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Microbiology 2 > Microbial cultures and metabolic engineering > Flashcards

Microbial cultures and metabolic engineering Flashcards

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1
Q

what is growth?

A

Growth is the coordinated synthesis of macromolecules
- macromolecular synthesis leads to cell division by binary fission and an increase in cell numbers

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2
Q

what is growth rate?

A

the change in cell number/cell mass per unit time

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3
Q

how can microbial growth be measured?

A
  1. cell dry weight
  2. cell count
  3. optical density
  4. measure a specific cell component e.g. protein, chlorophyll
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4
Q

what is the cell dry weight measurement of microbial growth?

A
  1. take known volume of cell culture
  2. pellet cells by centrifugation
  3. wash cells
  4. dry the pellet
  5. weigh the pellet
  6. subtract the weight of the tube from the weight of the pellet

limitations: time consuming, no indication of cell viability (live/dead cells)

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5
Q

what is the cell count measurement of microbial growth?

A
  1. Total count - count both live and dead cells
  2. Viable count - counts live cells
    - use serial dilutions (10^-6), culture on agar plate and count no. colonies and then back-calculate no. live cells
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6
Q

what is the optical density measurements of microbial growth?

A
  • light scatter shows cell growth
  • 595/600/610nm typically used
  • requires a standard curve to interpret
  • optical density is proportional to cell number
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7
Q

what is microbial batch culture?

A
  • culture of a fixed volume in a flask/culture vessel
  • closed system: no more culture is added during growth and no media is removed so culture volume remains the same
  • culture is inoculated and incubated in favourable conditions e.g. 37C
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8
Q

how do microbes in batch culture divide?

A

cells divide by binary fission, leading to exponential growth
- cell number doubles in each division
- 1 generation is a group of cells that has divided and doubled
- the time to form generations is known as generation time (g) or doubling time (tD)
- generation time varies between organisms and different culture environments

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9
Q

what is the generation time of E. coli?

A

E. coli divide every 20 minutes
- after 24 hours, would give 2^72 cells

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10
Q

is unrestrained growth in batch culture possible?

A

no, as it is a closed system:
1. an essential nutrient will eventually be depleted, as no new media is added, so they will get used up
2. metabolism leads to an accumulation of end products, leading to autoinhibition of growth, as media isn’t removed and replaced
- growth can cease due to a change in culture pH
- build-up of metabolic toxins e.g. lactic acid

eventually reaches stationary phase

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11
Q

what are the 4 main phases in the bacterial growth curve?

A
  1. Lag phase: bacteria prepare cell machinery for growth
    - this time period can vary: older cultures or cells from rich media that are placed in minimal media. cells need to adjust metabolism so lag phase may be longer
  2. Log phase: growth approximates an exponential curve due to division by binary fission
    - rapid increase in population - cells grow at max rate in these conditions
    - generation time depends on factors such as media richness, temp, osmolarity, pH
  3. stationary phase - cells stop growing and shut down their growth machinery due to autoinhibition
    - cells remain metabolically active and stress responses are switched on to retain viability
    - cell division is cancelled out by cell death
  4. death phase - no input of new nutrients leads to decrease in cell number
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12
Q

what are the growth kinetics in batch culture?

A

the increase in cell number during exponential growth is a geometric progression of the number 2
- e.g. 1 cell doubles to become 2, 2 cells become 4, 4 cells become 8

starting with any number of cells (N0), the number after n generations will be N0 x 2^n
e.g. starting with 1 cell (N0 = 1), after 4 generations, there will be 1x2^4 = 16 cells

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13
Q

what are the limitations of batch culture?

A
  • growth curve is a laboratory artefact
  • cells grow at their max rate in batch cultivation due to nutrients being provided in excess, so physiology cannot be studied at submaximal growth rates under nutrient-limited conditions
  • difficult to compare between 2 samples as the properties of cells continuously vary throughout the growth curve due to change in medium composition e.g. pH, oxygen tension, excretion products. samples must be taken at the exact same time in log phase, at the same optical density to compare in similar environments
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14
Q

what is continuous culture?

A

Fresh medium is continuously (chemostats) or periodically (turbidostats) added to the culture
- rate of addition of fresh media is matched to the growth rate of the culture

An equal volume of spent culture (including cells) is removed
- rate of dilution is matched to the rate at which the population doubles

allows precise control of the conditions that the cells are growing in

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15
Q

what does continuous culture involve?

