Strain improvement involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of strain improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Traditional methods of strain improvement include mutagenesis, classical genetics, and rational selection. Modern techniques utilize genetic engineering and recombinant DNA technology to introduce desired mutations for traits like increased thermostability or altered substrate range.

2. Strain improvement
The Science and technology of manipulating and improving microbial strains,
in order to enhance their metabolic capacities for biotechnological
applications, are referred to as strain improvement.

3. T
argets of strain improvement
 Rapid growth
 Genetic stability
 Non-toxicity to humans
 Large cell size, for easy removal from the culture fluid
 Ability to use cheaper substrates
 Elimination of the production of compounds that may interfere
with downstream processing
 Increase productivity.
 T
o improve the use of carbon and nitrogen sources.
 Reduction of cultivation cost
 -lower price in nutrition.
 -lower requirement for oxygen.
 Production of
 -additional enzymes.
 -compounds to inhibit contaminant
microorganisms.

4. – Candida utilis
marxianus
Proper strain used in Industry
Genetically regarded as safe
GRAS
– Bacillus subtilis
– Lactobacillus
bulgaricus
– Lactococcus lactis
Bacteria
– Leuconostoc oenos
Yeasts
– Kluyveromyces
– Kluyveromyces lactis
– Saccharomyces
cerevisiae
•Filamentous fungi
– Aspergillus niger
– Aspergillus oryzae
– Mucor javanicus
– Penicillium roqueforti

5. Traditional Method of Strain Improvement
• There are several methods by which genetic alterations of producer microbial strains can be
done for maximization of products or metabolites.
A. Use of mutagenesis:
• In recent years, efforts have been devoted to miniaturize and automate the screening
procedures and to enhance the frequency of improved strains by selection procedures, e.g.,
the isolation of anti-metabolite-resistant mutants in cases where the natural metabolite is a
precursor, an inhibitor or a co-repressor of a biosynthetic pathway.
• The remarkable increases in antibiotic productivity and the resulting decreases in costs have
resulted due to mutation and screening for higher producing microbial strains.
• Mutation has also served to shift the proportion of metabolites production in a fermentation
broth to a more favorable distribution, to elucidate the pathways of secondary metabolism,
and to yield new antibiotics.
• Targeted mutagenesis:
• It involves introduction of mutations at a specific location in DNA. As many antibiotics,
growth hormones, regulatory factors production genes have now been cloned, targeted
mutagenesis of the cloned DNA can be performed in vitro, followed by transformation of the
recipient organism.
• The major problems of the classic strain improvement procedure based on random
mutagenesis were the very low probability of introducing mutations into relevant genes and
high rate of unwanted mutations in other, unrelated genes.
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6. MUTAGEN MUTA
TION
INDUCED
IMPACT ON
DNA
RELA
TIVE
EFFECT
Ionizing
Radiations-X
Rays,gamma
rays
Single or double
strand bearkage
of DNA
Deletion/structura
l changes
high
UV
rays,chemicals
Pyrimidine
dymerisation
Trnsversion,deleti
on,frameshift
transitions from
GC AT
Medium
Hydroxylamine(NH2
OH
Deamination of
cytosine
GC AT
transitions
low
N-Methyl –N’-
Nitro N-
Nitrosoguanidine
Methylation of
bases and high
pH
GC AT
transitions
high
Nitrous
acid(HNO2)
Deamination of
A,C & G
Bidirectional
transitions,deletio
n,AT GC/GC
AT
Medium
Phage,plasmid,D
NA transposing
Base
substitution,break
age.
Deletion,duplicati
on,insertion.
high

