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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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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.
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.
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.
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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