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WO2001000845A1 - Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid - Google Patents

Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid Download PDF

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Publication number
WO2001000845A1
WO2001000845A1 PCT/EP2000/005864 EP0005864W WO0100845A1 WO 2001000845 A1 WO2001000845 A1 WO 2001000845A1 EP 0005864 W EP0005864 W EP 0005864W WO 0100845 A1 WO0100845 A1 WO 0100845A1
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Prior art keywords
polypeptide
folic acid
seq
amino acids
deletion
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PCT/EP2000/005864
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German (de)
French (fr)
Inventor
Matthias Mack
Karin Herbster
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Basf-Lynx Bioscience Ag
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Priority to AU59782/00A priority Critical patent/AU5978200A/en
Priority to KR1020017016565A priority patent/KR20020026469A/en
Priority to CA002377458A priority patent/CA2377458A1/en
Priority to EP00945815A priority patent/EP1194565A1/en
Publication of WO2001000845A1 publication Critical patent/WO2001000845A1/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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1235—Diphosphotransferases (2.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system

Definitions

  • the present invention relates to the production process for folic acid by fermentation using a genetically modified organism.
  • This invention consists of the nucleotide sequences of four genes (folE, folP, folB and folK) from Corynebacterium glutamicum for folic acid biosynthesis and their use for the microbial production of folic acid. These four genes form an operon and are transcribed in the following order: folE, folP, folB, folK.
  • Folic acid is essential for animal organisms. Its derivative tetrahydrofolate is a very versatile carrier of activated single-carbon units in cells of the animal organism. Folic acid consists of three groups: a substituted pteridine ring, p-aminobenzoate and glutamate. Mammals cannot synthesize a pteridine ring. They take in folic acid from food and from microorganisms in their intestinal tract. Folic acid deficiency mainly leads to lesions in the mucous membranes.
  • Folic acid is mainly used as a food additive.
  • Microorganisms can be used for the fermentative production of folic acid. They can be optimized in their folic acid biosynthesis performance by genetically modifying the biosynthetic pathway of folic acid.
  • genetic engineering means increasing the number of copies and / or the rate of transcription of the genes of the biosynthetic pathway for folic acid.
  • the proportion of gene product and thus also the intracellular enzyme activity increases.
  • Increased enzyme activity leads to an increased rate of conversion of food (eg glucose) to folic acid and thus to an increased product concentration.
  • the nucleotide sequences of the genes of the folic acid biosynthetic pathway must be identified.
  • This invention is concerned with four new gene sequences for the folic acid biosynthesis from Corynebacterium glutamicum and with their use for the microbial production of folic acid.
  • Part of the invention is the folE gene product.
  • SEQ ID NO. 2 describes a polypeptide sequence.
  • the folE gene product encodes a polypeptide of 202 amino acids with a molecular weight of 22029 Da.
  • the present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 2 one or more amino acids, preferably up to 25% of the amino acids, preferably up to 15% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution.
  • the term functional derivative means that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 2.
  • Another part of the invention is the folP gene product.
  • SEQ ID NO. 4 describes a polypeptide sequence.
  • the folP gene product encodes a polypeptide of 285 amino acids with a molecular weight of 29520 Da.
  • the present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 4 one or more amino acids, preferably up to 40% of the amino acids, preferably up to 25% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution.
  • the term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 4th
  • SEQ ID NO. 6 describes a polypeptide sequence.
  • the folB gene product encodes a polypeptide of 131 amino acids with a molecular weight of 14020 Da.
  • the present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 6 one or more amino acids, preferably up to 30% of the amino acids, preferably up to 20% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution.
  • the term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID R. 6.
  • SEQ ID NO. 8 describes a polypeptide sequence.
  • the folK gene product encodes a polypeptide of 160 amino acids with a molecular weight of 18043 Da.
  • the present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 8 by deletion, insertion or substitution or by a combination of deletion, Insertion and substitution of one or more amino acids, preferably replacing up to 40% of the amino acids, preferably up to 30% of the amino acids.
  • functional derivative it is meant that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 8th.
  • 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, 3, 5 and 7) by modifying these sequences by site-directed mutagenesis or after back-translating the corresponding polypeptide with genetic code carries out a total chemical synthesis.
  • polynucleotide sequences can preferably be used for the transformation of host organisms, and preferably of microorganisms, in the form of gene constructs which contain at least one copy of one of these 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 Coryneibacterium and Bacillus species. Any eukaryotic microorganism can also be used, preferably yeast strains of the genus Ashbya, Candlda, Plchla, Saccharomyces and Hansenula.
  • Another part of the invention consists in the process for the preparation of folic acid by culturing a host organism which is transformed in the manner described above and in the subsequent isolation of the folic acid.
  • the trained personnel are familiar with the processes and procedures for cultivating microorganisms and isolating folic acid from a microbial production.
  • DNA from the genome of Corynebacterium glutamlcum 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 (e.g. Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, e.g. pBluescript II KS- (Stratagene) or ZAP Express TM (Stratagene). Any fragment size can be used, preferably 5'au3AI fragments with a length of 2-9 kb, which can be integrated into cloning vectors with digested BamHI.
  • 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), after which the plasmid DNA can be isolated. If one clones genome fragments from the DNA of Corynebacterium glutamlcum in pBluescript II KS- (see Example 1), the DNA can be sequenced with the help of the oligonucleotides 5 '-AATTAACCCTCACTAAAGGG-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 The analysis of the E. coli clones, as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in a sequence as described with SEQ ID NO. 3 is described.
  • this sequence showed similarity to dihydropteroate synthases (FolP; EC 2.5.1.15) from different organisms. The greatest similarity was with the dihydropteroate synthase (FolP) from Mycobacterlum tuberculosls (NRDB 006274; 53% agreement at the amino acid level).
  • 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. 5 is described.
  • this sequence showed similarity to dihydroneopterin aldolases (FolB; EC 4.1.2.25) from different organisms. The greatest similarity 5 was with the dihydroneopterin aldolase (FolB) from Mycobacterlum tuberculosis (NRDB 006275; 61% agreement at the amino acid level).
  • Example 5 The analysis of the E. coli clones as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in one Sequence as shown with SEQ ID NO. 7 is described.
  • this sequence showed similarity with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinases (FolK; EC 2.7.6.3) from different organisms. The greatest similarity was with that
  • GTP cyclohydrolase I for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum for the production of folic acid
  • the genes for the GTP cyclohydrolase I, for the dihydropteroate synthase, for the dihydroneopterin aldolase and for the 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum can be obtained with the aid of suitable cloning and expression systems introduce into Corynebacterium glutamicum or into any other microorganism. Genetically modified microorganisms can be produced which differ from the wild-type organism with regard to the activity or the number of gene copies. These new, genetically modified strains can be used to produce folic acid.
  • SEQ ID NO. 1 DNA (folE)
  • SEQ ID NO. 2 amino acid (FolE)
  • SEQ ID NO. 3 DNA (folP)
  • SEQ ID NO. 4 amino acid (FolP)
  • SEQ ID NO. 6 amino acid (FolB)
  • SEQ ID NO. 7 DNA (folK)
  • SEQ ID NO. 8 amino acid (FolK)

