exam 1

Question 1

pasteur effect

Answer 1

low [glucose] and high [o2] repress fermentation and induce TCA and oxidative phosphorylation

Question 2

Transcriptional activators on TCA, ETC gene promoters

Answer 2

regulate synthesis to control concentration of enzyme

Question 3

Transcriptional repressors on fermentation gene promoters

Answer 3

regulate synthesis to control concentration of enzyme

Question 4

PTMs that increase their activity of the enzymes of TCA

Answer 4

regulate activity of enzymes

Question 5

Crabtree effect

Answer 5

high glucose concentrations
(regardless of O2 concentration) repress oxidative phosphorylation and induce fermentation

Question 6

Carbon catabolite repression

Answer 6

Expression of genes for the use of alternate carbon sources (other sugars, fats, methanol, etc) is repressed by the presence of significant level of glucose (high enough levels)

extension of Crabtree, common regulatory mechanism for many industrial microbes

Question 7

Peroxisomes (P)

Answer 7

needed for metabolism of methanol (MeOH) and catalase expression

Question 8

Can you get catalase production on glucose?

Answer 8

no

glucose represses transcription of catalase gene —> little catalase protein made since little catalase mRNA is made

repressor on catalase promoter

Question 9

grow methanol (MeOH) only

Answer 9

peroxisomes + catalase

Question 10

grow on only glucose

Answer 10

no catalase

Question 11

grow on methanol (MeOH) + glucose

Answer 11

no catalase (bc of repressors)

Question 12

catalase activity is activated by

Answer 12

PTMS in peroxisomes

Question 13

Peroxisome

Answer 13

organelle containing
100’s of proteins that are mainly
repressed by carbon catabolite

Question 14

crabtree effects mechanisms

Answer 14

• repressors on TCA/ETC gene promoters
• activators on Fermentation gene promoters
• PTM (post translational modifications ie. phosphorylation) on Fermentation enzymes to activate them

Question 15

normal catalase production

Answer 15

catalase mRNA is translated in
cytoplasm, signal sequence on protein for import into peroxisome

Question 16

result of carbon catabolite repression

Answer 16

Catalase is unstable in cytoplasm, yeast cell degrades it if it is loitering in cytoplasm if not imported efficiently into peroxisome

Question 17

Imagine get catalase mRNA from strong constitutive promoter in glucose
• Catalase mRNA made in glucose conditions, translated but since there are no peroxisomes to
“shelter” it, it gets __________.

Answer 17

degraded

Question 18

Can’t get active catalase made in

Answer 18

glucose-grown yeast cells

Question 19

why secondary metabolites only made after trophophase/primary phase is over?

Answer 19

lots of regulatory mechanisms

Question 20

where are the building blocks of secondary metabolites made?

Answer 20

made during making of primary metabolites

Question 21

hitch in making secondary metabolite

Answer 21

HITCH: Dependence on primary
metabolism since many secondary
metabolites derived from substrates
which are primary metabolites

Question 22

Regulation of secondary metabolites affected by

Answer 22

Presence of nonribosomal peptide
synthetases — enzymes that
polymerize peptides (branched and
cyclic) without ribosomes or mRNA (think ice cube tray)

Question 23

Nonribosomal peptide synthetases

Answer 23

made only at end of primary metabolism for secondary metabolism but use amino acids made mainly during trophophase

Question 24

why cyclosporin (2ndary metabolism, immunosuppressant
peptide) made in certain fungus made?

Answer 24

surpress insect immune system when attacking it, hurt other fungi

Question 25

Other hitches: regulation of secondary metabolites affected by

Answer 25

- inducers (ex. heavy metal) - repressors ex repressors — high level of a) nitrogen (ammonia) b) phosphate c) ATP d) carbon catabolite repression of secondary metabolites e) glucose (can only be fed at low rates during idiophase) & need FED BATCH fermenter

Question 26

objective approach

Answer 26

go to natural site where it is expected to be found ex. look for high ethanol resistant yeast in wine that is contaminated, open to the environment; look for a sludge degrader in dump sites

Question 27

Shotgun approach

Answer 27

just randomly get samples of microbes from places where there are lots of microbes (biofilms, soil, sewer systems, etc) and try them out

Question 28

How to obtain the production microbe from nature?

Answer 28

1. objective approach 2. shotgun approach

Question 29

how to start with coming up with a production microbe?

Answer 29

- First do a literature search - Find out what the competitors use - Talk to consultants/experts in the field

Question 30

after picking source, what are the next steps for getting product microbe?

Answer 30

enrichment (try to get it to improve) and isolation of pure culture, microbe must give competitive edge

Question 31

enrichment

Answer 31

try to get better microbe via spontaneous mutation & selection; getting rid of nondesired microbes EX Isolate some ethanol resistant yeasts from source • Grow mixed culture in fermenter and slowly increase ethanol concentration What you don’t want ---dies off • Take some growers, dilute in new medium, repeat in increased EtOH—weeks, months, etc • Survivors will be high ethanol tolerant—enriching in the liquid HOW SO?? Some SPONTA NEOUS mutation and selection going on while growing • Purify your microbe finally—usually on solid medium

Question 32

isolation methods

Answer 32

shotgun or objective

Question 33

another way to get production microbe - less work

Answer 33

request characterized specimen from culture collection (storehouses for microbes, cataloged traits)

Question 34

main drawback of requesting characterized specimen from a culture collection compared to getting microbe on your own

Answer 34

anyone can get it so it’s bad if want to be competitive and make money

Question 35

what should ideal production microbe be?

Answer 35

1. safe, nonpathogenic 2. cheap, robust (low cost growth medium, smthing grow cheaply on cheap medium) 3. efficient w well characterized biosynthetic pathway (know inducers and repressors that can be in medium) 4. adaptable to fermenter (don’t make foam and doesn’t flocculate unless you want it to) 5. genetically stable (doesnt do smthing to its dna while growing it such as rearrange dna, methylate dna (turn off genes for fun), start of as haploid then fuse and MATE and become diploid or more) 6. not dimorphic (yeast to mold) 7. make few contaminating byproduct (smtimes when growing make byproduct that makes stuff harder to purify) = want easy purification or less downstream processing 8. amenable to genetic manipulation (can send dna into microbe and transform) 9. GRAS (Generally Recognized As Safe) = FDA approved, less testing needed