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WO2001000847A1 - Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine - Google Patents

Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine Download PDF

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WO2001000847A1
WO2001000847A1 PCT/EP2000/005850 EP0005850W WO0100847A1 WO 2001000847 A1 WO2001000847 A1 WO 2001000847A1 EP 0005850 W EP0005850 W EP 0005850W WO 0100847 A1 WO0100847 A1 WO 0100847A1
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pyrimidine
corynebacterium glutamicum
dihydroorotate dehydrogenase
sequence
polypeptide
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PCT/EP2000/005850
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German (de)
French (fr)
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Matthias Mack
Karin Herbster
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Basf-Lynx Bioscience Ag
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Priority to KR1020017016566A priority Critical patent/KR20020026470A/en
Priority to EP00942126A priority patent/EP1196602A1/en
Priority to CA002377482A priority patent/CA2377482A1/en
Priority to AU56854/00A priority patent/AU5685400A/en
Publication of WO2001000847A1 publication Critical patent/WO2001000847A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)

Definitions

  • the present invention is concerned with the production process of pyrimidines by fermentation using a genetically modified organism.
  • This invention consists in the sequence of the dihydroorotate dehydrogenase from Corynebac erium glutamicum and its use for the microbial production of pyrimidines and / or compounds related to pyrimidine.
  • the pyrimidine nucleotides are pyrimidine derivatives and as such activated precursors of DNA and KNA and for many biosynthetic pathways
  • a pyrimidine base is present in the pyrimidine nucleosides cytidine, uridine, deoxycytidine and deoxythyidine linked to a pentose, the pyridine nucleotides are the phosphate esters of the pyrimidine nucleosides.
  • Pyrimidine nucleosides and pyrimidine nucleotides and their derivatives are also important starting compounds for the synthesis of valuable drugs, such as CDP-choline, orotic acid or UMP (a review of this is available from Kuninaka, A. in Biotechnology, Vol. 6 (Ed .: Rehm, H.-J. and Reed, G.), VCH, Weinheim, Deutsch-la d, 1996, pp. 561-612)
  • microorganisms can synthesize their pyrimidine nucleotides both de novo and from pyrimidine bases and pyrimidine nucleosides offered from outside. Pyrimidine bases and / or pyrimidine nucleosides do not normally occur intracellularly. However, they can be formed in excess under some growth conditions and are then excreted in the culture medium. Therefore, microorganisms can be used for the fermentative production of pyrimidine nucleotides and / or related compounds.
  • the biosynthetic performance of the microorganisms for pyrimidine nucleotides can be optimized by genetic modification of the pyrimidine biosynthetic pathway. Genetic modification means that the number of gene copies and / or the speed of transcription of the genes for the pyrimidine synthesis pathway is increased. As a result, the proportion of gene product and the intracellular enzymatic activity. An increased enzymatic activity leads to an increased conversion of compounds offered in the nutrient medium to pyrimidine nucleotides and / or related compounds and thus increases the synthesis performance.
  • the invention is concerned with the new pyrD gene for the dihydroorotate dehydrogenase of the pyrimidine biosynthetic pathway from Coryne bacterium glutamicum and its use for the production of pyrimidine nucleotides and / or compounds related to pyrimidine.
  • the SEQ ID NO. 2 describes a polypeptide sequence.
  • the pyrD gene encodes a polypeptide of 322 amino acids with a molecular weight of 33953.
  • the present invention also relates to functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 2 by deletion, insertion or substitution or by a combination of deletion, insertion and substitution one or more amino acids, preferably replaced up to 25% of the amino acids, preferably up to 15%.
  • the term functional derivative means that the enzyme activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. Second
  • polynucleotide sequences which encode the polypeptides described above.
  • the polynucleotide sequences can be generated starting from sequences which are isolated from Corynebacterium glutamicum (ie SEQ ID NO. 1) by modifying these sequences by site-directed mutagenesis or after retranslating the corresponding polypeptide with the genetic one Code to perform a total chemical synthesis.
  • polynucleotide sequences can best be used in the form of gene constructs for the transformation of host organisms, preferably microorganisms.
  • These gene constructs consist of at least one copy of one of the polynucleotides together with at least one regulatory sequence. Regulatory sequences include promoters, terminators, enhancers and ribosomal binding sites.
  • Preferred host organisms for the transformation with these gene constructs are Corynebacterium and Bacillus species, any eukaryotic microorganism can also be used for this, preferably yeast strains of the genus Ashbya, Candida, Pichia, Saccharomyces and Hansenula.
  • a pyrimidine derivative is a compound with a pyrimidine ring, which can be prepared by transforming a host organism with one of the polynucleotides corresponding to the present invention.
  • the trained personnel are familiar with the processes and procedures for cultivating microorganisms and isolating pyrimidines from a microbial production.
  • DNA from the genome of Corynejbacterium glutamicum ATCC 13032 can be obtained by standard methods which have already been described, e.g. B. by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet. 195: 134-138).
  • the genome library can be prepared according to standard regulations (for example: Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, for example pBluescript II KS- (Stratagene) or ZAP Express TM (Stratagene). Any fragment size can be used, preferably Sau3AI fragments with a length of 2-9 kb, which can be integrated into cloning vectors with digested BamEl.