7. B. Use of classical genetics
• The most effective use of classical genetics in the past was the backcrossing of
overproducing strains with parent strains to improve the vigor of mutant strains.
• After backcrossing such a strain with the wild type, progeny cells are produced that
have inherited the overproducing traits from the mutant parent and the wild-type
hardiness and vigor from the wild-type parent.
• Priorly this was not possible with Penicillium, which lacks a true sexual cycle.
• However, a parasexual cycle resulting in the production of heterokaryons was
discovered in Penicillium in 1958 and was used to improve the strains.
• Protoplast fusion is relatively a new versatile technique to induce or promote
genetic recombination in a variety of prokaryotic and eukaryotic cell especially,
industrially useful microorganisms such as Streptomyces ambofaciens, Micro-
monsporae chinospora, because it breaks the barriers to genetic exchange.
• Another use of protoplast fusion has been the recombination of different strains
from the same or different species to yield new antibiotics such as anthracyclines,
aminoglycosides and rifamycins.
• Protoplast fusion has also been useful in elimination of an undesirable component
from penicillin broths imposed by conventional mating systems
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8. C. Rational selection :
• It involves selecting an improved producer out of a very large population of progeny. For example,
some antibiotics, notably penicillin and tetracyclines, are chelators of heavy metal ions.
• The more of these antibiotics an organism produces, the more resistant it will be to heavy metals in
the medium.
• Thus, selection for mutants resistant to heavy metals was used in the improvement of the penicillin
producers.
• Cloning of the candidate genes recombinant DNA technology can be used to introduce genes coding
for antibiotic synthetases into producers of other antibiotics or into non-producing strains to obtain
modified or hybrid antibiotics.
• The use of recombinant DNA technology in antibiotic improvement and discovery has been enhanced
by the finding that some Streptomyces's antibiotic biosynthetic pathways are coded by plasmid
genes, eg. methylenomycin
• A. Even when the antibiotic biosynthetic pathway genes of streptomycetes are chromosomal, they
appear to be clustered into operons which facilitate transfer of an entire pathway in a single
manipulation.
• The genes encoding individual enzymes of antibiotic biosynthesis which have already been cloned
include those of the cephalosporin, clavulanic acid, prodigiosin, undecylprodigiosin, actinomycin, and
candicidin pathways.
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9. The isopenicillin N synthetase ("CyClaSe") gene of Cephalosporium acremonium
has been cloned in Escherichia coli and expressed at a level of 20% of total cell
protein.
Cyclase gene of Penicillium chrysogenum and Streptomyces clavuligerus has also
been cloned in E.coli system.
The expandase/ hydroxylase gene of C. acremonium has been cloned in E. coli. The
protein accumulated as inclusion bodies in E. coli near to 15% of total cell protein
Similar to E.coli, now a days Bacillus subtilis, Pichia pastoris, Saccharomyces
cerevisiae have also emerged as a promising heterologous expression system for
prokaryotic and eukaryotic candidate genes.
There are several factors which govern the production of recombinant therapeutic
proteins in B. subtilis.
The factors are as follows: a) Well understood transcription and translation
machinery including different regulatory factors responsible for extracellular
product. b) Generation of stable recombinant plasmids. c) Development of novel
recombinant B. subtilis strains with reduced nuclease and protease contents. d)
Better systematic understanding of the protein secretion method to elucidate the
factors responsible for the secretion of intracellular proteins.
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10. To get the soluble extracellular protein product from the gene, it can be
linked to a DNA fragment coding for B. subtilis signal peptide for extra-
cellular secretion.
The signal sequence may be preceded by a efficient translation initiator
sequence or ribosome binding site (RBS) which is followed by mRNA
stability-enhancing sequences (SES) at 5’ end of mRNA.
The ‘strong’ promoter for this gene consist a cluster of other efficient
promoters which would be regulated temporally by the growth condition or
growth medium components.
Thus genes possess suitable properties not only for efficient transcription,
translation but would be under temporal control avoiding other exogenous
induction.
Thus generated mRNA would be stabilized by 3’SES which protects mRNA
from degradation by 3’ exonucleases.
 The protein product consists of a typical B. subtilis signal peptide linked to
N-terminus and have to be further processed with proteases to recover only
the desired portion.
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11. Strain improvement
by Genetic Engineering of MO
• The potential productivity of the organisms is
controlled by its genes and hence their genome
must be altered for the maximum production of
various proteins product such as enzymes.
• The techniques involved are
* Mutations
* Recombination – Protoplast fusion
* Recombinant DNA technology
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12. Mutations
• One of the most successful approaches for strain improvement.
• A mutation is any change in the base sequence of DNA - deletion,
insertion, inversion, substitution.
• The types include
- Spontaneous mutation
- Induced mutation
- Site directed mutation
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13. Types of mutations
1.Spontaneous mutation:
 Occur spontaneously at the rate of 10-10 and 10-15 per generation and per
gene.
 Occur at low frequency and hence not used much in industrial strain
improvement.
2. Induced mutation:
 The rate of mutation can be increased by various factors and agents called
mutagens.
 ionizing radiations (e.g. X-rays, gamma rays)
 non-ionizing radiations (e.g. ultraviolet radiations)
 various chemicals (e.g. mustard gas, benzene, ethidium bromide,
Nitrosoguanidine-NTG)
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14. 3. Site directed mutations(SDM) (site-specific mutagenesis ):
Change in the base sequence of DNA
changing the codon in the gene coding for that amino acid.
Can be done by protein engineering method
Desired improvements might be
*increased thermostability
*altered substrate range
*reduction in negative feedback inhibition
*altered pH range, etc.,
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15. Steps involved in site directed mutagenesis
Isolate required enzyme gene, e.g. via mRNA and its conversion into cDNA.
Sequence the DNA of the gene (in order to decide on change required for primer in stage
5).
Splice gene into M13 vector dsDNA and transduce E. coli host cells.
Isolate ssDNA in phage particles released from host cells.
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Synthesize an oligonucleotide primer with the same sequence as part of the
gene but with altered codon (mismatch/mispair) at desired point(s).
For example, one of the codons in DNA coding for the amino acid Alanine is
CGG. If the middle base is changed by SDM from G to C the codon sequence
becomes CCG which codes for a different amino acid (Glycine).

