This document discusses techniques for strain improvement in microbiology. It begins by explaining why strain improvement is important to increase product yields from natural isolates. The main approaches discussed are natural variants, mutant selection, recombination, and recombinant DNA technology. Mutant selection involves using mutagens like chemicals or radiation to induce mutations and isolating improved mutants. Recombination involves combining genes from different strains. Recombinant DNA technology uses genetic engineering to modify microbial genomes and metabolic pathways to improve strains. The document provides examples of industrial applications and concludes that continued development of new genetic techniques like metabolic engineering and genome shuffling holds promise for further strain improvement.

1. STRAIN IMPROVEMENT Techniques

2. Introduction
Why strain improvement?
 Natural isolates usually produce commercially important
products in very low concentrations
 Increased yield may be achieved by optimizing the culture
medium and growth conditions
 Productivity of the organism is controlled by genome.
Therefore, the genome must be modified to increase the
product yield
 Cultural requirements of the modified organism would be
examined to provide suitable conditions to increase the yield.
 Strain improvement process involves continual genetic
modifications of the culture followed by reappraisals of its
culture requirements.

3. INTRODUCTION
Strain- A Strain is a group of species with one/ more
characteristics that distinguish it from other sub groups of
the same species of the strain. Each strain is identified by a
name, number or letter. Example:- E.coli Strain K12
Strain Improvement- The Science and Technology of
manipulating and improving microbial strains in order to
enhance their metabolic capacities is known as Strain
Improvement

4. Ideal Characteristics of Strain
⚫ Rapid growth
⚫ Genetic stability
⚫ Non-toxicity to humans
⚫ Ability to use cheaper substrates
⚫ Elimination of the production of compounds that may
interfere with downstream processing
⚫ To improve the use of carbon and nitrogen sources.
⚫ Reduction of cultivation cost
⚫ Shorter fermentation time.

5. Purpose of Strain Improvement
⚫ Increase the productivities
⚫ Regulating the activity of the enzymes
⚫ Introducing new genetic properties into the
organism by Recombinant DNAtechnology /
Genetic engineering.

6. Approaches for Strain Improvement
 Natural variants
 Mutant Selection
 Recombination
 Recombinant DNATechnology

7. Natural variants
 Generally a microbial culture will undergo a vast
number of divisions. There is a small probability of
genetic change occurring each time a cell divides.
 The microbial culture becomes heterogenous.
 The heterogeneity of some cultures can present serious
problems of yield degeneration because the variants are
the inferior producers compared with the original
culture.
 Therefore, periodically plate out the producing culture
and screen a proportion of the progeny for productivity.
Selection of natural variants may result in increased
yields.

8. MUTANT SELECTION
⚫ AMUTATION is a Sudden and Heritable change in the traits
of an organism.
⚫ Application of Mutagens to Induce mutation is called
MUTAGENESIS.
⚫ Agents capable of inducing mutations are called MUTGENS
 Chemical mutagens–Alkylating agents,Acridine Dyes, etc.
⚫ Mutation occurring without any specific treatment are called
“ Spontaneous Mutation.”
⚫ Mutation are resulting due to a treatment with certain agents
are known as “Induced Mutation.”

9. Many Mutations bring about marked changes in the
Biochemical Characters of practical interest these are called
Major Mutations – these can be used in Strain Improvement
Ex: Streptomyces griseus-Streptomycin-Mannosidostreptomycin
Ex: Streptomyces aurofaciens(S-604) –
Produce 6-demethyl tetracycline in place of Tetracycline
In contrast, most improvements in biochemical production
have been due to the Stepwise accumulation of so called
Minor genes.
Ex: Pencillium chrysogenum – Strain E15-1 was obtained which
yield 55% more penicillin than original strain

10. Reports on strain improvement by mutation-
• Karana and Medicherla (2006)- lipase from Aspergillus
japonicus MTCC 1975- mutation using UV, HNO2, NTG
showed 127%, 177%, 276% higher lipase yield than parent
strain respectively.
• First superior penicillin producing mutant, Penicillium
chrysogenum X-1612,was isolated after X ray mutagenesis.

11. Isolation of mutants
⚫ The following points highlight the four methods to detect and
isolate mutants. The methods are: 1. Replica Plating
Technique 2. Resistance Selection Method 3. Substrate
Utilization Method 4. Carcinogenicity Test.
1. Replica Plating Technique:
⚫ Lederberg and Lederberg (1952) have given replica plating
technique. This technique is used to detect auxotrophic
mutants which differentiates between mutants and wild type
strains on the basis of ability to grow in the absence of an
amino acid.

12. 2. Resistance Selection Method:
It is the other approach for isolation of mutants. Generally the
wild type cells are not resistant either to antibiotics or
bacteriophages. Therefore, it is possible to grow the bacterium
in the presence of the agent (antibiotics or bacteriophage) and
look for survivors.
3. Substrate Utilization Method:
This method is employed in the selection of bacteria. Several
bacteria utilize only a few primary carbon sources. The
cultures are plated onto medium containing an alternate carbon
sources.Any colony that grows on medium can use the substrate
and are possibly mutants. These can be isolated.
4. Carcinogenicity Test:
to identify the environmental carcionogens that cause mutation
and induce cancer in organisms. It’s based on detecting potential
of carcinogens and testing for mutagenicity in bacteria.

