This document discusses microbial strain improvement techniques. It describes how raising gene dose can increase yields for products involving one or few genes, benefiting biomass or primary metabolite production. Mutation techniques like spontaneous and induced mutation are explored, noting their mutation rates and limitations. Selection methods for auxotroph mutants are covered, including replica plating, penicillin selection, and enrichment procedures to isolate mutants unable to grow in minimal medium. Overall, the document provides an overview of genetic engineering methods for enhancing microbial production of metabolites.

1. BTE 302: MICROBIAL BIOTECHNOLOGY
Microbial Strain Improvement
1

2. 2

3. 3

4. 4

5. 5

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. 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. Mutation
8

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

10. 10

11. 11

12. 12

13. 13

14. 14

15. 15

16. 16

17. 17

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.  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.  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
Optimizing mutagenesis

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.  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.  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.  Gene activity:
 Become lost through mutation
 Can be restored through a second
mutation (suppressor mutation)
25
Factors affecting appearance of
mutants

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.  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
Selection of mutants

29. 29

30. 30

31. 31

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. 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.

34. 34

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

36. 36

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. 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

39. 39

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

41. 41

42. 42

43. 43

44. 44

45. 45

46. 46

47. 47

48. 48

49. 49

50. 50

51. 51

52. 52

53. 53

54. 54

55. 55

56. 56

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.  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