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Abstract

The invention relates to nucleotide sequences of four genes (folE, folP, folB and folK) from Corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid.

Description

Gene aus Corynebacterium glutami cum für die Folsäurebiosynthese und ihr Einsatz zur mikrobiellen Herstellung von FolsäureGenes from Corynebacterium glutami cum for folic acid biosynthesis and their use in the microbial production of folic acid
Beschreibungdescription
Die vorliegende Erfindung befaßt sich mit dem Herstellungsverfahren für Folsäure durch Fermentation mit Hilfe eines gentechnisch veränderten Organismus. Diese Erfindung besteht aus den Nucleo- tidsequenzen von vier Genen ( folE, folP, folB und folK) aus Corynebacterium glutamicum für die Folsäurebiosynthese und deren Einsatz zur mikrobiellen Herstellung von Folsäure. Diese vier Gene bilden ein Operon und werden in der folgenden Reihenfolge transkribiert: folE, folP, folB, folK.The present invention relates to the production process for folic acid by fermentation using a genetically modified organism. This invention consists of the nucleotide sequences of four genes (folE, folP, folB and folK) from Corynebacterium glutamicum for folic acid biosynthesis and their use for the microbial production of folic acid. These four genes form an operon and are transcribed in the following order: folE, folP, folB, folK.
Folsäure ist essentiell für tierische Organismen. Ihr Derivat Tetrahydrofolat ist in Zellen des tierischen Organismus ein sehr vielseitiger Carrier von aktivierten Einkohlenstoffeinheiten. Folsäure besteht aus drei Gruppen: einem substituierten Pteridin- ring, p-Aminobenzoat und Glutamat. Säuger können einen Pteridin- ring nicht synthetisieren. Sie nehmen Folsäure mit der Nahrung und von Mikroorganismen in ihrem Darmtrakt auf . Folsäuremangel führt hauptsächlich zu Läsionen in den Schleimhäuten.Folic acid is essential for animal organisms. Its derivative tetrahydrofolate is a very versatile carrier of activated single-carbon units in cells of the animal organism. Folic acid consists of three groups: a substituted pteridine ring, p-aminobenzoate and glutamate. Mammals cannot synthesize a pteridine ring. They take in folic acid from food and from microorganisms in their intestinal tract. Folic acid deficiency mainly leads to lesions in the mucous membranes.
Die kommerzielle Bedeutung der Folsäure liegt im Futtermittel- und Lebensmittelmarkt. Folsäure wird hauptsächlich als Nahrungsmittelzusatz eingesetzt.The commercial importance of folic acid lies in the feed and food market. Folic acid is mainly used as a food additive.
Mikroorganismen können zur fermentativen Herstellung von Folsäure eingesetzt werden. Man kann sie durch gentechnische Veränderung des Biosynthesewegs der Folsäure in ihrer Folsäurebiosyntheselei- stung optimieren. Gentechnische Veränderung bedeutet in diesem Zusammenhang, die Anzahl der Kopien und/oder die Transkriptionsgeschwindigkeit der Gene des Biosynthesewegs für die Folsäure zu erhöhen. Als Folge davon steigt der Anteil an Genprodukt und damit auch die intrazelluläre Enzymaktivität. Erhöhte Enzymaktivität führt zu einer vermehrten Umwandlungsgeschwindigkeit der Nahrung (z.B. Glucose) zu Folsäure und damit auch zu einer erhöhten Produktkonzentration. Zur gentechnischen Veränderung müssen die Nucleotidsequenzen der Gene des Folsäurebiosynthese- wegs identifiziert werden. Diese Erfindung befaßt sich mit vier neuen Gensequenzen für die Folsäurebiosynthese aus Corynebacterium glutamicum und mit ihrem Einsatz zur mikrobiellen Herstellung von Folsäure . Ein Teil der Erfindung besteht im folE-Genprodukt . SEQ ID NR. 2 beschreibt eine Polypeptidsequenz . Das folE-Genprodukt kodiert ein Polypeptid aus 202 Aminosäuren mit einem Molekulargewicht von 22029 Da. Die vorliegende Erfindung befaßt sich auch mit funktio- nellen 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 Aminosäuren. Mit dem Ausdruck funktionelles Derivat ist gemeint, daß die enzy- matische Aktivität des Derivats noch in der gleichen Größenordnung liegt wie die des Polypeptids mit der Sequenz SEQ ID NR. 2. Ein weiterer Teil der Erfindung besteht in dem folP-Genprodukt . SEQ ID NR. 4 beschreibt eine Polypeptidsequenz. Das folP-Genpro- dukt kodiert ein Polypeptid aus 285 Aminosäuren mit einem Molekulargewicht von 29520 Da. Die vorliegende Erfindung befaßt sich auch mit funktioneilen Derivaten dieses Polypeptids, die man erhalten kann, wenn man in der SEQ ID NR. 4 durch Deletion, Insertion oder Substitution oder durch eine Kombination von Deletion, Insertion und Substitution eine oder mehrere Aminosäuren, vorzugsweise bis zu 40% der Aminosäuren ersetzt, am besten bis zu 25% der Aminosäuren. Mit