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BIOCHEMISTRY BIOLOGY BIOTECHNOLOGY By Professor, Dr. Jim Mattoon PowerPoint PPT Presentation


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MBAA-Rocky Mountain District Meeting YEAST. BIOCHEMISTRY BIOLOGY BIOTECHNOLOGY By Professor, Dr. Jim Mattoon Director, Center for Biotechnology and Bioinformatics 1090 Garlock Lane, Colorado Springs, Colorado 80918 (719) 599-7992; [email protected]

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BIOCHEMISTRY BIOLOGY BIOTECHNOLOGY By Professor, Dr. Jim Mattoon

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Biochemistry biology biotechnology by professor dr jim mattoon

MBAA-Rocky Mountain District Meeting

YEAST

BIOCHEMISTRY

BIOLOGY

BIOTECHNOLOGY

By

Professor, Dr. Jim Mattoon

Director, Center for Biotechnology and Bioinformatics

1090 Garlock Lane, Colorado Springs, Colorado 80918

(719) 599-7992; [email protected]

www.uccs.edu/~biology/programs/biotech.htm

September 15, 2007


Yeast biochemistry

YEAST BIOCHEMISTRY

Eduard Buchner 1897

Alcoholic fermentation by

cell-free extracts of yeast.

“zymase”


Zymase

ZYMASE

  • NOT A SINGLE ENZYME

  • A MULTIENZYME SYSTEM

  • EACH ENZYME CATALYZES A SINGLE CHEMICAL CHANGE IN A CHAIN OF REACTIONS NOW CALLED GLYCOLYSIS


Glycolysis

GLYCOLYSIS

  • INVOLVES MANY ENYMES

  • AIDED BY COENZYMES DERIVED FROM VITAMINS

  • SIMILAR ENZYMES AND COENZYMES FOUND IN MUSCLE EXTRACTS

  • MUCH OF BIOCHEMISTRY IS UNIVERSAL IN LIVING ORGANISMS.


Compare yeast muscle glycolysis

YEAST

START: SUGAR

END: ETHANOL + CO2

MUSCLE

START: SUGAR

END: LACTIC ACID

COMPARE YEAST & MUSCLEGLYCOLYSIS


Fathers of microbiology

FATHERS OF MICROBIOLOGY

  • LOUIS PASTEUR – FRANCE

    • ALCOHOLIC FERMENTATION CAUSED BY LIVING YEAST CELLS

  • ROBERT KOCH – GERMANY

    • MANY DISEASES CAUSED BY LIVING CELLS OF BACTERIA


Yeast biology

YEAST BIOLOGY

  • SINGLE CELL

  • REPRODUCES BY BUDDING

  • GROWS WITH OR WITHOUT AIR

  • HAS A TRUE NUCLEUS WITH MULTIPLE LINEAR CHROMOSOMES

  • HAS A TOUGH CELL WALL

  • MUCH LARGER THAN BACTERIA


Vitamin discovery with yeast

RIBOFLAVIN

FOLIC ACID

BIOTIN

COENZYME FOR RESPIRATION

CURE FOR LARGE CELL ANEMIA

BIOASSAY - ALLOWED PURIFICATION OF FOLIC ACID

VITAMIN DISCOVERY WITH YEAST


Yeast biology1

YEAST BIOLOGY

  • ANATOMY & CELL BIOLOGY

  • REPRODUCTION

  • NUTRITION

  • LIFE CYCLE

  • GENETICS

  • TRANSFORMATION


Functions of cell components

FUNCTIONS OF CELL COMPONENTS

  • NUCLEUS: CHROMOSOMES [Strings of genes that control inheritance-DNA].

  • VACUOLE: Recycling center

  • CYTOPLASM:

    • Enzymes: Catalytic proteins controlling metabolism, including fermentation

    • Ribosomes: Protein factories


Yeast genetics i

YEAST GENETICS I

  • Founded by Øjvind Winge working at the laboratories of the Carlsberg Brewery in Copenhagen in the late 1930s and early 1940s.

  • He was interested in genes that controlled fermentation, particularly genes that controlled maltose fermentation.

  • During mashing of malt, the main sugar produced is maltose.


Life cycle ploidy

LIFE CYCLE - PLOIDY


Yeast genetics ii

YEAST GENETICS II

  • He first had to work out the yeast life-cycle & micro dissection methods.

  • Sexual reproduction

    • Double chromosomes [Diploids 2n--->4n]

    • Pairs separate, then separate again. 4n---> n+n+n+n. 4 haploid spores

    • Spores germinate & budding follows

    • Genes segregate in pairs.

    • For example mating type;2 α : 2 αlpha.

    • Different forms of same gene also 2:2.


Yeast genetics iii

YEAST GENETICS III

  • During the next 50 years over 1000 genes were studied and it was established the they were scattered over 16 chromosomes.

  • In the early 90s, André Goffeau organized about 30 labs who sequenced the entire yeast genome, over 6,200 genes. The sequences were published in 1996.

  • Many of these genes have now been cloned.


Yeast biotechnology

YEAST BIOTECHNOLOGY


Creating brewing yeast that ferments starch directly

CREATING BREWING YEAST THAT FERMENTS STARCH DIRECTLY

  • A wild yeast, Saccharomyces diastaticus, secretes glucoamylase, an enzyme that digests starch to form glucose.

  • We have cloned the STA gene that encodes glucoamylase and used the STA DNA to transform both lab strains and a brewing yeast strain so that they can ferment starch directly.


Psta plasmid

pSTA PLASMID


Transforming brewing yeast

TRANSFORMING BREWING YEAST

  • TREAT CELLS WITH LiAc

  • ADD STA PLASMID DNA

  • PLATE ON HIGH COPPER MEDIUM

  • SELECT Cu-RESISTANT COLONIES

  • TEST FOR STARCH DIGESTION


Transformation and expression of sta gene

TRANSFORMATION AND EXPRESSION OF STA GENE

  • PLASMID DNA IS REPLICATED AS A MINI-CHROMOSOME.

  • RNA POLYMERASE MAKES AN RNA COPY OF STA GENE.

  • RNA COPY IS TRANSLATED INTO GLUCOAMYLASE PROTEIN.

  • SECRETED ENZYME DIGESTS STARCH.

  • CELLS FERMENT STARCH!


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