  • E. coli clones can be selected from the genome library shown in Example 1.
  • E. coli cells are cultivated according to standard ancestors in suitable media (e.g. LB supplemented with 100 mg / 1 ampicillin), and the plasmid DNA can then be isolated. If one clones genome fragments from the DNA of Corynebacterium glutamicum in pBluescript II KS- (see Example 1), the DNA can be sequenced with the help of the oligonucleotides 5 '-AATTAACCCTCAC-TAAAGGG-3' and 5 '-GTAATACGACTCACTATAGGGC-3'.
  • nucleotide sequences can e.g. using the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410). In this way, one can discover new sequences and elucidate the function of these new genes.
  • Example 3 When the E. coli clones were analyzed as described in Example 2, followed by the analysis of the sequences obtained in Example 3, a sequence was obtained as described with SEQ ID NO. 1 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity with the dihydroorotate dehydrogenase (PyrD; EC 1.3.3.1) from different organisms. The greatest similarity was with the dihydroorotate dehydrogenase from .Mycoiacterium leprae (SWISSPROT P46727; 67% agreement at the amino acid level).
  • the gene for the dihydroorotate dehydrogenase from Corynebacterium glutamicum can be inserted into the Corynebacterium glutamicum or in with the aid of suitable cloning and / or expression systems introduce any other microorganism. Genetically modified microorganisms can be produced that differ from the wild type in the activity or the number of copies of the genes. These novel, genetically modified strains can be used to produce pyrimidine and / or compounds related to pyrimidine.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The invention relates to nucleotide sequences of a gene (pyrD) for biosynthesis of pyrimidine from Corynebacterium glutamicum and to the use thereof in microbial production of pyrimidine.

Description

Die Sequenz der Dihydroorotat-Dehydrogenase aus Corynebacterium glutamicum und deren Einsatz bei der mikrobiellen Produktion von Pyrimidinen und/oder mit Pyrimidin verwandten VerbindungenThe sequence of the dihydroorotate dehydrogenase from Corynebacterium glutamicum and its use in the microbial production of pyrimidines and / or compounds related to pyrimidine
Beschreibungdescription
Die vorliegende Erfindung befaßt sich mit dem Produktionsprozeß von Pyrimidinen durch Fermentation mit Hilfe eines gentechnisch veränderten Organismus . Diese Erfindung besteht in der Sequenz der Dihydroorotat-Dehydrogenase aus Corynebac erium glutamicum und deren Einsatz zur mikrobiellen Produktion von Pyrimidinen und/oder von mit Pyrimidin verwandten Verbindungen.The present invention is concerned with the production process of pyrimidines by fermentation using a genetically modified organism. This invention consists in the sequence of the dihydroorotate dehydrogenase from Corynebac erium glutamicum and its use for the microbial production of pyrimidines and / or compounds related to pyrimidine.
Der Biosyntheseweg für Pyrimidine ist für alle lebenden Organismen essentiell (ein Übersichtsartikel hierzu findet sich von Switzer, R. L. und Quinn, C. L. in Bacillus subtilis (Hrsg.: Sonenshein, A. L . , Hoch, J. A. und Losick, R. , American Society for Microbiology, Washington, D.C.), 1993, S. 343-358. Die Pyri- midinnucleotide sind Pyrimidinderivate und als solche aktivierte Vorstufen der DNA und KNA und für viele Biosynthesewege. In den Pyrimidinnucleosiden Cytidin, Uridin, Deoxycytidin und Deoxythy- idin ist eine Pyrimidinbase an eine Pentose gebunden, die Pyri- idinnucleotide sind die Phosphatester der Pyrimidinnucleoside. Pyrimidinnucleoside und Pyrimidinnucleotide und deren Derivate sind auch wichtige AusgangsVerbindungen zur Synthese wertvoller Arzneimittel, wie z.B. CDP-Cholin, Orotsäure oder UMP (ein Über- sichtsartikel hierzu gibt es von Kuninaka, A. in Biotechnology, Vol. 6 (Hrsg.: Rehm, H.-J. und Reed, G. ) , VCH, Weinheim, Deutsch- la d, 1996, S. 561-612)The biosynthetic pathway for pyrimidines is essential for all living organisms (an overview article can be found by Switzer, RL and Quinn, CL in Bacillus subtilis (ed .: Sonenshein, A. L., Hoch, JA and Losick, R., American Society for Microbiology, Washington, DC), 1993, pp. 343-358 The pyrimidine nucleotides are pyrimidine derivatives and as such activated precursors of DNA and KNA and for many biosynthetic pathways A pyrimidine base is present in the pyrimidine nucleosides cytidine, uridine, deoxycytidine and deoxythyidine linked to a pentose, the pyridine nucleotides are the phosphate esters of the pyrimidine nucleosides. Pyrimidine nucleosides and pyrimidine nucleotides and their derivatives are also important starting compounds for the synthesis of valuable drugs, such as CDP-choline, orotic acid or UMP (a review of this is available from Kuninaka, A. in Biotechnology, Vol. 6 (Ed .: Rehm, H.-J. and Reed, G.), VCH, Weinheim, Deutsch-la d, 1996, pp. 561-612)
Viele, aber nicht alle Mikroorganismen können ihre Pyrimidinnucleotide sowohl de novo als auch aus von außen angebotenen Pyri- midinbasen und Pyrimidinnucleosiden synthetisieren. Pyrimidin- basen und/oder Pyrimidinnucleoside kommen normalerweise nicht intrazellulär vor. Sie können jedoch unter manchen Wachstumsbedingungen im Überschuß gebildet werden und werden dann in das Kulturmedium ausgeschieden. Darum lassen sich Mikroorganismen zur fermentativen Produktion von Pyrimidinnucleotiden und/oder ver- wandten Verbindungen einsetzen.Many, but not all, microorganisms can synthesize their pyrimidine nucleotides both de novo and from pyrimidine bases and pyrimidine nucleosides offered from outside. Pyrimidine bases and / or pyrimidine nucleosides do not normally occur intracellularly. However, they can be formed in excess under some growth conditions and are then excreted in the culture medium. Therefore, microorganisms can be used for the fermentative production of pyrimidine nucleotides and / or related compounds.