dem Ausdruck funktionelles Derivat ist gemeint, daß die enzymatische Aktivität des Derivats noch in der gleichen Größenordnung liegt wie die des Polypeptids mit der Se- quenz SEQ ID NR. 4.Microorganisms can be used for the fermentative production of folic acid. They can be optimized in their folic acid biosynthesis performance by genetically modifying the biosynthetic pathway of folic acid. In this context, genetic engineering means increasing the number of copies and / or the rate of transcription of the genes of the biosynthetic pathway for folic acid. As a result, the proportion of gene product and thus also the intracellular enzyme activity increases. Increased enzyme activity leads to an increased rate of conversion of food (eg glucose) to folic acid and thus to an increased product concentration. For genetic modification, the nucleotide sequences of the genes of the folic acid biosynthetic pathway must be identified. This invention is concerned with four new gene sequences for the folic acid biosynthesis from Corynebacterium glutamicum and with their use for the microbial production of folic acid. Part of the invention is the folE gene product. SEQ ID NO. 2 describes a polypeptide sequence. The folE gene product encodes a polypeptide of 202 amino acids with a molecular weight of 22029 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 2 one or more amino acids, preferably up to 25% of the amino acids, preferably up to 15% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 2. Another part of the invention is the folP gene product. SEQ ID NO. 4 describes a polypeptide sequence. The folP gene product encodes a polypeptide of 285 amino acids with a molecular weight of 29520 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 4 one or more amino acids, preferably up to 40% of the amino acids, preferably up to 25% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 4th
Ein weiterer Teil der Erfindung besteht in dem folB-Genprodukt . SEQ ID NR. 6 beschreibt eine Polypeptidsequenz. Das folB-Genpro- dukt kodiert ein Polypeptid aus 131 Aminosäuren mit einem Moleku- largewicht von 14020 Da. Die vorliegende Erfindung befaßt sich auch mit funktioneilen Derivaten dieses Polypeptids, die man erhalten kann, wenn man in der SEQ ID NR. 6 durch Deletion, Insertion oder Substitution oder durch eine Kombination von Deletion, Insertion und Substitution eine oder mehrere Aminosäuren, vorzugsweise bis zu 30% der Aminosäuren ersetzt, am besten bis zu 20% der Aminosäuren. Mit dem Ausdruck funktionelles Derivat ist gemeint, daß die enzymatische Aktivität des Derivats noch in der gleichen Größenordnung liegt wie die des Polypeptids mit der Sequenz SEQ ID R. 6.Another part of the invention is the folB gene product. SEQ ID NO. 6 describes a polypeptide sequence. The folB gene product encodes a polypeptide of 131 amino acids with a molecular weight of 14020 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 6 one or more amino acids, preferably up to 30% of the amino acids, preferably up to 20% of the amino acids, are replaced by deletion, insertion or substitution or by a combination of deletion, insertion and substitution. The term functional derivative means that the enzymatic activity of the derivative is still of the same order of magnitude as that of the polypeptide with the sequence SEQ ID R. 6.
Ein weiterer Teil der Erfindung besteht in dem folfC-Genprodukt . SEQ ID NR. 8 beschreibt eine Polypeptidsequenz. Das folK-Genpro- dukt kodiert ein Polypeptid aus 160 Aminosäuren mit einem Molekulargewicht von 18043 Da. Die vorliegende Erfindung befaßt sich auch mit funktionellen Derivaten dieses Polypeptids, die man erhalten kann, wenn man in der SEQ ID NR. 8 durch Deletion, Insertion oder Substitution oder durch eine Kombination von Deletion, Insertion und Substitution eine oder mehrere Aminosäuren, vorzugsweise bis zu 40% der Aminosäuren ersetzt, am besten bis zu 30% der Aminosäuren. Mit dem Ausdruck funktionelles Derivat ist gemeint, daß die enzymatische Aktivität des Derivats noch in der gleichen Größenordnung liegt wie die des Polypeptids mit der Sequenz SEQ ID NR . 8.Another part of the invention is the folfC gene product. SEQ ID NO. 8 describes a polypeptide sequence. The folK gene product encodes a polypeptide of 160 amino acids with a molecular weight of 18043 Da. The present invention is also concerned with functional derivatives of this polypeptide, which can be obtained if one in SEQ ID NO. 8 by deletion, insertion or substitution or by a combination of deletion, Insertion and substitution of one or more amino acids, preferably replacing up to 40% of the amino acids, preferably up to 30% of the amino acids. By the term functional derivative it is meant that the enzymatic activity of the derivative is still in the same order of magnitude as that of the polypeptide with the sequence SEQ ID NO. 8th.
