This document discusses strain improvement and preservation in biotechnology. It defines a strain as a group of species with distinguishing characteristics. The main approaches to strain improvement discussed are mutant selection, recombination, and recombinant DNA technology. Mutant selection involves applying mutagens to induce beneficial mutations for traits like increased productivity. Recombination generates new combinations of genes between strains. Recombinant DNA technology transfers genes to modify metabolic activities or products. Proper strain preservation methods are also outlined, including freezing, lyophilization, and storage in glycerol or liquid nitrogen. Applications include production of vaccines, enzymes, and other industrial biomolecules.

1. PRESENTED BY
SACHIN .B .H
STRAIN IMPROVEMENT AND PRESERVATION
Biotechnology Skill Enhancement Program (BiSEP)
Domain: Fermentation and Bioprocessing
Department of Biotechnology, GUK

2. CONTENTS
 Introduction
 Ideal Characteristics of Strain
 Purpose of Strain Improvement
 Approaches for Strain Improvement
1.Mutant Selection
2.Recombination
3.Recombinant DNA Technology
 Strain preservation
 Applications
 Conclusion
 Reference

3. INTRODUCTION
 Strain- A Strain is group of species with one or
more characteristics that distinguish it from other
sub group of the species of the strain .
-each strain identified by a name , number or
letter .
Ex:-E. coli Strain k 12
 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 that cultivation cost.
 Shorter fermentation time.

5. PURPOSE OF STRAIN IMPROVEMENT
 Increase the productivities.
 Regulating the activity of the enzymes.
 Increasing the permeability.
 To change un used co-Metabolites.
 Introducing new genetic properties into the organism by
Recombination DNA technology/genetic engineering.

6. APPROACHES FOR STRAIN IMPROVEMENT
 Mutant Selection.
 Recombination.
 Recombinant DNA Technology.

7. MUTANT SELECTION
 A MUTATION is a sudden Heritable change in the
traits of an organism
 Application of Mutagens to Induce mutation is
called MUTAGENSIS.
 Agents capable of inducing mutations are called
MUTAGENS
Physical- Particulate and Non-Particulate
Chemical-Base analog, Deamine and
Alkylating agents Acridine Dyes.
 Mutations 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 in Strain
Improvement.
Ex: Streptomycin griseus- Streptomycin-
Mannsidostreptomycin
Ex: Streptomycin aurofaciens(S- 604)-Produce 6-
demethyl tetracycline in place of Tetracycline
 In contrast, most improvement in biochemical production
have been due to the accumulation of so called Minor
genes.
Ex: Pencillium chrysogenum- Strain E15-1 was
obtained which yield 55% more than original strain.

10. ISOLATION OF MUTANTS
 1. Isolation of Auxtrophic Mutants:- it has a defect in one its
biosynthetic pathways ,so it require a specific Bio-molecule for
normal growth and development.
 Ex: Phenylalanine mutant of C.glutamicus –require Phe for growth
so, it accumulates Tyrosine.
 2.Analogue- Resistant Mutant:- it have feed back insensitive
enzymes of the biosynthetic pathway.
 Feed –back inhibition- Tyrosine mutant of C.glutamics were
selected for resistance to 50mg/L of p- flurophenylalanine
(analogue of phenylalanine).
 3.Revertants from non producing mutants:- of a Strain are high
producer. Mutant mutate back to original phenotype is called
Reversion and mutant is called Revertant.
 Ex: Reversion mutant Streptomyces viridifaciens showed
over 6- fold increase in chlortetracycline production over
the original strain.

11.  4. Selection of Resistance to antibiotics:-
produced by the organism itself may lead to increased yields.
Ex: Streptomyces aurefaciens mutants selected for
resistance to 200- 400mg/L chlortetracycline
showed for 4 fold increase in the production of
antibiotics.
 5 . Mutants with altered cell membrane
permeability- Show high production of some metabolites
 Ex: A mutantE.coli strain has defective Lysine transport; it
actively excretes L-Lysine into the medium to 5 times as high
concentration as that with it cells.
 6. Mutants have been selected to produce altered metaboliteds,
especially in case of Aminoglycoside antibiotic.
 Ex:Pseudomonas aurofaciens produces the antibiotic
Pyrrolnitrin:: a mutant of this organisms 4’- fluropyrrolnitrin.

