This slide is about the different types and importance of metabolites. How we can do screening of strains and use different techniques, especially rDNA technology for strain improvement.
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Screening of metabolite and various rDNA technology for strain improvement.pptx
1. SCREENING OF METABOLITE AND VARIOUS
RECOMBINANT DNA TECHNOLOGY FOR STRAIN
IMPROVEMENT
AINDRILA MITRA BBT3
2. INTRODUCTION:
Biotechnology industries are based on harnessing the metabolic
activities of microbes, plants and animal cells to produce wide variety
of diverse compounds which are used by other industries such as
chemical, food, pharmaceutical , healthcare and the technology of
manipulating and improving microbial strain in order to enhance their
metabolic capabilities for biotechnological application is known as
strain improvement. It helps in high yield of target product. Apart from
modifying the strains the success of procuring the strains depend on
the efficient way of screening, testing and confirming the improved and
high yielding status of the mutants against a background of non
improved strain.
3. METABOLITE:
• They are low molecular weight, small compounds or molecules
produced as an intermediate or end product of metabolism.
There are two types of metabolite:
PRIMARY METABOLITE: are essential for the proper growth of micro
organisms.
Eg: consist of vitamins, amino acid, nucleoside and organic acids.
SECONDARY METABOLITE: are formed near the stationary phase of
growth and are not involved in growth, reproduction and
development.
Eg: Antibiotic- Penicilin: P.notatum
Vitamins: B2- Ashbya gossypii
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5. Screening of microbial culture for metabolites is essential for the discovery
of new biologically active compounds. This can be used in the industries to
develop vaccines, antibiotics, isolate chemicals for organic synthesis and to
obtain amino acids.
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6. Screening:
• The use of highly selective procedures to allow the detection and
isolation of only those microorganism which are of interest from a
large microbial population.
• It is the next step after enrichment and isolation.
Importance of screening :
The pure cultures must be screened for the desired property-
production of a specific enzyme, inhibitory compound etc.
The isolates must also be screened for other important features such
as stability, non toxicity.
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7. Criteria of organism chosen for screening:
• It should grow well in an ambient temperature preferably
at 30-40 C. This reduces cooling cost.
• It should yield high quality of the end product.
• It should posses stable biochemical characteristics.
• It should posses optimum growth rate so that it can be
easily cultivated on a large scale.
• It should posses minimum reaction time with the
equipment in a fermentation process.
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8. Types of screening:
Screening
Primary Screening
Organic acid
producing Mos
(By using dye)
Antibiotic
producing Mos
(By using crowded
plate technique)
Extracellular
metabolite
producing Mos(By
Auxanography
technique)
Enrichment
culture
techniques( by
defined media)
Secondary Screening
Giant colony
technique
Liquid media
technique Filtration
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9. Primary screening:
• It is a process of isolation, detection and separation of micro-
organism of our interest. It determines which micro organisms are
able to produce a compound. It does not provide much idea about
the production or yield potential of micro organisms.
• TYPES: 1. SCREENING FOR ORGANIC ACID PRODUCING MICROBES:
The ph indicating dyes can be used for detecting microbes able to
produce organic acids.
Eg: dye used- neutral red, bromophenol blue.
Also CaCO3 can be used for screening of organic acid producing
microorganism.
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10. 2. SCREENING FOR ANTIBIOTIC ( CROWDED PLATE TECHNIQUE):
Dilutions are made of samples ( which gives 300-400 colonies/plate)
and pour-plate/ spread-plate is done. Colonies which are able to
produce antibiotics, have a zone of inhibition. Such colonies are
selected and sub cultured for further studies. The purified cultures are
then tested to find the microbial inhibition spectrum.( in secondary
screening).
3. Enrichment culture technique: It is used to isolate desired
microorganism from heterogeneous microbial population. Enrichment
culture technique allows targeting of specific metabolic groups by using
selective nutrients and incubation condition.
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11. 4. SCREENING FOR EXTRACELLULAR METABOLITE PRODUCING
MICROBES: ( AUXANOGRAPHY TECHNIQUE)
It is used for detection of micro-organisms able to produce growth factors,
vitamins, amino acid etc extracellularly.
There are two steps:
• PREPARATION OF PLATE 1:
A filter paper strip is put across the bottom
of petri dish.
