DE10135053A1 - Preparing L-amino acids, e.g. L-..threonine by fermenting microorganisms of Enterobactericeae family in which at least the malE gene is enhanced, in particular overexpressed, and isolating the desired amino acid - Google Patents

Preparing L-amino acids, e.g. L-..threonine by fermenting microorganisms of Enterobactericeae family in which at least the malE gene is enhanced, in particular overexpressed, and isolating the desired amino acid

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DE10135053A1
DE10135053A1 DE2001135053 DE10135053A DE10135053A1 DE 10135053 A1 DE10135053 A1 DE 10135053A1 DE 2001135053 DE2001135053 DE 2001135053 DE 10135053 A DE10135053 A DE 10135053A DE 10135053 A1 DE10135053 A1 DE 10135053A1
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threonine
gene coding
coding
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Mechthild Rieping
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Evonik Operations GmbH
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Degussa GmbH
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Priority to DE2001135053 priority Critical patent/DE10135053A1/en
Priority to DK09156036.7T priority patent/DK2083080T3/en
Priority to US10/484,162 priority patent/US7332309B2/en
Priority to DK02743259T priority patent/DK1407027T3/en
Priority to DK02743258T priority patent/DK1407022T3/en
Priority to DE60232725T priority patent/DE60232725D1/en
Priority to AU2002354852A priority patent/AU2002354852A1/en
Priority to AU2002325865A priority patent/AU2002325865A1/en
Priority to PCT/EP2002/007356 priority patent/WO2003008607A2/en
Priority to PCT/EP2002/007355 priority patent/WO2003008606A2/en
Priority to AU2002354853A priority patent/AU2002354853A1/en
Priority to DE60232726T priority patent/DE60232726D1/en
Priority to PCT/EP2002/007368 priority patent/WO2003008610A2/en
Priority to DE60226239T priority patent/DE60226239T2/en
Priority to EP02787110A priority patent/EP1407026B1/en
Priority to AT02787110T priority patent/ATE407210T1/en
Priority to EP09156036.7A priority patent/EP2083080B1/en
Priority to US10/484,198 priority patent/US20050059124A1/en
Priority to AT02758304T priority patent/ATE434664T1/en
Priority to DE60233573T priority patent/DE60233573D1/en
Priority to AU2002319281A priority patent/AU2002319281A1/en
Priority to AU2002345084A priority patent/AU2002345084A1/en
Priority to US10/483,417 priority patent/US20040241814A1/en
Priority to ES02758304T priority patent/ES2328230T3/en
Priority to AU2002325294A priority patent/AU2002325294A1/en
Priority to PCT/EP2002/007373 priority patent/WO2003008613A2/en
Priority to EP02743259A priority patent/EP1407027B1/en
Priority to EP02758304A priority patent/EP1407028B1/en
Priority to PCT/EP2002/007369 priority patent/WO2003008611A2/en
Priority to AT02743258T priority patent/ATE441714T1/en
Priority to EP02751102A priority patent/EP1407024B1/en
Priority to US10/483,416 priority patent/US20050170472A1/en
Priority to DE60228715T priority patent/DE60228715D1/en
Priority to PCT/EP2002/007366 priority patent/WO2003008608A2/en
Priority to EP02743258A priority patent/EP1407022B1/en
Priority to DK02758304T priority patent/DK1407028T3/en
Priority to AT02751102T priority patent/ATE386121T1/en
Priority to PCT/EP2002/007374 priority patent/WO2003008614A2/en
Priority to AU2002345083A priority patent/AU2002345083A1/en
Priority to AT02758301T priority patent/ATE393226T1/en
Priority to AU2002321165A priority patent/AU2002321165A1/en
Priority to DE60225011T priority patent/DE60225011T2/en
Priority to PCT/EP2002/007367 priority patent/WO2003008609A2/en
Priority to PCT/EP2002/007354 priority patent/WO2003008605A2/en
Priority to EP02758301A priority patent/EP1407025B1/en
Priority to AU2002354854A priority patent/AU2002354854A1/en
Priority to AU2002325293A priority patent/AU2002325293A1/en
Priority to AT02743259T priority patent/ATE434663T1/en
Priority to PCT/EP2002/007370 priority patent/WO2003008612A2/en
Priority to AU2002354855A priority patent/AU2002354855A1/en
Priority to ES02743259T priority patent/ES2328229T3/en
Priority to PCT/EP2002/007375 priority patent/WO2003008615A2/en
Publication of DE10135053A1 publication Critical patent/DE10135053A1/en
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

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Abstract

Preparing (M1) L-amino acids, in particular L-threonine (L-Thr) by fermenting microorganisms of Enterobacteriaceae family which produce L-Thr and in which at least malE gene or nucleotide sequence which codes for this, is enhanced, in particular over-expressed; concentrating L-Thr in medium or in cells of microorganism; isolating L-Thr, constituents of fermentation broth and/or biomass wholly or partly, optionally remaining in product, is new.

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 malE, phoA, phoB, phoR, phoE, phnC, phnD phnE, phnF, phnG, phnJ, phnK, phhL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, 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 malE, phoA, phoB, phoR, phoE, phnC, phnD phnE, phnF, phnG, phnJ, phnK, phhL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, 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 L-Aminosäuren durch Fermentation von Stämmen der Enterobacteriaceae, insbesondere Escherichia coli (E. coli) und Serratia marcescens, herzustellen. 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 from fermentation of strains the Enterobacteriaceae, especially Escherichia coli (E. coli) and Serratia marcescens. Because of the great importance is constantly attached to the improvement of 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.

Aufgabe der ErfindungObject of the invention

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) malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, 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 malE, phoA, phoB, coding for the genes, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, 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 von Mikroorganismen der Familie Enterobacteriaceae, in denen eines oder mehrere der Gene, ausgewählt aus der Gruppe malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, verstärkt wird (werden),
  • b) Anreicherung der entsprechenden L-Aminosäure im Medium oder in den Zellen der Mikroorganismen der Familie Enterobacteriaceae, 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.
The process is characterized in that the following steps are carried out:
  • a) Fermentation of microorganisms of the Enterobacteriaceae family in which one or more of the genes selected from the group malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN , phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseApGC , sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, is (will) be strengthened,
  • b) enrichment of the corresponding L-amino acid in the medium or in the cells of the microorganisms of the Enterobacteriaceae family, and
  • c) Isolation of the desired L-amino acid, where appropriate components of the fermentation broth and / or the biomass in their entirety or in portions (> 0 to 100%) thereof remain in the product.