12. RECOMINATION
 Defined as formation of new gene combinations among
those present in different strains.
 Recombination used for genetic analysis as well as
strain improvement
 To generate new products
 Recombination may be based on:-
-Cross over
-Transformation
-Conjugation
-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 antibiotics.

13. RECOMINATION 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 protein
 Metabolic Engineering

14.  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 transgene, which affect enzymatic
,transport and as Metabolic Engineering.
 Ex- Over production of the amino acid Isolucine in
C. glutamicum and Ethonal by E.coli.

15.  Product modification include the new enzymes which
modifies the products of existing biosynthetic
pathway
Ex: Conversion of Cephalosporin C into 7 – amino
cephalosporanic acid by D-amino acid oxdidase (in
A.chryosgenum).
 Completely new metabolite formation include in which
all the genes of a new pathway transferred.
 Ex: E.coli , transfer 2 genes for
polyhydroxybutyrate synthesis from Alcaligenes
eutrophus.
 Enhance growth include enhanced substrate utilization .
 Ex; E. coli , glutamatye dehydrogenase into
M.methylotrophus carbon conversion increased from
4% to 7%.

16. PROPER STRAIN USED IN INDUSTRY
GENETICALLY REGARDED AS SAFE [GRAS]
 BACTERIA - Bacillus subtillis
-Lactobacillus bulgaricus
-Lactococcus lactis
-Leuconostock oenos
 Yeasts - Canidia utilis
-Klyuveromyces m axrianus
-Klyveronomyces lactis
-Sacharomyces cerevesiae
 Filamentous fungi - Aspergillus niger
-Aspergillus oryazae
-Mucor javanicus
-Penicillium roqueforti

17. STRAIN PRESERVATION
 Industrial Microbiology or Industrial Biotechnology
continuously uses specific Microorganisms isolates /
strains as research , assay, development and production
of cultures .
 These strains are highly valuable and must be
preserved over long without genetic and phenotypic
change
- Research culture
-Assay culture
-Development culture
-Production culture

18. APPROACHES OF STRAIN PRESERVATION
 Low Temperature Storage:- 2-6⁰c ( 2-6 months)
 Storage as Frozen Culture:- -20 to -100⁰c.
 Storage as Lyophilized cells:- Under high Vacuum at low
temperature ( 5/ even -20 to -70⁰c)
 Storage of Vegetative cells/spores in Liquid Nitrogen:- -
196⁰c / -167⁰c .
 Air dried at room temperature on sterile loam sand or on
 other natural substrate:- Like maize seed, rice, bran, etc.,
 ( bacterial culture may remain viable up to 70-80 years)
 Storage in Glycerin Stabs:- 0.85ml of cell suspension
 mixed with0.15ml of sterile glycerol and stored at - 70 or -75⁰c.

19. APPLICATIONS
 Large scale Production of vaccines, Enzymes,
Interferons, growth factors, blood clotting factors.
 In the field of Microbiology improve the microbe’s
productivities or characteristics.
 Treatment of Genetic diseases like SCID by rDNA
technology
 Production of medically useful biological like insulin

20.  • Ex- Streptomyces albus was sequentially treated
with mutagen ( UV, NTG, Nitrogen mustard etc.,) to
form a strain
 SAM-X which produces 10mg/ml of salinomycin as
compared to 250μg/ml in original strain.
 •Ex- The enhancement of production of
Asperenone, an inhibitor of lipoxygenase and
human platelet aggregation from Aspergillus niger
, was achieved by UV and nitrous acid mutagenesis

21. CONCLUSION
 These steps have been taken by firms in order to gap the
bridge between basic knowledge and industrial application.
 The task of broth 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 cost 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
- genomic shuffling
 The choice of approaches which should be taken driven by
the economics of the biotechnological process and the genetic
tools available for the strain of interest.

22. REFERENCE
 A text book of industrial microbiology by J. wates
 A text book of Molecular Biology, Genetic Engineering
and Industrial Biotechnology by B.D Singh
 A text book of Biotechnology by V. kumaresan
(SaraS publication).