phänotypischen Merkmale ausgewählt aus der Gruppe: Resistenz gegen α-Amino-β- Hydroxyvaleriansäure, Resistenz gegen Thialysin, Resistenz gegen Ethionin, Resistenz gegen α-Methylserin, Resistenz gegen Diaminobernsteinsäure, Resistenz gegen α- Aminobuttersäure, Resistenz gegen Borrelidin, Resistenz gegen Rifampicin, Resistenz gegen Valin-Analoga wie beispielsweise Valinhydroxamat, Resistenz gegen Purinanaloga wie beispielsweise 6-Dimethylaminopurin, Bedürftigkeit für L-Methionin, gegebenenfalls partielle und kompensierbare Bedürftigkeit für L-Isoleucin, Bedürftigkeit für meso-Diaminopimelinsäure, Auxotrophie bezüglich Threonin-haltiger Dipeptide, Resistenz gegen L-Threonin, Resistenz gegen L-Homoserin, Resistenz gegen L-Lysin, Resistenz gegen L-Methionin, Resistenz gegen L- Glutaminsäure, Resistenz gegen L-Aspartat, Resistenz gegen L-Leucin, Resistenz gegen L-Phenylalanin, Resistenz gegen L-Serin, Resistenz gegen L-Cystein, Resistenz gegen L- Valin, Empfindlichkeit gegenüber Fluoropyruvat, defekte Threonin-Dehydrogenase, gegebenenfalls Fähigkeit zur Saccharose-Verwertung, Verstärkung des Threonin-Operons, Verstärkung der Homoserin-Dehydrogenase I-Aspartatkinase I bevorzugt der feed back resistenten Form, Verstärkung der Homoserinkinase, Verstärkung der Threoninsynthase, Verstärkung der Aspartatkinase, gegebenenfalls der feed back resistenten Form, Verstärkung der Aspartatsemialdehyd- Dehydrogenase, Verstärkung der Phosphoenolpyruvat- Carboxylase, gegebenenfalls der feed back resistenten Form, Verstärkung der Phosphoenolpyruvat-Synthase, Verstärkung der Transhydrogenase, Verstärkung des RhtB-Genproduktes, Verstärkung des RhtC-Genproduktes, Verstärkung des YfiK- Genproduktes, Verstärkung einer Pyruvat-Carboxylase, und Abschwächung der Essigsäurebildung. L-threonine-producing strains from the Enterobacteriaceae preferably have, among others or more of the genetic or phenotypic traits selected from the group: resistance to α-amino-β- Hydroxyvaleric acid, resistance to thialysine, resistance against ethionine, resistance against α-methylserine, resistance against diamino succinic acid, resistance against α- Aminobutyric acid, resistance to borrelidine, resistance against rifampicin, resistance to valine analogues such as for example valine hydroxamate, resistance to Purine analogs such as 6-dimethylaminopurine, Need for L-methionine, partial and if necessary Compensable need for L-isoleucine, need for meso-diaminopimelic acid, auxotrophy related Dipeptides containing threonine, resistance to L-threonine, Resistance to L-homoserine, resistance to L-lysine, Resistance to L-methionine, resistance to L- Glutamic acid, resistance to L-aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, resistance to L- Valine, sensitivity to fluoropyruvate, defective Threonine dehydrogenase, optionally ability to Sucrose utilization, enhancement of the threonine operon, Enhancement of homoserine dehydrogenase I aspartate kinase I prefers the feed back resistant form, reinforcement of the Homoserine kinase, enhancement of threonine synthase, Enhancement of the aspartate kinase, possibly the feed back resistant form, reinforcement of aspartate semialdehyde Dehydrogenase, enhancement of phosphoenolpyruvate Carboxylase, optionally the feed-back-resistant form, Enhancement of phosphoenolpyruvate synthase, enhancement transhydrogenase, enhancement of the RhtB gene product, Enhancement of the RhtC gene product, enhancement of the YfiK Gene product, enhancement of a pyruvate carboxylase, and Attenuation of acetic acid formation.
Es wurde gefunden, dass Mikroorganismen der Familie Enterobacteriaceae nach Verstärkung, insbesondere Überexpression mindestens eines oder mehrere der Gene, ausgewählt aus der Gruppe lpd, aceE und aceF, in verbesserter Weise L-Aminosäuren, insbesondere L-Threonin produzieren. It has been found that family microorganisms Enterobacteriaceae after reinforcement, in particular Overexpression of at least one or more of the genes, selected from the group lpd, aceE and aceF, in improved way L-amino acids, especially L-threonine to produce.