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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, in improved way L-amino acids, especially L-threonine to produce.

Die Nukleotidsequenzen der Gene von Escherichia coli gehören zum Stand der Technik und können ebenfalls der von Blattner et al. (Science 277, 1453-1462 (1997)) publizierten Genomsequenz von Escherichia coli entnommen werden.
malE-Gen:
Bezeichnung: Periplasmatisches Maltose-Bindeprotein
Referenz: Duplay et al., Journal of Biological Chemistry 259(16): 10606-13 (1984)
Accession No.: AE000476
Alternativer Genname: malB
phoA-Gen:
Bezeichnung: Alkalische Phosphatase
EC-Nr.: 3.1.3.1
Referenz: Berg; Journal of Bacteriology 146(2), 660-7 (1981)
Accession No.: AE000145
phoB-Gen:
Bezeichnung: Positiver Regulator des pho Regulons, Zwei- Komponenten System
Referenz: Makino et al.; Journal of Molecular Biology 190(1), 37-44 (1986)
Accession No.: AE000146
Alternative Gennamen: phoRc, phoT
phoR-Gen:
Bezeichnung: Positives und negatives Sensorprotein des pho Regulons, Sensor des Zwei-Komponenten Systems
EC-Nr.: 2.7.3.-
Referenz: Makino et al.; Journal of Molecular Biology 192(3), 549-56 (1986)
Accession No.: AE000146
Alternative Gennamen: R1pho, nmpB, phoR1
phoE-Gen:
Bezeichnung: Protein der äusseren Zellmembran E (E, Ic, NmpAB)
Referenz; Overbeeke et al.; Journal of Molecular Biology 163(4), 513-32 (1983)
Accession No.: AE000132
Alternativer Genname: ompE
phnC-Gen:
Bezeichnung; ATP-Bindedomäne des Phosphonat Transporters
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnD-Gen:
Bezeichnung: Periplasmatisches Bindeprotein des Phosphonat Transporters
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
Alternativer Genname: psiD
phnE-Gen:
Bezeichnung: Integrale Membrankomponente des Phosphonat Transporters
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnF-Gen:
Bezeichnung: Putativer Regulator des Phosphonat Transporters
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnG-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnJ-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnK-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnL-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnM-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnN-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnO-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
phnP-Gen:
Bezeichnung: Kohlenstoff-Phosphor Lyase Komplex Untereinheit (Phosphonat Metabolismus)
Referenz: Makino et al. Journal of Bacteriology, 173(8), 2665-12 (1991)
Accession No.: AE000482
pykF-Gen:
Bezeichnung: Pyruvat Kinase I (ehemals F), Fructose stimuliert
EC-Nr.: 2.7.1.40
Referenz: Ponce et al.; Journal of Bacteriology 177(19): 5719-22 (1995)
Accession No.: AE000262
pfkB-Gen:
Bezeichnung: 6-Phosphofructokinase II; Suppressor von pfkA
Referenz: Daldal; Gene 28(3), 337-42 (1984)
Accession No.: AE000267
eda-Gen:
Bezeichnung: 2-Keto-3-Desoxygluconat 6-Phosphat Aldolase und 2-Keto-4-Hydroxyglutarat Aldolase (Entner-Doudoroff Aldolase)
EC-Nr.: 4.1.2.14 4.1.3.16
Referenz: Carter et al.; Gene 130(1), 155-6 (1993)
Accession No.: AE000279
Alternative Gennamen: kga, kdgA
talB-Gen:
Bezeichnung: Transaldolase B
EC-Nr.: 2.2.1.2
Referenz: Sprenger et al.; Journal of Bacteriology 177(20), 5930-6 (1995)
Accession No.: AE000111
rpiB-Gen:
Bezeichnung: Ribose 5-Phosphat Isomerase B
Referenz: Sorensen und Hove-Jensen; Journal of Bacteriology 178(4), 1003-11 (1996)
Accession No.: AE000482
zwf-Gen:
Bezeichnung: Glucose-6-Phosphat Dehydrogenase (Zwischenferment)
EC-Nr.: 1.1.1.49
Referenz: Rowley und Wolf; Journal of Bacteriology 173(3), 968-77 (1991)
Accession No.: AE000279
mopA-Gen:
Bezeichnung: GroEL, Chaperon Hsp60, Peptid-abhängig
Referenz: Chandrasekhar et al.; Journal of Biological Chemistry 261(26), 12414-9 (1986)
Accession No.: AE000487
Alternative Gennamen: groE, groEL, hdh, tabB; 60 kD chaperonin (CPN60)
pstA-Gen:
Bezeichnung: Hoch-affines Phosphat Transport System
Referenz: Surin et al.; Journal of Bacteriology 161(1), 189-98 (1985)
Amemura et al.; Journal of Molecular Biology 184(2), 241-50 (1985)
Accession No.: AE000449
Alternative Gennamen: R2pho, phoR2b, phoT
pstB-Gen:
Bezeichnung: ATP-Bindekomponente des hoch-affinen Phosphat Transport Systems
Referenz: Surin et al.; Journal of Bacteriology 161(1), 189-98 (1985)
Amemura et al.; Journal of Molecular Biology 184(2), 241-50 (1985)
Accession No.: AE000449
Alternativer Genname: phoT
pstC-Gen:
Bezeichnung: Hoch-affines Phosphat Transport System, Cytoplasmamembran Komponente
Referenz: Surin et al.; Journal of Bacteriology 161(1), 189-98 (1985)
Accession No.: AE000449
Alternativer Genname: phoT
pstS-Gen:
Bezeichnung: Hoch-affines Phosphat Transport System; Periplasmatisches Phosphatbindeprotein
Referenz: Surin et al., Journal of Bacteriology 157, 772-8 (1984);
Magota et al., Journal of Bacteriology 157, 909-17 (1984)
Accession No.: AE000449
Alternative Gennamen: R2pho, nmpA, phoR2a, phoS
ugpB-Gen:
Bezeichnung: sn-Glycerin-3-Phosphat Transport System; Periplasmatisches Bindeprotein
Referenz: Overduin et al.; Molecular Microbiology 2(6), 767-75 (1988)
Accession No.: AE000421
Alternative Gennamen: psiB, psiC,
ugpA-Gen:
Bezeichnung: sn-Glycerin-3-Phosphat Transport System, Integrales Membran Protein
Referenz: Overduin et al.; Molecular Microbiology 2(6), 767-75 (1988)
Accession No.: AE000421
Alternative Gennamen: psiB, psiC
ugpE-Gen:
Bezeichnung: sn-Glycerin-3-Phosphat Transport System, Integrales Membran Protein
Referenz: Overduin et al.; Molecular Microbiology 2(6), 767-75 (1988)
Accession No.: AE000421
ugpC-Gen:
Bezeichnung: ATP-Bindekomponente des sn-Glycerin-3- Phosphat Transport Systems
Referenz: Overduin et al.; Molecular Microbiology 2(6), 767-75 (1988)
Accession No.: AE000421
ugpQ-Gen:
Bezeichnung: Glycerinphosphodiester Phosphodiesterase, cytosolisch
EC-Nr.: 3.1.4.46
Referenz: Kasahara et al.; Nucleic Acids Research 17(7), 2854 (1989)
Accession No.: AE000421
dnaK-Gen:
Bezeichnung: Chaperon Hsp70; DNA Biosynthese;
autoreguliertes Hitzeschockprotein
Referenz: Bardwell und Craig; Proceedings of the National Academy of Sciences of the USA 81(3), 848-52 (1984)
Accession No.: AE000112
Alternative Gennamen: gro, groP, groPAB, groPC, groPF, grpC, grpF, seg
dnaJ-Gen:
Bezeichnung: Chaperon; Hitzeschockprotein