Ein weiterer Teil der Erfindung besteht in den Polynucleotidse- quenzen, die die oben beschriebenen Polypeptide kodieren. Die Po- lynucleotidsequenzen lassen sich ausgehend von Sequenzen, die man aus Corynebacterium glutamicum isoliert (d.h. SEQ ID NR. 1, 3, 5 und 7) , erzeugen, in dem man diese Sequenzen durch ortsgerichtete Mutagenese modifiziert oder nach Rückübersetzung des entsprechenden Polypeptids mit dem genetischen Code eine chemische Total- synthese ausführt.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, 3, 5 and 7) by modifying these sequences by site-directed mutagenesis or after back-translating the corresponding polypeptide with genetic code carries out a total chemical synthesis.
Diese Polynucleotidsequenzen lassen sich vorzugsweise einsetzen zur Transformation von Wirtsorganismen, und hierbei vorzugsweise von Mikroorganismen, und zwar in Form von Genkonstrukten, die zu- mindest eine Kopie eines dieser Polynucleotide zusammen mit mindestens einer regulatorischen Sequenz enthalten. Regulatorische Sequenzen beinhalten Promotoren, Terminatoren, Verstärker und ri- bosomale Bindungsstellen.These polynucleotide sequences can preferably be used for the transformation of host organisms, and preferably of microorganisms, in the form of gene constructs which contain at least one copy of one of these polynucleotides together with at least one regulatory sequence. Regulatory sequences include promoters, terminators, enhancers and ribosomal binding sites.
Bevorzugte Wirtsorganismen für die Transformation mit diesen Gen- konstrukten sind Coryneibacterium- und Bacillus-Arten. Auch jeden beliebigen eukaryontisehen Mikroorganismus kann man einsetzen, vorzugsweise Hefestämme der Gattung Ashbya , Candlda , Plchla , Sac- charomyces und Hansenula .Preferred host organisms for the transformation with these gene constructs are Coryneibacterium and Bacillus species. Any eukaryotic microorganism can also be used, preferably yeast strains of the genus Ashbya, Candlda, Plchla, Saccharomyces and Hansenula.
Ein weiterer Teil der Erfindung besteht in dem Verfahren zur Herstellung von Folsäure durch Kultivierung eines Wirtsorganismus, der in der oben beschriebenen Art transformiert ist, und in der nachfolgenden Isolierung der Folsäure.Another part of the invention consists in the process for the preparation of folic acid by culturing a host organism which is transformed in the manner described above and in the subsequent isolation of the folic acid.
Die Verfahren und die Vorgehensweisen zur Kultivierung von Mikroorganismen und zur Isolierung von Folsäure aus einer mikrobiellen Produktion sind dem geschulten Personal geläufig.The trained personnel are familiar with the processes and procedures for cultivating microorganisms and isolating folic acid from a microbial production.
In den folgenden Beispielen wird die Erfindung genauer beschrieben, ebenso ihre Anwendung zur gentechnischen Veränderung von Mikroorganismen zur Steigerung der Syntheseleistung von Folsäure. Beispiel 1The invention is described in more detail in the following examples, as is its application for the genetic modification of microorganisms to increase the synthesis performance of folic acid. example 1
Darstellung einer Genombibliothek aus Corynebacterium glutamlcum ATCC 13032Representation of a genome library from Corynebacterium glutamlcum ATCC 13032
DNA aus dem Genom von Corynebacterium glutamlcum 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 Standard- Vorschriften (z.B.: Sambrook, J. et al . (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) mit jedem beliebigen Klonierungsvektor herstellen, z.B. pBluescript II KS- (Stratagene) oder ZAP Express™ (Stratagene) . Dabei kann man jede beliebige Fragmentgröße benutzen, vorzugsweise 5'au3AI-Frag- mente mit einer Länge von 2-9 kb, die sich in Klonierungsvektoren mit verdautem BamHI einbinden lassen.DNA from the genome of Corynebacterium glutamlcum 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 (e.g. Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press) with any cloning vector, e.g. pBluescript II KS- (Stratagene) or ZAP Express ™ (Stratagene). Any fragment size can be used, preferably 5'au3AI fragments with a length of 2-9 kb, which can be integrated into cloning vectors with digested BamHI.