Die Biosyntheseleistung der Mikroorganismen für Pyrimidinnucleotide läßt sich durch gentechnische Veränderung des Pyrimidinbio- synthesewegs optimieren. Gentechnische Veränderung bedeutet hier- bei, daß die Anzahl der Genkopien und/oder die Geschwindigkeit der Transkription der Gene für den Pyrimidinsyntheseweg erhöht wird. Als Folge hiervon steigt der Anteil an Genprodukt und die intrazelluläre enzymatische Aktivität. Eine erhöhte enzymatische Aktivität führt zu einer vermehrten Umwandlung von im Nährmedium angebotenen Verbindungen zu Pyrimidinnucleotiden und/oder verwandten Verbindungen und steigert so die Syntheseleistung. So konnte man zeigen, daß z.B. ein Anstieg der Aktivität der Dihydroorotat-Dehydrogenase, die die Oxidation von (_?) -Dihydroorotat zu Orotat katalysiert - dies ist die vierte Stufe bei der de novo Pyri idinbiosynthese für Pyrimidinnucleotide - die UMP-Synthese- leistung in Corynebacterium ammoniagenes erhöht (Nudler, A. A., Garibyan, A. G. und Bourd, G. I. (1991) FEMS Microbiol. Lett. 82:263-266) .The biosynthetic performance of the microorganisms for pyrimidine nucleotides can be optimized by genetic modification of the pyrimidine biosynthetic pathway. Genetic modification means that the number of gene copies and / or the speed of transcription of the genes for the pyrimidine synthesis pathway is increased. As a result, the proportion of gene product and the intracellular enzymatic activity. An increased enzymatic activity leads to an increased conversion of compounds offered in the nutrient medium to pyrimidine nucleotides and / or related compounds and thus increases the synthesis performance. It has been shown, for example, that an increase in the activity of dihydroorotate dehydrogenase, which catalyzes the oxidation of (_?) Dihydroorotate to orotate - this is the fourth step in de novo pyridine synthesis for pyrimidine nucleotides - the UMP synthesis performance in Corynebacterium ammoniagenes (Nudler, AA, Garibyan, AG and Bourd, GI (1991) FEMS Microbiol. Lett. 82: 263-266).
Die Erfindung befaßt sich mit dem neuen pyrD-Gen für die Dihydroorotat-Dehydrogenase des Pyrimidinbiosynthesewegs aus Coryne- bacterium glutamicum und seinem Einsatz zur Herstellung von Pyrimidinnucleotiden und/oder mit Pyrimidin verwandten Verbindungen.The invention is concerned with the new pyrD gene for the dihydroorotate dehydrogenase of the pyrimidine biosynthetic pathway from Coryne bacterium glutamicum and its use for the production of pyrimidine nucleotides and / or compounds related to pyrimidine.
Ein Teil der Erfindung besteht in dem pyrD-Genprodukt . Die SEQ ID NR. 2 beschreibt eine Polypeptidseguenz . Das pyrD-Gen kodiert ein Polypeptid aus 322 Aminosäuren mit einem Molekulargewicht von 33953. Die vorliegende Erfindung befaßt sich aber auch mit funk- tionellen Derivaten dieses Polypeptids, die man erhalten kann, wenn man in der SEQ ID NR. 2 durch Deletion, Insertion oder Substitution oder durch eine Kombination von Deletion, Insertion und Substitution eine oder mehrere Aminosäuren, vorzugsweise bis zu 25% der Aminosäuren ersetzt, am besten bis zu 15%. Der Ausdruck funktionelles Derivat bedeutet, daß die Enzymaktivität des Derivats noch in der gleichen Größenordnung liegt wie die des Polypeptids mit der Sequenz SEQ ID NR. 2.Part of the invention is the pyrD gene product. The SEQ ID NO. 2 describes a polypeptide sequence. The pyrD gene encodes a polypeptide of 322 amino acids with a molecular weight of 33953. However, the present invention also relates to functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 2 by deletion, insertion or substitution or by a combination of deletion, insertion and substitution one or more amino acids, preferably replaced up to 25% of the amino acids, preferably up to 15%. The term functional derivative means that the enzyme activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. Second
Ein weiterer Teil der Erfindung besteht in den Polynucleotidse- quenzen, die die oben beschriebenen Polypeptide kodieren. Die Po- l nucleotidsequenzen lassen sich ausgehend von Sequenzen, die man aus Corynebacterium glutamicum isoliert (d.h. SEQ ID NR. 1) , er- zeugen, in dem man diese Sequenzen durch ortsgerichtete Muta- genese modifiziert oder nach Rückübersetzung des entsprechenden Polypeptids mit dem genetischen Code eine chemische Totalsynthese aus führ .Another part of the invention consists in the polynucleotide sequences which encode the polypeptides described above. The polynucleotide sequences can be generated starting from sequences which are isolated from Corynebacterium glutamicum (ie SEQ ID NO. 1) by modifying these sequences by site-directed mutagenesis or after retranslating the corresponding polypeptide with the genetic one Code to perform a total chemical synthesis.
