Culture & Metabolic Engineering

Question 1

Describe 4 ways to measure microbial growth

Answer 1

1. Cell dry weight, pellet, wash, dry and weigh. Provides mass/unit volume but doesn’t indicate cell viability
2. Cell number. Either as a total count of dead and live cells or as a viable count: Perform a serial dilution, usually upto 10^6, plate and count the number of colonies/volume used
3. Optical Density (OD), 595/600/610nm wavelength usually used, requires a standard curve to relate OD to cell count, photosynthetic bacteria can skew the results
4. Specific cell components, measuring the mass of chloroplasts, mitochondria, or specific proteins, this doesn’t account for biological variation.

Question 2

Tell me about batch culture

Answer 2

It is a culture of a fixed volume in a flask or culture vessel. Unrestrained growth isn’t possible due to the depletion of nutrients and accumulation of autoinhibitory waste products.

Question 3

Describe the phases of a batch culture growth curve

Answer 3

1. Lag phase, cells prepare machinery for growth, the length depends on the change in environment.
2. Log phase, exponential growth
3. Stationary phase, cells stop growing, growth machinery shuts down and stress responses turn on
4. Death phase, cells die with a half life.

Question 4

What are the limitations of batch culture?

Answer 4

It is an artefact of lab growth, it doesn’t represent a natural growth curve.
It is in a closed system, there are massive variations in the physiochemical environment of pH, number of cells, [O2], [CO2] and medium composition.
There is significant variation between 2 points in log phase.

Question 5

What’s the main difference between chemostats and turbidostats?

Answer 5

Chemostats involve spent medium being removed and fresh medium being added continuously whereas this happens periodically in turbidostats

Question 6

What are the benefits of continuous culture?

Answer 6

Substrates and nutrients are added for maintained growth.
Autoinhibitory waste products are diluted.
Bacterial populations can be maintained at a constant OD in log phase.
Growth rate and cell density can be controlled independently.
Chemostats allow the reproducible cultivation of microbes at submaximal growth rates at different growth limitations.

Question 7

Tell me about common chemostat/Turbidostat properties and limitations

Answer 7

Common properties:
Fresh media is added with the culture volume remaining constant
It is well-mixed, there is aeration and agitation to ensure the even distribution of cells, nutrients and oxygen tension.
pH is kept within a predetermined level alongside a constant temperature.
Limitations:
Wall growth & foaming, usually remedied with anti-foam chemicals.
Mutations can affect growth rate.

Question 8

Describe steady-state establishment in chemostats

Answer 8

1. Initially, the growth rate > dilution rate. The cell number increases
2. As the concentration of the growth-limiting nutrient decreases, the growth rate < dilution rate.
3. Eventually, growth rate = dilution rate and the steady state has been achieved.

Question 9

What is the chemostat steady state?

Answer 9

Specific growth rate (µ), cell density and growth-limiting nutrient concentrations are constant. Varying the dilution rate can vary the growth rate while maintaining a constant OD, cell density.

Question 10

When does the chemostat steady state break down?

Answer 10

At low and high dilution rates:
a) At low dilution rates, an increasing proportion of the growth-limiting nutrient is used in basic house-keeping processes reduces the amount available for growth taking the culture out of log phase
b) At high dilution rates, more cells are siphoned away than divide leading to a washout

Question 11

Tell me about turbidostat culture

Answer 11

Turbidity is maintained; there is a preset OD level, monitored by a spectrophotometer that feedbacks into the system to add and release medium to return to the preset turbidity/OD when it reaches a threshold. There is no growth-limiting nutrient.

Question 12

What is industrial microbiology and microbial biotechnology?

Answer 12

Industrial microbiology is the large-scale production of commercial products by microorganisms.
Microbial biotechnology is the engineering of microbes to produce non-native compounds.

Question 13

Give some examples of microbes that produce antibiotics

Answer 13

Penicillin by Penicillium chrysogenum.
Tetracycline by Streptomyces

Question 14

Give some examples of microbes that produce enzymes

Answer 14

Lipase by Candida cylindracae
Amylases by Bacillus subtilis
Lactase by Kluyveromyces lactis

Question 15

Give some examples of microbes that produce food additives

Answer 15

Vitamin riboflavin by Ashbya gossypii and Bacillus subtilis
Amino acids by Corynebacterium glutamicum

Question 16

Give some examples of microbes that produce chemicals

Answer 16

Citric acid by Aspergillus niger
Bioethanol by Saccharomyces cerevisiae
Butanediols by Escherichia coli

Question 17

Give some examples of microbes that produce terpenes

Answer 17

Artemisinin by Saccharomyces cerevisiae
Carotenoids by Chlorella

Question 18

List some useful properties of industrial microbes

Answer 18

a) Produces substance of interest in a high yield
b) Can grow rapidly in a reproducible manner, produces product in a short period of time
c) Can grow and produce product in large scale culture or under bioreactor conditions
d) Metabolically flexible and adaptable
e) Doesn’t produce toxins or toxic by-products, not pathogenic to humans or animals
f) Amenable to genetic engineering & is genetically stable
g) Can be stocked or stored
h) Secretes the product into the media or is easy to handle or break

Question 19

Tell me about fermentation, fermenters and fermentors

Answer 19

Fermentation refers to the growth of large quantities of fermenters (microbes) in a vessel called a fermentor or a bioreactor for the production of commodity chemicals, biofuels, pharmaceuticals, enzymes etc.
Most industrial fermentations are aerobic, the process relies on scalability with fermentors often being 10,000 to 500,000 Litre capacity where the fermenters can be subject to harsh conditions.