Beispiel 2Example 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) , danach läßt sich die Plasmid-DNA isolieren. Klont man Genomfragmente aus der DNA von Corynebacterium glutamlcum in pBluescript II KS- (siehe Beispiel 1) , läßt sich die DNA mit Hilfe der Oligonucleotide 5 ' -AATTAACCCTCACTAAAGGG-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), after which the plasmid DNA can be isolated. If one clones genome fragments from the DNA of Corynebacterium glutamlcum in pBluescript II KS- (see Example 1), the DNA can be sequenced with the help of the oligonucleotides 5 '-AATTAACCCTCACTAAAGGG-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- gorithmus (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. coii-Klons, der eine Nucleotidsequenz des Gens für die GTP-Cyclohydrolase I (EC 3.5.4.16) enthält Bei der Analyse der E . coli-Klone , wie sie im Beispiel 2 be- schrieben wurde, an die sich die im Beispiel 3 beschriebeneIdentification of an E. coii clone that contains a nucleotide sequence of the gene for GTP cyclohydrolase I (EC 3.5.4.16). When analyzing the E. coli clones as described in Example 2, to which the one described in Example 3 relates
Analyse der dabei erhaltenen Sequenzen anschloß, ergab sich eine Sequenz, wie sie mit SEQ ID NR. 1 beschrieben ist. Bei der Anwen- düng des BLASTX-Algorithmus (siehe Beispiel 3) ergab diese Sequenz Ähnlichkeit mit GTP-Cyclohydrolasen I (FolE; EC 3.5.4.16) aus verschiedenen Organismen. Die größte Ähnlichkeit war mit der GTP-Cyclohydrolase-I (FolE) aus Mycobacterlum tuberculoslε (NRDB 5 006273; 72% Übereinstimmung auf der Stufe der Aminosäuren).Analysis of the sequences obtained in this way resulted in a sequence as described with SEQ ID NO. 1 is described. When using Using the BLASTX algorithm (see Example 3), this sequence showed similarity to GTP cyclohydrolases I (FolE; EC 3.5.4.16) from different organisms. The greatest similarity was with GTP cyclohydrolase-I (FolE) from Mycobacterlum tuberculoslε (NRDB 5 006273; 72% agreement at the amino acid level).
Beispiel 5Example 5
Identifizierung eines E. coIi-Klons, der eine Nucleotidsequenz 0 des Gens für die Dihydropteroat-Synthase (EC 2.5.1.15) enthältIdentification of an E. coli clone which contains a nucleotide sequence 0 of the gene for the dihydropteroate synthase (EC 2.5.1.15)
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 5 Sequenz, wie sie mit SEQ ID NR. 3 beschrieben ist. Bei der Anwendung des BLASTX-Algorithmus (siehe Beispiel 3) ergab diese Sequenz Ähnlichkeit mit Dihydropteroat-Synthasen (FolP; EC 2.5.1.15) aus verschiedenen Organismen. Die größte Ähnlichkeit war mit der Dihydropteroat-Synthase (FolP) aus Mycobacterlum tu- 0 berculosls (NRDB 006274; 53% Übereinstimmung auf der Stufe der Aminosäuren) .The analysis of the E. coli clones, as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in a sequence as described with SEQ ID NO. 3 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity to dihydropteroate synthases (FolP; EC 2.5.1.15) from different organisms. The greatest similarity was with the dihydropteroate synthase (FolP) from Mycobacterlum tuberculosls (NRDB 006274; 53% agreement at the amino acid level).
Beispiel 6Example 6
5 Identifizierung eines E. coli-Klons, der eine Nucleotidsequenz des Gens für die Dihydroneopterin-Aldolase (EC 4.1.2.25) enthält5 Identification of an E. coli clone that contains a nucleotide sequence of the gene for dihydroneopterin aldolase (EC 4.1.2.25)
Bei der Analyse der E. coli-Klone, wie sie im Beispiel 2 beschrieben wurde, an die sich die im Beispiel 3 beschriebene 0 Analyse der dabei erhaltenen Sequenzen anschloß, ergab sich eine Sequenz, wie sie mit SEQ ID NR. 5 beschrieben ist. Bei der Anwendung des BLASTX-Algorithmus (siehe Beispiel 3) ergab diese Sequenz Ähnlichkeit mit Dihydroneopterin-Aldolasen (FolB; EC 4.1.2.25) aus verschiedenen Organismen. Die größte Ähnlichkeit 5 war mit der Dihydroneopterin-Aldolase (FolB) aus Mycobacterlum tuberculosis (NRDB 006275; 61% 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. 5 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity to dihydroneopterin aldolases (FolB; EC 4.1.2.25) from different organisms. The greatest similarity 5 was with the dihydroneopterin aldolase (FolB) from Mycobacterlum tuberculosis (NRDB 006275; 61% agreement at the amino acid level).
Beispiel 7 0Example 7 0
Identifizierung eines E. coli-Klons, der eine Nucleotidsequenz des Gens für die 2-Amino-4-hydroxy-6-hydroxymethyldihydropteri- din-pyrophosphokinase (EC 2.7.6.3) enthältIdentification of an E. coli clone which contains a nucleotide sequence of the gene for the 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3)