Referenz: Ohki et al.; Journal of Biological Chemistry 261(4), 1778-81 (1986)
Accession No.: AE000112
Alternative Gennamen: groP, grpC
clpB-Gen:
Bezeichnung: Hitzeschockprotein (caseinolytische Protease)
EC-Nr.: 1.17.4.-3.4.21.-
Referenz: Kitagawa et al.; Journal of Bacteriology 173(14), 4247-53 (1991)
Accession No.: AE000345
rpoE-Gen:
Bezeichnung: RNA Polymerase, Sigma-E Faktor; Hitzeschock und oxidativer Stress
Referenz: Raina et al.: EMBO Journal 14 (5), 1043-55 (1995)
Accession No.: AE000343
rseA-Gen:
Bezeichnung: sigma-E Faktor, negativer Regulator (Membranprotein Regulator der σE Aktivität)
Referenz: Missiakas et al.; Molecular Microbiology 24(2), 355-71 (1997)
Accession No.: AE000343
Alternative Gennamen: mclA
rseC-Gen:
Bezeichnung: sigma-E Faktor, globaler Regulator
Referenz: Missiakas et al.; Molecular Microbiology 24(2), 355-71 (1997)
Accession No.: AE000343
htpG-Gen:
Bezeichnung: Chaperon Hsp90, Hitzeschockprotein C 62.5
Referenz: Spence und Georgopoulos; Journal of Biological Chemistry 264(8), 4398-403 (1989)
Accession No.: AE000153
sodA-Gen:
Bezeichnung: Superoxiddismutase
Referenz: Touati; Journal of Bacteriology 155(3): 078-87 (1983)
Accession No.: AE000465
ompF-Gen:
Bezeichnung: Äusseres Membranprotein 1a (Ia; b; F)
Referenz: Inokuchi et al.; Nucleic Acids Research 10(21), 6957-68 (1982)
Accession No.: AE000195
Alternative Gennamen: cmlB, coa, cry, tolF
ompC-Gen:
Bezeichnung: Äusseres Membranprotein 1b (Ib; c)
Referenz: Mizuno et al.; Journal of Biological Chemistry 258(11), 6932-40 (1983)
Accession No.: AE000310
Alternative Gennamen: meoR, par
sucA-Gen:
Bezeichnung: 2-Ketoglutarat Dehydrogenase (Decarboxylase Untereinheit)
EC-Nr.: 1.2.4.2
Referenz: Darlison et al.; European Journal of Biochemistry 141(2), 351-9 (1984)
Accession No.: AE000175
Alternative Gennamen: lys, met
sucB-Gen:
Bezeichnung: 2-Ketoglutarat Dehydrogenase (Dihydrolipoyltranssuccinase E2 Untereinheit)
EC-Nr.: 2.3.1.61
Referenz: Spencer et al.; European Journal of Biochemistry 141(2), 361-74 (1984)
Accession No.: AE000175
Alternative Gennamen: lys, met
sucC-Gen:
Bezeichnung: Succinyl-CoA Synthetase, β-Untereinheit
EC-Nr. 6.2.1.5
Referenz: Buck et al.; Biochemistry 24(22), 6245-52 (1985)
Accession No.: AE000176
sucD-Gen:
Bezeichnung: Succinyl-CoA Synthetase, α-Untereinheit
EC-Nr. 6.2.1.5
Referenz: Buck et al.; Biochemistry 24(22), 6245-52 (1985)
Accession No.: AE000176
aspA-Gen:
Bezeichnung: Aspartat Ammonium-Lyase (Aspartase)
EC-Nr.: 4.3.1.1
Referenz: Takagi et al.; Nucleic Acids Research 13(6), 2063-74 (1985)
Accession No.: AE000486
gltA-Gen:
Bezeichnung: Citratsynthase
EC-Nr.: 4.1.3.7
Referenz: Spencer und Guest, Journal of Bacteriology 151(2), 542-52 (1982)
Accession No.: AE000175
Alternative Gennamen: gluT, icdB
sdhB-Gen:
Bezeichnung: Succinatdehydrogenase
EC-Nr.: 1.3.99.1
Referenz: Darlison und Guest, Biochemical Journal 223(2), 507-17 (1984)
Accession No.: AE000175
aceB-Gen:
Bezeichnung: Malatsynthase A
EC-Nr.: 4.1.3.2
Referenz: Byrne et al.; Nucleic Acids Research 16(19), 9342 (1988)
Accession No.: AE000474
Alternativer Genname: mas
aceK-Gen:
Bezeichnung: Isocitratdehydrogenase Kinase/Phosphatase
EC-Nr.: 2.7.1.116
Referenz: Cortay et al.; Journal of Bacteriology 170(1), 89-97 (1988)
Klumpp et al.; Journal of Bacteriology 170(6), 2763-9 (1988)
Accession No.: AE000474 The nucleotide sequences of the genes of Escherichia coli belong to the prior art and can also be that of Blattner et al. (Science 277, 1453-1462 (1997)) published genome sequence of Escherichia coli.
malE gene:
Name: Periplasmic maltose binding protein
Reference: Duplay et al., Journal of Biological Chemistry 259 (16): 10606-13 (1984)
Accession No .: AE000476
Alternative name: malB
phoA gene:
Name: Alkaline phosphatase
EC No .: 3.1.3.1
Reference: mountain; Journal of Bacteriology 146 (2), 660-7 (1981)
Accession No .: AE000145
phoB gene:
Description: Positive regulator of the pho regulon, two-component system
Reference: Makino et al .; Journal of Molecular Biology 190 (1), 37-44 (1986)
Accession No .: AE000146
Alternative names: phoRc, phoT
phoR gene:
Name: Positive and negative sensor protein of the pho regulon, sensor of the two-component system
EC No .: 2.7.3.-
Reference: Makino et al .; Journal of Molecular Biology 192 (3), 549-56 (1986)
Accession No .: AE000146
Alternative names: R1pho, nmpB, phoR1
phoE gene:
Name: Protein of the outer cell membrane E (E, Ic, NmpAB)
Reference; Overbeeke et al .; Journal of Molecular Biology 163 (4), 513-32 (1983)
Accession No .: AE000132
Alternative name: ompE
PhNC gene:
Description; ATP binding domain of the phosphonate transporter
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnD gene:
Name: Periplasmic binding protein of the phosphonate transporter
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
Alternative name: psiD
phne gene:
Name: Integral membrane component of the phosphonate transporter
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phNF gene:
Name: Putative regulator of the phosphonate transporter
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnG gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnJ gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnK gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
pHNL gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnM gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnN gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
PhNO gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
phnP gene:
Name: Carbon-phosphorus lyase complex subunit (phosphonate metabolism)
Reference: Makino et al. Journal of Bacteriology, 173 (8), 2665-12 (1991)
Accession No .: AE000482
pykF gene:
Name: Pyruvate Kinase I (formerly F), stimulated fructose
EC No .: 2.7.1.40
Reference: Ponce et al .; Journal of Bacteriology 177 (19): 5719-22 (1995)
Accession No .: AE000262
pfkB gene:
Name: 6-phosphofructokinase II; Suppressor from pfkA
Reference: Daldal; Gene 28 (3), 337-42 (1984)
Accession No .: AE000267
eda gene:
Name: 2-keto-3-deoxygluconate 6-phosphate aldolase and 2-keto-4-hydroxyglutarate aldolase (Entner-Doudoroff aldolase)
EC No .: 4.1.2.14 4.1.3.16
Reference: Carter et al .; Gene 130 (1), 155-6 (1993)
Accession No .: AE000279
Alternative names: kga, kdgA
Talb gene:
Name: Transaldolase B
EC No .: 2.2.1.2