Question 20

What’s the difference between batch, continuous and fed-batch fermentation?

Answer 20

a) Batch fermentation is where you add all the nutrients at the start, once they have been consumed, growth ceases and the fermentation has ended.
b) Continuous fermentation is essentially a large chemostat; culture is constantly added and removed
c) Fed-batch fermentation is where you provide nutrients in a batch culture medium, once consumed, a feed is initiated to provide the culture with additional nutrients allowing for further growth.

Question 21

Describe the process of fed-batch fermentation with Pencillium chrysogenum

Answer 21

1. Initial growth phase in a small fermentor inoculated with freeze-dried spores
2. Scaled-up via 2 successively larger fermentors to provide a large enough inoculum for the production phase.
3. Fermentation production phase is now a fed-batch process. High O2 levels and C/N levels are maintained.
4. Monitored to keep Penicillium in production phase for 120-200 hours
5. Penicillin is excreted into the medium & recovered at the end of the process.

Question 22

What is bioprospecting?

Answer 22

The search for organisms, enzymes and natural compounds with potential for commercial application. Typically occurs in extreme environments as extremophiles are more likely to be tolerant of harsh culturing environments.

Question 23

What is metagenomics?

Answer 23

The study of genes/genetic samples from environmental samples. Involves recovering nucleic acids, cloning them into BAC libraries, introducing BACs into E. coli and screening for phenotypes. Positive clones are then sequenced and analysed.

Question 24

What is gene-mining?

Answer 24

The process of identifying and isolating genes from environmental samples without having to culture the organism.

Question 25

List some methods used to increase product yield

Answer 25

1. Mutation & selection
2. Metabolic engineering/synthetic biology
3. Nutritional/physiological approaches
4. Optimising fermentation approaches

Question 26

How can metabolic engineering improve product yield?

Answer 26

1. Modify metabolic pathways to redirect existing metabolism to specific products
2. Enhancing precursor/cofactor supply to the pathway via engineering central metabolism
3. Engineering transport systems (substrate uptake, product secretion etc)
4. Increasing cellular tolerance to the product or substrate
5. Considering the regulatory effects such as product feedback inhibition
6. Decoupling growth from product formation

Question 27

Tell me about Corynebacterium glutamicum

Answer 27

It is an aerobic gram positive soil bacterium. It can grow on a simple salt-medium with glucose.
The genome has been fully sequenced, a wide range of tools are available for mutagenesis, cloning and transformation.
It secretes lysine as it cannot break it down.
Lysine production occurs via batch-fed fermentation in 500,000L fermentors. Produces 180g/L.

Question 28

Tell me about the elimination of allosteric feedback inhibition in C. glutamicum

Answer 28

Lysine and threonine inhibit aspartate kinase (LysC). C. glutamicum was cultured in the presence of Lysine analogue Aminoethyl-L-cysteine, bacteria was forced to mutate resistance to LysC inhibition to survive. Lysine no longer inhibits LysC.

Question 29

Tell me about promoter engineering in C. glutamicum

Answer 29

Promoter engineering was used to improve metabolic flux at the aspartate semi-aldehyde branch point.
DapA is the first lysine-specific synthesis enzyme. Point mutations in DapA’s promoter increased DapA expression promoting Lysine synthesis.
Led to a 1.3fold increase in Lysine production.

Question 30

Tell me about increasing cofactor supply in C. glutamicum

Answer 30

1 mol of lysine production requires 4 mols of NADPH. Increased production of lysine disrupts the redox balance leading to a decrease in cellular NADPH.
Transhydrogenase enzyme was overexpressed (pntAB gene), it catalyses the reaction: NADH + NADP+ ⇌ NAD+ + NADPH.
Led to a 1.2fold increase in lysine production.

Question 31

Tell me about the overproduction of LysE in C. glutamicum

Answer 31

LysE is a lysine exporter. It is a symport, secreting lysine with 2OH- groups.
In high concentrations, lysine is toxic to the cell.

Question 32

Tell me about vitamin B12 production in E. coli

Answer 32

E. coli does not normally produce vitamin B12. 28 genes were added to E.coli for de novo B12 synthesis. B12 aka cobalamin is a corrin ring with a central cobalt ion. It is only synthesized by prokaryotes and has a very complicated synthesis pathway. E. coli initially had a yield of 1-2µg/g which was pushed up to 530µg/g through many improvements

Question 33

How was vitamin B12 production increased in E. coli?

Answer 33

1. Optimised gene expression
2. Enhancing the uptake and chelation of cobalt
3. Increased metabolic flux to the uroporphyrinogen III starting substrate
4. Downregulation in competing heme and siroheme biosynthetic pathways
5. Optimised fermentation process