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     STRAIN IMPROVEMENT




                          1
INDE
                               X
•Introduction
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•Process involved
    Selection of natural variants
    Selection of induced mutants
    The use of recombination systems
•Important characteristics for strain improvement
    The selection of stable strains
    The selection of strains resistant to infection
    The selection of non-foaming strains
    The Selection of strains which are resistant to components in the
    medium
    The selection of morphologically favourable strains
    The selection of strains which are tolerant of low oxygen tension
    The elimination of undesirable Products from a production strain
    The development of strains producing New fermentation products
•Conclusion
•Reference


                                                                    2
Introductio
n:
Strain: A strain is a subgroup of a species with one or more
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characteristics that distinguish it from other subgroups of
the same species. Each strain is identified by a name, number,
or letter.

For example: E. coli strain K12, E. coli strain 0157:H7
              [Ref: Jacquelyn G. Black (pg no. 242), Tortora (Pg. no. 18)]


Strain improvement:
The science and technology of manipulating and improving
microbial strains, in order to enhance their metabolic
capacities is known as Strain Improvement.
                                             [Ref:
                                             www.indianscience.in]


                                                                       3
Process      of                   strain
improvement:
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                                 Selection of
               Selection of
                                   induced
             natural variants
                                   mutants

                           Use of
                        recombinant
                         technology

                                [Ref: Stanbury, Principles of fermentation
                                                                technology



                                                                       4
Selection        of       natural
variants
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Keywords:
    Genetic changes
    Cell division
    Variants
    Mycelial organisms
    Heterokaryons
    Homokaryons




                               [Ref: Stanbury, Principles of fermentation
                                                               technology



                                                                      5
Selection of induced mutants
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• The selection of induced mutants synthesizing
improved levels of primary metabolites:
The levels of primary metabolites in micro-organisms are
regulated by Feedback control systems.

The major systems involved are feedback            inhibition and
feedback repression.



                              [Ref: Stanbury, Principles of fermentation
                                                              technology



                                                                     6
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            FIG.1.1     The control of a biosynthetic pathway
            converting precursor A to end product E via the
            intermediates B, C and D.

                                    [Ref: Stanbury, Principles of fermentation
                                                                    technology


                                                                           7
Concerted
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             or   multivalent      feedback
control:




      FIG.1.2 The control of a biosynthetic pathway by the
      concerted effects of products D and F on the first
      enzyme of the pathway.
                               [Ref: Stanbury, Principles of fermentation
                                                               technology



                                                                      8
Co-operative feedback control
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 FIG. 1.3 The control of a biosynthetic pathway by the co-
 operative control by end products D and F.

                               [Ref: Stanbury, Principles of fermentation
                                                               technology



                                                                      9
Cumulative feedback control
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--- 50% ----         Inhibition of 50% of the activity of the enzyme
------        Total inhibition of enzyme activity
FIG.1.4 The control of a biosynthetic pathway by the cumulative control of
products D and F.
                                              [Ref: Stanbury, Principles of fermentation
                                                                              technology


                                                                                     10
Sequential feedback control
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  FIG.1.5 The control of a biosynthetic pathway by sequential
  feedback control.
                                [Ref: Stanbury, Principles of fermentation
                                                                technology


                                                                       11
Isoenzyme control
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    Fig.1.6 The control of two isoenzymes (catalysing the conversion of A to
    B) by end products D and F.

                                        [Ref: Stanbury, Principles of fermentation
                                                                        technology



                                                                               12
•The isolation of mutants which do not produce
feedback inhibitors or repressors:
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 Fig.1.7
 Overproduction
 of       primary
 metabolites by
 decreasing the
 concentration
 of a repressing
 or     inhibiting
 end product.




                                             13
•Examples of the use of auxotrophs for the
production of primary metabolites:
  Your logo




  FIG. 1.8. The control of
  the aspartate family of
  amino acids in C.
  glutamicum.




                                        14
The use of recombination systems for the
improvement of industrial micro-organisms
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• Recombinant DNA techniques – In simple words, rDNA
technique can be explained as, bringing together in one
organism, genes from several organisms, has the potential for
not only increasing yields but also for producing entirely new
substances.

• Recombinant DNA technology has resulted in organisms
producing compounds which they were not able to produce
previously.

                                [Ref: Stanbury, Principles of fermentation
                                                                technology]



                                                                        15
The logo
 Your
      application of the parasexual cycle

•Many industrially important fungi do not possess a sexual
stage and therefore it would appear difficult to achieve
recombination in these organisms.

