introduction of biotechnology -...
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Graduate School of Engineering, Osaka UniversityGraduate School of Information Science, Osaka UniversityInternational Center for Biotechnology, Osaka University
Graduate School of Engineering, Osaka UniversityGraduate School of Information Science, Osaka UniversityInternational Center for Biotechnology, Osaka University
Introduction of BiotechnologyNo.7: Yeast Genetics and
Biotechnology
Introduction of BiotechnologyNo.7: Yeast Genetics and
Biotechnology
Handai Cyber University
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Hello! My name is Satoshi Harashima.
Scientific interest: Yeast Genetics, Yeast GenomicsHobby: Listening and playing Jazz music
Date of birth: 21 May 1949 ; Birthplace: Ehime, Japan
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No.7 Yeast Genetics and Biotechnology
• Yeast biotechnology and breeding techniques
• Development of dominant selectable markers for transformation of industrial strains
• Regulation of mating-type in yeast
• A novel breeding technique for industrial strains of yeast
• Regulation of sporulation in yeast
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I. Yeast biotechnology and breeding techniques
• Saccharomyces cerevisiae as a simple eukaryotic model
• Breeding techniques in yeast
• Characteristics of industrial strains of yeast
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Saccharomycescerevisiae cells
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Saccharomyces cerevisiae as a simple eukaryotic model
A variety of techniques for genetics and molecular biology
1) Its ease and rapidity of growth
2) Ready isolation and selection of mutants
3) Small genome size with whole sequence determined
4) Haploid and diploid life cycle
are established
5) Host-vector system established
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1) Traditional biotechnology
2) Modern biotechnology
Production of heterologous proteins of higher eukaryotes by genetic engineering
Beer、 Wine、 Sake、Bread、 Fermented foodEthanol、 Other useful cell constituents
Yeast traditional and modern biotechnology
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Mutagenesis
Mating and sporulation
Cell fusion (Cytoplasmic fusion)
Breeding Techniques for Yeast
Transformation
(Recombinant DNA technique)
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Characteristics of S. cerevisiae industrial strains and relevant obstacles in breeding
No genetic markers Difficulty in selection of cell fusants and transformants
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No mating ability Impossible to perform sexual hybridization
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Polyploidy Inefficient mutagenesis -----
Poor sporulation ability difficult to obtainmeiotic segregants
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Aneuploidy Poor spore germination-----
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An efficient method for direct selection of
mating-competent clones
Industrial strains A
a mating-type clone α mating-type clone
Hybridization
Industrial strains B
Creation of a variety of new strains
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II. Development of dominant selectable markers for transformation of
industrial strains
• Cerulenine
• Antibiotics G418
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Drugs which inhibit growth of S. cerevisiae industrial strains
1) Cerulenine:
I nhibitor for β-subunit of fatty acid synthase
2) G418:
Inhibitor of some ribosomal proteins
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Development of dominant selective markers for transformation of S. cerevisiae industrial strains
A putative transcriptional activator for genes functioning to pump out various drugs
Amynoglycoside-3’- phosphotransferasewhich inactivates G418 by phosphorylation
1) Cerulenine resistant gene PDR4
2) G418 resistant gene
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Application of PDR4 as dominant selectable marker for transformation of brewing yeasts
Whiskey yeast
YPDA+ Cerulenine (2 μg/ml)
Vector Vector + PDR4
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III. Regulation of mating-typein yeast
• Mating type and life cycle of S. cerevisiae
• α2 repression and a1-α2 repression
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Mating reaction between a and α cells
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Sporulation of a/α diploid cells
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α cell
MATα1MATα2
α1
α2MATα mating-type locus
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a cell
MATa1MATa2
a1
a2MATa mating-type locus
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a-specific genes ------------ asg
α-sepcific genes ----------- αsg
Non-specific genes --------- nsg
Three classes of mating-type related genes
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Gene Gene product or function
MFα1, MFα2 —α -pheromoneSTE3 — a-pheromone receptorKEX2, STE13 — Processing for α-pheromone
MFa1, MFa2 — a-pheromoneSTE2 —α -pheromone receptorSTE6 — a-pheromone secretionSTE14, STE16 — Processing for a-pheromone
STE5, STE7 — component of pheromone signalFUS1 — nuclear fusionSTE12 — transcriptional activator
Examples of genes involved in mating-type determination
3) non a-specific genes
1) α-specific genes
2) a-specific genes
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a/α cellα2 repression
Sporulation
nsg
a1/α2 repression
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IV. A method for direct selection of mating competent clones from industrial
strains
• mating-type specific reporter gene
• Efficient selection of a and a mating type clones using the reporter gene
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a cell
α cell
APase
Expression of STE6p-PHO5 and MFa1-pPHO5 reporter genes
STE6p-PHO5
MFα1p-PHO5
APase
STE6p-PHO5
MFα1p-PHO5
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STE6p-PHO5
PDR4
2μm Origin
pNN63
MFα1p-PHO5
PDR4
2μm Origin
pNN64
Plasmids harboring STE6p-PHO5 and MFα1-PHO5 reporter
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Isolation of clones showing mating ability from Sake yeastby the use of STE6p-PHO5 reporter gene
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Breeding of a hybrid by mating between K-a and S-α clones
K-a S-α K-a x S-α(Hybrid)
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Characteristics of S. cerevisiae industrial strains and relevant obstacles in breeding
No genetic markers Difficulty in selection of cell fusants and transformants
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No mating ability Impossible to perform sexual hybridization
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Polyploidy Inefficient mutagenesis -----
No sporulation ability Impossible to obtainmeiotic segregants
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Aneuploidy Poor spore germination-----
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V. Regulation of sporulationin yeast
• IME1 gene as activator for sporulation
• Restoration of sporulation ability by overexpression of IME1 gene
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Meiosis
Sporulation
IME1
Nutritional repressiona1 α2
Regulatoty system of meiosis and sporulation
RME1
(a1-α2 repression)
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2μm Origin
pIGZ1
2μm Origin
pIGZ2
Multicopy plasmid harboring IME1
G418rG418r
IME1
Vector Vector + IME1
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Vector IME1
a/a
α/α
Sakeyeast
Restoration of sporulation ability by introduction of multicopy IME1
Meiotic segregants were obtained
A laboratory scale Sake brewing
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An efficient method for direct selection of
mating-competent clones
Industrial strains A
a mating-type clone α mating-type clone
Hybridization
Industrial strains B
Creation of a variety of new strains
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Summary and conclusion of this lecture
1) A novel breeding method which allows interbreedingbetween industrial strains was developed.
2) With this method, a variety of industrial strains fortraditional biotechnology could be created.
3) Genetics and molecular biology is important for development of biotechnology of yeast
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Graduate School of Engineering, Osaka UniversityGraduate School of Information Science, Osaka UniversitInternational Center for Biotechnology, Osaka University
Graduate School of Engineering, Osaka UniversityGraduate School of Information Science, Osaka UniversitInternational Center for Biotechnology, Osaka University
Handai Cyber UniversityNo.7 : Yeast Genetics and Biotechnology
END
Handai Cyber UniversityNo.7 : Yeast Genetics and Biotechnology
ENDThank you very much for your attention!
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