Reference: Sprenger et al .; Journal of Bacteriology 177 (20), 5930-6 (1995)
Accession No .: AE000111
rpiB gene:
Name: Ribose 5-phosphate isomerase B
Reference: Sorensen and Hove-Jensen; Journal of Bacteriology 178 (4), 1003-11 (1996)
Accession No .: AE000482
zwf gene:
Name: Glucose-6-phosphate dehydrogenase (intermediate ferment)
EC No .: 1.1.1.49
Reference: Rowley and Wolf; Journal of Bacteriology 173 (3), 968-77 (1991)
Accession No .: AE000279
MOPA gene:
Name: GroEL, Chaperon Hsp60, peptide dependent
Reference: Chandrasekhar et al .; Journal of Biological Chemistry 261 (26), 12414-9 (1986)
Accession No .: AE000487
Alternative names: groE, groEL, hdh, tabB; 60 kD chaperonin (CPN60)
PSTA gene:
Description: Highly affine phosphate transport system
Reference: Surin et al .; Journal of Bacteriology 161 (1), 189-98 (1985)
Amemura et al .; Journal of Molecular Biology 184 (2), 241-50 (1985)
Accession No .: AE000449
Alternative names: R2pho, phoR2b, phoT
Pstb gene:
Name: ATP binding component of the high-affinity phosphate transport system
Reference: Surin et al .; Journal of Bacteriology 161 (1), 189-98 (1985)
Amemura et al .; Journal of Molecular Biology 184 (2), 241-50 (1985)
Accession No .: AE000449
Alternative name: phoT
PSTC-gene:
Name: High-affine phosphate transport system, cytoplasmic membrane component
Reference: Surin et al .; Journal of Bacteriology 161 (1), 189-98 (1985)
Accession No .: AE000449
Alternative name: phoT
pstS gene:
Name: High-affinity phosphate transport system; Periplasmic phosphate binding protein
Reference: Surin et al., Journal of Bacteriology 157, 772-8 (1984);
Magota et al., Journal of Bacteriology 157, 909-17 (1984)
Accession No .: AE000449
Alternative names: R2pho, nmpA, phoR2a, phoS
ugpB gene:
Name: sn-glycerin-3-phosphate transport system; Periplasmic binding protein
Reference: Overduin et al .; Molecular Microbiology 2 (6), 767-75 (1988)
Accession No .: AE000421
Alternative names: psiB, psiC,
ugpA gene:
Name: sn-glycerin-3-phosphate transport system, integral membrane protein
Reference: Overduin et al .; Molecular Microbiology 2 (6), 767-75 (1988)
Accession No .: AE000421
Alternative names: psiB, psiC
ugpE gene:
Name: sn-glycerin-3-phosphate transport system, integral membrane protein
Reference: Overduin et al .; Molecular Microbiology 2 (6), 767-75 (1988)
Accession No .: AE000421
ugpC gene:
Name: ATP binding component of the sn-glycerin-3-phosphate transport system
Reference: Overduin et al .; Molecular Microbiology 2 (6), 767-75 (1988)
Accession No .: AE000421
ugpQ gene:
Name: Glycerin phosphodiester phosphodiesterase, cytosolic
EC No .: 3.1.4.46
Reference: Kasahara et al .; Nucleic Acids Research 17 (7), 2854 (1989)
Accession No .: AE000421
dnaK gene:
Name: Chaperon Hsp70; DNA biosynthesis;
autoregulated heat shock protein
Reference: Bardwell and Craig; Proceedings of the National Academy of Sciences of the USA 81 (3), 848-52 (1984)
Accession No .: AE000112
Alternative names: gro, groP, groPAB, groPC, groPF, grpC, grpF, seg
dnaJ gene:
Name: chaperone; Heat shock protein
Reference: Ohki et al .; Journal of Biological Chemistry 261 (4), 1778-81 (1986)
Accession No .: AE000112
Alternative names: groP, grpC
clpB gene:
Name: heat shock protein (caseinolytic protease)
EC No .: 1.17.4.-3.4.21.-
Reference: Kitagawa et al .; Journal of Bacteriology 173 (14), 4247-53 (1991)
Accession No .: AE000345
RpoE gene:
Name: RNA polymerase, Sigma-E factor; Heat shock and oxidative stress
Reference: Raina et al .: EMBO Journal 14 (5), 1043-55 (1995)
Accession No .: AE000343
rSEA gene:
Name: sigma-E factor, negative regulator (membrane protein regulator of σE activity)
Reference: Missiakas et al .; Molecular Microbiology 24 (2), 355-71 (1997)
Accession No .: AE000343
Alternative names: mclA
RSEC gene:
Name: sigma-E factor, global regulator
Reference: Missiakas et al .; Molecular Microbiology 24 (2), 355-71 (1997)
Accession No .: AE000343
HtpG gene:
Name: Chaperon Hsp90, heat shock protein C 62.5
Reference: Spence and Georgopoulos; Journal of Biological Chemistry 264 (8), 4398-403 (1989)
Accession No .: AE000153
sodA gene:
Name: Superoxide dismutase
Reference: Touati; Journal of Bacteriology 155 (3): 078-87 (1983)
Accession No .: AE000465
ompF gene:
Name: Outer membrane protein 1a (Ia; b; F)
Reference: Inokuchi et al .; Nucleic Acids Research 10 (21), 6957-68 (1982)
Accession No .: AE000195
Alternative names: cmlB, coa, cry, tolF
ompC gene:
Name: Outer membrane protein 1b (Ib; c)
Reference: Mizuno et al .; Journal of Biological Chemistry 258 (11), 6932-40 (1983)
Accession No .: AE000310
Alternative names: meoR, par
sucA gene:
Name: 2-ketoglutarate dehydrogenase (decarboxylase subunit)
EC No .: 1.2.4.2
Reference: Darlison et al .; European Journal of Biochemistry 141 (2), 351-9 (1984)
Accession No .: AE000175
Alternative names: lys, met
sucB gene:
Name: 2-ketoglutarate dehydrogenase (Dihydrolipoyltranssuccinase E2 subunit)
EC No .: 2.3.1.61
Reference: Spencer et al .; European Journal of Biochemistry 141 (2), 361-74 (1984)
Accession No .: AE000175
Alternative names: lys, met
sucC gene:
Name: succinyl-CoA synthetase, β-subunit
EC-No. 6.2.1.5
Reference: Buck et al .; Biochemistry 24 (22), 6245-52 (1985)
Accession No .: AE000176
sucD gene:
Name: Succinyl-CoA synthetase, α subunit
EC-No. 6.2.1.5
Reference: Buck et al .; Biochemistry 24 (22), 6245-52 (1985)
Accession No .: AE000176
aspA gene:
Name: aspartate ammonium lyase (aspartase)
EC No .: 4.3.1.1
Reference: Takagi et al .; Nucleic Acids Research 13 (6), 2063-74 (1985)
Accession No .: AE000486
gltA gene:
Name: Citrate synthase
EC No .: 4.1.3.7
Reference: Spencer and Guest, Journal of Bacteriology 151 (2), 542-52 (1982)
Accession No .: AE000175
Alternative names: gluT, icdB
sdhB gene:
Name: succinate dehydrogenase
EC No .: 1.3.99.1
Reference: Darlison and Guest, Biochemical Journal 223 (2), 507-17 (1984)
Accession No .: AE000175
aceB gene:
Name: Malate synthase A
EC No .: 4.1.3.2
Reference: Byrne et al .; Nucleic Acids Research 16 (19), 9342 (1988)
Accession No .: AE000474
Alternative name: mas
AceK gene:
Name: Isocitrate dehydrogenase kinase / phosphatase
EC No .: 2.7.1.116
Reference: Cortay et al .; Journal of Bacteriology 170 (1), 89-97 (1988)
Klumpp et al .; Journal of Bacteriology 170 (6), 2763-9 (1988)
Accession No .: AE000474