Diese Polynucleotidsequenzen können am besten in Form von Genkon- strukten zur Transformation von WirtsOrganismen, vorzugsweise von Mikroorganismen, eingesetzt werden. Diese Genkonstrukte bestehen zumindest aus einer Kopie eines der Polynucleotide zusammen mit zumindest einer regulatorischen Sequenz. Regulatorische Sequenzen umfassen Promotoren, Terminatoren, Verstärker und ribosomale Bindungsstellen . Bevorzugte Wirtsorganismen für die Transformation mit diesen Gen- konstrukten sind Corynebacterium- und Bacillus-Arten, auch jeden eukaryontischen Mikroorganismus kann man dafür einsetzen, vorzugsweise Hefestämme der Gattung Ashbya, Candida , Pichia , Saccharomyces und Hansenula .These polynucleotide sequences can best be used in the form of gene constructs for the transformation of host organisms, preferably microorganisms. These gene constructs consist of at least one copy of one of the polynucleotides together with at least one regulatory sequence. Regulatory sequences include promoters, terminators, enhancers and ribosomal binding sites. Preferred host organisms for the transformation with these gene constructs are Corynebacterium and Bacillus species, any eukaryotic microorganism can also be used for this, preferably yeast strains of the genus Ashbya, Candida, Pichia, Saccharomyces and Hansenula.
Ein weiterer Teil der Erfindung besteht im Herstellungsprozeß für Pyrimidine und Pyrimidinderivate mit Hilfe der Kultivierung eines Wirtsorganismus, der in der oben beschriebenen Art transformiert ist, und in der nachfolgenden Isolierung der Pyrimidine. Unter einem Pyrimidinderivat versteht man eine Verbindung mit einem Pyrimidinring, das sich dadurch herstellen läßt, daß man einen Wirtsorganismus mit einem der der hier vorliegenden Erfindung entsprechenden Polynucleotide transformiert.Another part of the invention consists in the production process for pyrimidines and pyrimidine derivatives with the aid of the cultivation of a host organism which is transformed in the manner described above, and in the subsequent isolation of the pyrimidines. A pyrimidine derivative is a compound with a pyrimidine ring, which can be prepared by transforming a host organism with one of the polynucleotides corresponding to the present invention.
Die Verfahren und Vorgehensweisen zur Kultivierung von Mikroorganismen und zur Isolierung von Pyrimidinen aus einer mikrobiellen Produktion sind dem geschulten Personal geläufig.The trained personnel are familiar with the processes and procedures for cultivating microorganisms and isolating pyrimidines from a microbial production.
Die folgenden Beispiele beschreiben, wie die Erfindung entstand und ihre Anwendung bei der gentechnischen Veränderung von Mikroorganismen zur erhöhten Produktionsleistung von Pyrimidinnucleo- tiden und/oder verwandten Verbindungen.The following examples describe how the invention originated and its use in the genetic engineering of microorganisms to increase the production output of pyrimidine nucleotides and / or related compounds.
Beispiel 1example 1
Darstellung einer Genombibliothek aus Corynebacterium glutamicum ATCC 13032Representation of a genome library from Corynebacterium glutamicum ATCC 13032
DNA aus dem Genom von Corynejbacterium glutamicum ATCC 13032 läßt sich nach Standardmethoden gewinnen, die bereits beschrieben sind, z. B. von J. Altenbuchner und J. Cullum (1984, Mol. Gen. Genet. 195:134-138). Die Genombibliothek läßt sich nach Standardvorschriften (z.B.: Sambrook, J. et al . (1989) Molecular cloning: a laboratory manual , Cold Spring Harbor Laboratory Press) mit einem beliebigen Klonierungsvektor herstellen, z.B. pBluescript II KS- (Stratagene) oder ZAP Express™ (Stratagene) . Dabei kann man jede beliebige Fragmentgröße benutzen, vorzugsweise Sau3AI- Fragmente mit einer Länge von 2-9 kb, die sich in Klonierungsvek- toren mit verdautem BamEl einbinden lassen. Beispiel 2DNA from the genome of Corynejbacterium glutamicum ATCC 13032 can be obtained by standard methods which have already been described, e.g. B. by J. Altenbuchner and J. Cullum (1984, Mol. Gen. Genet. 195: 134-138). The genome library can be prepared according to standard regulations (for example: Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, for example pBluescript II KS- (Stratagene) or ZAP Express ™ (Stratagene). Any fragment size can be used, preferably Sau3AI fragments with a length of 2-9 kb, which can be integrated into cloning vectors with digested BamEl. Example 2
Analyse der Nucleinsäuresequenz der GenombibliothekAnalysis of the nucleic acid sequence of the genome library
Einzelne E. coli-Klone kann man aus der im Beispiel 1 dargestellten Genombibliothek auswählen. E. coli-Zellen werden nach Standardvorfahren in geeigneten Medien kultiviert (z.B. LB ergänzt mit 100 mg/1 Ampicillin) , und danach läßt sich die Plasmid-DNA isolieren. Klont man Genomfragmente aus der DNA von Corynebacte- rium glutamicum in pBluescript II KS- (siehe Beispiel 1) , läßt sich die DNA mit Hilfe der Oligonucleotide 5 ' -AATTAACCCTCAC- TAAAGGG-3 ' und 5 ' -GTAATACGACTCACTATAGGGC-3 ' sequenzieren.Individual E. coli clones can be selected from the genome library shown in Example 1. E. coli cells are cultivated according to standard ancestors in suitable media (e.g. LB supplemented with 100 mg / 1 ampicillin), and the plasmid DNA can then be isolated. If one clones genome fragments from the DNA of Corynebacterium glutamicum in pBluescript II KS- (see Example 1), the DNA can be sequenced with the help of the oligonucleotides 5 '-AATTAACCCTCAC-TAAAGGG-3' and 5 '-GTAATACGACTCACTATAGGGC-3'.
