STRAIN IMPROVEMENT

Techniques

Presented by
Ranjusha v.p
Assistant professor
 CONTENS

• Introduction
• Ideal Characteristics of Strain
• Purpose of Strain Improvement
• Approaches for Strain Improvement
1. Mutant Selection
2. Recombination
3. Recombinant DNA Technology
• Novel technologies
• Applications
• Conclusion
• Reference
 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
 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.
 Purpose of Strain
Improvement
 Increase the productivities
 Regulating the activity of the enzymes
 Introducing new genetic properties into the
organism by Recombinant DNA technology /
Genetic engineering.
Approaches for Strain
Improvement
 Mutant Selection
 Recombination
 Recombinant DNA
Technology
 MUTANT SELECTION
 A MUTATION 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.”
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
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.
 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.
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.
• Ames (1973) developed a method for deletion of mutagenicity
of carcinogens which is commonly known as Ames test.
• It is widely used to detect the carcinogens.
• The Ames 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.
• 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.
• A mammalian 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 DNA
molecule.
• 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.
 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.
Protoplast
fusion
 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
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
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
Novel genetic technologies

Novel genetic
tech.

Metabolic Genome shuffling

engineering
 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.
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.
 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
rDNA technology
 Production of medically useful biological
products like insulin
 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
DNA technology 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.
 REFERENCES
 http://www.yourarticlelibrary.com/micro-biology/strain-

improvement
 https://www.researchgate.net/.../226497441_Strain_impr
o 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
THANK YOU MY
STUDENTS