Die Nukleinsäuresequenzen können den Datenbanken 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 be found in the databases of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA), the European Molecular Biologies nucleotide sequence database 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. 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.

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. 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. 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. 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 to overexpress the genes in question Change in media composition and culture management can be achieved.

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 (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)), by Hamilton (Journal of Bacteriology 171: 4617-4622 (1989)) Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998) and in well-known 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, oder dafür codierende Nucleotidsequenzen 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, ein oder mehrere Enzyme des bekannten Threonin-Biosyntheseweges oder Enzyme des anaplerotischen Stoffwechsels oder Enzyme für die Produktion von reduziertem Nicotinamid-Adenin-Dinukleotid- Phosphat zu verstärken. 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, 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 To reinforce phosphate.

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: 332 (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 kodierende thrE-Gen von Corynebacterium glutamicum (DE 100 26 494.8), und
  • - das für die Glutamat-Dehydrogenase kodierende gdhA-Gen (Nucleic Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983))
verstärkt, insbesondere überexprimiert werden. For example, one or more of the genes selected from the group can be used simultaneously
  • the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase (US Pat. No. 4,278,765),
  • the pyc gene coding for the pyruvate carboxylase (DE-A-198 31 609),
  • the pps gene coding for phosphoenolpyruvate synthase (Molecular and General Genetics 231: 332 (1992)),
  • the ppc gene coding for 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 rhtB gene which mediates resistance to homoserine (EP-A-0 994 190),
  • the mqo gene coding for the malate: quinone oxidoreductase (DE 100 34 833.5),
  • the rreC gene which mediates resistance to threonine (EP-A-1 013 765),
  • - The thrE gene from Corynebacterium glutamicum (DE 100 26 494.8) coding for threonine export, and
  • - the gdhA gene coding for glutamate dehydrogenase (Nucleic Acids Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983))
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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceR und aceK, eines oder mehrere der Gene ausgewählt aus der Gruppe

