STRAIN IMPROVEMENT Techniques

INTRODUCTION
Strain- A Strain is a sub group of species with one or
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 K12
Saccharomyces boulardii CNCM I-745
Lactobacillus plantarum 299v

Strain Improvement- The Science and Technology of

manipulating and improving microbial strains in order to
enhance their metabolic capacities is known as Strain
Improvement
Targets 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
 To 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.
 Approaches for Strain

Improvement
Mutation
- Replica Plating Technique
- Resistance Selection Method
- Substrate Utilization Method
- Activity on agar media
 Recombinant DNA Technology/Genetic engineering
- Production of Recombinant proteins
- Metabolic Engineering
- Genome shuffling
 Recombination
- Transformation
- Conjugation
- Transduction
- others like protoplast fusion
 MUTATION
 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, mustard gas etc.

 Physical agents – UV rays, X rays

 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

Reports on strain improvement by mutation:

Karana and Medicherla (2006)- lipase from Aspergillus japonicus
MTCC 1975- mutation using UV, HNO2, Nitrosoguanidine 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 which yield 55% more
penicillin than original strain
 ISOLATION OF MUT ANTS:
 Mutation occurring in microorganism can be detected and efficiently
isolated
 While studying we must be aware of wild type characters of an
organism ,so the mutants can easily detected.
 In bacteria and other haploid microorganism, the detection system are
straight forward because any new allele should be observed
immediately.

The following points highlight the four methods to detect and isolate
mutants
1. Replica Plating Technique
2. Resistance Selection Method
3. Substrate Utilization Method
4. Activity on agar media
 1. REPLICA PLATING TECHNIQUE:

 Joshua and Esther Ledgerberg (1952) developed a

technique called replica plating .
 This technique is used to detect auxotrophic mutants
and wild type strains on the basis of ability to grow in the
absence of amino acids.
STEPS INVOLVED
 Generate the mutants by treating a culture with a mutagen
e.g.nitrosoguanidine .
 Inoculate a plate containing complete growth medium and incubate it at proper
temperature. Both wild type and mutant survivors will form complete medium.
 This plate containing complete medium is called master plate.
 Prepare a piece of sterile velvet and gently place on the upper surface of the
master plate to pick up bacterial cell from each colony.
 As pressed the master plate, again gently press the velvet on the replica plates
containing complete medium in one set and lacking only leucine in the other set
(Minimal medium)
 Thus, the bacterial cells are transferred in replica plates in the same position as
in master plate.
 Incubate the plates and compare the replica plate with master plate for
bacterial colony not growing on replica plate.
lacking only leucine
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 several
bacteria that 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. Activity on plate:
• By checking the activity of enzymes or antibiotics on the agar plate

Ex. Colonies which shows more intense zones on skim milk agar
plates will be selected as mutants for production of protease

Zones of casein degradation on skim milk agar plates

 Genetic Engineering/
Recombinant DNA technology
 Genetic engineering, also known as recombinant DNA technology,
molecular cloning or gene cloning.
 Recombinant DNA Technology enables isolation of genes from an
organism, this gene can be amplified, studied, altered & put into
another organism

 This technique has been used to achieve 3 broad objectives:

- Production of Recombinant proteins

- Metabolic Engineering

- Genome shuffling
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

 Recombinant DNA procedure:

i.Cutting of donor DNA : Restriction endonucleases cut DNA

molecule at specific sites and desired fragment is isolated by gel
electrophoresis.

ii.Cloning of a gene : DNA fragment, which wanted to be cloned,

is joined to one of vectors (plasmid, phage, cosmid). For this purpose,
vector and donor DNA are first cleaved with the same restriction
endonuclease, or with the ones producing the same ends

iii.Then by using DNA ligase, DNA fragment and vector DNA is

joined.

iv.Transformed into fresh bacterium

v.Isolation of mutants
Metabolic engineering
When metabolic activities of an organism are modified, 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

Product Modification
Completely new metabolite
 Enhance growth include enhanced substrate utilization.
 Genome Shuffling
 Itis 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.
 RECOMBINATION
 Defined as formation of new gene combinations among
those present in different strains.
 Recombination is used for strain improvement and to
generate new products
 Recombination may be based on:-
- Transformation
- Conjugation
- Transduction
- others like protoplast fusion
 Protoplast fusion
• Protoplasts are the cells of which cell walls are removed

• 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.