industrial microbiology organisms: selection and improvement
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Industrial Microbiology
Organisms: Selection and Improvement
Recap on Thursday’s lectureLarge and Small Scale Processes Improving the Process- Titre, Yield and
VPPrimary and Secondary MetabolitesThe Necessity for Growth
Lecture 2The Organism and Mutants
Outline Properties of useful industrial microorganisms
Finding and selecting your microorganism
Improving the microorganism’s properties Conquering the cell’s control systems
Storing industrial micro-organisms – the culture collection
Properties of a Useful Industrial Microorganism It must Produce the product!
But yield and titre may need subsequent improvement. Get the product on the market first and then improve!
Grows fast and produces product in large scale culture. Resulting requirements for growth factors etc.
usually acceptable. Sometimes can only get biomass / product yield required in small scale due to aeration difficulties in larger fermenter.
Properties of a Useful Industrial Microorganism Compatibility with substrates.
May require subsequent modification of medium or organism e.g. v. low iron levels are required for citric acid production by Aspergillus.
Ease of genetic manipulation. Genome known. Gene transfer systems available.
Genetically stable. Safe….Bacillus anthricis? Well known industrially.
Could take genes for product formation and insert them into an industrial “workhorse” (Saccharomyces, Bacillus etc.).
Also Worth Considering: Yeasts and fungi can withstand higher initial
concentrations of carbon substrates especially sugars
Product tolerance…will acid build up kill the organism?
Product location – is product excreted? Excretion e.g. amylases
Can improve product tolerance(higher titres and yields). Easier purification (especially proteins). Essential for correct form of some recombinant products.
i.e. folding of protein Retention inside the cell e.g. B-glucosidase in
yeast Can assist product concentration.
Ease of microorganism/medium separation vis a vis viscosity or organism density (brewing)
Sources of Potential Industrial Microorganisms Culture collections.
Public e.g. NCCLS Private i.e. within industry
Existing processes often yield hyper-producing strains due to self mutation…these may appear different on plates.
The natural environment – Biodiscovery.
Biodiscovery To “strike it rich” try
environments that: Have high biodiversity Are extreme Are unexplored Encourage the
dominance of suitable organisms
Biodiscovery: DNA Route Collect isolates or go the
“DNA route”: Make total community DNA
extracts – can screen at this level or:
Put fragments (random or selected) into a suitable host.
Screen these recombinant organisms.
Artificial chromosomes (BACs and YACs) can carry whole pathways.
Screening Selecting the useful organisms/genes from a
vast number of possibilities during process development or improvement
Can operate at the cell or gene (DNA) level Make task easier by
Keeping initial assays simple or capable of high throughput
Eliminate the useless before working on the useful Get rid of duplicates (especially when working with
DNA)
Screening
Decreasing No. of
Isolates
Simple/High throughput assays
More complex studies. Medium/process optimisation, genetic stability etc.
High Throughput screening Use of cell sorters,
multiwell plates, DNA chips and robotics
System shown can handle 3,000-10,000 assays per day
www.degussa.com/en/innovations/ highlights_extremophile.html -
Strain Improvement Essential when setting up a new process or
maintaining the competitiveness of an existing one. Strive to improve growth or yield of the strains you use.
NoteOrganisms, medium and process will be discussed separately during this course, but they must always be considered TOGETHER when developing or improving an industrial process.
Improvement in Antibiotic Titre
Titre
Year
Obtaining improved strains Select from existing populations
Mutation using chemicals or radiation
“Classical” Genetics: conjugation, Transposon, transduction, etc.
Genetic Engineering….strain construction, plasmid vectors, temperature sensitive promoters, gene shuffling using cassettes etc.
Conquering Cell Control Systems
Cells normally have control mechanisms which avoids unnecessary production of enzymes and metabolic intermediates.
We must manipulate or destroy these to ensure overproduction of the desired enzyme.
Substrate EnzymeImmediate or final product
InductionInhibition/Repression stops or reduces enzyme activity
Induction
Enzyme is only produced in the presence of an inducer (usually the substrate).
Our strategy: Use constitutive mutants. Supply an inducer in the medium (discussed later).
Substrate EnzymeImmediate or subsequent product
InductionInhibition/Repression
Constitutive Mutants Produce an inducible enzyme in the absence
of its inducer thus the enzyme is never switched off. Lactose induces the Lac operon producing B-Gal. Glucose switches off the operon. In a constitutive mutant glucose never switches off B-Gal production.
Lactose ---------------------------> Glucose + Galactose ß-galactosidase
Enrich populations for constitutive mutants by:
Chemostat cultures where the enzyme substrate is the limiting nutrient (e.g. lactose)
The Chemostat
Enrich populations for constitutive mutants by:
Sequential batch cultures alternating use of the inducing substrate as a nutrient with use of an alternate nutrient. Example: sequential cultures of
Escherichia coli alternating lactose and glucose will enrich for mutants constitutive for beta galactosidase.