Die Nukleotidsequenzen der Gene von Escherichia coli gehören zum Stand der Technik (siehe nachfolgende Textstellen) und können ebenfalls der von Blattner et al. (Science 277: 1453-1462 (1997)) publizierten Genomsequenz von Escherichia coli entnommen werden. The nucleotide sequences of the genes from Escherichia coli belong to the state of the art (see following Passages) and can also that of Blattner et al. (Science 277: 1453-1462 (1997)) published genome sequence from Escherichia coli.
Die Gene lpd, aceE und aceF werden unter anderem durch
folgende Angaben beschrieben:
lpd-Gen
Bezeichnung: Dihydrolipoamid-Dehydrogenase
(NADH-abhängig), Komponente der
2-Oxodehydrogenase und E3-Komponente des
Pyruvat-Dehydrogenase-Komplexes,
L-Protein des Glycin Spaltungskomplexes.
EC-Nr.: 1.8.1.4.
Referenz: Stephens et al.; European Journal of Bio-
Chemistry 135(3): 519-527 (1983)
Steiert et al.; Journal of Bacteriology
172: 6142-6144 (1990)
Accession No.: AE000121
Alternative Gennamen: dldH, lpdA
aceE-Gen
Bezeichnung: Pyruvat-Dehydrogenase, E1-Komponente des
Pyruvat-Dehydrogenase-Komplexes,
Decarboxylase-Komponente.
EC-Nr.: 1.2.4.1
Referenz: Stephens et al.; European Journal of
Biochemistry 133(1): 155-162 (1983)
Guest et al.; Annals of the New York
Academy of Sciences 573: 76-99 (1989)
Acession No.: AE000120
aceF-Gen
Bezeichnung: Dihydrolipoamid-Acetyltransferase,
E2 Komponente des Pyruvat-Dehydrogenase-
Komplexes.
EC-Nr.: 2.3.1.12
Referenz: Stephens et al.; European Journal of
Biochemistry 133 (3): 48: 1-489 (1983)
Guest et al.; Annals of the New York
Academy of Sciences 573: 76-99 (1989)
Acession No.: AE000120.
The genes lpd, aceE and aceF are described by the following information, among others:
lpd gene
Name: Dihydrolipoamide dehydrogenase (depending on NADH), component of 2-oxodehydrogenase and E3 component of the pyruvate dehydrogenase complex, L-protein of the glycine cleavage complex.
EC No .: 1.8.1.4.
Reference: Stephens et al .; European Journal of Bio-Chemistry 135 (3): 519-527 (1983) Steiert et al .; Journal of Bacteriology 172: 6142-6144 (1990)
Accession No .: AE000121
Alternative names: dldH, lpdA
AceE gene
Name: Pyruvate dehydrogenase, E1 component of the pyruvate dehydrogenase complex, decarboxylase component.
EC No .: 1.2.4.1
Reference: Stephens et al .; European Journal of Biochemistry 133 (1): 155-162 (1983) Guest et al .; Annals of the New York Academy of Sciences 573: 76-99 (1989)
Acession No .: AE000120
ACEF gene
Name: Dihydrolipoamide acetyltransferase, E2 component of the pyruvate dehydrogenase complex.
EC No .: 2.3.1.12
Reference: Stephens et al .; European Journal of Biochemistry 133 (3): 48: 1-489 (1983) Guest et al .; Annals of the New York Academy of Sciences 573: 76-99 (1989)
Acession No .: AE000120.
Die Nukleinsäuresequenzen können den Dagenbanken des National Center for Biotechnology Information (NCBI) der National Library of Medicine (Bethesda, MD, USA), der Nukleotidsequenz-Datenbank der European. Molecular Biologies Laboratories (EMBL, Heidelberg, Deutschland bzw. Cambridge, UK) oder der DNA Datenbank von Japan (DDBJ, Mishima, Japan) entnommen werden. The nucleic acid sequences can the Dagenbanken of National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA), the European nucleotide sequence database. Molecular Biologies Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK) or the DNA database of Japan (DDBJ, Mishima, Japan) be removed.
Die in den angegebenen Textstellen beschriebenen Gene können erfindungsgemäß verwendet werden. Weiterhin können Allele der Gene verwendet werden, die sich aus der Degeneriertheit des genetischen Codes oder durch funktionsneutrale Sinnmutationen ("sense mutations") ergeben. Die Verwendung endogener Gene wird bevorzugt. The genes described in the specified passages can be used according to the invention. Can continue Alleles of the genes that are derived from the Degeneracy of the genetic code or through Functionally neutral sense mutations result. The use of endogenous genes is preferred.