5 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. 7 beschrieben ist. Bei der Anwendung des BLASTX-Algorithmus (siehe Beispiel 3) ergab diese Sequenz Ähnlichkeit mit 2-Amino-4-hydroxy-6-hydroxymethyldihydrop- teridin-pyrophosphokinasen (FolK; EC 2.7.6.3) aus verschiedenen Organismen. Die größte Ähnlichkeit war mit der5 The analysis of the E. coli clones as described in Example 2, which was followed by the analysis of the sequences obtained in Example 3, resulted in one Sequence as shown with SEQ ID NO. 7 is described. When using the BLASTX algorithm (see Example 3), this sequence showed similarity with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinases (FolK; EC 2.7.6.3) from different organisms. The greatest similarity was with that
2-Amino-4-hydroxy-6-hydroxymethyldihydropteridin-pyrophosphoki- nase (FolK) aus Nyco acteriurn leprae (EMBL AL023093; 43% Übereinstimmung auf der Stufe der Aminosäuren) .2-Amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (FolK) from Nyco acteriurn leprae (EMBL AL023093; 43% agreement at the amino acid level).
Beispiel 8Example 8
Einsatz der Gene für die GTP-Cyclohydrolase I, für die Dihydropteroat-Synthase, für die Dihydroneopterin-Aldolase und für die 2-Amino-4-hydroxy-6-hydroxymethyldihydropteridin-pyrophosphoki- nase aus Corynebacterium glutamicum zur Herstellung von FolsäureUse of the genes for GTP cyclohydrolase I, for dihydropteroate synthase, for dihydroneopterin aldolase and for 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum for the production of folic acid
Die Gene für die GTP-Cyclohydrolase I, für die Dihydropteroat- Synthase, für die Dihydroneopterin-Aldolase und für die 2-Amino-4-hydroxy-6-hydroxymethyldihydropteridin-pyrophosphoki- nase aus Corynebacterium glutamicum lassen sich mit Hilfe geeigneter Klonierungs- und Expressionssysteme in Corynebacterium glutamicum oder in jeden beliebigen anderen Mikroorganismus einbringen. Man kann gentechnisch veränderte Mikroorganismen herstellen, die sich vom Wildtyp-Organismus hinsichtlich der Aktivität oder der Anzahl der Genkopien unterscheiden. Diese neuartigen, gentechnisch veränderten Stämme lassen sich zur Herstellung von Folsäure einsetzen.The genes for the GTP cyclohydrolase I, for the dihydropteroate synthase, for the dihydroneopterin aldolase and for the 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase from Corynebacterium glutamicum can be obtained with the aid of suitable cloning and expression systems introduce into Corynebacterium glutamicum or into any other microorganism. Genetically modified microorganisms can be produced which differ from the wild-type organism with regard to the activity or the number of gene copies. These new, genetically modified strains can be used to produce folic acid.
Sequenzlistesequence Listing
(I) Allgemeine Angaben(I) General information
[1) Anmelder:[1) Applicant:
(A) Name: BASF-LYNX Bioscience AG(A) Name: BASF-LYNX Bioscience AG
(B) Straße: Im Neuenheimer Feld 515(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(F) Phone: 06221/4546
(G) Telefax: 06221/454770(G) Fax: 06221/454770
(2) Titel: Gene aus Corynebacterium glutamicum für die Biosynthese der Folsäure und ihr Einsatz zur mikrobiellen Herstellung von Fol säure (3) Anzahl der Sequenzen: 8(2) Title: Genes from Corynebacterium glutamicum for the biosynthesis of folic acid and its use in the microbial production of folic acid (3) Number of sequences: 8
SEQ ID NR. 1: DNA ( folE)SEQ ID NO. 1: DNA (folE)
ATGAAGGAGACAACCGTGGATAACCACGCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC TGCGATTCGTGAGTTGCTCATCGCTGTGGGTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC CAGCTCGAGTGGCTAGGGCGTACAAGGAAACTTTCGCGGGTCTGCATGAGGATCCCACCACTGTG CTGGAGAAGACGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCACATTGGTTACATTCCGGGTAAGT CCGGCAAGGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTAAGCGACCTCAG GTTCAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT GGCCGTGGTGATTGAAGCTGAGCACCTGTGCATGGCCATGCGCGGAATCCGTAAGCCTGGTGCTG TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGAACAACGCTGCCTCCCGCGCTGAGGTGTTC TCCCTGATTCGGGGGCACTAAATGAAGGAGACAACCGTGGATAACCACGCTGCAGTTCGCGAGTTCGATGAGGAGCGCGCAACAGC TGCGATTCGTGAGTTGCTCATCGCTGTGGGTGAGGATCCAGATCGCGAAGGCCTGTTGGAAACCC CAGCTCGAGTGGCTAGGGCGTACAAGGAAACTTTCGCGGGTCTGCATGAGGATCCCACCACTGTG CTGGAGAAGACGTTCTCTGAGGGCCATGAAGAGTTGGTTCTGGTTCGTGAGATCCCGATTTACTC CATGTGTGAGCACCACTTGGTGCCGTTCTTTGGCGTGGCGCACATTGGTTACATTCCGGGTAAGT CCGGCAAGGTGACTGGCCTGTCCAAGCTGGCGCGTTTAGCGGATATGTTTGCTAAGCGACCTCAG GTTCAGGAGCGCTTGACCTCCCAAATTGCGGATGCTCTCGTCGAAAAGCTTGATGCCCAGGCCGT GGCCGTGGTGATTGAAGCTGAGCACCTGTGCATGGCCATGCGCGGAATCCGTAAGCCTGGTGCTG TGACCACGACGTCTGCGGTGCGCGGCGGTTTTAAGAACAACGCTGCCTCCCGCGCTGAGGTGTTC TCCCTGATTCGGGGGCACTAA
SEQ ID NR. 2: Aminosäure (FolE)SEQ ID NO. 2: amino acid (FolE)
MKETTVDNHAAVREFDEERATAAIRELLIAVGEDPDREGLLΞTPARVARAYKETFAGLHEDPTTV LEKTFSEGHEELVLVREIPIYSMCEHHLVPFFGVAHIGYIPGKSGKVTGLSKLARLADMFAKRPQ VQERLTSQIADALVEKLDAQAVAWIEAEHLCMAMRGIRKPGAVTTTSAVRGGFKNNAASRAEVF SLIRGHMKETTVDNHAAVREFDEERATAAIRELLIAVGEDPDREGLLΞTPARVARAYKETFAGLHEDPTTV LEKTFSEGHEELVLVREIPIYSMCEHHLVPFFGVAHIGYIPGKSGKVTGLSKLARLADMFAKRPQ VQERLTSQIADALVKRGGHASKL
