EP0951538A2 - SEQUENCES PARTIELLES DE GENES DE BIOSYNTHESE DE PURINE DE L$i('ASHBYA GOSSYPII) ET LEUR UTILISATION DANS LA SYNTHESE DE RIBOFLAVINE PAR DES MICROBES - Google Patents

SEQUENCES PARTIELLES DE GENES DE BIOSYNTHESE DE PURINE DE L$i('ASHBYA GOSSYPII) ET LEUR UTILISATION DANS LA SYNTHESE DE RIBOFLAVINE PAR DES MICROBES

Info

Publication number
EP0951538A2
EP0951538A2 EP97953928A EP97953928A EP0951538A2 EP 0951538 A2 EP0951538 A2 EP 0951538A2 EP 97953928 A EP97953928 A EP 97953928A EP 97953928 A EP97953928 A EP 97953928A EP 0951538 A2 EP0951538 A2 EP 0951538A2
Authority
EP
European Patent Office
Prior art keywords
seq
nucleotide sequence
microorganism
ashbya gossypii
particularly preferably
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97953928A
Other languages
German (de)
English (en)
Inventor
Peter Philippsen
Markus Pompejus
Harald Seulberger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP0951538A2 publication Critical patent/EP0951538A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • 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
    • C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin

Definitions

  • the present invention relates to partial sequences of genes of purine biosynthesis from Ashbya gossypii and their use in riboflavin synthesis.
  • Vitamin B2 also called riboflavin, is essential for humans and animals.
  • Vitamin B2 deficiency inflammation of the mucous membranes of the mouth and throat, itching and inflammation in the skin folds and similar skin damage, conjunctivitis, reduced visual acuity and clouding of the cornea occur. Growth and weight loss may occur in infants and children.
  • Vitamin B2 is therefore of economic importance, especially as a vitamin supplement in the case of a vitamin deficiency and as a feed additive. It is also used as a food coloring, for example in mayonnaise, ice cream, pudding, etc.
  • Vitamin B2 is produced either chemically or microbially (see e.g. Kurth et al. (1996) Riboflavin, in: Ulimann's Encyclopedia of industrial chemistry, VCH Weinheim).
  • riboflavin is usually obtained as a pure end product in multi-stage processes, with relatively expensive starting products, such as D-Ribose, must be used.
  • An alternative to the chemical synthesis of riboflavin is the production of this substance by microorganisms. Renewable raw materials such as sugar or vegetable oils are used as starting materials.
  • riboflavin by fermentation of fungi such as Eremothecium ashbyii or Ashbya gossypii is known (The Merck Index, Windholz et al., Eds. Merck & Co., page 1183, 1983), but also yeasts, e.g. Candida, Pichia and Saccharomyces or bacteria, e.g.
  • Bacillus, clostridia or corynebacteria are described as riboflavin producers.
  • EP 405370 describes riboflavin-overproducing bacterial strains obtained by transforming the riboflavin biosynthesis genes from Bacillus subtilis.
  • the genes described there and other genes from prokaryotes involved in vitamin B2 biosynthesis are unsuitable for a recombinant riboflavin production process with eukaryotes, such as, for example, Saccharomyces cerevisiae or Ashbya gossypii.
  • DE4420785 describes six riboflavin biosynthesis genes from Ashbya gossypii, as well as microorganisms which have been transformed with these genes and the use of such microorganisms for riboflavin synthesis.
  • the invention relates to new partial sequences for new genes of
  • the invention relates to a gene fragment which contains the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence obtainable from SEQ ID NO: 1 by substitution, insertion or deletion of up to 20% of the nucleotides.
  • Another object of the invention is a gene fragment which contains the nucleotide sequence shown in SEQ ID NO: 2 or a nucleotide sequence obtainable from SEQ ID NO: 2 by substitution, insertion or deletion of up to 20% of the nucleotides.
  • Another object of the invention is a gene fragment which contains the nucleotide sequence shown in SEQ ID NO: 3 or a nucleotide sequence obtainable from SEQ ID NO: 3 by substitution, insertion or deletion of up to 15% of the nucleotides.
  • Another object of the invention is a gene fragment which contains the nucleotide sequence shown in SEQ ID NO: 4 or a nucleotide sequence obtainable from SEQ ID NO: 4 by substitution, insertion or deletion of up to 20% of the nucleotides.
  • Another object of the invention is the use of the nucleic acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 for the construction of genetically modified microorganisms.
  • microorganisms can be any bacteria, but preferably bacteria of the genera Bacillus and Corynebacterium.
  • These microorganisms can be any eukaryotic microorganisms, preferably fungi of the genera Ashbya or Eremothecium, in particular Ashbya gossypii or yeasts, but preferably yeasts of the genera Candida, Pichia and Saccharomyces.