  • - das für die Threonin-Dehydrogenase kodierende tdh-Gen (Ravnikar und Somerville, Journal of Bacteriology 169, 4716-4721 (1987)),
  • - das für die Malat-Dehydrogenase (E. C. 1.1.1.37) kodierende mdh-Gen (Vogel et al., 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, MD, USA),
  • - das für das Enzym Phosphoenolpyruvat-Carboxykinase kodierende pckA-Gen (Medina et al. (Journal of Bacteriology 172, 7151-7156 (1990)),
  • - das für die Pyruvat-Oxidase kodierende poxB-Gen (Grabau und Cronan (Nucleic Acids Research 14(13), 5449-5460 (1986)),
  • - das für das Enzym Isocitrat-Lyase kodierende aceA-Gen (Matsuoko und McFadden, Journal of Bacteriology 170, 4528-4536 (1988)),
  • - das für den DgsA-Regulator des Phosphotransferase- Systems kodierende dgsA-Gen (Hosono et al., Bioscience, Biotechnology and Biochemistry 59, 256-251 (1995)), das auch unter der Bezeichnung mlc-Gen bekannt ist, und
  • - das für den Fructose-Repressor kodierende fruR-Gen (Jahreis et al., Molecular and General Genetics 226, 332-336 (1991)), das auch unter der Bezeichnung cra-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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG , phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQJ, dna , clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceR and aceK, one or more of the genes selected from the group
  • the tdh gene coding for threonine dehydrogenase (Ravnikar and Somerville, Journal of Bacteriology 169, 4716-4721 (1987)),
  • the mdh gene coding for malate dehydrogenase (EC 1.1.1.37) (Vogel et al., Archives in Microbiology 149, 36-42 (1987)),
  • - the gene product of the open reading framework (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, MD, USA),
  • the pckA gene coding for the enzyme phosphoenolpyruvate carboxykinase (Medina et al. (Journal of Bacteriology 172, 7151-7156 (1990)),
  • the poxB gene coding for pyruvate oxidase (Grabau and Cronan (Nucleic Acids Research 14 (13), 5449-5460 (1986)),
  • the aceA gene coding for the enzyme isocitrate lyase (Matsuoko and McFadden, Journal of Bacteriology 170, 4528-4536 (1988)),
  • - The dgsA gene coding for the DgsA regulator of the phosphotransferase system (Hosono et al., Bioscience, Biotechnology and Biochemistry 59, 256-251 (1995)), which is also known under the name mlc gene, and
  • the fruR gene coding for the fructose repressor (Jahreis et al., Molecular and General Genetics 226, 332-336 (1991)), which is also known under the name cra 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 one. 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, 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. 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 culture medium to be used must be in a suitable manner The requirements of the respective tribes meet. descriptions of culture media of various microorganisms are in "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, 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 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. Organic nitrogen-containing can be used as 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 also contain 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.

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. Basic compounds are used to control the pH of the culture such as 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, (1958), 1190) beschrieben, oder sie kann durch reversed phase HPLC erfolgen, so wie bei Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174) 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, (1958), 1190), or it can be done by reversed phase HPLC, such as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

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 (7)

