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
Q
Example of protoplast fusion
A
Two strains of fungus Acremonium chrysogenum combined for increased growth + increased production of cephalosporin
26
Q
Heterologous gene expression
A
- 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
Q
Example of heterologous gene expression
A
Human insulin produced by E.coli
28
Q
Metagenomics (gene mining)
A
- 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
Q
Synthetic biology
A
- Use of genetic engineering to create novel biological systems from parts term biobricks
30
Q
Biobricks
A
Promoters, enhancers, operators, riboswitches, regulatory proteins etc
31
Q
Advantages of synthetic biology
A
- 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
Q
Disadvantages of synthetic biology
A
No instructions (lots of planning and trouble shooting required)
33
Q
Antibiotics
A
- Secondary compounds (produced during stationary growth phase)
- May be used medically in same form as produced or modified (semi-synthetic)
34
Q
Penicillin production
A
- 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
Q
Amino acids
A
- 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
Q
Glutamic acid
A
- 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
Q
Organic acids
A
- 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
Q
Specific fermentation conditions to produce citric acid
A
- 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
Q
Enzymes
A
- Used in pharmaceutical, agriculture, food, textile
- Most are hydrolases (break down polymers like proteins)
40
Q
Examples of enzymes
A
- Proteases (biggest category)
- Lipases
- Amylases (starch; glycogen)
- Taq polymerase
41
Q
Proteases
A
- Used in food industry, cleaning (in laundry detergents), biofuels
- Many produced by Bacillus species
42
Q
Lipases
A
Used in cleaning and waste treatment
43
Q
Amylases
A
- Produced from bacteria or fungi
- Used in cleaning and food industries
44
Q
Mammalian proteins
A
- 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
Q
Somatotrophin (growth hormone)
A
- 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
Q
Two binding sites of somatotropin
A
- Somatotropin receptor (growth)
- Prolactin receptor (milk production)
47
Q
Biofuels
A
- E.g. Ethanol and hydrogen
- Many different biofuels or biofuel precursors produced (broad range of organisms involved)
48
Q
Two steps in ethanol production that involves microbes
A
- Enzymatic hydrolysis (lignocellulose breakdown)
- Fermentation
49
Q
Enzymatic hydrolysis
A
- Cellulase, mannanase, xylanase, redox enzymes
- Enzymes cleave polysaccharides into simple sugars
50
Q
Fermentation in ethanol production
A
- Sugars converted to (bio)ethanol
- Range of microbes used (e.g. Saccharomyces cerevisiae)
51
Q
Why is lignocellulose and cellulose difficult for most organisms to digest
A
As they lack the enzymes
52
Q
Feedstocks
A
- Enzyme hydrolysis step depends on what is being used as “feedstock”
- Potential to use ‘waste products’ as feedstock for making biofuels
53
Q
Most common feedstocks for bioethanol
A
- Wheat
- Molasses
- Sorghum
- Barley
54
Q
Microbial plastics (biopolymers)
A
- 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
Q
Microbes as food
A
- 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
Q
Example of microbes as food
A
- Spirulina (cyanobacteria)
- Mycoprotein from Fusarium venenatum