A
  • addition of substrates/nutrients for growth
  • removal of autoinhibitory products by an overflow device
  • bacterial population is maintained in exponential phase at a constant cell density: adding/removing media matched to growth rate
  • growth rate and cell density can be controlled independently
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16
Q

why is continuous culture desirable?

A
  • it allows reproducible cultivation of microorganisms at submaximal growth rates at different growth limitations (chemostats)
  • culture conditions remain constant, in a steady state (dynamic equilibrium), over extended periods of time
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17
Q

what are common properties of turbidostats and chemostats?

A
  • fresh media is added and old volume leaves, so culture volume is constant
  • must be well mixed for uniform aeration and distribution of cells and nutrients
  • pH and temperature kept constant
  • silanizing agents and antifoam chemicals ensure the surface of the culture is hydrophobic to prevent microbes growing on them - stable

everything is kept in steady state so that the growth of organisms can be studied under tightly controlled physiochemical conditions

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18
Q

what is the chemostat culture?

A
  • fresh medium is supplied at constant rate (flow rate constant), spent culture is removed at the same constant rate so culture volume is constant
  • defined growth medium is used
  • medium contains a limiting conc of one essential nutrient - the growth rate of culture has to adjust to the supply of this limiting substrate until steady state is reached (prevents growth at max rate)
  • control of growth rate over wide submaximal range
  • control growth rate and cell density independently of one another, so can keep flow rate constant but increase substrate conc, so growth rate stays constant but steady state biomass increases
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19
Q

what is the process of chemostat culture?

A
  1. initially growth rate > dilution rate, so cell no. increases
  2. As cell number increases, conc of substrate decreases, so
    growth rate is < dilution rate and cell no. decreases
    - there is a reduction in the amount of growth-limiting substrate
    - cells are lost in overflow device quicker than they grow
  3. addition of new media reaches equilibrium so growth rate = dilution rate and cell no. is constant
    - this is the steady state
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20
Q

what are the key principles of chemostat culture?

A
  • once steady state is established at given dilution rate, the specific growth rate (mu), cell density and limiting-substrate conc in culture are constant
  • enables a fixed doubling time
  • cells removed from the vessel dilution = increase in cell number due to growth supported by input of limiting nutrient
  • by varying the dilution rate (rate at which limiting nutrient is added to culture), growth rate can be varied whilst keeping cell density the same

enables a range of steady states with submaximal growth rates without changing cell density

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21
Q

How can steady state cell density be increased?

A

Alternatively, can increase the steady state cell density without changing the dilution/growth rate by increasing the concentration of the limiting substrate in the influent media – would cause an increase in bacterial concentration

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22
Q

why does the steady state chemostat model break down at low dilution rates?

A

At low dilution rates, bacterial concentration is decreased due to the requirement of maintenance energy:
- Maintenance energy is the use of the growth-limiting substrate for essential cellular functions other than growth e.g. maintaining PMF, motility
- At low dilution/growth rates the percentage of the total consumed substrate used for cell maintenance compared to that used for growth is more significant (assumes maintenance energy independent of growth rate)
- substrate is used for other processes rather than growth, so cell density decreases

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23
Q

why does the steady state chemostat model break down at high dilution rates?

A

When the dilution rate is increased above the maximum specific growth rate, cells will quickly “washout” of the chemostat quicker than they can divide, leaving you with just media:
- Cells cannot grow any faster even with the limiting nutrient no longer being limiting and bacterial concentration cannot be maintained
- Bacterial concentration decreases and limiting nutrient increases to the concentration in the input media
- media volume is too high

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24
Q

what is turbidostat culture?

A

a continuous culture with a growth-dependent feedback system, in which the dilution rate is controlled by monitoring cell density (turbidity)
- Turbidity is maintained at a constant set level thus the population density (cell number/cell mass) is constant
- Feedback is between the density (turbidity) of the culture and the dilution rate (the rate at which fresh media is added)
- no growth limiting substrate so bacteria grow at max rate

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25
Q

what is the process of turbidostat culture?

A
  • Spectrophotometer constantly detects turbidity - any increase above desired value and the pumping rate is adjusted to add fresh medium into the culture vessel (i.e., the dilution rate is variable)
  • Fresh medium is only added in response to increase in cell density to dilute the culture
  • Simple overflow device keeps culture volume constant (i.e. when fresh media added the same volume of spent media/cells is removed)
  • there is no growth limiting substrate so cells grow at max rate at constant population density under constant conditions
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26
Q

what are the differences between chemostat culture and turbidostat culture?