13. • Ames (1973) developed a method for deletion of mutagenicity
of carcinogens which is commonly known asAmes test.
• It is widely used to detect the carcinogens.
• TheAmes test is a mutational reversion assay in which several
special strains of Salmonella typhimurium are employed.
• Each strain contains a different mutation in the operon of
histidine biosynthesis.

14. • The number of spontaneous reverants is low, whereas the
number of reverants induced by the test mutagen is quite high.
• In order to estimate the relative mutagenicity of the mutagenic
substance the visible colonies are counted and compared with
control.
• The high number of colonies represents the greater
mutagenicity.
• Amammalian liver extract is added to the above molten top
agar before plating.
• The extract converts the carcinogens into electrophilic
derivatives which will soon react with DNAmolecule.
• The liver extract is added to this test, just to promote the
transformation.
• The Ames test has now been used with thousands of
substances and mixtures such as the industrial chemicals, food
additives, pesticides, hair dyes and cosmetics.

15. RECOMBINATION
⚫ Defined as formation of new gene combinations among
those present in different strains.
⚫ Recombination is used for both genetic analysis as well as
strain improvement
⚫ To generate new products
⚫ Recombination may be based on:-
- Transformation
- Conjugation
- others like cross over and transduction
-protoplast fusion – The fusion between non producing
strains of two species ( Streptomyces griseus and
Streptomyces tenjimariensis) has yielded a strain that
produces indolizomycin, a new Indolizine antibiotic.

18. Protoplast
fusion

19. RECOMBINATION DNA TECHNOLOGY
⚫ rDNA Technology or Genetic Engineering
involves the isolation and cloning of genes of
interest, production of the necessary gene
constructs using appropriate enzymes and then
transfer and expression of these genes into an
suitable host organism.
⚫ This technique has been used to achieve 2 broad
objectives:
- Production of Recombinant proteins
- Metabolic Engineering

20. 1. Recombinant proteins:- These are the proteins
produced by the transferred gene / transgene ; they
themselves are of commercial value.
Ex: Insulin, Interferons etc.. are produced in
Bacteria
2. Metabolic Engineering :- When metabolic
activities of an organism are modified by
introducing into it transgenes, which affect
enzymatic, transport and /or regulatory function of
its cells its known as Metabolic Engineering.
Ex: Over production of the amino acid Isoleucine
in Corynebacterium glutamicum & Ethanol by
E.coli .

21. Product Modification include the new enzymes which
modifies the product of existing biosynthetic pathway
e.g. Conversion of Cephalosporin C into 7- amino
cephalosporanic acid by D-amino acid oxidase
(in A. chrysogenum).
Completely new metabolite formation include in which
all the genes of a new pathway are transferred
e.g. E.coli, transfer of 2 genes for polyhydroxybutyrate
synthesis from Alcaligenes eutrophus.
 Enhance growth include enhanced substrate utilization.
e.g. E.coli , glutamate dehydrogenase into
M.methylotrophus carbon conversion increased from 4% to
7%

22. Novel genetic technologies
Novel genetic
tech.
Metabolic
engineering
Genome shuffling

23. Metabolic engineering-
⚫The existing pathways are modified, or entirely new ones are
introduced through the manipulation of the genes so as to
improve the yields of the microbial product, eliminate or reduce
undesirable side products or shift to the production of an entirely
new product.
⚫It has been used to over-produce the amino acid isoluecine in
Corynebacterium glutamicum, & ethanol by E. coli and has been
employed to introduce the gene for utilizing lactose into
Corynebacterium glutamicum thus making it possible for the
organism to utilize whey which is plentiful and cheap.

24. Genome Shuffling
⚫ It is a novel technique for strain improvement that
allows for recombination between multiple parents at
each generation and several rounds of recursive genome
fusion were carried out resulting in the final improved
strain involving genetic trait from multiple initial
strains.

26. APPLICATIONS
⚫Large scale Production of vaccines, Enzymes,
Interferon, growth factors, blood clotting factors.
⚫In the field of Microbiology to improve the
microbe’s productivities or characteristics.
⚫Treatment of Genetic diseases like SCID by
rDNAtechnology
⚫Production of medically useful biological
products like insulin

27. CONCLUSION
⚫ These steps have been taken by firms in order to gap the bridge
between basic knowledge and industrial application.
⚫ The task of both discovering new microbial compounds and
improving the synthesis of known ones have become more and
more challenging.
⚫ The tremendous increase in fermentation productivity and
resulting decreases in costs have come about mainly by using
mutagenesis. In recent years,recombinant DNAtechnology has
also been applied.
⚫ The promise of the future is via extensive of new genetic
techniques-Metabolic engineering and Genomic shuffling.
⚫ The choice of approaches which should be taken will be driven
by the economics of the biotechnological process and the genetic
tools available for the strain of interest.

28. REFERENCES
⚫ http://www.yourarticlelibrary.com/micro-biology/strain-
improvement
⚫ https://www.researchgate.net/.../226497441_Strain_impro
vement
⚫ https://www.jic.ac.uk/.../Marinelli%20Lecture%202%20pa
rt%201.pdf
⚫ http://technologyinscience.blogspot.in/2012/08/strain-
improvement-importance-of-pure.html#
⚫ http://www.cabri.org/guidelines/micro-
organisms/M300.html
⚫ A text book of Molecular Biology, Genetic Engineering
and Industrial Biotechnology by B.D Singh

29. Presented to,
Prof.Prasanna Srinivas
Dept. of Microbiology
RCASC