BIOCHEMISTRY BIOLOGY BIOTECHNOLOGY By Professor, Dr. Jim Mattoon

1. 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

2. YEAST BIOCHEMISTRY Eduard Buchner 1897 Alcoholic fermentation by cell-free extracts of yeast. “zymase”

3. ZYMASE • NOT A SINGLE ENZYME • A MULTIENZYME SYSTEM • EACH ENZYME CATALYZES A SINGLE CHEMICAL CHANGE IN A CHAIN OF REACTIONS NOW CALLED GLYCOLYSIS

4. 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.

5. YEAST START: SUGAR END: ETHANOL + CO2 MUSCLE START: SUGAR END: LACTIC ACID COMPARE YEAST & MUSCLEGLYCOLYSIS

6. FATHERS OF MICROBIOLOGY • LOUIS PASTEUR – FRANCE • ALCOHOLIC FERMENTATION CAUSED BY LIVING YEAST CELLS • ROBERT KOCH – GERMANY • MANY DISEASES CAUSED BY LIVING CELLS OF BACTERIA

7. 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

8. RIBOFLAVIN FOLIC ACID BIOTIN COENZYME FOR RESPIRATION CURE FOR LARGE CELL ANEMIA BIOASSAY - ALLOWED PURIFICATION OF FOLIC ACID VITAMIN DISCOVERY WITH YEAST

9. YEAST BIOLOGY • ANATOMY & CELL BIOLOGY • REPRODUCTION • NUTRITION • LIFE CYCLE • GENETICS • TRANSFORMATION

11. 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

12. 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.

13. LIFE CYCLE - PLOIDY

15. 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.

16. 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.

17. YEAST BIOTECHNOLOGY

18. 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.

19. pSTA PLASMID

20. TRANSFORMING BREWING YEAST • TREAT CELLS WITH LiAc • ADD STA PLASMID DNA • PLATE ON HIGH COPPER MEDIUM • SELECT Cu-RESISTANT COLONIES • TEST FOR STARCH DIGESTION

21. 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!