Unter "endogenen Genen" oder "endogenen Nukleotidsequenzen" versteht man die in der Population einer Art vorhandenen Gene oder Allele beziehungsweise Nukleotidsequenzen. Under "endogenous genes" or "endogenous nucleotide sequences" one understands those existing in the population of a species Genes or alleles or nucleotide sequences.
Zur Erzielung einer Verstärkung können beispielsweise die Expression der Gene oder die katalytischen Eigenschaften der Proteine erhöht werden. Gegebenenfalls können beide Maßnahmen kombiniert werden. To achieve reinforcement, for example Expression of the genes or the catalytic properties of the proteins are increased. If necessary, both can Measures are combined.
Zur Erzielung einer Überexpression kann die Kopienzahl der entsprechenden Gene erhöht werden, oder es kann die Promotor- und Regulationsregion oder die Ribosomenbindungsstelle, die sich stromaufwärts des Strukturgens befindet, mutiert werden. In gleicher Weise wirken Expressionskassetten, die stromaufwärts des Strukturgens eingebaut werden. Durch induzierbare Promotoren ist es zusätzlich möglich die Expression im Verlaufe der fermentativen L-Threonin-Produktion zu steigern. Durch Maßnahmen zur Verlängerung der Lebensdauer der m-RNA wird ebenfalls die Expression verbessert. To achieve overexpression, the number of copies of the corresponding genes can be increased, or it can Promoter and regulatory region or the Ribosome binding site located upstream of the Structural gene located to be mutated. In the same way expression cassettes act upstream of the Structural gene can be installed. By inducible It is also possible for promoters to express in History of fermentative L-threonine production increase. Through measures to extend the lifespan expression of the m-RNA is also improved.
Weiterhin wird durch Verhinderung des Abbaus des Enzymproteins ebenfalls die Enzymaktivität verstärkt. Die Gene oder Genkonstrukte können entweder in Plasmiden mit unterschiedlicher Kopienzahl vorliegen oder im Chromosom integriert und amplifiziert sein. Alternativ kann weiterhin eine Überexpression der betreffenden Gene durch Veränderung der Medienzusammensetzung und Kulturführung erreicht werden. Furthermore, by preventing the degradation of the Enzyme protein also increases enzyme activity. The Genes or gene constructs can either be found in plasmids different number of copies are present or in the chromosome be integrated and amplified. Alternatively, you can continue overexpression of the genes in question by change media composition and culture management become.
Anleitungen hierzu findet der Fachmann unter anderem bei Chang und Cohen (Journal of Bacteriology 134: 1141-1156 (1978)), bei Hartley und Gregori (Gene 13: 347-353 (1981)), bei Amann und Brosius (Gene 40: 183-190 (1985)), bei de Broer et al. (Proceedings of the National Academy of Sciences of the United States of America 80: 21-25 (1983)), bei LaVallie et al. (BIO/TECHNOLOGY 11: 187-193 (1993)), in PCT/US 97/13359, bei Llosa et al. (Plasmid 26: 222-224 (1991)), bei Quandt und Klipp (Gene 80: 161-169 (1989)), bei Hamilton et al. (Journal of Bacteriology 171: 4617-4622 (1989)), bei Jensen und Hammer (Biotechnology and Bioengineering 58: 191-195 (1998)) und in bekannten Lehrbüchern der Genetik und Molekularbiologie. The expert can find instructions on this among others Chang and Cohen (Journal of Bacteriology 134: 1141-1156 (1978)), by Hartley and Gregori (Gene 13: 347-353 (1981)), in Amann and Brosius (Gene 40: 183-190 (1985)), in de Broer et al. (Proceedings of the National Academy of Sciences of the United States of America 80: 21-25 (1983)), in LaVallie et al. (BIO / TECHNOLOGY 11: 187-193 (1993)), in PCT / US 97/13359, by Llosa et al. (Plasmid 26: 222-224 (1991)), by Quandt and Klipp (Gene 80: 161-169 (1989)), in Hamilton et al. (Journal of Bacteriology 171: 4617-4622 (1989)), with Jensen and Hammer (Biotechnology and Bioengineering 58: 191-195 (1998)) and in known ones Textbooks of genetics and molecular biology.
