Strain improvt25 (2)

BTE 302: MICROBIAL BIOTECHNOLOGY

Microbial Strain Improvement

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Strain improvement

Metabolite concentration produced by wild strains are usually too low for economic processes. That is why strain improve is needed.

Success of strain improvement depends greatly on the target product.

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Strain improvement

Simply raising the gene dose can increase the yield from products involving activity of one or few genes.

This is beneficial if the product is cell biomass or primary metabolites.

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Mutation

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Spontaneous mutation

Rate depends on growth condition of organisms

Between 10-10 to 10-5 per generation per gene

All mutant types are found although deletions are frequent

Not cost effective because of low frequency of mutation

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Induced mutation

Mutation frequency is significantly increased

10-5 to 10-3 for secondary metabolite producers

10-2 to 10-1 for auxotrophic mutants

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Genome mutation may cause: Change in no. of chromosomes

Chromosome mutation may change: Order of genes by deficiency, deletion,

inversion, duplication or translocation

Gene or point mutation

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Changes due to mutation

Less lethal and mutagenic effects than short wavelength UV

Exposure of cells in the presence of various dyes causes interaction of DNA with UV with greater rates which results in increased frequency of mutation

Effective activators are psoralen derivatives (e.g. 8-methoxypsoralen)

Mechanism of action: Biadduct formation between complementary strands which results in crosslinks

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Long wavelength UV

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Optimizing mutagenesis

Molecular mode of action of some mutagens is well known but what can never be predicted is:

Effect of mutagen on specific gene Effect of mutation on a complex process (e.g. biosynthesis of secondary metabolite)

Appearance of mutants depends on several factors Base sequence of gene Repair system of cell Gene activity

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Mode of action of mutagens

Base sequence of gene to be mutated:

Mutation are not evenly distributed

There are areas of high mutation frequency known as hot spots

Different mutagens cause hot spots at different sites in the genome

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Factors affecting appearance of mutants

Strains with partially defective repair mechanisms:

Organisms may be killed without having induced mutation

Specific mutagen may be ineffective

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Gene activity:

Become lost through mutation

Can be restored through a second mutation (suppressor mutation)

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Act in several different ways

Occurs in the same gene that already carries primary mutation (intragenic suppressors)

Compensated through exchange of amino acid or additional insertion or deletion which corrects primary mutation

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Suppressor mutation

Occurs in another gene (extragenic suppressor)

Compensate primary mutation at the level of translation by formation of mutant tRNA or ribosome

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Suppressor mutation

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Selection of mutants

Isolation of auxotroph32

By using certain blocked mutants, desired products such as amino acids and nucleotides may be formed via branching biosynthetic pathways.

The isolation of auxotrophs is done by plating of the mutagenized population on a complete agar medium, on which the biochemically deficient mutants can also grow.

Isolation of auxotroph33

The antibiotic resistance character can not only be used as a genetic marker, but mutants isolated may also have increased cell permeability or a protein synthesis, making them useful for industrial purposes.

By means of Lederberg’s well-known replica plating technique, the clones are transferred to minimal medium where the auxotrophic colonies cannot grow.

These mutants are picked up from the master plates and their defect is characterized.

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Isolation of auxotroph

Since in this method a large number havof plates must be

observed, various procedures e been developed to enrich for

auxotrophic mutants by removing or killing prototrophic

organisms.

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Isolation of auxotroph

After mutagenesis the spores of filamentous organisms (actinomycetes, fungi) are allowed to develop in a liquid minimal medium.

The developing micro colonies of prototrophs are then separated by filtration, leaving behind in the filtrate spores of auxotrophs, which have been unable to grow.

The filtrate is then plated and the resulting colonies are checked for auxotrophic

characteristics.

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Penicillin selection method Penicillin kills growing cells but not non-

growing cells. In this procedure, growing cells are selectively killed by antibiotic treatment, thus enriching for auxotrophs, which cannot grow on minimal medium.

Several inhibitors other than penicillin can also be used in this procedure: dihydrostreptomycin for Pseudomonas aeruginosa, nalidixic acid for Salmonella typhimurium.

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An enrichment procedure with sodium pentachlorophenolate makes use of the greater toxicity of this compound against germinating spores than against vegetative cells.

The method has been successfully applied with Penicillium chrysogenum, Streptomyces aureofaciens, S. olivaceus, and Bacillus subtilis.

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Enrichment method

By these methods, enrichments for auxotrophs of 10- to100-fold can be attained, thus increasing the probability of obtaining mutants.

However, it should be remembered

that the types of mutants present in the original population may be shifted; for instance, an increased proportion of proline auxotrophs has been found in E. coli after auxotroph enrichment.

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Enrichment method

Strain improvt25 (2)

Technology

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