Beispiel 3Example 3
Computeranalyse der Sequenzen der isolierten NukleinsäurenComputer analysis of the sequences of the isolated nucleic acids
Die Nucleotidsequenzen lassen sich z.B. mit Hilfe des BLASTX-Al- gorith us (Altschul et al . (1990) J. Mol. Biol . 215: 403-410) aneinanderfügen. Auf diesem Weg kann man neuartige Sequenzen entdecken und die Funktion dieser neuartigen Gene aufklären.The nucleotide sequences can e.g. using the BLASTX algorithm (Altschul et al. (1990) J. Mol. Biol. 215: 403-410). In this way, one can discover new sequences and elucidate the function of these new genes.
Beispiel 4Example 4
Identifizierung eines E. coli-Klons, der das Gen für die Dihydroorotat-Dehydrogenase (EC 1.3.3.1) enthältIdentification of an E. coli clone that contains the gene for dihydroorotate dehydrogenase (EC 1.3.3.1)
Bei der Analyse der E. coli-Klone, wie sie im Beispiel 2 beschrieben wurde, an die sich die im Beispiel 3 beschriebene Analyse der dabei erhaltenen Sequenzen anschloß, ergab sich eine Sequenz, wie sie mit SEQ ID NR. 1 beschrieben ist. Bei der Anwendung des BLASTX-Algorithmus (siehe Beispiel 3) ergab diese Sequenz Ähnlichkeit mit der Dihydroorotat-Dehydrogenase (PyrD; EC 1.3.3.1) aus verschiedenen Organismen. Die größte Ähnlichkeit war mit der Dihydroorotat-Dehydrogenase aus .Mycoiacterium leprae gegeben (SWISSPROT P46727; 67% Übereinstimmung auf der Stufe der Aminosäuren) .When the E. coli clones were analyzed as described in Example 2, followed by the analysis of the sequences obtained in Example 3, a sequence was obtained as described with SEQ ID NO. 1 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity with the dihydroorotate dehydrogenase (PyrD; EC 1.3.3.1) from different organisms. The greatest similarity was with the dihydroorotate dehydrogenase from .Mycoiacterium leprae (SWISSPROT P46727; 67% agreement at the amino acid level).
Beispiel 5Example 5
Der Einsatz des Gens für die Dihydroorotat-Dehydrogenase (pyrD) aus Corynebacteri um glutamicum zur Produktion von Pyrimidin und/ oder mit Pyrimidin verwandten VerbindungenThe use of the gene for dihydroorotate dehydrogenase (pyrD) from Corynebacteri um glutamicum for the production of pyrimidine and / or compounds related to pyrimidine
Das Gen für die Dihydroorotat-Dehydrogenase aus Corynebacterium glutamicum kann man mit Hilfe geeigneter Klonierungs- und/oder Expressions Systeme in das Corynebacterium glutamicum oder in einen beliebigen anderen Mikroorganismus einführen. Es lassen sich gentechnisch veränderte Mikroorganismen herstellen, die sich vom Wildtyp in der Aktivität oder der Anzahl der Kopien der Gene unterscheiden. Diese neuartigen, gentechnisch veränderten Stämme kann man zur Produktion von Pyrimidin und/oder mit Pyrimidin verwandten Verbindungen einsetzen.The gene for the dihydroorotate dehydrogenase from Corynebacterium glutamicum can be inserted into the Corynebacterium glutamicum or in with the aid of suitable cloning and / or expression systems introduce any other microorganism. Genetically modified microorganisms can be produced that differ from the wild type in the activity or the number of copies of the genes. These novel, genetically modified strains can be used to produce pyrimidine and / or compounds related to pyrimidine.
