1. Industrial Microbiology
Organisms: Selection and Improvement

2. 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

3. Lecture 2
The Organism and Mutants

4. 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

5. 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.

6. 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.).

7. 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)

8. 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.

9. Biodiscovery To “strike it rich” try environments that:
Have high biodiversity
Are extreme
Are unexplored
Encourage the dominance of suitable organisms

10. 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.

11. 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)
                                                                

12. Screening
More complex studies. Medium/process optimisation, genetic stability etc.
Simple/High throughput assays
Decreasing No. of
Isolates

13. 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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14. 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.

15. Improvement in Antibiotic Titre
Year

16. 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.

17. 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.

18. Induction Immediate or subsequent Enzyme Substrate 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).

19. 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

20. Enrich populations for constitutive mutants by:
Chemostat cultures where the enzyme substrate is the limiting nutrient (e.g. lactose)

21. The Chemostat

22. 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.

23. 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

24. Inhibition/Repression
Substrate
Enzyme
Immediate or 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.

25. 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

26. 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

27. 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.

28. Strain A Cannot convert Meso DAP to l-lysine
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

29. 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.

30. 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.

31. 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

32. 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.

33. The Lysine Pathway in Corynebacterium glutamicum
Aspartate
Aspartate semi-aldehyde
Lysine
Homoserine
Methionine
Threonine
Isoleucine
CONCERTED
CONTROL

34. NOTE
No switch off occurs unless BOTH lysine and threonine build up

35. 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

36. 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

37. 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

38. 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

39. Catabolite Repression (Glucose Effect)
Time (hr)
galactosidase
+ lactose
Glucose added

40. 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

41. Your Strains
How to Maintain them so they do not mutate

42. The “In House” Culture Collection
Source material for R & D.
Strain preservation during screening and optimisation.
Starter cultures for production.

43. 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.

44. 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.

45. 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