Introduction of Biotechnology - 大阪大学ocw.osaka-u.ac.jp/engineering-jp/biotechnology-fundamentals-jp/dr... · International Center for Biotechnology, Osaka University Introduction

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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

Deleted based on copyright concern.

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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

-----

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

-----

No mating ability Impossible to perform sexual hybridization

-----

Polyploidy Inefficient mutagenesis -----

No sporulation ability Impossible to obtainmeiotic segregants

-----

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!

Don’t forget to check the next slide, please!

Introduction of Biotechnology - 大阪大学ocw.osaka-u.ac.jp/engineering-jp/biotechnology-fundamentals-jp/dr... · International Center for Biotechnology, Osaka University Introduction

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