Microbial strain selection..

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Microbial Strain Selection Techniques

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Strain Strain is a genetic variant or subtype of a microorganism, plant and

rodents.

A group of organisms of same species, sharing some certain characteristics not typical of the entire species but minor enough not to permit classification as a separate breed or variety.

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Sources of Strain

Bacteria

– Lactobacillus bulgaricus

– Bacillus subtilis

Yeasts

– Candida utilis

– Saccharomyces

cerevisiae

Filamentous fungi

– Penicillium roqueforti

– Aspergillus niger

Microorganisms:

Microbial strains can be manipulated in order to improve their technological properties. The classical examples are improved product yield and/or improved growth characteristics.

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Sources of Strain Plants:

Strain is designated group of off springs that are descendant from a modified plant produced by conventional breeding by biotechnological means or result from genetic mutation.

Rodents:

A mouse or rat strain is a group of animals that is genetically uniform.

Microbes are preferred to plants and animals because:

- They are generally cheaper to produce.

- Their enzyme contents are more predictable and controllable.

- Plant and animal tissues contain more potentially harmful materials than microbes, including phenolic compounds (from plants).

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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.

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Purpose of Strain Development

To relate individual cases to an outbreak of infectious disease.

To establish an association between an outbreak of food poisoning and a specific food vehicle.

To study variations in the pathogenicity, virulence and antibiotic resistance of individual strains within a species.

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Purpose of Strain Development

To trace the source of contaminants within a manufacturing process

To study the microbial ecology of complex communities, such as biofilms

To characterize microorganisms with important industrial applications

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Targets of Strain Improvement

Rapid growth

Genetic stability

Non-toxicity to humans

Large cell size, for easy removal from the culture fluids

Ability to use cheaper substrates

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Targets of Strain Improvement

Increase productivity

To improve the use of carbon and nitrogen sources

Reduction of cultivation cost

Production of - Additional enzymes- Compounds to inhibit contaminant microorganisms

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Methods for strain selection

Examples for strain selection methods

Bio-typing (picking up differences in biochemical reactions. Strains

"biotypes“)

Bacteriocin-Typing (anti-bacterial products)

Protein-Typing (protein synthesis by different strains)

Phage-Typing

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Optimization of Microbial Activity

It can be done by

Optimizing environmental conditions

Optimizing nutrition of microorganisms

Other includes1. Method not involving foreign DNA- mutagenesis2. Methods involving foreign DNA- recombination

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Optimizing of Environmental Conditions

Modification of physical parameter (temperature, agitation etc)

Modification of chemical parameter (pH,O2 concentration)

Modification of biological parameter (enzymes )

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Optimization of nutrition of microorganisms

Carbon sources

Nitrogen sources

Mineral sources

Precursor

Enzymes

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Methods not involving foreign DNA -Mutagenesis

Mutagenesis is a process of treatment given to microorganism which will cause an improvement in their genotypic and phenotypic performances

Mutagenesis

Spontaneous Mutation

Direct mutation (addition, deletion, substitution, point)

Induced Mutation

Site Directed Mutation

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Methods not involving foreign DNA -Mutagenesis

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Selection Procedure

Exposing organisms to the mutagen

The organism undergoing mutation should be in the haploid stage during the exposure.

The use of haploid is essential because many mutant genes are recessive in comparison to the parent or wild-type gene.

Bacterial cells are haploid; in fungi and actinomycetes the haploid stage is found in the spores.

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Screening of Microbial Strain

Random ScreeningAfter inducing the mutations ,survivors from the population are randomly picked and tested for their ability to produce the metabolite of interest.

A very large number of colonies must be tested

Advantage-

Minimal startup time and sustaining for years

Disadvantage-

Non-targeted and non-specific.

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Rational Screening

Rational screening requires some basic understanding of product metabolism and pathway regulation which gives information about metabolic check points and suggest ways to isolate mutants with specific traits.

Environmental conditions i.e. pH, temperature, aeration can be manipulated or chemicals can be incorporated in the culture media to select mutants with desired traits.

