Industrial Microbiology

Organisms: Selection and

Improvement

Recap on Thursdays lecture

Large

and Small Scale Processes

Improving the Process- Titre, Yield and
VP
Primary and Secondary Metabolites
The Necessity for Growth

Lecture 2
The

Organism and Mutants

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

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 tolerancewill acid build up kill the
organism?
Product location is product excreted?

Excretion e.g. amylases

Retention inside the cell e.g. B-glucosidase in

yeast

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

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 hyperproducing strains due to self mutationthese

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
More complex studies.
Medium/process
optimisation, genetic
stability etc.

Decreasing No. of
Isolates

Simple/High
throughput assays

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/inno
vations/
highlights_extremophile.ht
ml -

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.

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

Substrate

Induction

Enzyme

Immediate or final
product

Inhibition/Repression
stops or reduces enzyme activity

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.

Induction
Substrate

Induction

Enzyme

Immediate or
subsequent
product

Inhibition/Repression

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).

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
-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 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

Inhibition/Repression
Substrate

Enzyme

Induction

Inhibition/Repression

Build up of enzyme product (or another

intermediate or end product further down the
metabolic pathway):

Immediate or
subsequent
product

Switches off enzyme activity (inhibition).

Switches off enzyme production (repression).

Our strategy:

Avoid build-up of inhibitor/repressor.

Find mutants lacking inhibition/repression control.

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

Simple Pathway: The Lysine

Pathway in Aerobacter
aerogenes
Glycerol

L,L DAP

Meso DAP

L-lysine + CO2

Feedback
Control

In

normal cells, feedback control stops

the build up of lysine by acting at an
early stage in the pathway

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.

Strain A
Glycerol

Cannot

L,L DAP

Meso DAP

L-lysine + CO2

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

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.

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

Lysine

Homoserine

Methionine
CONCERTED
CONTROL

Threonine
Isoleucine

NOTE

No

switch off occurs unless BOTH

lysine and threonine build up

Lysine production using

Corynebacterium glutamicum
Aspartate
Aspartate semi-aldehyde

Lysine

Homoserine
Methionine

Threonine
Isoleucine

Use

a mutant that cannot convert

aspartate semi-aldehyde to homoserine

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

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 repression
When

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)

galactosidase

- glucose

Glucose
added
+ glucose

Time (hr)
+ lactose

Avoiding Problems with

Catabolite Repression
Use
Use

fed batch cultures (discussed later)

mutants which lack catabolite

repression i.e. can grow in high levels of
glucose and still express galactosidase

Your Strains
How

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

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.

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