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MICR3310 > 7 - Products from Microbes > Flashcards

7 - Products from Microbes Flashcards

1
Q

Examples of products from microbes

A

Beer, wine, vinegar, dairy

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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
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3
Q

Biomanufacturing

A

Use of systems incorporating biological agents (such as microbes)

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4
Q

Four major categories that microbes are involved in producing

A
  • Industrial products
  • Food additives
  • Medical products
  • Biofuels
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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)
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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
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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
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8
Q

Fermentation in industrial microbiology

A

Mass culture of microbes

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9
Q

Fermentation in physiology

A

Type of metabolism

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10
Q

Submerged fermentation

A
  • Culture is in contact with liquid
  • Most common type
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11
Q

Solid state fermentation

A
  • Culture is on a surface
  • e.g. cereal grains (rice, wheat), legume, seeds, straw etc
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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
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13
Q

Types of culture systems

A
  • Continuous culture
  • Batch culture
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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
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15
Q

Batch culture

A
  • Closed system (no new nutrients added)
  • Will observe lag, log, stationary and death phases
  • Best for producing secondary metabolites
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16
Q

Primary metabolites

A
  • Produced during exponential growth phase
  • Compounds related to the
    synthesis of microbial cells / growth
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17
Q

Examples of primary metabolites

A
  • Enzymes
  • Amino acids
  • Organic acids
  • Vitamins
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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
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19
Q

Examples of secondary metabolites

A
  • Pigments
  • Antibiotics
  • Toxins
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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)
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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
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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
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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

MICR3310 (28 decks)

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