Applications – Selection of mutants resistant to the antibiotic produced

Selection of morphological variants

Selective detoxification

Selection of overproducers of a biosynthetic precursor

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Screening Tests for Mutants Cells should be suitably diluted and plated out to yield 50 – 100 colonies

per plate.

The selection of mutants is greatly facilitated by relying on the morphology of the mutants or on some selectivity in the medium.

Morphology:

When morphological mutants are selected, the desired mutation may be pleotropic (i.e., a mutation in which change in one property is linked with a mutation in another character).

The classic example of a pleotropic mutation is to be seen in the development of penicillin-yielding strains of Penicillium chrysogenum.

After irradiation, strains of Penicillium chrysogenum with smaller colonies and were better producers of penicillin.

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Screening Tests for Mutants

Selectivity by Media:

It is desired to select for mutants able to stand a higher concentration of alcohol, an antibiotic, or some other chemical substance, then the desired level of the material is added to the medium on which the organisms are plated. Only mutants able to survive the higher concentration will develop

Most of bacteria might well grow on 1-2% concentration of this substance. However, as the concentration increase, the number of surviving bacteria will decrease.

The concentration of the toxic pollutant could be gradually increased in the growth medium thus selecting the most resistant ones. This method is called acclimatization.

Screening must be carefully carried out with statistically organized experimentation to enable one to accept with confidence any apparent improvement in a producing organism.

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Methods involving foreign DNA-Recombination

Method involving Foreign DNA

(recombination)

Transduction

Protoplast fusion

Transformation

Genetic engineering

Conjugation

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Transduction

Transduction is the transfer of bacterial DNA from one bacterial cell to another by means of a bacteriophage.

Two types:

• General Transduction

• Specialized Transduction.

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Transformation

• When foreign DNA is absorbed by, and integrates with the genome of the donor cell.

• Cells in which transformation can occur are ‘competent’ cells.

• The method therefore has good industrial potential.

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Conjugation

• Conjugation involves cell to cell contact or through sex pili and the transfer of plasmids.

• Plasmids play an important role in the formation of some industrial products, including many antibiotics.

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Protoplast FusionFusion (Hybridization)

The fusion of two cells in tissue culture.

The two different strains after removal of cell wall are forced to fuse using Polyethylene Glycol (PEG).

The method has great industrial potential and experimentally has been used to achieve higher yields of antibiotics through fusion with protoplasts from different fungi.

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Genetic EngineeringVectors

Plasmids small, circular, dispensable genetic elements, found in most prokaryotic.

Phages viruses of bacteria, consist of a molecule of DNA or RNA and protein coat. bind to receptors on bacteria and transfer genetic material into the cell for reproduction.

Cosmids are artificial vectors prepared by DNA segments from plasmids and phages.

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Novel Genetic Techniques

Novel genetic techniques

Metabolic engineering

Genome shuffling

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Metabolic Engineering:

The existing pathways are modified, or entirely new ones 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.

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Genome Shuffling

Genome Shuffling– is a novel tech for strain improvement allow 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.

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Applications of Strain Selection Its basic purpose is to bridge basic knowledge and industrial

application. The tremendous increase in fermentation productivity and resulting decreases in

cultivation costs.

Discovering new microbial compounds and improving the synthesis of known ones. (amylase, protease)

Recombinant DNA technology has also been applied in medicine (insulin, HGH, hepatitis B vaccine), agriculture (golden rice, insect resistant crops)

Improve thermotolerance and ethanol tolerance in S.cerevisiae.

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Applications of Strain SelectionGenomic shuffling:

The yeild of biochemically products such as ethanol and bioinsectisides is successfully improved by genomic shuffling.

Metabolic engineering:

It has been used to overproduce the amino acid isoluecine in Corynebacterium glutamicum

It has been also implied 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.

The Strain of E.coli has been engineered for the production of lycopene amino acids and alcohols through metabolic engineering methods.

The improvement of Saccharomyces cerevisiae for the production of ethanol by metabolic engineering method

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Presented By:

Mujahid IqbalTHANK YOU!