A

chemostat:
- fresh medium supplied at constant rate and spent culture removed at constant rate
- influent medium contains a limiting conc of one essential substrate
- growth rate is kept submaximal
- fixed volume, fixed flow rate, fixed dilution rate

turbidostat:
- turbidity is monitored and if it is too high, only then is fresh media added and spent media removed (not constant rate)
- there is no growth-limiting substrate
- growth rate is at its max
- dilution rate varies

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27
Q

what is industrial microbiology?

A

the large-scale production of commercial products by microorganisms
- using microorganisms to produce a compound they already make
- engineer organisms and optimise growth conditions to enhance the process
- products are low in value but high in yield

28
Q

what is microbial biotechnology?

A

microbes are genetically engineered to produce non-native compounds e.g. insulin, somatotropin)
- biotechnological products are typically high value and are produced in lower yields

29
Q

give examples of microbial products:

A
  1. Antibiotics e.g. penicillin (Pencillium chrysogenum), tetracycline (Streptomyces spp.)
  2. Enzymes e.g. lipases (Candida cylindraceae), amylases (Bacillus subtilis), lactase (Kluyveromyces lactis)
  3. Food additives e.g. vitamins (e.g. riboflavin; Ashbya gossypii, Bacillus subtilis), amino acids (Corynebacterium glutamicum)
  4. Chemicals e.g. citric acid (Aspergillus niger), bioethanol (Saccharomyces cerevisiae), butanediols (E. coli)
  5. Terpenes e.g. artemisinin (Saccharomyces cerevisiae), carotenoids
  6. Alcoholic drinks e.g. beer, wine, spirits (yeasts)
30
Q

what are useful properties of industrial microbes?

A
  • they produce substance of interest in high yield
  • they grow rapidly and reproducibly to produce product in short period
  • grow and make product on large scale in bioreactors
  • secrete product straight into media
  • they are robust
  • grow in simple and inexpensive media without complex nutritional requirements
  • metabolic flexibility/adaptability
  • do not produce toxic byproducts and non-pathogenic
  • can be genetically engineered and are genetically stable
  • can be stocked/stored
31
Q

what is the definition of fermentation in industrial settings?

A

In industrial settings, fermentation refers to growth of large quantities of cells (the fermenters) within a vessel called a fermentor (or bioreactor) for production of commodity chemicals, biofuels, pharmaceuticals, enzymes, etc
- This is the case whether or not the microbial process is biochemically a fermentation - most industrial fermentations are actually aerobic

32
Q

what is scaling-up?

A

how to get a lab culture of a few litres to an industrial scale where fermentors can be 10,000-500,000 litres

Lab 1-10L
industrial fermentor 300-3000L
commercial fermentor 10,000-500,000L

33
Q

in what 3 ways can industrial fermentations be carried out?

A
  1. batch fermentations
  2. continuous fermentations
  3. fed-batch fermentations - typical method
34
Q

what is batch fermentations in microbial industry?

A

In batch fermentations, all of the nutrients required for the fermentation are provided in the initial culture medium.
- Once these nutrients have been consumed, growth of the organism ceases and the fermentation is ended.

35
Q

what is continuous fermentations in microbial industry?

A
  • Continuous fermentations are performed by continually supplying fresh medium to the culture with the subsequent removal of the same amount of culture, resulting in a steady state being reached in the fermentor.
36
Q

what is fed-batch fermentations in microbial industry?

A

In fed-batch fermentations, nutrients are provided in the batch culture medium for growth phase to build up the biomass.
- Once consumed, a feed is initiated to provide the culture with additional nutrients and thus allow for further growth of the culture.
- Typically, industrial fermentations are fed-batch fermentations

37
Q

Give an example of a fed-batch fermentation:

A

Penicillium chrysogenum fed-batch fermentation for penicillin production - carried out by 300,000L bioreactors

38
Q

what is the process of P. chrysogenum fed-batch fermentation for penicillin?

A
  1. Initial growth phase in a small fermenter inoculated with freeze-dried spores
    - Lactose is used for carbon source
    - Cells grow exponentially
    - Not making much penicillin
  2. Scaled up through two further growth stages in successively larger fermenters to provide a large enough inoculum for the production phase

3.The fermentation production phase is a fed-batch culture with high oxygen levels maintained and C and N feeding
- Once lactose has ran out and biomass has increased, culture is fed with glucose and a source of nitrogen to continue growth of biomass
- More penicillin is produced

  1. Carefully monitored to keep the fermentation in optimal penicillin production mode during production phase, which lasts for 120 to 200 hours
  2. Penicillin is excreted into the medium and is recovered at the end of the fermentation
39
Q

what criteria does an organism have to meet in order to be used industrially?