In Enterobacteriaceae replizierbare Plasmidvektoren wie z. B. von pACYC184 abgeleitete Kloniervektoren (Bartolomé et al.; Gene 102: 75-78 (1991)), pTrc99A (Amann et al.; Gene 69: 301-315 (1988)) oder pSC101-Derivate (Vocke und Bastia; Proceedings of the National Academy of Sciences USA 80(21): 6557-6561 (1983)) können verwendet werden. In einem erfindungsgemäßen Verfahren kann man einen mit einem Plasmidvektor transformierten Stamm einsetzen, wobei der Plasmidvektor mindestens eines oder mehrere der Gene ausgewählt aus der Gruppe lpd, aceE und. aceF, oder dafür kodierende Nukleotidsequenzen trägt. Plasmids vectors replicable in Enterobacteriaceae such as z. B. cloning vectors derived from pACYC184 (Bartolomé et al .; Gene 102: 75-78 (1991)), pTrc99A (Amann et al .; Gene 69: 301-315 (1988)) or pSC101 derivatives (Vocke and Bastia; Proceedings of the National Academy of Sciences USA 80 (21): 6557-6561 (1983)) can be used. In one The method according to the invention can be used with a Use plasmid vector transformed strain, the Plasmid vector of at least one or more of the genes selected from the group lpd, aceE and. face, or for that encoding nucleotide sequences.
Es ist ebenfalls möglich, Mutationen, die die Expression der jeweiligen Gene betreffen, durch Sequenzaustausch (Hamilton et al.; Journal of Bacteriology 171: 4617-4622 (1989)), Konjugation oder Transduktion in verschiedene Stämme zu überführen. It is also possible to mutate the expression of the relevant genes, by sequence exchange (Hamilton et al .; Journal of Bacteriology 171: 4617-4622 (1989)), conjugation or transduction into different To transfer tribes.
Weiterhin kann es für die Produktion von L-Aminosäuren, insbesondere L-Threonin mit Stämmen der Familie Enterobacteriaceae vorteilhaft sein, zusätzlich zur Verstärkung eines oder mehrere der Gene, ausgewählt aus der Gruppe lpd, aceE und aceF, ein oder mehrere Enzyme des bekannten Threonin-Biosyntheseweges oder Enzyme des anaplerotischen Stoffwechsels oder Enzyme für die Produktion von reduziertem Nicotinamid-Adenin-Dinukleotid- Phosphat oder Enzyme der Glykolyse oder PTS-Enzyme oder Enzyme des Schwefelstoffwechsels zu verstärken. Die Verwendung endogener Gene wird im allgemeinen bevorzugt. It can also be used for the production of L-amino acids, especially L-threonine with strains of the family Enterobacteriaceae may be beneficial in addition to Amplification of one or more of the genes selected from the Group lpd, aceE and aceF, one or more enzymes of the known threonine biosynthetic pathway or enzymes of anaplerotic metabolism or enzymes for the Production of reduced nicotinamide adenine dinucleotide Phosphate or enzymes of glycolysis or PTS enzymes or Enhance enzymes of sulfur metabolism. The Use of endogenous genes is generally preferred.
So können beispielsweise gleichzeitig eines oder mehrere
der Gene, ausgewählt aus der Gruppe
das für die Aspartatkinase, die Homoserin-Dehydrogenase,
die Homoserinkinase und die Threoninsynthase kodierende
thrABC-Operon (US-A-4,278,765);
das für die Pyruvat-Carboxylase kodierende pyc-Gen (DE-
A-198 31 609);
das für die Phosphoenolpyruvat-Synthase kodierende pps-
Gen (Molecular and General Genetics 231(2): 332-336
(1992));
das für die Phosphoenolpyruvat-Carboxylase kodierende
ppc-Gen (Gene 31: 279-283 (1984));