SEQ ID NR. 3: DNA ( folP)SEQ ID NO. 3: DNA (folP)
ATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAATTGTCAATGTCACTGA GGATTCCTTTTCGGACGGTGGCAAGTACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT TGGTGGCTGCTGGCGCCGACATGATTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC GTCGACGCGTCCGTGGAACGGGACCGGGTTGTGCCGGTCATTAAGGCGCTTCACGACGCCGGCAT CCACACTTCCGTAGACACCATGCGGGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGGCGGAAGCGCAA ATTCCCGTGTGTTTGATGCACTGGCGCACCCTCCAATTCGGTGATGCCGCAGGTCAGGCAGATCA CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTCGCCCGCGCCACCGCTG CTGGTGTGGCCGAAAACCAGATCGTGCTTGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC AACTGGCGTTtGCTGCAAGCACTGCCCGAGTTTATTTCTGGACCTTTCCCCATCCTGGTGGGAGC ATCCCGGAAGCGATTCCTGGCTGGCGTGCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTGTGCGCGTG CACGATGTCCCAGTATCAAGGGACGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA CCATGGCTGAATGAACGTATCCTCTTTGACCATCCCGGGACGCTGTTTGGTCATGGGAATTGTCAATGTCACTGA GGATTCCTTTTCGGACGGTGGCAAGTACATTGACGTTGATCAGGCGATCGCGCATGCCAAGGAAT TGGTGGCTGCTGGCGCCGACATGATTGATGTCGGCGGCGAGTCCACCCGGCCTGGGGCAGTGCGC GTCGACGCGTCCGTGGAACGGGACCGGGTTGTGCCGGTCATTAAGGCGCTTCACGACGCCGGCAT CCACACTTCCGTAGACACCATGCGGGCCTCCGTGGCGCAGGCTGCCGCGGGCGCTGGCGTCTCCA TGATCAACGACGTCTCTGGCGGTTTGGCTGATCCTGAGATGTTTTCTGTCATGGCGGAAGCGCAA ATTCCCGTGTGTTTGATGCACTGGCGCACCCTCCAATTCGGTGATGCCGCAGGTCAGGCAGATCA CGGTGGAGACGTTGTAGCCGATGTGCACGCAGTGCTTGATGATCTTGTCGCCCGCGCCACCGCTG CTGGTGTGGCCGAAAACCAGATCGTGCTTGATCCAGGTTTGGGTTTTGCCAAATCACGTGAAGAC AACTGGCGTTtGCTGCAAGCACTGCCCGAGTTTATTTCTGGACCTTTCCCCATCCTGGTGGGAGC ATCCCGGAAGCGATTCCTGGCTGGCGTGCGCAAAGACCGTGGCCTAGATGTCACCCCCATTGATG CCGACCCAGCAACCGCAGCGGTGACCGCAGTGTCTGCACATATGGGAGCATGGGGTGTGCGCGTG CACGATGTCCCAGTATCAAGGGACGCTGTTGATGTTGCCGCATTGTGGCGAAGTGGAGGAACTCA CCATGGCTGA
SEQ ID NR. 4: Aminosäure (FolP)SEQ ID NO. 4: amino acid (FolP)
MNVSSLTIPGRCLVMGIVNVTEDSFSDGGKYIDVDQAIAHAKELVAAGADMIDVGGESTRPGAVR VDASVERDRWPVIKALHDAGIHTSVDTMRASVAQAAAGAGVSMINDVSGGLADPEMFSVMAEAQ IPVCLMHWRTLQFGDAAGQADHGGDWADVHAVLDDLVARATAAGVAENQIVLDPGLGFAKSRED 'NWRLLQALPEFISGPFPILVGASRKRFLAGVRKDRGLDVTPIDADPATAAVTAVSAHMGAWGVRV HDVPVSRDAVDVAALWRSGGTHHG SEQ ID NR . 5 : DNA ( folB)MNVSSLTIPGRCLVMGIVNVTEDSFSDGGKYIDVDQAIAHAKELVAAGADMIDVGGESTRPGAVR VDASVERDRWPVIKALHDAGIHTSVDTMRASVAQAAAGAGVSMINDVSGGLADPEMFSVMAEAQ IPVCLMHWRTLQFGDAAGQADHGGDWADVHAVLDDLVARATAAGVAENQIVLDPGLGFAKSRED 'NWRLLQALPEFISGPFPILVGASRKRFLAGVRKDRGLDVTPIDADPATAAVTAVSAHMGAWGVRV HDVPVSRDAVDVAALWRSGGTHHG SEQ ID NO. 5: DNA (folB)
ATGGCTGATCGTATTGAACTTAAAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG CCAGCGATGACCTTTCCGACACCGTAGATTACGGCGCGTTGGCATTGTTGGTTGCTGAAATCGTG GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGCCCAAAGCACCGATCCCACGTACTT TTGCTGACGTCGCGGTGGTTGCCCGACGTTCCAGGAAATCCATGGCTGCTGGAAGGAGCAACGCC TAAATGGCTGATCGTATTGAACTTAAAGGCCTTGAATGCTTCGGACACCACGGTGTGTTCGACTTTGA AAAAGAGCAAGGCCAGCCCTTCATTGTGGATGTCACCTGCTGGATGGATTTCGATGCCGCAGGTG CCAGCGATGACCTTTCCGACACCGTAGATTACGGCGCGTTGGCATTGTTGGTTGCTGAAATCGTG GAAGGCCCATCCAGGGATTTGATCGAGACGGTGGCCACGGAATCTGCGGATGCTGTGATGGCTAA ATTTGATGCGCTTCATGCGGTGGAAGTAACCATCCATAAGCCCAAAGCACCGATCCCACGTACTT TTGCTGACGTCGCGGTGGTTGCCCGACGTTCCAGGAAATCCATGGCTGCTGGAAGGAGCAACGCC TAA
SEQ ID NR. 6: Aminosäure (FolB)SEQ ID NO. 6: amino acid (FolB)
MADRIELKGLECFGHHGVFDFEKEQGQPFIVDVTCWMDFDAAGASDDLSDTVDYGALALLVAEIV EGPSRDLIETVATESADAVMAKFDALHAVEVTIHKPKAPIPRTFADVAWARRSRKSMAAGRSNAMADRIELKGLECFGHHGVFDFEKEQGQPFIVDVTCWMDFDAAGASDDLSDTVDYGALALLVAEIV EGPSRDLIETVATESADAVMAKFDALHAVEVTIHKPKAPIPRTFADVAWARRSRKSMAAGRSNA
SEQ ID NR. 7 : DNA ( folK)SEQ ID NO. 7: DNA (folK)
ATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTACTCAACCCCACCGTGGGGCATTG AGGATCAGGATGAATTCCTCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTTGATCCCTTGGTTGGAAGCA GAACCTGATGCCGTCCTGCACGGCACGACCATTGCAGAACATGTGGATAATCTTGATCCCACAGA CATTGAAGGTGTCACCAAGATTTAAATGCATGCAGTTTTGTCCATCGGTTCCAACATGGATGATCGCTACGCGCTGCTCAACACAGTGAT CGAGGAATTCAAAGATGAGATCGTGGCGCAGTCTGCGATCTACTCAACCCCACCGTGGGGCATTG AGGATCAGGATGAATTCCTCAACGCAGTGCTCGTTGTTGAGGTTGAAGAAACCCCCATCGAGTTG CTGCGCCGTgGCCAAAAACTCGAAGAAGCCGCCGAGCGGGTCCGCGTCCGCAAATGGGGGCCACG CACCCTCGATGTGGATATCGTGCAGATCATTAAAGATGGGGAAGAGATCCTTTCTGAGGATCCCG AACTGACCTTGCCACACCCTTGGGCTTGGCAGCGTGCCTTCGTGTTGATCCCTTGGTTGGAAGCA GAACCTGATGCCGTCCTGCACGGCACGACCATTGCAGAACATGTGGATAATCTTGATCCCACAGA CATTGAAGGTGTCACCAAGATTTAA
SEQ ID NR. 8: Aminosäure (FolK)SEQ ID NO. 8: amino acid (FolK)
MHAVLSIGSNMDDRYALLNTVIEEFKDEIVAQSAIYSTPPWGIEDQDEFLNAVLWEVEETPIEL LRRGQKLEEAAERVRVRKWGPRTLDVDIVQIIKDGEEILSEDPELTLPHPWAWQRAFVLIPWLEA EPDAVLHGTTIAEHVENLDPTDIEGVTKI MHAVLSIGSNMDDRYALLNTVIEEFKDEIVAQSAIYSTPPWGIEDQDEFLNAVLWEVEETPIEL LRRGQKLEEAAERVRVRKWGPRTLDVDIVQIIKDGEEILSEDPELTLPHPWAWQRAFVLIPWLEA EPDAVLVGTI

