exam 1 Flashcards
pasteur effect
low [glucose] and high [o2] repress fermentation and induce TCA and oxidative phosphorylation
Transcriptional activators on TCA, ETC gene promoters
regulate synthesis to control concentration of enzyme
Transcriptional repressors on fermentation gene promoters
regulate synthesis to control concentration of enzyme
PTMs that increase their activity of the enzymes of TCA
regulate activity of enzymes
Crabtree effect
high glucose concentrations
(regardless of O2 concentration) repress oxidative phosphorylation and induce fermentation
Carbon catabolite repression
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
Peroxisomes (P)
needed for metabolism of methanol (MeOH) and catalase expression
Can you get catalase production on glucose?
no
glucose represses transcription of catalase gene —> little catalase protein made since little catalase mRNA is made
repressor on catalase promoter
grow methanol (MeOH) only
peroxisomes + catalase
grow on only glucose
no catalase
grow on methanol (MeOH) + glucose
no catalase (bc of repressors)
catalase activity is activated by
PTMS in peroxisomes
Peroxisome
organelle containing
100’s of proteins that are mainly
repressed by carbon catabolite
crabtree effects mechanisms
• repressors on TCA/ETC gene promoters
• activators on Fermentation gene promoters
• PTM (post translational modifications ie. phosphorylation) on Fermentation enzymes to activate them
normal catalase production
catalase mRNA is translated in
cytoplasm, signal sequence on protein for import into peroxisome
result of carbon catabolite repression
Catalase is unstable in cytoplasm, yeast cell degrades it if it is loitering in cytoplasm if not imported efficiently into peroxisome
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 __________.
degraded
Can’t get active catalase made in
glucose-grown yeast cells
why secondary metabolites only made after trophophase/primary phase is over?
lots of regulatory mechanisms
where are the building blocks of secondary metabolites made?
made during making of primary metabolites
hitch in making secondary metabolite
HITCH: Dependence on primary
metabolism since many secondary
metabolites derived from substrates
which are primary metabolites
Regulation of secondary metabolites affected by
Presence of nonribosomal peptide
synthetases — enzymes that
polymerize peptides (branched and
cyclic) without ribosomes or mRNA (think ice cube tray)
Nonribosomal peptide synthetases
made only at end of primary metabolism for secondary metabolism but use amino acids made mainly during trophophase
why cyclosporin (2ndary metabolism, immunosuppressant
peptide) made in certain fungus made?
surpress insect immune system when attacking it, hurt other fungi
Other hitches: regulation of secondary metabolites affected by
- 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
objective approach
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
Shotgun approach
just randomly get samples of microbes from places where there
are lots of microbes (biofilms, soil, sewer systems, etc) and try them out
How to obtain the production microbe from nature?
- objective approach
- shotgun approach
how to start with coming up with a production microbe?
- First do a literature search
- Find out what the competitors use
- Talk to consultants/experts in the field
after picking source, what are the next steps for getting product microbe?
enrichment (try to get it to improve) and isolation of pure culture, microbe must give competitive edge
enrichment
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
isolation methods
shotgun or objective
another way to get production microbe - less work
request characterized specimen from culture collection (storehouses for microbes, cataloged traits)
main drawback of requesting characterized specimen from a culture collection compared to getting microbe on your own
anyone can get it so it’s bad if want to be competitive and make money
what should ideal production microbe be?
- safe, nonpathogenic
- cheap, robust (low cost growth medium, smthing grow cheaply on cheap medium)
- efficient w well characterized biosynthetic pathway (know inducers and repressors that can be in medium)
- adaptable to fermenter (don’t make foam and doesn’t flocculate unless you want it to)
- 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)
- not dimorphic (yeast to mold)
- make few contaminating byproduct (smtimes when growing make byproduct that makes stuff harder to purify) = want easy purification or less downstream processing
- amenable to genetic manipulation (can send dna into microbe and transform)
- GRAS (Generally Recognized As Safe) = FDA approved, less testing needed