die für die Transhydrogenase kodierenden Gene pntA und
pntB (European Journal of Biochemistry 158: 647-653
(1986));
das Homoserinresistenz vermittelnde Gen rhtB
(EP-A-0 994 190);
das für die Malat:Chinon Oxidoreduktase kodierende mqo-
Gen (DE 100 34 833.5);
das Threoninresistenz vermittelnde Gen rhtC
(EP-A-1 013 765);
das für den Threoninexport-Protein kodierende thrE-Gen
von Corynebacterium glutamicum (DE 100 26 494.8);
das für die Glutamat-Dehydrogenase kodierende gdhA-Gen
(Nucleic Acids Research 11: 5257-5266 (1983); Gene 23:
199-209 (1983));
das für das DNA-Bindeprotein HLP-II kodierende hns-Gen
(Molecular and General Genetics 212: 199-202 (1988));
das für die Phosphoglucomutase kodierende pgm-Gen
(Journal of Bacteriology 176: 5847-5851 (1994));
das für die Fructose Biphosphat Aldolase kodierende fba-
Gen (Biochemical Journal 257: 529-534 (1989));
das für die Phosphohistidin-Protein-Hexose-
Phosphotransferase des Phosphotransferase-Systems PTS
kodierende ptsH-Gen des ptsHIcrr-Operons (Journal of
Biological Chemistry 262: 16241-1625 3 (1987));
das für das Enzym I des Phosphotransferase-Systems PTS
kodierende ptsI-Gen des ptsHIcrr-Operons (Journal of
Biological Chemistry 262: 16241-1625 3 (1987));
das für die Glucose-spezifische IIA Komponente des
Phosphotransferase-Systems PTS kodierende crr-Gen des
ptsHIcrr-Operons (Journal of Biological Chemistry 262:
16241-16253 (1987));
das für die Glucose-spezifische IIBC Komponente
kodierende ptsG-Gen (Journal of Biological Chemistry
261: 16398-16403 (1986));
das für den Regulator des Leucin-Regulons kodierende
lrp-Gen (Journal of Biological Chemistry 266:
10768-10774 (1991));
das für den globalen Regulator Csr kodierende csrA-Gen
(Journal of Bacteriology 175: 4744-4755 (1993));
das für den Regulator des fad-Regulons kodierende fadR-
Gen (Nucleic Acids Research 16: 7995-8009 (1988));
das für den Regulator des zentralen
Intermediärstoffwechsels kodierende iclR-Gen (Journal of
Bacteriology 172: 2642-2649 (1990));
das für das 10 Kd Chaperon kodierende mopB-Gen (Journal
of Biological Chemistry 261: 12414-12419 (1986)), das
auch unter der Bezeichnung groES bekannt ist;
das für die kleine Untereinheit der Alkyl Hydroperoxid
Reduktase kodierende ahpC-Gen des ahpCF-Operons
(Proceedings of the National Academy of Sciences USA 92:
7617-7621 (1995));
das für die große Untereinheit der Alkyl Hydroperoxid
Reduktase kodierende ahpF-Gen des ahpCF-Operons
(Proceedings of the National Academy of Sciences USA 92:
7617-7621 (1995));
das für die Cystein-Synthase A kodierende cysK-Gen
(Journal of Bacteriology 170: 3150-3157 (1988));
das für den Regulator des cys-Regulons kodierende cysB-
Gen (Journal of Biological Chemistry 262: 5999-6005
(1987));
das für das Flavoprotein der NADPH-Sulfit-Reduktase
kodierende cysJ-Gen des cysJIH-Operons (Journal of
Biological Chemistry 264: 15796-15806 (1989), Journal of
Biological Chemistry 264: 15726-15737 (1989));
das für das Hämoprotein der NADPH-Sulfit-Reduktase
kodierende cysI-Gen des cysJIH-Operons (Journal of
Biological Chemistry 264: 15796-15808 (1989), Journal of
Biological Chemistry 264: 15726-15731 (1989)) und;
das für die Adenylylsulfat-Reduktase kodierende cysH-Gen
des cysJIH-Operons (Journal of Biological Chemistry 264:
15796-15808 (1989), Journal of Biological Chemistry 264:
15726-15737 (1989));
verstärkt, insbesondere überexprimiert werden.
