7 - Products from Microbes Flashcards
1
Q
Examples of products from microbes
A
Beer, wine, vinegar, dairy
2
Q
Products from microbes
A
- May be originally from microbes themselves (e.g. antibiotics)
- Or not microbial in origin but are now used to produce them
3
Q
Biomanufacturing
A
Use of systems incorporating biological agents (such as microbes)
4
Q
Four major categories that microbes are involved in producing
A
- Industrial products
- Food additives
- Medical products
- Biofuels
5
Q
Industrial microbiology
A
- Processes where microbes are used in the production of important substances
- All stages of production must be optimised before start, then controlled during production
- If possible limit feedback inhibition (where accumulation of end product inhibits cycle)
6
Q
Variables that must be optimised in industrial microbiology
A
- Optimise production strain
- Optimise conditions (temperature, pH, aeration, trace elements and others)
- Optimise feed type
7
Q
Scale up
A
- Transfer of small scale technologies to large scale
- Conditions must be maintained when scaling up to ensure same end result
- Large scale production usually achieved via fermentation
8
Q
Fermentation in industrial microbiology
A
Mass culture of microbes
9
Q
Fermentation in physiology
A
Type of metabolism
10
Q
Submerged fermentation
A
- Culture is in contact with liquid
- Most common type
11
Q
Solid state fermentation
A
- Culture is on a surface
- e.g. cereal grains (rice, wheat), legume, seeds, straw etc
12
Q
Stirred fermenter
A
- Up to 100,000 L
- can be run under oxic or anoxic conditions
- May need foam control agents for high protein culture media
- Impellers assist with stirring, spargers are for air
- Sensors used to monitor
13
Q
Types of culture systems
A
- Continuous culture
- Batch culture
14
Q
Continuous culture
A
- Open system (new nutrients added at constant rate and spent medium removed)
- Known as continuous feed
- After equilibrium established, culture attains steady state
- Organisms can be maintained in logarithmic phase-
- Best for producing primary metabolites
- commonly achieved using a chemostat (to allow control of growth rate and cell density
15
Q
Batch culture
A
- Closed system (no new nutrients added)
- Will observe lag, log, stationary and death phases
- Best for producing secondary metabolites
16
Q
Primary metabolites
A
- Produced during exponential growth phase
- Compounds related to the
synthesis of microbial cells / growth
17
Q
Examples of primary metabolites
A
- Enzymes
- Amino acids
- Organic acids
- Vitamins
18
Q
Secondary metabolites
A
- Typically produced during stationary growth phase
- Produced when waste accumulates or nutrients
become limiting - Produced from primary metabolites
- Sometimes considered part of a microbial stress
response
19
Q
Examples of secondary metabolites
A
- Pigments
- Antibiotics
- Toxins
20
Q
Production strains
A
- Many microbes that produce useful compounds are originally from natural environments and don’t grow well under lab conditions
- Original strain can be modified to overproduce the compound, grow faster or grow using different substrates (called production strain)
21
Q
Methods of production strain optimisation
A
- Mutagenesis via chemicals, UV light or X rays
- Directed evolution
- Protoplast fusion
- Heterologous gene expression
- Metagenomics
- Synthetic biology
22
Q
Mutagenesis via chemicals, UV light or X rays
A
- Generates population with random mutations then screen mutants for desired outcome
- Also known as “brute force” mutagenesis
- Used before gene editing techniques were developed
23
Q
Direct evolution
A
- Genes of interest are targeted for mutagenesis
- Altered in vitro then cloned back into original strain or heterologous host
- Uses CRISPR/Cas
24
Q
Protoplast fusion
A
- Cell walls removed
- Protoplasts (cell without walls) co-incubated and protoplasts fuse
- Chromosomes of two cells combine within a single recombinant cell
- Recombinant cells grows new cell wall
- Organisms must be very closely related
25
Example of protoplast fusion
Two strains of fungus Acremonium chrysogenum combined for increased growth + increased production of cephalosporin
26
Heterologous gene expression
- Gene of interest is cloned from one organism into another (GOI not from host strain)
- Then transcribed and translated into protein
- BUT production of non-native proteins disrupts the energy balance (redox power) of the production cell
- Metabolic engineering used to balance and optimize metabolic activities
27
Example of heterologous gene expression
Human insulin produced by E.coli
28
Metagenomics (gene mining)
- Culture independent
- Collect DNA from environmental source
- Sequence DNA, compare to already sequenced genes
- Identify novel genes of interest
- Clone into vector
- Express in host
- Observe phenotype
29
Synthetic biology
- Use of genetic engineering to create novel biological systems from parts term biobricks