  • Another object of the invention is the use of such genetically modified microorganisms for the production of riboflavin.
  • Genomic DNA from Ashbya gossypii ATCC10895 can be prepared by conventional methods, for example as described in EP9703208.
  • the genomic library, based on this DNA can be prepared using conventional methods (e.g. Sambrook, J. et al. (1989) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press or Ausubel, FM et al. (1994) Current protocols in molecular biology, John Wiley and sons) in any plasmids, such as, for example, in pRS316 and other plasmids of the pRS series (Sikorski and Hieter (1989) Genetics, 19-27).
  • the size of the fragment of the Ashbya gossypii genomic DNA used is arbitrary, but Sau3A fragments between 2 and 9 kb in length are preferred, which can be cloned into a BamHI site.
  • Individual clones can be selected from E. coli clones which carry the library described in Example 1.
  • the cells can be cultured in suitable media (for example LB with 100 mg / 1 ampicillin) using conventional methods and plasmid DNA can be isolated from these cells.
  • suitable media for example LB with 100 mg / 1 ampicillin
  • the fragments of the genomic DNA from Ashbya gossypii are cloned into an interface of the polylinker of pRS plasmids (described in Example 1)
  • the nucleotide sequence of partial regions of the inserts can then be determined using suitable oligonucleotides and standard methods.
  • Sequence comparisons of newly identified sequences with existing DNA and protein databases can e.g. with BLAST algorithms (Altschul et al. (1990) J. Mol. Biol. 215, 403-410) or the Clustal algorithm with the help of the PAM250 weighting table or the Wilbur-Lipman DNA alignment algorithm (as for example in the program package MegAlign 3.06 from the company DNAstar implemented).
  • BLAST algorithms Altschul et al. (1990) J. Mol. Biol. 215, 403-410
  • the Clustal algorithm with the help of the PAM250 weighting table or the Wilbur-Lipman DNA alignment algorithm (as for example in the program package MegAlign 3.06 from the company DNAstar implemented).
  • Example 3 If one examines clones as described in Example 2 and the sequences obtained as analyzed in Example 3, one finds sequences as described in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3.
  • these sequences show the following similarities to partial regions of ADE4 genes, in particular to partial regions of the ADE4 gene from Saccharomyces kluyveri (gene bank entry SKU32992) and to partial regions of the ADE4 gene from Saccharomyces cerevisiae (gene bank entry YSCADE4) ): Nucleotide sequence according to SEQ ID NO: 1: 74% or 69% similarity, nucleotide sequence according to SEQ ID NO: 2: 78% or 73% Similarity and nucleotide sequence according to SEQ ID NO: 3: 81% and 75%
  • Glamin aminophosphosylpyrophosphate amidotransferase AgADE4
  • inosine 5 'monophosphate dehydrogenase AgGUAl
  • the nucleic acid sequences shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 can be used as probes to clone the complete gene for glutamine-phosphoribosylpyrophosphate-amidotransferase (AgADE4) from Ashbya gossypii.
  • the nucleic acid sequence shown in SEQ ID NO: can be used as a probe to clone the complete gene for inosine 5i monophosphate dehydrogenase (AgGUAl) from Ashbya gossypii.
  • AgGUAl inosine 5i monophosphate dehydrogenase
  • AgADE4 glutamine phosphoribosyl pyrophosphate amidotransferase
  • AgGUAl inosine 5i monophosphate dehydrogenase
  • microorganisms such as Saccharomyces cerevisiae or other yeasts, as shown in WO9703208 or DE4420785.
  • genetically modified microorganisms can be constructed which differ from the natural situation in terms of the activity of genes or the number of copies of genes.
  • the microorganisms constructed in this way can be used for the biosynthesis of riboflavin.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • GAGTGTGCCA ACCATTTAAA CAAACCTTAT CGCGAAGGAT TTGTCAAGAA CAGATATGTT 120
  • MOLECULE TYPE DNA (genomic)
  • GAGTGTGCCA ACCATTTAAA CAAACCTTAT CGCGAAGGAT TTGTCAAGAA CAGATATGTT 120
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des séquences partielles des gènes de biosynthèse de purine de l'Ashbya gossypii, leur production et leur utilisation.
EP97953928A 1996-12-31 1997-12-29 SEQUENCES PARTIELLES DE GENES DE BIOSYNTHESE DE PURINE DE L$i('ASHBYA GOSSYPII) ET LEUR UTILISATION DANS LA SYNTHESE DE RIBOFLAVINE PAR DES MICROBES Withdrawn EP0951538A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CH1697 1996-12-31
CH1697 1996-12-31
PCT/EP1997/007312 WO1998029539A2 (fr) 1996-12-31 1997-12-29 Sequences partielles de genes de biosynthese de purine de l'ashbya gossypii et leur utilisation dans la synthese de riboflavine par des microbes

