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

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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.

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