lecture 10: metabolic engineering

fermentation in industry

the growth of microbes with a bioreactor for production of commodity chemicals, biofuels etc.

batch fermentations

• all nutrients required for the fermentation are provided in the initial culture medium

• once all the nutrients has been consumed, growth of the organism ceases and the fermentation has ended

continuous fermentations

• performed by continually supplyinh fresh medium to the culture with the subsequent removal of the same amount of culture, so a steady state is reached in the fermenter

fed-batch fermentations

• nutrients are provided in the batch culture medium

• once consumed, a feed is initiated to provide the culture with additional nutrients and thus allows for further growth of the culture

penicillin chysogenum fed-batch fermentation for penicilin production

1. initial growth phase in a small fermenter inoculated with freeze-dried spores of penicillin chysogenum

2. once full, it is scaled-up through 2 further growth stages in successively large fermenters to provide a large enough inoculum for the production phase

3. the production phase is a fed-batch culture with high levels of oxygen maintains and carbon and nitrogen feeding

4. monitored to keep the fermentation in optimal penicillin production mode, during the production mode during the production phase

5. penicillin is excreted into the medium and is recovered at the end

useful properties of industrial microbes

• high yield in a relatively short time period

• grows rapidly and in simple growth media

• does not produce toxins

• amenable to genetic engineering

• genetically stable so it can be stocked/ stored

metabolic engineering

the deliberate redesign of cellular biochemical pathways to enhance production of a product

process of metabolic engineering

1. modifying metabolic pathway to redirect existing metabolism to specific products

2. enhancing precursors and energy/ cofactor supply

3. engineering transport systems

4. increasing cellular tolerance to the product/ substrate

5. consideration of regulatory effects

6. decoupling of growth and production formation

corynebacterium glutamicum

an aerobic, gram-positive soil bacterium, capable of growing on a simple mineral salt medium wuth glucose. it cannot breakdown lysine so it excretes it

what does LysC do in corynebacterium glutamicum and how can it be optimised by engineering?

• when lysine is high, it binds to the enzyme LysC and shuts down production

• by engineering LysC gene, the enzyme becomes insensitive to high lysine levels

what is DapA in corynebacterium glutamicum and how is it engineered to be optimised?

• aspartate semi-aldehyde can either be converted into lysine (via DapA enzyme) or it can divert into other amino acids

• by optimising the expression of the DapA gene, it increases the flux towards lysine

what is PntAB in corynebacterium glutamicum and how can it be optimised?

• the reaction requires lots of NADPH, which is produced via PntAB and NADH

• by overexpressing PntAB along with NADH, this reaction converts NADP+ back itno NADPH, ensuring the cell has a continuous, abundant supply

what is LysE in corynebacterium glutamicum and how can it be optimised?

• lysE is an exporter protein that pumps lysine out of the cell (so it does not trigger negative feedback mechanisms)

• by overexpressing LysE, it allows the lysine to move into the fermentation broth

vitamin B12 (cobalumin)

essential role in the coenzymes in animals, but is only synthesised by some prokaryotes (must be taken in via consumption of animal products)

why are natural B12 producers not used in industrial fermentation of B12? give examples of natural B12 producers

• e.g. Pseudomonas denitrificans, Propionibacterium friednereich

• they are slow and difficult to engineer

how does industry produce B12

uses engineered (28 genes added) E.coli, produces low yield but at a fast rate