1. Verfahren zur fermentativen Herstellung von L- Aminosäuren, insbesondere L-Threonin, 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, 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. 1. A process for the fermentative production of L-amino acids, in particular L-threonine, characterized in that the following steps are carried out: 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 malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, clnaP, dnaJ rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, or nucleotide sequences coding for them 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 proportions (> 0 to 100) thereof remaining in the product, if appropriate. 2. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man Mikroorganismen einsetzt, in denen man zusätzlich weitere Gene des Biosyntheseweges der gewünschten L-Aminosäure verstärkt. 2. The method according to claim 1, characterized characterized that microorganisms uses, in which one also additional genes of Biosynthetic pathway of the desired L-amino acid strengthened. 3. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man Mikroorganismen einsetzt, in denen die Stoffwechselwege zumindest teilweise ausgeschaltet sind, die die Bildung der gewünschten L-Aminosäure verringern. 3. The method according to claim 1, characterized characterized that microorganisms uses in which the metabolic pathways at least are partially turned off, which is the formation of the reduce the desired L-amino acid. 4. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man die Expression des (der) Polynukleotides (e), das (die) für eines oder mehrere der Gene, ausgewählt aus der Gruppe malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, kodiert (kodieren) erhöht. 4. The method according to claim 1, characterized characterized in that the expression of polynucleotide (s) for one or several of the genes selected from the group malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, coded (coding) elevated. 5. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man die regulatorischen und/oder katalytischen Eigenschäften der Polypeptide (Proteine) verbessert oder erhöht, für die die Polynukleotide malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB und aceK, kodieren. 5. The method according to claim 1, characterized characterized that one the regulatory and / or catalytic properties the polypeptides (proteins) are improved or increased, for which the polynucleotides malE, phoA, phoB, phoR, phoE, phnC, phnD, phnE, phnF, phnG, phnJ, phnK, phnL, phnM, phnN, phnO, phnP, pykF, pfkB, eda, talB, rpiB, zwf, mopA, pstA, pstB, pstC, pstS, ugpB, ugpA, ugpE, ugpC, ugpQ, dnaK, dnaJ, clpB, rpoE, rseA, rseC, htpG, sodA, ompF, ompC, sucA, sucB, sucC, sucD, aspA, gltA, sdhB, aceB and aceK, code. 6. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man zur Herstellung von L-Aminosäuren Mikroorganismen der Familie Enterobacteriaceae fermentiert, in denen man zusätzlich gleichzeitig eines oder mehrere der Gene, ausgewählt aus der Gruppe: 1. 6.1 das für die Aspartatkinase, die Homoserin- Dehydrogenase, die Homoserinkinase und die Threoninsynthase kodierende thrABC-Operon, 2. 6.2 das für die Pyruvat-Carboxylase kodierende pyc- Gen, 3. 6.3 das für die Phosphoenolpyruvat-Synthase kodierende pps-Gen, 4. 6.4 das für die Phosphoenolpyruvat-Carboxylase kodierende ppc-Gen, 5. 6.5 die für die Transhydrogenase kodierenden Gene pntA und pntB, 6. 6.6 das Homoserinresistenz vermittelnde Gen rhtB, 7. 6.7 das für die Malat:Chinon Oxidoreduktase kodierende mqo-Gen, 8. 6.8 das Threoninresistenz vermittelnde Gen rhtC, und 9. 6.9 das für den Threoninexport kodierende thrE-Gen 10. 6.10 das für die Glutamat-Dehydrogenase kodierende gdhA-Gen verstärkt, insbesondere überexprimiert. 6. The method according to claim 1, characterized in that for the production of L-amino acids microorganisms of the Enterobacteriaceae family are fermented, in which one additionally one or more of the genes selected from the group: 1. 6.1 the thrABC operon coding for aspartate kinase, homoserine dehydrogenase, homoserine kinase and threonine synthase, 2. 6.2 the pyc gene coding for the pyruvate carboxylase, 3. 6.3 the pps gene coding for the phosphoenolpyruvate synthase, 4. 6.4 the ppc gene coding for the phosphoenolpyruvate carboxylase, 5. 6.5 the genes pntA and pntB coding for the transhydrogenase, 6. 6.6 the gene which mediates homoserine resistance rhtB, 7. 6.7 the mqo gene coding for the malate: quinone oxidoreductase, 8. 6.8 the gene mediating threonine resistance rhtC, and 9. 6.9 the thrE gene coding for threonine export 10. 6.10 the gdhA gene coding for glutamate dehydrogenase amplified, especially overexpressed. 7. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man zur Herstellung von L-Aminosäuren Mikroorganismen der Familie Enterobacteriaceae fermentiert, in denen man zusätzlich gleichzeitig eines oder mehrere der Gene, ausgewählt aus der Gruppe: 1. 7.1 das für die Threonin-Dehydrogenase kodierende tdh-Gen, 2. 7.2 das für die Malat-Dehydrogenase kodierende mdh- Gen, 3. 7.3 das Genprodukt des offenen Leserahmens (orf) yjfA, 4. 7.4 das Genprodukt des offenen Leserahmens (orf) ytfP, 5. 7.5 das für die Phosphoenolpyruvat-Carboxykinase kodierende pckA-Gen, 6. 7.6 das für die Pyruvat-Oxidase kodierende poxB- Gen, 7. 7.7 das für die Isocitrat-Lyase kodierende aceA- Gen, 8. 7.8 das für den DgsA-Regulator des Phosphotransferase-Systems kodierende dgsA-Gen, 9. 7.9 das für den Fructose-Repressor kodierende fruR- Gen, abschwächt, insbesondere ausschaltet oder die Expression verringert. 7. The method according to claim 1, characterized in that for the production of L-amino acids, microorganisms of the Enterobacteriaceae family are fermented, in which additionally one or more of the genes selected from the group: 1. 7.1 the tdh gene coding for the threonine dehydrogenase, 2. 7.2 the mdh gene coding for malate dehydrogenase, 3. 7.3 the gene product of the open reading frame (orf) yjfA, 4. 7.4 the gene product of the open reading frame (orf) ytfP, 5. 7.5 the pckA gene coding for the phosphoenolpyruvate carboxykinase, 6. 7.6 the poxB gene coding for the pyruvate oxidase, 7. 7.7 the aceA gene coding for the isocitrate lyase, 8. 7.8 the dgsA gene coding for the DgsA regulator of the phosphotransferase system, 9. 7.9 the fruR gene coding for the fructose repressor, attenuates, in particular switches off or the expression decreases.
DE2001135053 2001-07-18 2001-07-18 Preparing L-amino acids, e.g. L-..threonine by fermenting microorganisms of Enterobactericeae family in which at least the malE gene is enhanced, in particular overexpressed, and isolating the desired amino acid Withdrawn DE10135053A1 (en)