•However, Pontecorvo et al. (1953) demonstrated that
nuclear fusion and gene segregation could take place
outside, or in the absence of, the sexual organs.

•The process was termed the parasexual cycle and has
been demonstrated in the imperfect fungi, A. niger and P.
chrysogenum, as well as the sexual fungus A. nidulans.
                              [Ref: Stanbury, Principles of fermentation
                                                              technology]


                                                                      16
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FIG. 1.9. Diagrammatic representation of the mitotic division of a
eukaryotic cell containing two chromosomes. The nuclear membrane
has not been portrayed in the figure.
                                  [Ref: Stanbury, Principles of fermentation
                                                                  technology]


                                                                          17
•Mitotic crossing over involves the exchange of distal
segments between chromatids of homologous chromosomes
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shown in Fig. 1.10.




 FIG. 1.10.
 Diagrammatic
 representation of
 mitosis including mitotic
 crossing over.




                                                    18
• Haploidization is a process which results in the equal
distribution of chromatids between the progeny of a mitosis.
  Your logo
Fig. 1.11




 FIG. 1.11. Diagrammatic representation of mitosis involving
 haploidization.


                                                               19
The application of protoplast fusion
techniques are cells devoid of their cell walls and may be
  Your logo
• Protoplasts
prepared by subjecting cells to the action of wall degrading
enzymes in isotonic solutions. Protoplasts may regenerate
their cell walls and are then capable of growth as normal cells.

• Protoplast fusion has been demonstrated in a large number
of industrially important organisms including Streptomyces
spp. (Hopwood et al., 1977), Bacillus spp. (Fodor and Alfoldi,
1976)



                                 [Ref: Stanbury, Principles of fermentation
                                                                 technology]



                                                                         20
The improvement of industrial strains:
  Your logo

       Although a strain may produce a very high level of a
metabolite it would be unsuitable for a commercial process if
its productivity were extremely unstable, or if the organism's
oxygen demand were such that it could not be satisfied in the
industrial fermenter available for the process.

      Therefore, characteristics of the producing organism
which affect the process may be critical to its commercial
success. Thus, it may be desirable to modify such
characteristics of the producing organism which may be
achieved by selecting natural and induced variants and
recombinants.                  [Ref: Stanbury, Principles of fermentation
                                                                technology]



                                                                        21
Important characteristics for strain
• The selection of stable strains
improvement
  Your logo
• The selection of strains resistant to infection
• The selection of non-foaming strains
• The Selection of strains which are resistant to components
in the    medium
• The selection of morphologically favourable strains
• The selection of strains which are tolerant of low oxygen
tension
• The elimination of undesirable Products from a production
strain
• The development of strains producing New fermentation
products

                                [Ref: Stanbury, Principles of fermentation
                                                                technology]


                                                                        22
The logoConclusion:
 Your
      selection of stable strains:

•The ability of the producing strain to maintain its high
productivity duringgenetic and molecular genetic methods a
      A number of both culture maintenance and
fermentation is a very important fermentation product yields
      are available to improve quality.
      and other strain characteristics. The methods used for
Example: improvement of Johnson (1970)effective yet double
      the Woodruff and the strain are selected a a bit
auxotrophic mutant of Micrococcus glutamicus requiring both
      complicated too. The main reason of using such
homoserine and threonine and compared its lysine-producing
properties with thoseattainhomoserine auxotroph. strai that
      methods is to of a an improved and stable
      can be used at industrial or commercial level.


                               [Ref: Stanbury, Principles of fermentation
                                                               technology]



                                                                       23
Thelogo
 Your
      selection of strains resistant to infection:

•Bacterial fermentations may be affected very seriously by
phage infections, which may result in the lysis of the bacteria.

•A possible method for reducing of failure due to phage
contamination is to select bacterial strains which are resistant
to the phages isolated in the fermentation plant (Hongo et
al., 1972)




                                 [Ref: Stanbury, Principles of fermentation
                                                                 technology]



                                                                         24
•The selection of non-foaming strains:
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• Foaming during a fermentation may result in the loss of
broth cells and product via the air outlet as well as putting the
fermentation at risk from contamination.

• Thus, foaming is normally controlled either by the chemical
or mechanical means, but this task may be made easier if a
non-foaming strain of the commercial organism can be
developed.



                                  [Ref: Stanbury, Principles of fermentation
                                                                  technology]



                                                                          25
The selection of strains which are resistant to
components in the medium:
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Polya and Nyiri (1966) applied this approach to the isolation
of mutants of P. chrysogenum resistant to phenylacetic acid, a
precursor of penicillin and toxic to the organism at high
concentrations.