Sequenzlistesequence Listing
(I) Allgemeine Angaben(I) General information
(1) Anmelder:(1) Applicant:
(A) Name: BASF-LYNX Bioscience AG (B) Straße: Im Neuenheimer Feld 515(A) Name: BASF-LYNX Bioscience AG (B) Street: Im Neuenheimer Feld 515
(C) Stadt: Heidelberg(C) City: Heidelberg
(D) Land: Deutschland(D) Country: Germany
(E) Postleitzahl: 69120(E) Postcode: 69120
(F) Telephon: 06221/4546 (G) Telefax: 06221/454770(F) Telephone: 06221/4546 (G) Fax: 06221/454770
(2) Titel: Die Sequenz der Dihydroorotat-Dehydrogenase aus(2) Title: The sequence of the dihydroorotate dehydrogenase
Corynebacterium glutamicum und deren Einsatz zur mikrobiellen Produktion von Pyrimidinen und/oder mit Pyrimidin verwandten VerbindungenCorynebacterium glutamicum and their use for the microbial production of pyrimidines and / or compounds related to pyrimidine
(3) Anzahl der Sequenzen: 2(3) Number of sequences: 2
(4) Art der vom Computer lesbaren Form:(4) Type of computer readable form:
(A) Datenträger: Diskette(A) Disk: diskette
(B) Computer: IBM PC kompatibel(B) Computer: IBM PC compatible
(C) Betriebssystem: Windows NT(C) Operating system: Windows NT
(D) Software: Microsof ®word 97 SR-1(D) Software: Microsof ®word 97 SR-1
(I) Angaben zur SEQ ID NR. 1:(I) Information on SEQ ID NO. 1:
(1) Sequenzcharakteristika:(1) Sequence characteristics:
(A) Länge: 966(A) Length: 966
(B) Art: Nucleinsäure(B) Type: nucleic acid
(C) Strangtyp: Doppelsträng(C) Strand type: double strand
(D) Topologie: linear(D) Topology: linear
(2) Art des Moleküls: DNA(2) Type of molecule: DNA
(3) hypothetisch: nein(3) hypothetically: no
(4) Antisense: nein ( 5 ) Herkunft :(4) Antisense: no (5) Origin:
(A) Organismus: Corynebacterium glutamicum(A) Organism: Corynebacterium glutamicum
(6) Beschreibung der Sequenz: SEQ ID NR. 1:(6) Description of the sequence: SEQ ID NO. 1:
ATGGAAAAAATCATCGCAGTGCACGATGATTCCCTCTCCCAGGAAGTCTTCGGCGTCACCTTCCC ACGACCACTAGGCCTCGCCGCAGGTTTCGACAAAAACGCATCAATGGCTGATGCCTGGGGTGCCG TTGGATTCGGATACGCCGAACTTGGCACCGTCACCGCCTCCCCACAGCCAGGAAACCCCACCCCG CGCCTTTTCCGCCTGCCTGCCGACAAAGCTATCTTGAACCGCATGGGATTCAACAACCTGGGTGC AGCAGAAGTCGCAAAAAACCTGCGCAACCGGAAATCCACCGATGTCATCGGCATCAACATCGGTA AAACCAAAGTGGTTCCCGCTGAACACGCAGTAGATGACTACCGCCGTTCTGCATCTTTGTTAGGT GATCTTGCTGATTACCTGGTTGTCAACGTTTCCTCCCCCAACACTCCGGGTCTCCGCGATCTGCA GGCTGTGGAATCTTTGCGACCAATCCTCGCCGCAGTGCAGGAATCCACCACCGTCCCAGTCTTGG TGAAAATCGCACCAGACCTCTCCGACGAAGACATCGACGCCGTAGCTGACCTGGCAGTTGAGCTC AAACTCGCCGGAATCGTAGCCACCAATACCACCATTTCCCGCGAAGGCCTCAACACTCCTTCAGG TGAAGTCGAAGCCATGGGTGCTGGCGGAATCTCCGGTGCTCCAGTAGCAGCCCGATCTTTGGAGG TACTCAAGCGCCTCTACGCACGGGTAGGCAAAGAGATGGTGTTGATCTCTGTCGGTGGCATCAGC ACCCCTGAGCAAGCCTGGGAACGCATCACCTCCGGCGCAACCCTTCTGCAGGGATACACCCCATT CATCTACGGTGGCCCCGATTGGATCAGAGATATCCACCTTGGTATCGCCAAGCAGCTGAAAGCTC ACGGTCTGCGCAACATCGCTGACGCTGTGGGCAGCGAATTGGAGTGGAAGAACTAAATGGAAAAAATCATCGCAGTGCACGATGATTCCCTCTCCCAGGAAGTCTTCGGCGTCACCTTCCC ACGACCACTAGGCCTCGCCGCAGGTTTCGACAAAAACGCATCAATGGCTGATGCCTGGGGTGCCG TTGGATTCGGATACGCCGAACTTGGCACCGTCACCGCCTCCCCACAGCCAGGAAACCCCACCCCG CGCCTTTTCCGCCTGCCTGCCGACAAAGCTATCTTGAACCGCATGGGATTCAACAACCTGGGTGC AGCAGAAGTCGCAAAAAACCTGCGCAACCGGAAATCCACCGATGTCATCGGCATCAACATCGGTA AAACCAAAGTGGTTCCCGCTGAACACGCAGTAGATGACTACCGCCGTTCTGCATCTTTGTTAGGT GATCTTGCTGATTACCTGGTTGTCAACGTTTCCTCCCCCAACACTCCGGGTCTCCGCGATCTGCA GGCTGTGGAATCTTTGCGACCAATCCTCGCCGCAGTGCAGGAATCCACCACCGTCCCAGTCTTGG TGAAAATCGCACCAGACCTCTCCGACGAAGACATCGACGCCGTAGCTGACCTGGCAGTTGAGCTC AAACTCGCCGGAATCGTAGCCACCAATACCACCATTTCCCGCGAAGGCCTCAACACTCCTTCAGG TGAAGTCGAAGCCATGGGTGCTGGCGGAATCTCCGGTGCTCCAGTAGCAGCCCGATCTTTGGAGG TACTCAAGCGCCTCTACGCACGGGTAGGCAAAGAGATGGTGTTGATCTCTGTCGGTGGCATCAGC ACCCCTGAGCAAGCCTGGGAACGCATCACCTCCGGCGCAACCCTTCTGCAGGGATACACCCCATT CATCTACGGTGGCCCCGATTGGATCAGAGATATCCACCTTGGTATCGCCAAGCAGCTGAAAGCTC ACGGTCTGCGCAACATCGCTGACGCTGTGGGCAGCGAATTGGAGTGGAAGAACTAA
