Flashcards in Lecture 9 -Metabolic engineering of bacteria Deck (29):
What are the empirical approaches to improving yield?
can improve yeild of microbial processes through random mutation and seletion to give improvement, not directed
Give an example of the empiricle approaches to improving yield
yeild of penecillin from inital experiments 10 ug/l through mutation and seletion of strains led to greater yields 10g/l
Two approaches to improving yield
What is rationale design to improve yield?
being able to work out how to improve yield of certain cell processes by understanding all the processes it can do and how they are controlled
What do we know about E.coli and why is it a model bacterium?
-biochemical and genetic information for 500+ enxymes
-2000 proteins have known functions
-37C aerobic growth
-grow in simple defined medium doubling hourly
-high biosynthetic capability
-high metabolic capacity
-can use anaerobic respiration, aerobic respiration or fermentation
What is metabolic flux?
rate of turnover of molecules through a metabolic pathway, regulated by enzymes in a pathway
What 5 ways are there to increase flux through a particular metabolic pathway?
1- removal of rate limiting transcriptional and allosteric control
2-kinetic enhancement of rate limiting step
3-genetic blockage of competing pathways
4- enhanced carbon commitment to primary metabolic pathway from central metabolism
5-enhanced transport of substrate into cell
How can metabolism be directed for increased protein production?
In TCA cycle, protein synthesis becomes limited by amino acid supply.
-10 amino acids are derived from TCA cycle metabolites
Can INCREASE FLUX of carbon to amino acid biosynthesis by reducing wasted flux to acetate
-delete enzymes that make acetate
-add enzymes add more C to TCA cycle
-can delete genes for catabolic enzymes keep amino acid pool high
What is phenylalanine prododuction in E.coli used for? And problems with production pathway?
-one of 3 aromatic amino acids synthesised in E.coli, some parts pathway shared
-cell tightly regulates expression and activity of enzymes in pathway depending on level of products in the cell
How can phenylalanine expression be increased in E.coli?
1) remove feedback inhibition
-use phenylalanine analogoue that will always repress so that the cell mutates enzyme not to respond
2)Overexpress similar enzyme not inhibited by phenylalanine
-use promotor not regulated by tryptophan
3)Remove competing pathways
-delele genes for tyrosine and tryptophan syntheisis
4)Enhance carbon commitment to primary metabolic pathway from central metabolism
-increased supply of E4P and PEP precursors
How is indigo produced in E.coli?
1)napthalene dioxygenase from pseudomonas ezpressed in E.coli and E.coli grown in presence of tryptophan
2)E.coli expresses a catabolic tryptophase which makes indole as product
3)indole converted to indoxyl by napthalene dioxygenase
4)undergoes spontaneous oxidation to indigo
What is a problem with indigo production in E.coli?
Indoxyl also oxidised to isatin which forms completely different dye (deep burgandy) -> as this is a competing spontaneous pathway cannot be deleted genetically
What is indigo used for?
important for cotton and blue jeans
produced originally from sea snails
What is Sinorhizobium meliloti (RMBPC-2)?
an engineered microbe approved in USA for agricultural use
What dose Sinorhizobium meliloti do?
-forms a symbiotic relationship with legumus plants to fix nitrogen, modified bacteria can fix nitrogen at a greater rate
1-infects roots of allella plant and form roto nodules
2-differentiates into form that can fix N2 from air and convert to NH3
3-incorperated into plant biomass
What genes have been overexpressed in Sinorhizobium meliloti strain RMBPC-2?
1)Increased expression of DctA
-DctA encodes a membrane transport protein for dicarboxylates are the preferred C source for bacterium in nodule -> increases rate of metabolism in nodule and amount of N fixed
2)Increased expression of NifA
-NifA transcription factor activates gene required for nitrogen fixation leading to more copies of the protein and more nitrogen fixation
Positives and negatives of overexpression of DctA and NifA in Sinorhizobium meliloti strain RMBPC-2?
Increased yield by 10%
Carry antibiotic resistance genes
HOW MANY CASES OF MALARIA ARE THERE GLOBALLY PER YEAR
around 0.5 billion
Where is the malaria treatment Artemisinin obtained from?
from plant Artemisia annua
Features of arteminisin
Sesquiterpene lactone endoperoxide
machanism of action unknown
can be made synthetically but very expensive
What is arteminisin synthesised from in plants?
FPP (multiple uses in the cell)
How have microbes be engineered to make artemisinic acid
engineer S.cerevisae to make the precursor to artemisinic acid FPP through metabolic engineering
1-How can yeast be engineered to make more FPP?
1) Mutate the transcription factor that normally controls the synthesis of pathway, to be constituatively active. Increases synthesis of some enzymes
2) Alter genes that encode tHMGR and ERG20 to be under the control of a galactose inducible promotor
3)Add extra copies of tHMGR gene on the chromosome
THEN MADE MORE FPP AVAILABLE FOR MAKING DRUG
What does FPP stand for?
2-How did engineer yeast to make more FPP available to make malaria drug?
1) manipulated erg9 gene so expression was repressed by methionine in growth medium
-reducing expression of erg9 decreases amount of enzyme present and flux of reaction, cannot remove completely as needs flux though pathway to make sterols
3-How did scientists use the identification of genes in plant that catalysed the steps between FPP and artenisimic acid to increase yield?
1)cloned and manipulated gene encoding ADS reaction so expression was induced in presence of galactose
2)manipulated new genes isolated from plant so that their expression was induced with presence of galactose
What were the three ways FPP production to arteminisic acid was increased in yeast cells?
1-Engineered yeast to produce moreFPP
2- Enginnered so that more FPP was available for making drug
3-Identified genes that catalysed the steps between artenisimic acid and manipulated them
After genetic enginnering in yeast cells to produce FPP and arteminisic acid, how were yeast cells grown?
-grown in presence of galactose and methionine