For example, one or more of the genes selected from the group can be used simultaneously
thrABC-operon (US-A-4,278,765) encoding aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase;
the pyc gene coding for the pyruvate carboxylase (DE-A-198 31 609);
the pps gene coding for phosphoenol pyruvate synthase (Molecular and General Genetics 231 (2): 332-336 (1992));
the ppc gene encoding the phosphoenolpyruvate carboxylase (Gene 31: 279-283 (1984));
the genes pntA and pntB coding for the transhydrogenase (European Journal of Biochemistry 158: 647-653 (1986));
the homoserine resistance-mediating gene rhtB (EP-A-0 994 190);
the mqo gene coding for the malate: quinone oxidoreductase (DE 100 34 833.5);
the threonine resistance-mediating gene rhtC (EP-A-1 013 765);
the thrE gene from Corynebacterium glutamicum coding for the threonine export protein (DE 100 26 494.8);
the gdhA gene encoding glutamate dehydrogenase (Nucleic Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983));
the hns gene coding for the DNA binding protein HLP-II (Molecular and General Genetics 212: 199-202 (1988));
the pgm gene encoding the phosphoglucomutase (Journal of Bacteriology 176: 5847-5851 (1994));
the fba gene coding for the fructose biphosphate aldolase (Biochemical Journal 257: 529-534 (1989));
the ptsH gene of the ptsHIcrr operon coding for the phosphohistidine protein hexose phosphotransferase of the phosphotransferase system PTS (Journal of Biological Chemistry 262: 16241-1625 3 (1987));
the ptsI gene of the ptsHIcrr operon coding for the enzyme I of the phosphotransferase system PTS (Journal of Biological Chemistry 262: 16241-1625 3 (1987));
the crr gene of the ptsHIcrr operon coding for the glucose-specific IIA component of the phosphotransferase system PTS (Journal of Biological Chemistry 262: 16241-16253 (1987));
the ptsG gene coding for the glucose-specific IIBC component (Journal of Biological Chemistry 261: 16398-16403 (1986));
the lrp gene coding for the regulator of the leucine regulon (Journal of Biological Chemistry 266: 10768-10774 (1991));
the csrA gene coding for the global regulator Csr (Journal of Bacteriology 175: 4744-4755 (1993));
the fadR gene coding for the regulator of the fad regulon (Nucleic Acids Research 16: 7995-8009 (1988));
the iclR gene coding for the regulator of the central intermediate metabolism (Journal of Bacteriology 172: 2642-2649 (1990));
the mopB gene coding for the 10 Kd chaperone (Journal of Biological Chemistry 261: 12414-12419 (1986)), which is also known under the name groES;
the ahpC gene of the ahpCF operon coding for the small subunit of the alkyl hydroperoxide reductase (Proceedings of the National Academy of Sciences USA 92: 7617-7621 (1995));
the ahpF gene of the ahpCF operon coding for the large subunit of the alkyl hydroperoxide reductase (Proceedings of the National Academy of Sciences USA 92: 7617-7621 (1995));
the cysK gene encoding cysteine synthase A (Journal of Bacteriology 170: 3150-3157 (1988));
the cysB gene coding for the regulator of the cys regulon (Journal of Biological Chemistry 262: 5999-6005 (1987));
the cysJ gene of the cysJIH operon encoding the NADPH sulfite reductase flavoprotein (Journal of Biological Chemistry 264: 15796-15806 (1989), Journal of Biological Chemistry 264: 15726-15737 (1989));
the cysJIH operon cysI gene encoding the NADPH sulfite reductase hemoprotein (Journal of Biological Chemistry 264: 15796-15808 (1989), Journal of Biological Chemistry 264: 15726-15731 (1989)) and;
the cysH gene of the cysJIH operon encoding the adenylyl sulfate reductase (Journal of Biological Chemistry 264: 15796-15808 (1989), Journal of Biological Chemistry 264: 15726-15737 (1989));
amplified, especially overexpressed.
Weiterhin kann es für die Produktion von L-Aminosäuren,
insbesondere L-Threonin vorteilhaft sein, zusätzlich zur
Verstärkung eines oder mehrere der Gene, ausgewählt aus der
Gruppe lpd, aceE und aceF, eines oder mehrere der Gene
ausgewählt aus der Gruppe
das für die Threonin-Dehydrogenase kodierende tdh-Gen
(Journal of Bacteriology 169: 4716-4721 (1987));
das für die Malat-Dehydrogenase (E.C. 1.1.1.37)
kodierende mdh-Gen (Archives in Microbiology 149: 36-42
(1987));
das Genprodukt des offenen Leserahmens (orf) yjfA
(Accession Number AAC77180 des National Center for
Biotechnology Information (NCBI, Bethesda, MD, USA));
das Genprodukt des offenen Leserahmens (orf) ytfP
(Accession Number AAC77179 des National Center for
Biotechnology Information (NCBI, Bethesda, Na, USA));
das für das Enzym Phosphoenolpyruvat-Carboxykinase
kodierende pckA-Gen (Journal of Bacteriology 172:
7151-7156 (1990));
das für die Pyruvat-Oxidase kodierende poxB-Gen (Nucleic
Acids Research 14 (13): 5449-5460 (1986));
das für das Enzym Isocitrat-Lyase kodierende aceA-Gen
(Journal of Bacteriology 170: 4528-4536 (1988));
das für den DgsA-Regulator des Phosphotransferase-
Systems kodierende dgsA-Gen (Bioscience, Biotechnology
and Biochemistry 59: 256-251 (1995)), das auch unter der
Bezeichnung mlc-Gen bekannt ist;
das für den Fructose-Repressor kodierende fruR-Gen
(Molecular and General Genetics 226: 332-336 (1991));
das auch unter der Bezeichnung cra-Gen bekannt ist, und
das für den Sigma38-Faktor kodierende rpoS-Gen (WO 01/05939),
das auch unter der Bezeichnung katF-Gen
bekannt ist;
abzuschwächen, insbesondere auszuschalten oder die
Expression zu verringern.
