BIOCHEMISTRYBIOLOGY

BIOTECHNOLOGYBy

Professor, Dr. Jim MattoonDirector, Center for Biotechnology and

Bioinformatics1090 Garlock Lane, Colorado Springs, Colorado

80918(719) 599-7992; jmattoon@uccs.edu

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

September 15, 2007

MBAA-Rocky Mountain District Meeting

YEAST

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 & MUSCLEGLYCOLYSIS

YEAST

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

PLOIDYChromo-

some SetsChromo-some No.

MatingTypes

HAPLOID ONE 16a or

alpha(α)

DIPLOID TWO 32No

Mating

POLY-PLOID

Three orMore

16 X N ---

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!