biochemistry biology biotechnology by professor, dr. jim mattoon director, center for biotechnology...
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BIOCHEMISTRYBIOLOGY
BIOTECHNOLOGYBy
Professor, Dr. Jim MattoonDirector, Center for Biotechnology and
Bioinformatics1090 Garlock Lane, Colorado Springs, Colorado
80918(719) 599-7992; [email protected]
www.uccs.edu/~biology/programs/biotech.htm
September 15, 2007
MBAA-Rocky Mountain District Meeting
YEAST
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YEAST BIOCHEMISTRY
Eduard Buchner 1897
Alcoholic fermentation by cell-free extracts of yeast.
“zymase”
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ZYMASE
• NOT A SINGLE ENZYME
• A MULTIENZYME SYSTEM
• EACH ENZYME CATALYZES A SINGLE CHEMICAL CHANGE IN A CHAIN OF
REACTIONS NOW CALLED GLYCOLYSIS
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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.
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COMPARE YEAST & MUSCLEGLYCOLYSIS
YEAST
START: SUGAR
END: ETHANOL + CO2
MUSCLE
START: SUGAR
END: LACTIC ACID
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FATHERS OF MICROBIOLOGY
• LOUIS PASTEUR – FRANCE– ALCOHOLIC FERMENTATION CAUSED BY
LIVING YEAST CELLS
• ROBERT KOCH – GERMANY– MANY DISEASES CAUSED BY LIVING
CELLS OF BACTERIA
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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
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VITAMIN DISCOVERY WITH YEAST
• RIBOFLAVIN
• FOLIC ACID
• BIOTIN
• COENZYME FOR RESPIRATION
• CURE FOR LARGE CELL ANEMIA
• BIOASSAY - ALLOWED PURIFICATION OF FOLIC ACID
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YEAST BIOLOGY
• ANATOMY & CELL BIOLOGY
• REPRODUCTION
• NUTRITION
• LIFE CYCLE
• GENETICS
• TRANSFORMATION
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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
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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.
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LIFE CYCLE - PLOIDY
PLOIDYChromo-
some SetsChromo-some No.
MatingTypes
HAPLOID ONE 16a or
alpha(α)
DIPLOID TWO 32No
Mating
POLY-PLOID
Three orMore
16 X N ---
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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.
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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.
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YEAST BIOTECHNOLOGY
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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.
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pSTA PLASMID
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TRANSFORMING BREWING YEAST
• TREAT CELLS WITH LiAc
• ADD STA PLASMID DNA
• PLATE ON HIGH COPPER MEDIUM
• SELECT Cu-RESISTANT COLONIES
• TEST FOR STARCH DIGESTION
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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!