Lecture 9 -Metabolic engineering of bacteria

Question 1

What are the empirical approaches to improving yield?

Answer 1

can improve yeild of microbial processes through random mutation and seletion to give improvement, not directed

Question 2

Give an example of the empiricle approaches to improving yield

Answer 2

yeild of penecillin from inital experiments 10 ug/l through mutation and seletion of strains led to greater yields 10g/l

Question 3

Two approaches to improving yield

Answer 3

Empirical

Rational

Question 4

What is rationale design to improve yield?

Answer 4

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

Question 5

What do we know about E.coli and why is it a model bacterium?

Answer 5

• 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

Question 6

What is metabolic flux?

Answer 6

rate of turnover of molecules through a metabolic pathway, regulated by enzymes in a pathway

Question 7

What 5 ways are there to increase flux through a particular metabolic pathway?

Answer 7

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

Question 8

How can metabolism be directed for increased protein production?

Answer 8

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

Question 9

What is phenylalanine prododuction in E.coli used for? And problems with production pathway?

Answer 9

• making sweeteners
• 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

Question 10

How can phenylalanine expression be increased in E.coli?

Answer 10

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

Question 11

How is indigo produced in E.coli?

Answer 11

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

Question 12

What is a problem with indigo production in E.coli?

Answer 12

Indoxyl also oxidised to isatin which forms completely different dye (deep burgandy) -> as this is a competing spontaneous pathway cannot be deleted genetically

Question 13

What is indigo used for?

Answer 13

important for cotton and blue jeans
produced originally from sea snails
-plants
-chemically

Question 14

What is Sinorhizobium meliloti (RMBPC-2)?

Answer 14

an engineered microbe approved in USA for agricultural use

Question 15

What dose Sinorhizobium meliloti do?

Answer 15

-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

Question 16

What genes have been overexpressed in Sinorhizobium meliloti strain RMBPC-2?

Answer 16

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

Question 17

Positives and negatives of overexpression of DctA and NifA in Sinorhizobium meliloti strain RMBPC-2?

Answer 17

Increased yield by 10%

Carry antibiotic resistance genes

Question 18

HOW MANY CASES OF MALARIA ARE THERE GLOBALLY PER YEAR

Answer 18

around 0.5 billion

Question 19

Where is the malaria treatment Artemisinin obtained from?

Answer 19

from plant Artemisia annua

Question 20

Features of arteminisin

Answer 20

Malaria treatment
Sesquiterpene lactone endoperoxide
machanism of action unknown
can be made synthetically but very expensive

Question 21

What is arteminisin synthesised from in plants?

Answer 21

FPP (multiple uses in the cell)

Question 22

How have microbes be engineered to make artemisinic acid

Answer 22

engineer S.cerevisae to make the precursor to artemisinic acid FPP through metabolic engineering

Question 23

1-How can yeast be engineered to make more FPP?

Answer 23

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

Question 24

What does FPP stand for?

Answer 24

farnesyh pyrophosphate

Question 25

2-How did engineer yeast to make more FPP available to make malaria drug?

Answer 25

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

Question 26

3-How did scientists use the identification of genes in plant that catalysed the steps between FPP and artenisimic acid to increase yield?

Answer 26

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

Question 27

What were the three ways FPP production to arteminisic acid was increased in yeast cells?

Answer 27

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

Question 28

After genetic enginnering in yeast cells to produce FPP and arteminisic acid, how were yeast cells grown?

Answer 28

-grown in presence of galactose and methionine

Question 29

What are the main principles of genetic engineering to increase yield shown by the arteminisic acid production?

Answer 29

1) Increase flux through producing pathway
- Increase gene dosage, add more copies of key genes
- Upregulation of expression of genes in the pathway
2) Repressed flux of reactions that compete for desired precursor
- downregulate the expression of competing pathways
3) Express recombinant proteins to enable use of an endogenous precursor to synthesise novel metabolites
- Identified genes that were responsible in plant and managed to express in yeast