Biotechnology

Question 1

SUMMARY

Describe the characteristics that are required of a microbial process for it to be industrially useful.

Explain the difference between primary and secondary metabolites.

Describe the key steps in the industrial production of:
Penicillin
Insulin
QuornTM

Answer 1

…

Question 2

8 Characteristics required for industrial use

Answer 2

It is economically advantageous to do so (e.g. more economical than chemical processes).

1. Must produce large quantities of the required product (high yield)
2. Must grow rapidly in liquid culture
3. Must be suitable for growth in large batch or continuous fermentation systems
4. Must use nutrients which can be obtained readily and cheaply
5. Product must be easily separated from other compounds produced by the organism
6. Must be able to be scaled up
7. Must not be pathogenic
8. Beneficial if easily genetically engineered

Question 3

Primary metabolites

2 points

Answer 3

1. Products produced up to end of exponential phase of growth.
2. Often associated with essential energy metabolism, e.g. lactic acid, ethanol.

Question 4

Secondary metabolites

4 points

Answer 4

1. Produced at end of exponential and during stationary phase of growth.
2. Characteristic of specific organisms.
3. Not essential for growth.
4. Production varies depending on environmental conditions.

Question 5

Antibiotic production
Penicillin production
4 points

Answer 5

1. Produced by Penicillium chrysogenum.
2. Originally (1 mg/L), now (~50 g/L).
3. Grown in a fed-batch culture.
4. Harvesting - penicillin excreted into medium by cells.

Question 6

Production of insulin

7 points

Answer 6

1. Insulin regulates uptake of glucose from the blood into cells.
2. Type I diabetes is caused by lack of insulin -> high blood sugar.
3. Originally purified from pancreas of slaughtered pigs and cows.
4. Not identical to human insulin so not as effective and could potentially cause allergic reaction.
5. Genetic engineering enabled human insulin to be produced by microorganisms.
6. Insulin made of an A and B chain linked by disulphide bonds
   Proinsulin produced then C-peptide removed to produce insulin
7. Two options industrially (bacteria or yeast as host):
   Both chains produced separately then joined
   Proinsulin produced then cleaved

Question 7

production of insulin using bacteria E.coli as host

6 points

Answer 7

1. A DNA fragment containing the proinsulin gene is introduced into E. coli.
2. Grows in a fermenter and synthesises proinsulin.
3. The proinsulin precipitates in the form of inclusion bodies inside the cells, which are subsequently disrupted.
4. After being separated from the cell debris, the proinsulin is transformed into insulin and purified in several stages.
5. First, the intramolecular bonds (disulfide bridges) are disrupted and, later on, in a renaturation step, they are reformed.
6. Afterwards, the renatured proinsulin is purified and converted into insulin (by enzymatic and chemical reactions) and submitted to other purification steps such as ion-exchange, RP-HPLC and crystallization, leading to the insulin crystal which is then formulated.

Question 8

QuornTM

3 points

Answer 8

1. Meat substitute - mycoprotein
2. Consists of filamentous fungus
3. Fusarium species

Question 9

QuornTM challenges faced

3 points

Answer 9

1. Nucleic acid content (RNA) of product was 10%: needed <2% (WHO safe limit is 20 g/kg DM)
2. Associated with toxic effects
   Gout - ‘the disease of kings’
3. RNA is metabolised by humans to uric acid - excess uric acid in blood
   Arthritis, kidney problems.

Question 10

QuornTM
RNA reduction
5 points

Answer 10

1. Achieved by mild heat shock (65 oC).
2. Heated to 65 oC for 20-30 min – inactivates proteases but not RNAses (heat-stable).
3. Allows RNAses to degrade RNA.
4. RNA degraded (<2%).
5. Protein:RNA changed from ~5:1 to ~23:1

Question 11

QuornTM
Safety testing
5 points

Answer 11

1. Mycotoxins - secondary metabolites produced by many fungi.
2. Not produced by the Fusarium species used.
3. Difficult to prove at the time – now you could sequence the genome.
4. 15 years of pilot scale culture and food trials (animals and humans).
5. Ultimately proved to be a safe, nutritious food.

Question 12

production of quorn 5 points

Answer 12

1. Continuous aerobic pure culture.
2. 30 oC, pH 6.
3. Air lift fermentation.
4. Fermenters approx. 45 m high.
5. Run for 1000 hours.