Claims

Patentansprüche claims
1. Ein Polypeptid mit GTP-Cyclohydrolase-I-Aktivität , das aus folgender Gruppe ausgewählt ist:1. A polypeptide with GTP cyclohydrolase I activity, which is selected from the following group:
(a) ein Polypeptid mit der Aminosäuresequenz, die in der SEQ ID NR. 2 beschrieben ist(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 2 is described
(b) ein Polypeptid das im Vergleich zu dem in (a) durch Deletion, Insertion oder Substitution einer oder mehrerer Aminosäuren verändert ist.(b) a polypeptide which is modified compared to that in (a) by deletion, insertion or substitution of one or more amino acids.
2. Ein Polypeptid mit Dihydropteroat-Synthaseaktivität , das aus folgender Gruppe ausgewählt ist:2. A polypeptide with dihydropteroate synthase activity, which is selected from the following group:
(a) ein Polypeptid mit der Aminosäuresequenz, die in der SEQ ID NR. 4 beschrieben ist;(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 4 is described;
(b) ein Polypeptid das im Vergleich zu dem in (a) durch Deletion, Insertion oder Substitution einer oder mehrerer Aminosäuren verändert ist .(b) a polypeptide which is modified compared to that in (a) by deletion, insertion or substitution of one or more amino acids.
3. Ein Polypeptid mit Dihydroneopterin-Aldolaseaktivität , das aus folgender Gruppe ausgewählt ist:3. A polypeptide with dihydroneopterin aldolase activity, which is selected from the following group:
(a) ein Polypeptid mit der Aminosäuresequenz, die in der SEQ ID NR. 6 beschrieben ist(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 6 is described
(b) ein Polypeptid, das im Vergleich zu dem in (a) durch(b) a polypeptide compared to that in (a) by
Deletion, Insertion oder Substitution einer oder mehrerer Aminosäuren verändert ist .Deletion, insertion or substitution of one or more amino acids is changed.
4. Ein Polypeptid mit 2-Amino-4-hydroxy-6-hydroxymethyldihydrop- teridin-pyrophosphokinaseaktivität, das aus der folgenden4. A polypeptide with 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase activity, which results from the following
Gruppe ausgewählt ist:Group is selected:
(a) ein Polypeptid mit der Aminosäuresequenz, die in der SEQ ID NR. 8 beschrieben ist(a) a polypeptide with the amino acid sequence shown in SEQ ID NO. 8 is described
ein Polypeptid, das im Vergleich zu (a) durch Deletion, Insertion oder Substitution einer oder mehrerer Aminosäuren verändert ist . a polypeptide which is modified compared to (a) by deletion, insertion or substitution of one or more amino acids.
5. Ein Polynucleotid, das ein dem Anspruch 1, 2, 3 oder 4 entsprechendes Polypeptid kodiert.5. A polynucleotide encoding a polypeptide corresponding to claim 1, 2, 3 or 4.
6. Ein Genkonstrukt mit mindestens einer Kopie eines dem Anspruch 5 entsprechenden Polynucleotids zusammen mit mindestens einer regulatorischen Sequenz.6. A gene construct with at least one copy of a polynucleotide corresponding to claim 5 together with at least one regulatory sequence.
7. Ein Wirtsorganismus, der mit einem dem Anspruch 6 entsprechenden Genkonstrukt transformiert ist.7. A host organism which is transformed with a gene construct corresponding to claim 6.
Verfahren zur Herstellung von Folsäure durch Kultivieren eines dem Anspruch 7 entsprechenden Wirtsorganismus mit nachfolgender Isolierung der Folsäure. Process for the preparation of folic acid by cultivating a host organism corresponding to claim 7 with subsequent isolation of the folic acid.
PCT/EP2000/005864 1999-06-25 2000-06-23 Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid WO2001000845A1 (en)

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AU59782/00A AU5978200A (en) 1999-06-25 2000-06-23 Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid
KR1020017016565A KR20020026469A (en) 1999-06-25 2000-06-23 Genes from Corynebacterium Glutamicum for the Biosynthesis of Folic Acid and Their Use for the Microbial Production of Folic Acid
CA002377458A CA2377458A1 (en) 1999-06-25 2000-06-23 Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid
EP00945815A EP1194565A1 (en) 1999-06-25 2000-06-23 Genes from corynebacterium glutamicum for the biosynthesis of folic acid and their use for the microbial production of folic acid

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DE19929363A DE19929363A1 (en) 1999-06-25 1999-06-25 New Corynebacterium glutamicum GTP cyclohydrolase I, dihydropteroate synthase, dihydroneopterin aldolase and 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase polypeptides

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EP1262541A1 (en) * 2001-05-28 2002-12-04 Stichting Top-Instituut Voedselwetenschappen Production of bioavailable folic acid
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
US6812016B2 (en) 2000-09-02 2004-11-02 Degussa Ag Nucleotide sequences which code for the metY gene
US6812006B2 (en) 2000-08-10 2004-11-02 Degussa Ag Nucleotide sequences which code for the lysR3 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
US6893852B1 (en) 1999-07-02 2005-05-17 Ajinomoto Co., Inc. Dna encoding sucrose pts enzyme II
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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CN111235169A (en) * 2020-02-03 2020-06-05 昆明理工大学 GTP cyclohydrolase I gene folE and application thereof
CN112852844A (en) * 2021-03-05 2021-05-28 昆明理工大学 Application of hydroxymethyl dihydropterin pyrophosphokinase gene folK

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Cited By (21)

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US6893852B1 (en) 1999-07-02 2005-05-17 Ajinomoto Co., Inc. Dna encoding sucrose pts enzyme II
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
US6812006B2 (en) 2000-08-10 2004-11-02 Degussa Ag Nucleotide sequences which code for the lysR3 gene
US7173105B2 (en) 2000-08-10 2007-02-06 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
US6875586B2 (en) 2000-08-10 2005-04-05 Degussa Ag Nucleotide sequences coding for the luxR gene
US7105321B2 (en) 2000-08-26 2006-09-12 Degussa Ag Nucleotide sequences which code for the ccpA2 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
US7038034B2 (en) 2000-09-09 2006-05-02 Degussa Ag Nucleotide sequences coding for the Dep33 efflux protein
US6759224B2 (en) 2000-09-09 2004-07-06 Degussa Ag Nucleotide sequences which code for the sahH gene
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
EP1262541A1 (en) * 2001-05-28 2002-12-04 Stichting Top-Instituut Voedselwetenschappen Production of bioavailable folic acid
WO2002097063A1 (en) * 2001-05-28 2002-12-05 Campina B.V. Production of bioavailable folic acid
US7468262B2 (en) 2003-05-16 2008-12-23 Ajinomoto Co., Inc. Polynucleotides encoding useful polypeptides in corynebacterium glutamicum ssp. lactofermentum
US7695946B2 (en) 2003-05-16 2010-04-13 Ajinomoto Co., Inc. Polynucleotides encoding useful polypeptides in Corynebacterium glutamicum ssp. lactofermentum
US7696315B2 (en) 2003-05-16 2010-04-13 Ajinomoto Co., Inc. Polynucleotides encoding useful polypeptides in Corynebacterium glutamicum ssp. lactofermentum

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