Finding Constitutive MutantsSelect constitutive isolates by their ability
to grow:
When the sole carbon source (e.g. Lactose) is a substrate for the enzyme but does not induce it. Enzyme is switched on in presence of both Lactose and Glucose
Inhibition/Repression
Build up of enzyme product (or another intermediate or end product further down the metabolic pathway): Switches off enzyme activity (inhibition). Switches off enzyme production (repression).
Our strategy: Avoid build-up of inhibitor/repressor. Find mutants lacking inhibition/repression control.
Substrate EnzymeImmediate or subsequent product
InductionInhibition/Repression
Avoiding Build-up of Inhibitors and RepressorsModifying pathways to avoid
inhibitor/repressor build-up. Simple pathway example: lysine production
by Aerobacter aerogenes. Branched pathway example: lysine
production by Corynebacteium glutamicum and effect of progressive and concretive inhibition
Simple Pathway: The Lysine Pathway in Aerobacter
aerogenes
In normal cells, feedback control stops the build up of lysine by acting at an early stage in the pathway
Glycerol L,L DAP Meso DAP L-lysine + CO2
Feedback Control
Lysine Production using Aerobacter aerogenesA dual fermentation is used:
Cultures of two different strains (A & B) are grown up separately and then added together in the presence of acetone which breaks down permeability barriers and allows the cell contents to mix.
Strain A
Cannot convert Meso DAP to l-lysineGrow in medium with plenty of glycerol
and limiting amounts of lysineLarge amounts of L,L and Meso DAP
build up
Glycerol L,L DAP Meso DAP L-lysine + CO2
Strain BThe normal wild type strain.Growth does not produce build up of
lysine or intermediates.Cells contain all pathway enzymes
including that missing in strain A.
What happens when the cultures are mixed:The mixture contains:
Large amounts of L,L and Meso DAP (from strain A).
The enzymes necessary for their conversion to lysine (from strain B).
The resultant is the production of large quantities of lysine.
Feedback control in branched pathways: Progressive and Concerted Control
Product levels at the end of branches control the pathway at a point before branching occurs.
Control Point
Feedback control in branched pathways
Controls can be complex, but fall into two broad groups: Control is progressive – build up of one end product
causes partial switch off – further switch off occurs if there is build up at the end of another branch and so on.
Control is concerted – no switch off unless products at the end of several branches build up – complete switch off then occurs.
The Lysine Pathway in Corynebacterium glutamicum
Aspartate
Aspartate semi-aldehyde
LysineHomoserine
Methionine Threonine
Isoleucine
CONCERTEDCONTROL
NOTE
No switch off occurs unless BOTH lysine and threonine build up
Lysine production using Corynebacterium glutamicum
Use a mutant that cannot convert aspartate semi-aldehyde to homoserine
Aspartate
Aspartate semi-aldehyde
LysineHomoserine
Methionine Threonine
Isoleucine
Lysine production using Corynebacterium glutamicumMedium must contain limited amount of
homoserineThreonine levels will remain low, so no
control will be exercised when high levels of lysine build up
Finding Mutants which do not recognise Inhibitors & Repressors Isolate mutants which have lost an enzyme
and then screen these mutants for revertants e.g. Isolate a Lactose-negative E. coli and then look for mutants that can use lactose.
Select strains which can grow in the presence of a compound very similar to a product or intermediary (an analogue) which: Mimics its control properties Is not metabolised e.g. IPTG (isopropyl-B-D-thiogalactoside) turns on
lactose operon but cannot be used as a substrate by B-galactosidase
Catabolite repressionWhen readily utilised carbon sources
are available to organisms catabolite repression may occur
May override induction mechanisms Whole pathways my be switched off
Catabolite Repression (Glucose Effect)
+ glucose
- glucose
Time (hr)
gala
ctos
idas
e
+ lactose
Glucose added
Avoiding Problems with Catabolite RepressionUse fed batch cultures (discussed later)
Use mutants which lack catabolite repression i.e. can grow in high levels of glucose and still express galactosidase
Your StrainsHow to Maintain them so they
do not mutate
The “In House” Culture Collection
Source material for R & D.
Strain preservation during screening and optimisation.
Starter cultures for production.
The “In House” Culture Collection
Isolates must remain. Uncontaminated. True to their known
characteristics, both qualitative and quantitative.
Starters must be provided in a suitable and active form.
The “In House” Culture CollectionTo avoid changes due to mutation and
selection: Avoid excessive growth and subcuture. Store strains in an inactive state.
Keep adequate backup stocks.Keep full records of characteristics and
validate strains periodically.
Some storage methods. Lyophilisation (freeze
dried stocks) Glycerol suspensions at
–80oc to -196oc Freeze onto cryobeads
(The Protect system) Agar slope cultures
overlaid with mineral oil and stored at –20oc
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