(1) Angaben zur SEQ ID NR. 2:(1) Information on SEQ ID NO. 2:
(1) Sequenzcharakteristika:(1) Sequence characteristics:
(A) Länge: 322(A) Length: 322
(B) Art: Aminosäure(B) Type: amino acid
(C) Strangtyp: eine Kette (D) Topologie: linear(C) strand type: a chain (D) topology: linear
(2) Art des Moleküls: Aminosäure(2) Type of molecule: amino acid
(3) hypothetisch: nein(3) hypothetically: no
(4) Antisense: nein (5) Herkunft:(4) antisense: no (5) origin:
(B) Organismus: Corynebacterium glutamicum(B) Organism: Corynebacterium glutamicum
(6) Beschreibung der Sequenz: SEQ ID NR. 2:(6) Description of the sequence: SEQ ID NO. 2:
MEKIIAVHDDSLSQEVFGVTFPRPLGLAAGFDKNASMADAWGAVGFGYAELGTVTASPQPGNPTP RLFRLPADKAILNRMGFNNLGAAFVAKNLRNRKSTDVIGINIGKTKVVPAEHAVDDYRRSASLLG DLADYLWNVSSPNTPGLRDLQAVESLRPILAAVQESTTVPVLVKIAPDLSDEDIDAVADLAVEL KLAGIVATNTTISREGLNTPSGEVEAMGAGGISGAPVAARSLEVLKRLYARVGKEMVLISVGGIS TPEQAWERITSGATLLQGYTPFIYGGPDWIRDIHLGIAKQLKAHGLRNIADAVGSELEWKN MEKIIAVHDDSLSQEVFGVTFPRPLGLAAGFDKNASMADAWGAVGFGYAELGTVTASPQPGNPTP RLFRLPADKAILNRMGFNNLGAAFVAKNLRNRKSTDVIGINIGKTKVVPAEHAVDDYRRSASLLG DLADYLWNVSSPNTPGLRDLQAVESLRPILAAVQESTTVPVLVKIAPDLSDEDIDAVADLAVEL KLAGIVATNTTISREGLNTPSGEVEAMGAGGISGAPVAARSLEVLKRLYARVGKEMVLISVGGIS TPEQAWERITSGATLLQGYTPFIYGGPDWIRDIHLGIAKQLKAHGLRNIADAVGSELEWKN

Claims

Patentansprüche claims
1. Ein Polypeptid mit Dihydroorotat-Dehydrogenaseaktivität , das aus der folgenden Gruppe ausgewählt wurde:1. A polypeptide with dihydroorotate dehydrogenase activity selected from the following group:
(a) ein Polypeptid mit einer Aminosäuresequenz, wie sie in der SEQ ID NR. 2 beschrieben ist,(a) a polypeptide with an amino acid sequence as described in SEQ ID NO. 2 is described
(b) ein Polypeptid, das im Vergleich zu (a) durch Deletion, Insertion oder Substitution einer oder mehrerer Aminosäuren verändert ist.(b) a polypeptide which is modified compared to (a) by deletion, insertion or substitution of one or more amino acids.
2. Ein Polynucleotid, das ein dem Anspruch 1 entsprechendes Polypeptid kodiert.2. A polynucleotide encoding a polypeptide according to claim 1.
3. Ein Genkonstruk , das zumindest aus einer Kopie eines dem Anspruch 2 entsprechenden Polynucleotids zusammen mit zumindest einer regulatorischen Sequenz besteht.3. A gene construct consisting of at least one copy of a polynucleotide corresponding to claim 2 together with at least one regulatory sequence.
4. Ein Wir sOrganismus, der mit einem dem Anspruch 3 entsprechenden Gen transformiert ist.4. An organism that is transformed with a gene corresponding to claim 3.
5. Ein Prozeß zur Produktion von Pyrimidinen und Pyrimidin- derivaten, bei dem ein dem Anspruch 4 entsprechender Wirtsorganismus kultiviert und in der Folge das Pyrimidin oder das Pyrimidinderivat isoliert wird. 5. A process for the production of pyrimidines and pyrimidine derivatives, in which a host organism corresponding to claim 4 is cultivated and the pyrimidine or the pyrimidine derivative is subsequently isolated.