A
  • the organism must naturally produce the product
  • the organism should be appropriate for use in commercial processes
40
Q

if an organism is not suitable for commercial processes, how can the product be alternatively made?

A

if the organism is not commercially appropriate, an alternative pathway to make the product should be used via metabolic engineering and synthetic biology
- genes should be introduced to the host organism so that it is appropriate for use in commercial processes

41
Q

how can product yield of commercial organisms be improved?

A
  1. mutagen and selection e.g. UV mutagenesis
    - repeated rounds of mutation can cause 1000-fold increase in yield of product e.g. penicillin
  2. metabolic engineering/synthetic biology - if the pathway is simple
  3. nutritional/physiological approaches - growth conditions, pH, aeration
    - multi-omics approaches
  4. optimising fermentation conditions - type of bioreactor used, how it is scaled up
42
Q

what is bioprospecting?

A

Bioprospecting is the search for organisms, enzymes or natural products with potential commercial applications
- Often search for extremophiles which could survive in commercial conditions

43
Q

what is metagenomics? what is the process?

A

Metagenomics is the study of genes/genetic material from environmental samples (‘the genome of an environment’)

Process:
1. Recover nucleic acid from an environment e.g. soil
2. make library of DNA in bacterial artificial chromosome (BAC),
3. introduce BAC into a bacterium,
4. let colony form
5. screen library for reactive colonies - can then analyse and sequence positive clones

44
Q

what is gene mining?

A

Gene mining is the process of identifying and isolating genes from environmental samples without having to culture to the organism

45
Q

what is metabolic engineering?

A

Metabolic engineering is the deliberate redesign of cellular biochemical pathways to enhance production of a desired product or to produce a novel product
- Minimise production of products we don’t want while preventing damage to the host

46
Q

what are the 6 ways in which metabolic engineering is performed?

A
  1. modifying the metabolic pathway to redirect the metabolism to form specific products
  2. enhancing the precursor and energy/cofactor supply by engineering the central metabolism
  3. engineering transport systems
  4. increasing cellular tolerance to product or substrate
  5. consideration of regulatory effects such as product feedback inhibiton
  6. decoupling of growth and product formation via fed-batch culture
47
Q

how can metabolic engineering occur by modifying the metabolic pathway of the organism?

A
  • increase the yield of the desired product by boosting enzymes involved in the reaction
  • identifying branch points which lead to the formation of the products, and enhancing the activity of these branch points
48
Q

how can metabolic engineering occur by increasing energy/cofactor supply?

A
  • engineering the central metabolism to regenerate cofactors such as NADH and ATP
  • or could use gene mining to introduce enzymes to the metabolism that do not require these cofactors
49
Q

how can metabolic engineering occur by altering transport systems?

A
  • transport systems are ways to take things up into the cell
  • increasing the substrate concentration given to the cell will require a greater number of uptake transporters
  • exporter proteins are also essential to allow the product to leave the cell into the media
50
Q

how can metabolic engineering occur through increasing cellular tolerance to the substrate/product?

A
  • substrates/products at too high concs may be toxic to the organism
  • can use omics to analyse how the host changes its transcription/translation in response to high concs of substrates or products
  • can then engineer genes to help deal with these stresses
51
Q

how may considering product feedback inhibition aid metabolic engineering?

A
  • some byproducts of reactions may feedback to early enzymes in the pathway to inhibit overproduction of molecules and overuse of cofactors, and therefore slow production of the desired product
  • by inhibiting these feedback inhibitors, more of the product can be produced
52
Q

are large improvements required in metabolic engineering?

A

no - small improvements to the metabolic pathway of the organism can make a big difference to the economics of the process

53
Q

which bacteria is involved in Lysine production?

A

Corynebacterium glutamicum
- it is an aerobic, gram-positive soil bacterium
- it can grow on simple mineral salt medium with glucose
- its genome is fully sequenced, it can be mutagenised, cloned etc
- used for industrial synthesis of amino acids such as glutamine, lysine
- it cannot breakdown lysine and excretes it

Lysine production on an industrial scale by fed-batch fermentation (500,000 litre fermentors, 180 g L-1 lysine)

54
Q

how was C. glutamicum metabolically engineered to improve lysine production?