                                 [Ref: Stanbury, Principles of fermentation
                                                                 technology]



                                                                         26
The logo
 Your
      selection of morphologically favorable strains:

Backus and Stauffer (1955) recognized the influence of the
genetic of a strain on the morphology of P. chrysogenum in
submerged culture and its role in controlling foaming and
broth filtration characteristics




                               [Ref: Stanbury, Principles of fermentation
                                                               technology]



                                                                       27
The selection of strains which are tolerant of low
oxygen tension:
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Example, Mindlin and Zaitseva (1966) isolated a lysine-
producing strain which maintained its productivity under
aeration conditions which decreased the parental strain
productivity by almost a half.




                             [Ref: Stanbury, Principles of fermentation
                                                             technology]



                                                                     28
The elimination of undesirable products from a
production strain:
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• Athough an industrial micro-organism may produce large
quantities of a desirable metabolite it may also produce a large
amount of a metabolite which is not required, is toxic or may
interfere with the extraction process.

• An example in the penicillin-producing strains is the
elimination of the production of the yellow pigment,
chrysogenein, selection of non-pigmented mutants which made
the extraction of the antibiotic much simpler (Backus
Stauffer, 1955).
                                 [Ref: Stanbury, Principles of fermentation
                                                                 technology]



                                                                         29
The logo development of
 Your
                               strains         producing            new
fermentation products:

• The isolation of organisms from the natural environment
synthesizing commercially useful metabolites an expensive and
laborious process.
• Therefore, means of producing novel compounds which may
be some industrial significance have been attempted.




                                [Ref: Stanbury, Principles of fermentation
                                                                technology]



                                                                        30
Conclusion
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A number of genetic and molecular genetic methods are
available to improve fermentation product yields and other
strain characteristics. The methods used for              the
improvement of the strain are effective yet a bit complicated
too. The main reason of using such methods is to attain an
improved and stable strain that can be used at industrial or
commercial level.




                                                           31
Reference
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• Stanbury F. Peter , 2003, Strain Improvement, Principles of
fermentation technology, Great Britain by MPG Books Ltd, Bodmin,
Cornwall, second Edition, Pg. 43-82.
•Tortora J. Gerard, 2010, Strain, Pearson Benjamin Cummings, San
Francisco, USA, tenth edition, Pg. no. 18
•Prescott M. Lansing, 2007, Strain, The McGraw-Hill Companies, Inc.,
New York,
America, seventh edition, Pg. no. 425
•Black G. Jacquelyn, 2008, Strain, JohnWiley & Sons, Inc., pg no.
242

• Net   Source:
       www.cheric.org
       www.springerlink.com
       www.jhu.edu
       www.indianscience.in