PCT/EP2000/005850 1999-06-25 2000-06-23 Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine WO2001000847A1 (en)

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KR1020017016566A KR20020026470A (en) 1999-06-25 2000-06-23 Dihydroorotate Dehydrogenase Sequence of Corynebacterium Glutamicum and The Use Thereof in Microbial Production of Pyrimidine and/or Compounds Used With Pyrimidine
EP00942126A EP1196602A1 (en) 1999-06-25 2000-06-23 Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine
CA002377482A CA2377482A1 (en) 1999-06-25 2000-06-23 Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine
AU56854/00A AU5685400A (en) 1999-06-25 2000-06-23 Dihydroorotate dehydrogenase sequence of corynebacterium glutamicum and the use thereof in microbial production of pyrimidine and/or compounds used with pyrimidine

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DE19929364.3 1999-06-25
DE19929364A DE19929364A1 (en) 1999-06-25 1999-06-25 New Corynebacterium glutamicum dihydroorotate dehydrogenase polypeptide and corresponding DNA useful for creating pyrimidine-producing organisms

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US6680187B2 (en) 2000-09-13 2004-01-20 Degussa Ag Nucleotide sequences coding for the PTSI protein
US6689587B2 (en) 2000-11-10 2004-02-10 Degussa Ag Polynucleotides encoding the nadC gene and methods of producing nicotinic acid or nicotinic acid derivatives
US6692946B2 (en) 2000-11-10 2004-02-17 Degussa Ag Polynucleotides encoding the nadA gene and methods of producing nicotinic acid or nicotinic acid derivatives
US6759224B2 (en) 2000-09-09 2004-07-06 Degussa Ag Nucleotide sequences which code for the sahH gene
US6812006B2 (en) 2000-08-10 2004-11-02 Degussa Ag Nucleotide sequences which code for the lysR3 gene
US6812016B2 (en) 2000-09-02 2004-11-02 Degussa Ag Nucleotide sequences which code for the metY gene
US6815196B2 (en) 2000-09-02 2004-11-09 Degussa Ag Nucleotide sequences encoding o-succinylhomoserine sulfhydrylase
US6875586B2 (en) 2000-08-10 2005-04-05 Degussa Ag Nucleotide sequences coding for the luxR gene
US6902916B2 (en) 2000-08-10 2005-06-07 Degussa Ag Nucleotide sequences coding for the 1ysR1 gene
US6942996B2 (en) 2000-08-02 2005-09-13 Degussa Ag Isolated polynucleotide from Corynebacterium encoding a homocysteine methyltransferase
US6958228B2 (en) 2000-08-02 2005-10-25 Degussa Ag Nucleotide sequence which code for the metH gene
US7038034B2 (en) 2000-09-09 2006-05-02 Degussa Ag Nucleotide sequences coding for the Dep33 efflux protein
US7105321B2 (en) 2000-08-26 2006-09-12 Degussa Ag Nucleotide sequences which code for the ccpA2 gene

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6958228B2 (en) 2000-08-02 2005-10-25 Degussa Ag Nucleotide sequence which code for the metH gene
US6942996B2 (en) 2000-08-02 2005-09-13 Degussa Ag Isolated polynucleotide from Corynebacterium encoding a homocysteine methyltransferase
US6875586B2 (en) 2000-08-10 2005-04-05 Degussa Ag Nucleotide sequences coding for the luxR gene
US7173105B2 (en) 2000-08-10 2007-02-06 Degussa Ag Nucleotide sequences coding for the LuxR gene
US6812006B2 (en) 2000-08-10 2004-11-02 Degussa Ag Nucleotide sequences which code for the lysR3 gene
US6902916B2 (en) 2000-08-10 2005-06-07 Degussa Ag Nucleotide sequences coding for the 1ysR1 gene
US7105321B2 (en) 2000-08-26 2006-09-12 Degussa Ag Nucleotide sequences which code for the ccpA2 gene
US6815196B2 (en) 2000-09-02 2004-11-09 Degussa Ag Nucleotide sequences encoding o-succinylhomoserine sulfhydrylase
US6812016B2 (en) 2000-09-02 2004-11-02 Degussa Ag Nucleotide sequences which code for the metY gene
US6759224B2 (en) 2000-09-09 2004-07-06 Degussa Ag Nucleotide sequences which code for the sahH gene
US7038034B2 (en) 2000-09-09 2006-05-02 Degussa Ag Nucleotide sequences coding for the Dep33 efflux protein
US6680187B2 (en) 2000-09-13 2004-01-20 Degussa Ag Nucleotide sequences coding for the PTSI protein
US7160703B2 (en) 2000-09-14 2007-01-09 Degussa Ag Nucleotide sequences coding for the PtsI protein
US6692946B2 (en) 2000-11-10 2004-02-17 Degussa Ag Polynucleotides encoding the nadA gene and methods of producing nicotinic acid or nicotinic acid derivatives
US6689587B2 (en) 2000-11-10 2004-02-10 Degussa Ag Polynucleotides encoding the nadC gene and methods of producing nicotinic acid or nicotinic acid derivatives

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