Organisms

Organisms

Industrial Microbiology

Organisms: Selection and

Improvement

Recap on Thursday’s 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 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 hyperproducing 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

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

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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.

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

Enzyme

Immediate or final product

Induction

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

Enzyme

Induction

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

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 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

Immediate or

Substrate

Enzyme subsequent product

Induction

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.

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

L,L DAP Meso DAP

L-lysine + CO2

 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

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

CONCERTED

CONTROL

Aspartate semi-aldehyde

Lysine

Homoserine

Methionine

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)

- glucose

Glucose added

+ glucose

+ lactose

Time (hr)

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

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

–80 o c to -196 o c

 Freeze onto cryobeads

(The Protect system)

 Agar slope cultures overlaid with mineral oil and stored at

–20 o c

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