A
  1. elimination of allosteric feedback inhibition using anti-metabolites
  2. promoter engineering to improve metabolic flux at the aspartate semi-aldehyde branch point
  3. increasing cofactor supply by overproduction of transhydrogenase
  4. increasing lysine secretion by overproduction of lysine exporter LysE
55
Q

how did elimination of allosteric feedback inhibition improve C. glutamicum’s production of lysine?

A

Lys and Thr have an allosteric feedback inhibition on LysC (aspartate kinase)
- LysC is an ATP-requiring enzyme, if LysC is inhibited, less lysine is made - - The anti-metabolite aminoethyl-L-cysteine allosterically inhibits LysC

Used to select feedback-resistant LysC mutants which could be used to engineer organism with LysC enzyme that is no longer under control of feedback inhibition, helped to produce 36g/L lysine

56
Q

what is an anti-metabolite?

A

Anti-metabolite is a substrate analogue that cannot be broken down by organism

57
Q

how did promoter engineering at the aspartate semi-aldehyde branch point improve C. glutamicum’s production of lysine?

A

DapA is the first specific lysine biosynthesis enzyme at the branch point at aspartate semi-aldehyde
- point mutations in the dapA promoter resulted in increased expression of dapA and thus increased its activity
- resulted in a 1.3-fold increase in lysine production

58
Q

how did increasing cofactor supply by overproduction of transhydrogenase improve C. glutamicum’s production of lysine?

A

Synthesis of 1 mol L-lysine from oxaloacetate requires 4 mols of NADPH
- Need regeneration of NADPH to keep pathway active
- Transhydrogenase (PntAB) reaction: NADH + NADP+ ⇌ NADPH + NAD+
- Transhydrogenase interconverts NADH and NADPH
- Cellular NADH pool greater than cellular NADPH pool
- Overexpression of pntAB transhydrogenase increases NADPH levels

Resulted in a 1.2-fold increase in lysine production

59
Q

How did increasing lysine secretion by overproduction of exporter LysE improve C. glutamicum’s production of lysine?

A

Lysine is toxic to the cell at high concentration - C. glutamicum has a lysine exporter (LysE)
- Lysine is positively charged at neutral pH (pKa of ε-amino group = 10.28) - active export requires either symport with two hydroxyl ions or antiport with two protons

Overproduction of LysE can increase lysine excretion but this can be harmful to the cell membrane – have to use controlled over-expression of lysE gene to achieve the right balance

60
Q

what is vitamin B12?

A
  • Vitamin B12 (cobalamin) is one of the eight B vitamins and plays an essential role as a coenzyme in animals
  • A very complicated molecule - corrin ring with a central cobalt ion held in place by upper and lower ligand
  • Related to heme
  • Only synthesised by some prokaryotes - humans must acquire it through their diet (animal products)
61
Q

what does vitamin b12 deficiency lead to?

A

Deficiency in vitamin B12 is common amongst vegetarians and vegans - severe deficiency can lead to pernicious anaemia

62
Q

what does biosynthesis of vitamin b12 require?

A

around 30 enzymes:
- Pathway branches from the universal tetrapyrrole precursor uroporphyrinogen III - requires around 25 extra enzymes to convert it to vitamin B12
- By comparison only need 4 enzymes to make heme from uroporphyrinogen III
- E. coli does not naturally produce vitamin B12

63
Q

which organisms naturally produce vitamin B12?

A

Industrial fermentation by Pseudomonas denitrificans (produces 214 mg/L) and Propionibacterium freudenreichii (produces 206 mg/L)

But these strains grow slowly and are difficult to engineer - not suitable for metabolic engineering

64
Q

how was E. coli engineered to produce vitamin B12?

A

28 genes added to E. coli – strain made 1-2 µg/g dry cell weight
- 4 genetic modules added to make small amounts of vitamin B12
- Metabolic engineering and improvements to the fermentation process achieved 530 μg/g DCW (~1 mg/L)
- This is a 250-fold increase - it took ten years to improve yield 100-fold in P. denitrificans by rounds of random mutation
- Yield very low but is produced in quicker time (E. coli fermentation 24 hrs compared to 180 hrs in P. denitrificans)

65
Q

how did scientists optimise the production of vitamin B12 in E. coli?

A
  1. Optimised expression of genes
  2. Enhanced the uptake and chelation of cobalt
  3. Increased metabolic flux to the uroporphyrinogen III starting
    substrate
  4. Downregulated the competing heme and siroheme biosynthesis
    pathways
  5. Optimised the fermentation process

all of the above whilst maintaining optimal growth of E. coli

Microbiology 2 (10 decks)

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