30
Biobricks
Promoters, enhancers, operators, riboswitches, regulatory proteins etc
31
Advantages of synthetic biology
- Can construct what you want rather than to try to find it in nature (and modify it)
- May not need metabolic engineering to optimise metabolism
- Mix and match regulatory systems for gene expression (e.g. bacterial strain seeks out cancer cells)
32
Disadvantages of synthetic biology
No instructions (lots of planning and trouble shooting required)
33
Antibiotics
- Secondary compounds (produced during stationary growth phase)
- May be used medically in same form as produced or modified (semi-synthetic)
34
Penicillin production
- Batch culture (conditions controlled to maximise production)
- Lactose used as a food source
- Nitrogen is controlled (low levels)
- Glucose and nitrogen feeding also used
- Depletion of carbon source
- Specific precursors may be added to encourage formation of penicillin variants:
35
Amino acids
- E.g. lysine, glutamic acid (used in food industry)
- Typically produced from regulatory mutants (over produced a specific amino acid)
- Fermentation conditions require low biotin
- Production strain is a biotin auxotroph
- Low biotin inhibits ODHC and increases membrane permeability
36
Glutamic acid
- Produced from Corynebacterium glutamicum mutants
- Can’t process α-ketoglutarate to succinyl CoA in TCA cycle
- Instead convert isocitrate to 2-oxoglutarate
- Use glyoxalate cycle: produces glutamate
37
Organic acids
- E.g. Citric, acetic, lactic acids (used as preservatives
- Citric acid may be produced from fungus Aspergillus niger (via submerged fermentation, primary product is an intermediate of TCA cycle)
- Only accumulated in specific fermentation conditions
38
Specific fermentation conditions to produce citric acid
- Limit trace elements manganese and iron (stops fungal growth at a specific point)
- Low pH 1.6 – 2.2
- High sugar concentrations (15-18%) increases activity of glycolytic pathway, TCA cycle and citrate
synthase activity
- Citric acid accumulated then excreted by stressed fungi
39
Enzymes
- Used in pharmaceutical, agriculture, food, textile
- Most are hydrolases (break down polymers like proteins)
40
Examples of enzymes
- Proteases (biggest category)
- Lipases
- Amylases (starch; glycogen)
- Taq polymerase
41
Proteases
- Used in food industry, cleaning (in laundry detergents), biofuels
- Many produced by Bacillus species
42
Lipases
Used in cleaning and waste treatment
43
Amylases
- Produced from bacteria or fungi
- Used in cleaning and food industries
44
Mammalian proteins
- Mammalian proteins are present in only low amounts in normal tissue
- Some can be produced in cell culture but sometimes expensive and difficult
- Instead, production in microbes is easy
- Insulin first human protein produced by bacteria
45
Somatotrophin (growth hormone)
- Recombinant bovine somatotropin stimulates milk production in lactating cows
- Two binding sites
- Recombinant human somatotropin used to treat human growth hormone deficiency (site-directed mutagenesis used to change gene to alter the amino acids that bind to prolactin receptor)
46
Two binding sites of somatotropin
- Somatotropin receptor (growth)
- Prolactin receptor (milk production)
47
Biofuels
- E.g. Ethanol and hydrogen
- Many different biofuels or biofuel precursors produced (broad range of organisms involved)
48
Two steps in ethanol production that involves microbes
- Enzymatic hydrolysis (lignocellulose breakdown)
- Fermentation
49
Enzymatic hydrolysis
- Cellulase, mannanase, xylanase, redox enzymes
- Enzymes cleave polysaccharides into simple sugars
50
Fermentation in ethanol production
- Sugars converted to (bio)ethanol
- Range of microbes used (e.g. Saccharomyces cerevisiae)
51
Why is lignocellulose and cellulose difficult for most organisms to digest
As they lack the enzymes
52
Feedstocks
- Enzyme hydrolysis step depends on what is being used as “feedstock”
- Potential to use 'waste products' as feedstock for making biofuels
53
Most common feedstocks for bioethanol
- Wheat
- Molasses
- Sorghum
- Barley
54
Microbial plastics (biopolymers)
- Bacteria produce storage polymers (PHAs - linear polyester molecules)
- Properties resemble xenobiotic plastics BUT they are readily biodegradable
- PHA + poly beta-hydroxyvalerate is most commercially successful microbial plastic
- Ralstonia eutropha is the model organism for PHA production (genetically manipulable and produces PHA in high yield)
55
Microbes as food
- Microbes as food known as “single-cell protein”
- May be from yeasts, filamentous fungi, bacteria, algae
- Theoretically more green than agriculture or animal production
- Do not require large tracts of land, less water and ‘fertiliser’
- Can use a range of ‘waste’ products for feedstock
56
Example of microbes as food
- Spirulina (cyanobacteria)
- Mycoprotein from Fusarium venenatum