Furthermore, it can be advantageous for the production of L-amino acids, in particular L-threonine, in addition to amplifying one or more of the genes selected from the group lpd, aceE and aceF, one or more of the genes selected from the group
the tdh gene encoding threonine dehydrogenase (Journal of Bacteriology 169: 4716-4721 (1987));
the mdh gene coding for malate dehydrogenase (EC 1.1.1.37) (Archives in Microbiology 149: 36-42 (1987));
the gene product of the open reading frame (orf) yjfA (Accession Number AAC77180 of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA));
the gene product of the open reading framework (orf) ytfP (Accession Number AAC77179 of the National Center for Biotechnology Information (NCBI, Bethesda, Na, USA));
the pckA gene encoding the enzyme phosphoenolpyruvate carboxykinase (Journal of Bacteriology 172: 7151-7156 (1990));
the poxB gene encoding pyruvate oxidase (Nucleic Acids Research 14 (13): 5449-5460 (1986));
the aceA gene coding for the enzyme isocitrate lyase (Journal of Bacteriology 170: 4528-4536 (1988));
the dgsA gene coding for the DgsA regulator of the phosphotransferase system (Bioscience, Biotechnology and Biochemistry 59: 256-251 (1995)), which is also known under the name mlc gene;
the fruR gene encoding the fructose repressor (Molecular and General Genetics 226: 332-336 (1991)); which is also known as the cra gene, and
the rpoS gene coding for the Sigma 38 factor (WO 01/05939), which is also known under the name katF gene;
weaken, in particular switch off or reduce expression.
Der Begriff "Abschwächung" beschreibt in diesem Zusammenhang die Verringerung oder Ausschaltung der intrazellulären Aktivität eines oder mehrerer Enzyme (Proteine) in einem Mikroorganismus, die durch die entsprechende DNA kodiert werden, indem man beispielsweise einen schwachen Promotor oder ein Gen bzw. Allel verwendet, das für ein entsprechendes Enzym mit einer niedrigen Aktivität kodiert bzw. das entsprechende Enzym (Protein) oder Gen inaktiviert und gegebenenfalls diese Maßnahmen kombiniert. The term "weakening" describes in this Related to reducing or eliminating the intracellular activity of one or more enzymes (Proteins) in a microorganism caused by the corresponding DNA can be encoded, for example by uses a weak promoter or a gene or allele, that for a corresponding enzyme with a low Activity coded or the corresponding enzyme (protein) or gene inactivated and, if necessary, 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, herabgesenkt. Through the mitigation measures, the activity or concentration of the corresponding protein in the general to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type Protein, or the activity or concentration of the protein in the starting microorganism.
Weiterhin kann es für die Produktion von L-Aminosäuren, insbesondere L-Threonin vorteilhaft sein, zusätzlich zur Verstärkung eines oder mehrere der Gene, ausgewählt aus der Gruppe lpd, aceE und aceF, unerwünschte Nebenreaktionen auszuschalten (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). It can also be used for the production of L-amino acids, L-threonine in particular may be advantageous in addition to Amplification of one or more of the genes selected from the Group lpd, aceE and aceF, undesirable 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 können im batch-Verfahren (Satzkultivierung), im fed batch (Zulaufverfahren) oder im repeated fed batch-Verfahren (repetitives Zulaufverfahren) kultiviert werden. Eine Zusammenfassung über bekannte Kultivierungsmethoden sind 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 microorganisms produced according to the invention can in batch process (batch cultivation), in fed batch (Feed process) or in the repeated fed batch process (repetitive feed process) can be cultivated. A Summary of known cultivation methods are in the Textbook by Chmiel (Bioprocess Engineering 1. Introduction to Bioprocess engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (bioreactors and peripheral facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).
Das zu verwendende Kulturmedium muss in geeigneter Weise den Ansprüchen der jeweiligen Stämme genügen. The culture medium to be used must be suitable meet the requirements of the respective tribes.
