YEAST BIOCHEMISTRY BIOLOGY BIOTECHNOLOGY 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; jmattoon@uccs.edu www.uccs.edu/~biology/programs/biotech.htm September 15, 2007

Alcoholic fermentation by YEAST BIOCHEMISTRY Eduard Buchner 1897 Alcoholic fermentation by cell-free extracts of yeast. “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 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 START: SUGAR END: ETHANOL + CO2 MUSCLE START: SUGAR END: LACTIC ACID

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 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

YEAST BIOLOGY ANATOMY & CELL BIOLOGY REPRODUCTION NUTRITION LIFE CYCLE GENETICS TRANSFORMATION

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 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

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 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

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

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 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!