Industrial Microbiology

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Industrial Microbiology

• Organisms Selection and Improvement

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Recap on Thursdays lecture

• Large and Small Scale Processes
• Improving the Process- Titre, Yield and VP
• Primary and Secondary Metabolites
• The Necessity for Growth

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Lecture 2

• The Organism and Mutants

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Outline

• Properties of useful industrial microorganisms
• Finding and selecting your microorganism
• Improving the microorganisms properties
• Conquering the cells control systems
• Storing industrial micro-organisms the culture
  collection

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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.

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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.).

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Also Worth Considering

• Yeasts and fungi can withstand higher initial
  concentrations of carbon substrates especially
  sugars
• Product tolerancewill 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)

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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 mutationthese may appear
  different on plates.
• The natural environment Biodiscovery.

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Biodiscovery

• To strike it rich try environments that
• Have high biodiversity
• Are extreme
• Are unexplored
• Encourage the dominance of suitable organisms

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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.

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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)

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Screening
More complex studies. Medium/process
optimisation, genetic stability etc.
Simple/High throughput assays
Decreasing No. of Isolates
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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 -
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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.
• Note
• Organisms, medium and process will be discussed
  separately during this course, but they must
  always be considered TOGETHER when developing or
  improving an industrial process.

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Improvement in Antibiotic Titre
Titre
Year
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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.

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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.

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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).

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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 ---------------------------gt Glucose
Galactose ß-galactosidase
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Enrich populations for constitutive mutants by

• Chemostat cultures where the enzyme substrate is
  the limiting nutrient (e.g. lactose)

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The Chemostat
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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.

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Finding Constitutive Mutants

• Select 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

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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.

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Avoiding Build-up of Inhibitors and Repressors

• Modifying 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

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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

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Lysine Production using Aerobacter aerogenes

• A 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.

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Strain A

• Cannot convert Meso DAP to l-lysine
• Grow in medium with plenty of glycerol and
  limiting amounts of lysine
• Large amounts of L,L and Meso DAP build up

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Strain B

• The 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.

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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.

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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
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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.

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The Lysine Pathway in Corynebacterium glutamicum
CONCERTED CONTROL
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NOTE

• No switch off occurs unless BOTH lysine and
  threonine build up

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Lysine production using Corynebacterium glutamicum

• Use a mutant that cannot convert aspartate
  semi-aldehyde to homoserine

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Lysine production using Corynebacterium glutamicum

• Medium must contain limited amount of homoserine
• Threonine levels will remain low, so no control
  will be exercised when high levels of lysine
  build up

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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

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Catabolite repression

• When readily utilised carbon sources are
  available to organisms catabolite repression may
  occur
• May override induction mechanisms
• Whole pathways my be switched off

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Catabolite Repression (Glucose Effect)
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Avoiding Problems with Catabolite Repression

• Use fed batch cultures (discussed later)
• Use mutants which lack catabolite repression i.e.
  can grow in high levels of glucose and still
  express galactosidase

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Your Strains

• How to Maintain them so they do not mutate

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The In House Culture Collection

• Source material for R D.
• Strain preservation during screening and
  optimisation.
• Starter cultures for production.

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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.

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The In House Culture Collection

• To 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.

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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