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. Descriptions of cultural media from various Microorganisms are described in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981) included.
Als Kohlenstoffquelle können Zucker und Kohlehydrate wie z. B. Glucose, Saccharose, Lactose, Fructose, Maltose, Melasse, Stärke und gegebenenfalls 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. Sugar and carbohydrates such as z. B. glucose, sucrose, lactose, fructose, maltose, Molasses, starch and possibly cellulose, oils and fats such as B. soybean oil, sunflower oil, peanut oil and coconut oil, Fatty acids such as B. palmitic acid, stearic acid and Linoleic acid, alcohols such as B. glycerin and ethanol and organic acids such as B. acetic acid can be used. These substances can be used individually or as a mixture become.
Als Stickstoffquelle können organische Stickstoffhaltige 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. Organic nitrogenous substances can be used as the nitrogen source 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 be used individually 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 are used. The culture medium must continue to salts of metals included, such as B. magnesium sulfate or iron sulfate, the are necessary for growth. Finally, you can essential growth substances such as amino acids and vitamins in addition to the substances mentioned above. Suitable precursors can also be used in the culture medium be added. The feedstocks mentioned can be used for Culture added in the form of a unique approach or appropriately fed during cultivation become.
Die Fermentation wird im allgemeinen bei einem pH-Wert von 5,5 bis 9,0, insbesondere 6,0 bis 8,0, durchgeführt. 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 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 25°C bis 45°C und vorzugsweise bei 30°C bis 40°C. Die Kultur wird solange fortgesetzt, bis sich ein Maximum an L- Aminosäuren bzw. L-Threonin gebildet hat. Dieses Ziel wird normalerweise innerhalb von 10 Stunden bis 160 Stunden erreicht. The fermentation is generally at a pH of 5.5 to 9.0, in particular 6.0 to 8.0. to pH control of the culture will be basic compounds like Sodium hydroxide, potassium hydroxide, ammonia or Ammonia water or acidic compounds such as phosphoric acid or sulfuric acid used in a suitable manner. to Antifoam agents such as z. B. fatty acid polyglycol esters. to Maintaining the stability of plasmids can do that Medium suitable selectively acting substances such. B. Antibiotics to be added. To aerobic conditions will maintain oxygen or Oxygen-containing gas mixtures such as B. Air into culture entered. The temperature of the culture is usually at 25 ° C to 45 ° C and preferably at 30 ° C to 40 ° C. The Culture continues until a maximum of L- Has formed amino acids or L-threonine. That goal will usually within 10 hours to 160 hours reached.
Die Analyse von L-Aminosäuren kann durch Anionenaustauschchromatographie mit anschließender Ninhydrin Derivatisierung erfolgen, so wie bei Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)) beschrieben, oder sie kann durch reversed phase HPLC erfolgen, so wie bei Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)) beschrieben. The analysis of L-amino acids can be done by Anion exchange chromatography with subsequent Ninhydrin derivatization take place, as in Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)) described, or it can by reversed phase HPLC take place, as in Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)).
Das erfindungsgemäße Verfahren dient zur fermentativen Herstellung von L-Aminosäuren, wie beispielsweise L- Threonin, L-Isoleucin, L-Valin, L-Methionin, L-Homoserin und L-Lysin, insbesondere L-Threonin. The method according to the invention is used for fermentative purposes Production of L-amino acids, such as L- Threonine, L-isoleucine, L-valine, L-methionine, L-homoserine and L-lysine, especially L-threonine.
Claims (8)
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10210962A DE10210962A1 (en) | 2002-03-13 | 2002-03-13 | Preparation of amino acids, particularly threonine useful e.g. in animal nutrition, by growing Enterobacteriaceae having increased activity of e.g. lpd, aceE or aceF genes |
| EP03708150A EP1483393A1 (en) | 2002-03-13 | 2003-02-27 | Process for the preparation of l-amino acids using strains of the family enterobacteriaceae |
| PCT/EP2003/001992 WO2003076635A1 (en) | 2002-03-13 | 2003-02-27 | Process for the preparation of l-amino acids using strains of the family enterobacteriaceae |
| US10/937,598 US20050095688A1 (en) | 2002-03-13 | 2004-09-10 | Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10210962A DE10210962A1 (en) | 2002-03-13 | 2002-03-13 | Preparation of amino acids, particularly threonine useful e.g. in animal nutrition, by growing Enterobacteriaceae having increased activity of e.g. lpd, aceE or aceF genes |
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| Publication Number | Publication Date |
|---|---|
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Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE10210962A1 (en) |
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