16. Mix oligonucleotide with recombinant vector ssDNA.
Carried out at low temperature (0-10oC) and in high salt concentration to allow hybridization between
oligonucleotide and part of gene.
Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a primer for the DNA
synthesis).
Then add ligase to join primer and new strand
dsDNA molecule.
Transform E. coli cells and allow them to replicate recombinant vector molecule.
DNA replication is semi-conservative, therefore two types of clone are produced each of which excretes phage
particles containing ssDNA:
 Type 1: contain the wild-type gene (i.e. unaltered)
 Type 2: contain the mutated gene!!!
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17. Microbial Genetics and Molecular Biology 17

18. 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
• Sandana Mala et al., 2001- lipase from A.
niger - Nitrous acid induced mutation – showed
2.53 times higher activity.
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19. Recombination – Protoplast fusion
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20. Protoplast fusion
Protoplast fusion depends on the following criteria-
 Lytic enzyme
 Osmotic stability
 Age of the mycelia
 Inoculation period
 Regeneration medium
 Regeneration frequency
 PEG concentration
 Osmotic stability
 Fusant formation
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21. Types of protoplast fusion
• Intraspecific hybridization
• Interspecific hybridization
• Intergeneric hybridization
oThe organism selected for fusion should be genetically related.
oAs the distance increases in genetic relationship between the
two mating isolates, the less successful protoplast fusion will be
(Anne and Peberdy, 1976).
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22. Reports on strain improvement by
protoplast fusion
• Kim et al., 1998 did a comparative study on strain improvement of
Aspergillus oryzae for protease production by both mutation and
protoplast fusion.
• UV radiation – 14 times higher yield.
• Ethyl methanesulphonate – 39 times higher yield.
• Protoplast fusion – using PEG and CaCl2 – 82 times higher yield.
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23. Recombinant DNA technology
• The more advanced method
• to increase the yields and consistencies of enzymes.
• Genetic material derived from one species may be incorporated into another where
it is expressed
• Increases the production of heterologous proteins by
- increasing the gene expression using strong promoters
- deletion of unwanted genes from the genome
- manipulation of metabolic pathways.
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24. Objectives
1. to get multiple copies of specific gene
2. to get high amounts of specific protein or product
3. to integrate gene of interest of one organism into another
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25. Steps involved in rDNA process
Steps involved:
Preparation of desired DNA
Insertion of desired DNA into vector DNA
Introduction of recombinant DNAs into host cells
Identification of recombinants
Expression of cloned genes
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26. Steps involved
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27. Some examples for strain improvement
by rDNA technology
Sidhu et al., 1998 tried both mutagenesis and cloning in E.coli for
increased production by amylase in Bacillus sp. MK716.
Mutation – ethyl methane sulphonate – 40 times higher.
 Mutated gene – cloned in E.coli pBR322- 107 times higher yield than
parent strain.
Calado et al., 2004 – cutinase enzyme – Arthrobacter simplex - 205
fold higher.
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28. Drawbacks of rDNA technology
• Genetic instability
• Genetical information of the industrially employed organism is
unknown
• Costlier than other methods
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29. Conclusion
• The task of both discovering new microbial compounds and improving
the synthesis of known ones have become more and more challenging.
• Newer genetic methods have been developed to obtain higher yields.
• The basic genetic information for all the organisms used industrially is
not available
• The steps have been taken by firms in order to gap the bridge between
basic knowledge and industrial application.
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