                                                                  32

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Strain Improvement

  • 1. Your logo STRAIN IMPROVEMENT 1
  • 2. INDE X •Introduction Your logo •Process involved Selection of natural variants Selection of induced mutants The use of recombination systems •Important characteristics for strain improvement The selection of stable strains The selection of strains resistant to infection The selection of non-foaming strains The Selection of strains which are resistant to components in the medium The selection of morphologically favourable strains The selection of strains which are tolerant of low oxygen tension The elimination of undesirable Products from a production strain The development of strains producing New fermentation products •Conclusion •Reference 2
  • 3. Introductio n: Strain: A strain is a subgroup of a species with one or more Your logo characteristics that distinguish it from other subgroups of the same species. Each strain is identified by a name, number, or letter. For example: E. coli strain K12, E. coli strain 0157:H7 [Ref: Jacquelyn G. Black (pg no. 242), Tortora (Pg. no. 18)] Strain improvement: The science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities is known as Strain Improvement. [Ref: www.indianscience.in] 3
  • 4. Process of strain improvement: Your logo Selection of Selection of induced natural variants mutants Use of recombinant technology [Ref: Stanbury, Principles of fermentation technology 4
  • 5. Selection of natural variants Your logo Keywords:  Genetic changes  Cell division  Variants  Mycelial organisms  Heterokaryons  Homokaryons [Ref: Stanbury, Principles of fermentation technology 5
  • 6. Selection of induced mutants Your logo • The selection of induced mutants synthesizing improved levels of primary metabolites: The levels of primary metabolites in micro-organisms are regulated by Feedback control systems. The major systems involved are feedback inhibition and feedback repression. [Ref: Stanbury, Principles of fermentation technology 6
  • 7. Your logo FIG.1.1 The control of a biosynthetic pathway converting precursor A to end product E via the intermediates B, C and D. [Ref: Stanbury, Principles of fermentation technology 7
  • 8. Concerted Your logo or multivalent feedback control: FIG.1.2 The control of a biosynthetic pathway by the concerted effects of products D and F on the first enzyme of the pathway. [Ref: Stanbury, Principles of fermentation technology 8
  • 9. Co-operative feedback control Your logo FIG. 1.3 The control of a biosynthetic pathway by the co- operative control by end products D and F. [Ref: Stanbury, Principles of fermentation technology 9
  • 10. Cumulative feedback control Your logo --- 50% ---- Inhibition of 50% of the activity of the enzyme ------ Total inhibition of enzyme activity FIG.1.4 The control of a biosynthetic pathway by the cumulative control of products D and F. [Ref: Stanbury, Principles of fermentation technology 10
  • 11. Sequential feedback control Your logo FIG.1.5 The control of a biosynthetic pathway by sequential feedback control. [Ref: Stanbury, Principles of fermentation technology 11
  • 12. Isoenzyme control Your logo Fig.1.6 The control of two isoenzymes (catalysing the conversion of A to B) by end products D and F. [Ref: Stanbury, Principles of fermentation technology 12
  • 13. •The isolation of mutants which do not produce feedback inhibitors or repressors: Your logo Fig.1.7 Overproduction of primary metabolites by decreasing the concentration of a repressing or inhibiting end product. 13
  • 14. •Examples of the use of auxotrophs for the production of primary metabolites: Your logo FIG. 1.8. The control of the aspartate family of amino acids in C. glutamicum. 14
  • 15. The use of recombination systems for the improvement of industrial micro-organisms Your logo • Recombinant DNA techniques – In simple words, rDNA technique can be explained as, bringing together in one organism, genes from several organisms, has the potential for not only increasing yields but also for producing entirely new substances. • Recombinant DNA technology has resulted in organisms producing compounds which they were not able to produce previously. [Ref: Stanbury, Principles of fermentation technology] 15
  • 16. The logo Your application of the parasexual cycle •Many industrially important fungi do not possess a sexual stage and therefore it would appear difficult to achieve recombination in these organisms. •However, Pontecorvo et al. (1953) demonstrated that nuclear fusion and gene segregation could take place outside, or in the absence of, the sexual organs. •The process was termed the parasexual cycle and has been demonstrated in the imperfect fungi, A. niger and P. chrysogenum, as well as the sexual fungus A. nidulans. [Ref: Stanbury, Principles of fermentation technology] 16
  • 17. Your logo FIG. 1.9. Diagrammatic representation of the mitotic division of a eukaryotic cell containing two chromosomes. The nuclear membrane has not been portrayed in the figure. [Ref: Stanbury, Principles of fermentation technology] 17
  • 18. •Mitotic crossing over involves the exchange of distal segments between chromatids of homologous chromosomes Your logo shown in Fig. 1.10. FIG. 1.10. Diagrammatic representation of mitosis including mitotic crossing over. 18
  • 19. • Haploidization is a process which results in the equal distribution of chromatids between the progeny of a mitosis. Your logo Fig. 1.11 FIG. 1.11. Diagrammatic representation of mitosis involving haploidization. 19
  • 20. The application of protoplast fusion techniques are cells devoid of their cell walls and may be Your logo • Protoplasts prepared by subjecting cells to the action of wall degrading enzymes in isotonic solutions. Protoplasts may regenerate their cell walls and are then capable of growth as normal cells. • Protoplast fusion has been demonstrated in a large number of industrially important organisms including Streptomyces spp. (Hopwood et al., 1977), Bacillus spp. (Fodor and Alfoldi, 1976) [Ref: Stanbury, Principles of fermentation technology] 20
