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Strain improvt25 (2)

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Strain improvt25 (2)

  1. 1. BTE 302: MICROBIAL BIOTECHNOLOGY Microbial Strain Improvement 1
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  6. 6. 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. 6
  7. 7. 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. 7
  8. 8. Mutation 8
  9. 9. 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 9
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  18. 18. Induced mutation  Mutation frequency is significantly increased  10-5 to 10-3 for secondary metabolite producers  10-2 to 10-1 for auxotrophic mutants 18
  19. 19.  Genome mutation may cause:  Change in no. of chromosomes  Chromosome mutation may change:  Order of genes by deficiency, deletion, inversion, dup lication or translocation  Gene or point mutation 19 Changes due to mutation
  20. 20.  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 20 Long wavelength UV
  21. 21. 21 Optimizing mutagenesis
  22. 22.  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 22 Mode of action of mutagens
  23. 23.  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 23 Factors affecting appearance of mutants
  24. 24.  Strains with partially defective repair mechanisms:  Organisms may be killed without having induced mutation  Specific mutagen may be ineffective 24 Factors affecting appearance of mutants
  25. 25.  Gene activity:  Become lost through mutation  Can be restored through a second mutation (suppressor mutation) 25 Factors affecting appearance of mutants
  26. 26.  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 26 Suppressor mutation
  27. 27.  Occurs in another gene (extragenic suppressor)  Compensate primary mutation at the level of translation by formation of mutant tRNA or ribosome 27 Suppressor mutation
  28. 28. 28 Selection of mutants
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  32. 32. Isolation of auxotroph 32  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.
  33. 33. Isolation of auxotroph 33  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.
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  35. 35.  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. 35
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  37. 37. 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. 37
  38. 38. 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. 38
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  40. 40. 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. 40
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  57. 57.  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. 57 Enrichment method
  58. 58.  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. 58 Enrichment method

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