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DE2001135053 DE10135053A1 (en) 2001-07-18 2001-07-18 Preparing L-amino acids, e.g. L-..threonine by fermenting microorganisms of Enterobactericeae family in which at least the malE gene is enhanced, in particular overexpressed, and isolating the desired amino acid
DK09156036.7T DK2083080T3 (en) 2001-07-18 2002-07-03 Process for Preparation of L-Amino Acids Using Strains of the Enterobacteriaceae Family Containing an Increased rseA or rseC Gene
US10/484,162 US7332309B2 (en) 2001-07-18 2002-07-03 Process for the preparation of L-amino acids using strains of the enterobacteriaceae family which contain an enhanced sucC or sucD gene
DK02743259T DK1407027T3 (en) 2001-07-18 2002-07-03 Process for Preparation of L-Threonine Using Strains of the Enterobacteriaceae Family Containing Enhanced Suc and SucD Genes
DK02743258T DK1407022T3 (en) 2001-07-18 2002-07-03 Methods for Preparing L-Amino Acids Using Strains of the Enterobacteriaceae Family Containing an Enhanced rseA Gene
DE60232725T DE60232725D1 (en) 2001-07-18 2002-07-03 METHOD FOR THE PREPARATION OF L-THREONINE BY ENTEROBAKTERIACEAE STRAINS WITH INCREASED EXPRESSION OF THE SUCC AND SUCD GENES
AU2002354852A AU2002354852A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced phob or phor gene
AU2002325865A AU2002325865A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced talb gene
PCT/EP2002/007356 WO2003008607A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family
PCT/EP2002/007355 WO2003008606A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced phob or phor gene
AU2002354853A AU2002354853A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family
DE60232726T DE60232726D1 (en) 2001-07-18 2002-07-03 METHOD FOR THE PRODUCTION OF L-THREONINE BY ENTEROBAKTERIACEAE STRAINS WITH INCREASED EXPRESSION OF THE SUCA AND SUCB GENES
PCT/EP2002/007368 WO2003008610A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced pfkb gene
DE60226239T DE60226239T2 (en) 2001-07-18 2002-07-03 METHOD FOR THE PREPARATION OF L-THREONINE BY ENTEROBAKTERIACEAE STRAINS WITH INCREASED EXPOSURE OF THE MALE GENE
EP02787110A EP1407026B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-threonine using strains of the enterobacteriaceae family which contain enhanced phob and phor genes
AT02787110T ATE407210T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-THREONINE BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE PHOB AND PHOR GENES
EP09156036.7A EP2083080B1 (en) 2001-07-18 2002-07-03 Process for the preparation of L-threonine using strains of the Enterobacteriaceae family which contain an enhanced rseC gene
US10/484,198 US20050059124A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced suca or sucb gene
AT02758304T ATE434664T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-THREONINE BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE SUCA AND SUCB GENES
DE60233573T DE60233573D1 (en) 2001-07-18 2002-07-03 METHOD FOR THE PREPARATION OF L-AMINO ACIDS BY ENTEROBAKTERIACEAE STRAINS WITH INCREASED EXPRESSION OF THE RSEA GENE
AU2002319281A AU2002319281A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced pykf gene
AU2002345084A AU2002345084A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced succ or sucd gene
US10/483,417 US20040241814A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced rsea or rsec gene
ES02758304T ES2328230T3 (en) 2001-07-18 2002-07-03 PROCESS FOR THE PREPARATION OF L-TREONIN USING ENTEROBACTERIACEAS FAMILY STRAPS CONTAINING INTENSIFIED SUCA AND SUCB GENES.
AU2002325294A AU2002325294A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced suca or sucb gene
PCT/EP2002/007373 WO2003008613A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced soda gene
EP02743259A EP1407027B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-threonine using strains of the enterobacteriaceae family which contain enhanced succ and sucd genes
EP02758304A EP1407028B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-threonine using strains of the enterobacteriaceae family which contain enhanced suca and sucb genes
PCT/EP2002/007369 WO2003008611A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced talb gene
AT02743258T ATE441714T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-AMINO ACIDS BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE RSEA GENE
EP02751102A EP1407024B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-threonine using strains of the enterobacteriaceae family which contain an enhanced phoe gene
US10/483,416 US20050170472A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced soda gene
DE60228715T DE60228715D1 (en) 2001-07-18 2002-07-03 METHOD FOR THE PRODUCTION OF L-THREONINE BY ENTEROBAKTERIACEAE STRAINS WITH IMPROVED EXPOSURE OF THE PHOB AND PHOR GENES
PCT/EP2002/007366 WO2003008608A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced phoe gene
EP02743258A EP1407022B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced rsea gene
DK02758304T DK1407028T3 (en) 2001-07-18 2002-07-03 Process for Preparation of L-Threonine Using Strains of the Enterobacteriaceae Family Containing Enhanced SucA and SucB Genes
AT02751102T ATE386121T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-THREONINE BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE PHOE GENE
PCT/EP2002/007374 WO2003008614A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced suca or sucb gene
AU2002345083A AU2002345083A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced rsea or rsec gene
AT02758301T ATE393226T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-THREONINE BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE MALE GENE
AU2002321165A AU2002321165A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced pfkb gene
DE60225011T DE60225011T2 (en) 2001-07-18 2002-07-03 METHOD FOR THE PRODUCTION OF L-THREONINE BY ENTEROBAKTERIACEAE STRAINS WITH INCREASED EXPOSURE OF THE PHOE GENE
PCT/EP2002/007367 WO2003008609A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced pykf gene
PCT/EP2002/007354 WO2003008605A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced male gene
EP02758301A EP1407025B1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-threonine using strains of the enterobacteriaceae family which contain an enhanced male gene
AU2002354854A AU2002354854A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced phoe gene
AU2002325293A AU2002325293A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced male gene
AT02743259T ATE434663T1 (en) 2001-07-18 2002-07-03 METHOD FOR PRODUCING L-THREONINE BY ENTEROBACTERIACEAE STRAINS WITH ENHANCED EXPRESSION OF THE SUCC AND SUCD GENES
PCT/EP2002/007370 WO2003008612A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced rsea or rsec gene
AU2002354855A AU2002354855A1 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced soda gene
ES02743259T ES2328229T3 (en) 2001-07-18 2002-07-03 PROCESS FOR THE PREPARATION OF L-TREONIN USING ENTEROBACTERIACEAS FAMILY STRAPS CONTAINING INTENSIFIED SUCC AND SUCD GENES.
PCT/EP2002/007375 WO2003008615A2 (en) 2001-07-18 2002-07-03 Process for the preparation of l-amino acids using strains of the enterobacteriaceae family which contain an enhanced succ or sucd gene

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WO2004090149A1 (en) * 2003-04-09 2004-10-21 Degussa Ag Process for the production of l-amino acids using strains of the enterobacteriaceae family which contain an enhance yfidd orf and/or pfld gene
WO2005078113A1 (en) * 2004-02-12 2005-08-25 Ajinomoto Co., Inc. Method for producing l-threonine using bacteria belonging to the genus escherichia
US7575905B2 (en) 2004-02-06 2009-08-18 Evonik Degussa Gmbh Process for L-amino acid production using enterobacteriaceae strains with enhanced yibD
US7638313B2 (en) 2003-01-30 2009-12-29 Degussa Ag Processes for the fermentative preparation of L-threonine using strains of Escherichia in which the yjgF gene is inactivated
CN114729012A (en) * 2019-09-09 2022-07-08 Cj第一制糖株式会社 L-threonine exporter variants and methods of producing L-threonine using the same

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7638313B2 (en) 2003-01-30 2009-12-29 Degussa Ag Processes for the fermentative preparation of L-threonine using strains of Escherichia in which the yjgF gene is inactivated
WO2004090149A1 (en) * 2003-04-09 2004-10-21 Degussa Ag Process for the production of l-amino acids using strains of the enterobacteriaceae family which contain an enhance yfidd orf and/or pfld gene
US7211415B2 (en) 2003-04-09 2007-05-01 Degussa Ag Enterobacteriaceae strains over-expressing the yfiD gene for the fermentative production of L-amino acids
US7575905B2 (en) 2004-02-06 2009-08-18 Evonik Degussa Gmbh Process for L-amino acid production using enterobacteriaceae strains with enhanced yibD
WO2005078113A1 (en) * 2004-02-12 2005-08-25 Ajinomoto Co., Inc. Method for producing l-threonine using bacteria belonging to the genus escherichia
CN114729012A (en) * 2019-09-09 2022-07-08 Cj第一制糖株式会社 L-threonine exporter variants and methods of producing L-threonine using the same
CN114729012B (en) * 2019-09-09 2024-02-06 Cj第一制糖株式会社 L-threonine export protein variants and methods of producing L-threonine using the same

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