  • 21. The improvement of industrial strains: Your logo Although a strain may produce a very high level of a metabolite it would be unsuitable for a commercial process if its productivity were extremely unstable, or if the organism's oxygen demand were such that it could not be satisfied in the industrial fermenter available for the process. Therefore, characteristics of the producing organism which affect the process may be critical to its commercial success. Thus, it may be desirable to modify such characteristics of the producing organism which may be achieved by selecting natural and induced variants and recombinants. [Ref: Stanbury, Principles of fermentation technology] 21
  • 22. Important characteristics for strain • The selection of stable strains improvement Your logo • The selection of strains resistant to infection • The selection of non-foaming strains • The Selection of strains which are resistant to components in the medium • The selection of morphologically favourable strains • The selection of strains which are tolerant of low oxygen tension • The elimination of undesirable Products from a production strain • The development of strains producing New fermentation products [Ref: Stanbury, Principles of fermentation technology] 22
  • 23. The logoConclusion: Your selection of stable strains: •The ability of the producing strain to maintain its high productivity duringgenetic and molecular genetic methods a A number of both culture maintenance and fermentation is a very important fermentation product yields are available to improve quality. and other strain characteristics. The methods used for Example: improvement of Johnson (1970)effective yet double the Woodruff and the strain are selected a a bit auxotrophic mutant of Micrococcus glutamicus requiring both complicated too. The main reason of using such homoserine and threonine and compared its lysine-producing properties with thoseattainhomoserine auxotroph. strai that methods is to of a an improved and stable can be used at industrial or commercial level. [Ref: Stanbury, Principles of fermentation technology] 23
  • 24. Thelogo Your selection of strains resistant to infection: •Bacterial fermentations may be affected very seriously by phage infections, which may result in the lysis of the bacteria. •A possible method for reducing of failure due to phage contamination is to select bacterial strains which are resistant to the phages isolated in the fermentation plant (Hongo et al., 1972) [Ref: Stanbury, Principles of fermentation technology] 24
  • 25. •The selection of non-foaming strains: Your logo • Foaming during a fermentation may result in the loss of broth cells and product via the air outlet as well as putting the fermentation at risk from contamination. • Thus, foaming is normally controlled either by the chemical or mechanical means, but this task may be made easier if a non-foaming strain of the commercial organism can be developed. [Ref: Stanbury, Principles of fermentation technology] 25
  • 26. The selection of strains which are resistant to components in the medium: Your logo Polya and Nyiri (1966) applied this approach to the isolation of mutants of P. chrysogenum resistant to phenylacetic acid, a precursor of penicillin and toxic to the organism at high concentrations. [Ref: Stanbury, Principles of fermentation technology] 26
  • 27. The logo Your selection of morphologically favorable strains: Backus and Stauffer (1955) recognized the influence of the genetic of a strain on the morphology of P. chrysogenum in submerged culture and its role in controlling foaming and broth filtration characteristics [Ref: Stanbury, Principles of fermentation technology] 27
  • 28. The selection of strains which are tolerant of low oxygen tension: Your logo Example, Mindlin and Zaitseva (1966) isolated a lysine- producing strain which maintained its productivity under aeration conditions which decreased the parental strain productivity by almost a half. [Ref: Stanbury, Principles of fermentation technology] 28
  • 29. The elimination of undesirable products from a production strain: Your logo • Athough an industrial micro-organism may produce large quantities of a desirable metabolite it may also produce a large amount of a metabolite which is not required, is toxic or may interfere with the extraction process. • An example in the penicillin-producing strains is the elimination of the production of the yellow pigment, chrysogenein, selection of non-pigmented mutants which made the extraction of the antibiotic much simpler (Backus Stauffer, 1955). [Ref: Stanbury, Principles of fermentation technology] 29
  • 30. The logo development of Your strains producing new fermentation products: • The isolation of organisms from the natural environment synthesizing commercially useful metabolites an expensive and laborious process. • Therefore, means of producing novel compounds which may be some industrial significance have been attempted. [Ref: Stanbury, Principles of fermentation technology] 30
  • 31. Conclusion Your logo A number of genetic and molecular genetic methods are available to improve fermentation product yields and other strain characteristics. The methods used for the improvement of the strain are effective yet a bit complicated too. The main reason of using such methods is to attain an improved and stable strain that can be used at industrial or commercial level. 31
  • 32. Reference Your logo • Stanbury F. Peter , 2003, Strain Improvement, Principles of fermentation technology, Great Britain by MPG Books Ltd, Bodmin, Cornwall, second Edition, Pg. 43-82. •Tortora J. Gerard, 2010, Strain, Pearson Benjamin Cummings, San Francisco, USA, tenth edition, Pg. no. 18 •Prescott M. Lansing, 2007, Strain, The McGraw-Hill Companies, Inc., New York, America, seventh edition, Pg. no. 425 •Black G. Jacquelyn, 2008, Strain, JohnWiley & Sons, Inc., pg no. 242 • Net Source:  www.cheric.org  www.springerlink.com  www.jhu.edu  www.indianscience.in 32