Publications (1)

Publication Number Publication Date
EP0951538A2 true EP0951538A2 (fr) 1999-10-27

Family

ID=4177492

Family Applications (3)

Application Number Title Priority Date Filing Date
EP97811020A Withdrawn EP0866129A2 (fr) 1996-12-31 1997-12-24 Séquences d'ADN génomiques de Ashbya gossypii et leurs utilisations
EP97953928A Withdrawn EP0951538A2 (fr) 1996-12-31 1997-12-29 SEQUENCES PARTIELLES DE GENES DE BIOSYNTHESE DE PURINE DE L$i('ASHBYA GOSSYPII) ET LEUR UTILISATION DANS LA SYNTHESE DE RIBOFLAVINE PAR DES MICROBES
EP97954977A Withdrawn EP0953044A2 (fr) 1996-12-31 1997-12-29 Gene de l'adenylate cyclase et son utilisation

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP97811020A Withdrawn EP0866129A2 (fr) 1996-12-31 1997-12-24 Séquences d'ADN génomiques de Ashbya gossypii et leurs utilisations

Family Applications After (1)

Application Number Title Priority Date Filing Date
EP97954977A Withdrawn EP0953044A2 (fr) 1996-12-31 1997-12-29 Gene de l'adenylate cyclase et son utilisation

Country Status (6)

Country Link
US (2) US6239264B1 (fr)
EP (3) EP0866129A2 (fr)
JP (3) JP2001508289A (fr)
AU (2) AU6291698A (fr)
CA (2) CA2276093A1 (fr)
WO (2) WO1998029538A2 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001510983A (ja) * 1996-06-19 2001-08-07 ディベルサ コーポレーション 熱安定性ホスファターゼ
US6448003B1 (en) * 1998-06-10 2002-09-10 Dna Sciences Laboratories, Inc. Genotyping the human phenol sulfotransferbase 2 gene STP2
DE19839567A1 (de) * 1998-08-31 2000-03-09 Basf Ag Organismen zur extrazellulären Herstellung von Riboflavin
AU6090299A (en) * 1998-10-08 2000-05-01 Syngenta Participations Ag Fungal genes required for normal growth and development
US6291660B1 (en) 1998-10-08 2001-09-18 Syngenta Participations Ag Fungal genes required for normal growth and development
US20050053932A1 (en) * 2001-07-10 2005-03-10 Marvin Karos Novel stress-associated genetic products from ashbya gossypii
US20050069882A1 (en) * 2001-08-29 2005-03-31 Marvin Karos Novel genetic products obtained from ashbya gossypii, which are associated with transcription mechanisms, rna processing and/or translation
KR20040032999A (ko) * 2001-08-29 2004-04-17 바스프 악티엔게젤샤프트 전사 메카니즘, ｒｎａ 프로세싱 및(또는) 번역과 관련된,아쉬비아 고쉬피로부터의 신규 유전자 산물
DK2164975T3 (da) 2007-07-06 2012-05-29 Basf Se Fremgangsmåde til fremstilling af en koncentreret vandig glukoseopløsning af majs
CN103194524B (zh) * 2013-04-03 2014-12-24 山东省花生研究所 快速鉴定抗青枯病花生种质的方法
KR102583349B1 (ko) * 2014-11-19 2023-09-26 바스프 에스이 Gmp 신테타아제 활성 증가를 위한 에레모테시움의 유전적 변형
KR20220116504A (ko) 2019-12-19 2022-08-23 바스프 에스이 정밀 화학물의 제조에서 공시 수율, 탄소-전환-효율 및 탄소 기질 적응성의 증가

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2216010T3 (es) 1994-03-25 2004-10-16 Basf Aktiengesellschaft Biosintesis de riboflavina en hongos.
DE4420785A1 (de) * 1994-03-25 1995-10-05 Basf Ag Riboflavin-Biosynthese in Pilzen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9829539A2 *

Also Published As

Publication number Publication date
US6489147B1 (en) 2002-12-03
EP0866129A2 (fr) 1998-09-23
JPH11225770A (ja) 1999-08-24
JP2001509012A (ja) 2001-07-10
CA2276110A1 (fr) 1998-07-09
WO1998029538A3 (fr) 1998-12-10
JP2001508289A (ja) 2001-06-26
US6239264B1 (en) 2001-05-29
AU6291698A (en) 1998-07-31
EP0953044A2 (fr) 1999-11-03
WO1998029538A2 (fr) 1998-07-09
CA2276093A1 (fr) 1998-07-09
WO1998029539A2 (fr) 1998-07-09
WO1998029539A3 (fr) 1998-11-12
AU5764398A (en) 1998-07-31

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