The nutrient agar is prepared and poured
on the paper strip and allowed to solidify.
Soil sample is diluted and proper dilutions are inoculated.
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12. • PREPARATION OF PLATE2:
>A minimal media is lacking the growth is prepared and is seeded with the test organism.
>The seeded medium is poured onto the fresh petri plate and plate is allowed to set.
>The agar from plate1 is lifted and
placed on the second plate without
inverting.
>The growth factors produced on agar can
diffuse into the lower layer containing test
organism.
>The zone of stimulated growth of test organism
around colonies is an indication that it produces
growth factors extracellularly.
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13. SECONDARY SCREENING:
• Micro-organisms isolated in primary screening are critically evaluated in
secondary screening so that industrially important and viable potentialities can
be assessed.
• The techniques for secondary screening are-
Giant colony technique : This technique is used for isolation and detection of
those antibiotics, which diffuses through solid medium.
Filtration: This method is employed for testing those antibiotics which are poorly
soluble in water or do not diffuse through the solid medium.
Liquid medium method: This method is generally employed for further screening
to determine exact amount of antibiotics produced by microorganism.
After an organism producing a valuable product is identified, it become necessary
to increase the product yield from fermentation to minimize production costs. For
this reason strain improvement is necessary.
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14. Strain improvement:
• STRAIN: A strain is a subgroup of a species with one or more
characteristics that distinguish it from other sub group of the
same species. It can be characterized by serotyping, enzyme
type, functional trait etc.
Eg: E.coli strain K12 and B.
• STRAIN IMPROVEMENT :The science and technology of
manipulating and improving microbial strains in order to
enhance their metabolic capacity is known as strain
improvement.
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15. Purpose:
• High yield of the target product.
• Regulating the activity of the enzymes.
• Reduction of by product formation.
• Introducing new genetic properties into the organism by
recombinant DNA technology/ genetic engineering ( for
genetically stable and superior).
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16. Approaches :
Methods of strain
improvement
Mutagenesis
Cloning and
genetic
engineering
Recombinant
DNA tech
Site directed
mutagenesis
Precision
engineering
tech
Genetic recombination
Protoplast
fusion
Transformation Conjugation
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17. • MUTAGENESIS: Method not involving foreign DNA.
method: chemical and physical mutagenesis
• PERSCISION ENGINEERING TECHNOLOGY: Precision engineering
technology or integrated strain improvement is a newly developed
aspect that also involves strain improvement. Previous approaches
of strain improvement often ignores the negative effects on the
industrial microbes such as -formation of undesired products or
slow growth, substrate specificity. Therefore, the industrial
geneticists are shifting towards the metabolic engineering
approaches. Precision engineering technology involves the
integration of classical metabolic engineering and screening
methods with profiling technologies which give a more clear
understanding of genetics and physiology of metabolite production.
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18. • GENETIC RECOMBINATION: This technique is employed to get new
strains containing novel combinations of mutations and superior
microbial strains. It include those technique that combine two DNA
molecules having similar sequences( homologues).
methods: protoplast fusion, transformation and conjugation
• CLONING AND GENETIC ENGINEERING: Used to assemble new
combinations of DNA in vitro which are then reinserted into the genome
of the microbe, creating new varieties of microbe. A specific and desired
character of microbial cells can be introduced in the selected strain to
improve the yield of product.
methods: recombinant DNA technology, site directed mutagenesis.
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19. Recombinant DNA technology:
Recombinant DNA technology has played an enormously potential role
in microbial strain improvement of industrial micro-organisms.
It 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 is used to achieve the following two broad objectives:
Production of recombinant protein
Modification of the organisms metabolic pattern for the production
of new modified or more quantity of metabolites.
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20. APPROACHES:
• RECOMBINANT PROTEIN: these are the protein produced by the
transferred gene or transgene; they themselves are commercial value.
Eg: insulin, interferon etc produced in bacteria.
• METABOLIC ENGINEERING: when metabolic activities of an organisms
are modified by introducing into it transgenes, which affect enzymatic
transport or regulatory function of its cells is known as metabolic
engineering.
The existing pathways are modified 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 entirely
new product.
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21. Some of the common strategies used for metabolic engineering are
(1) overexpressing the gene encoding the rate-limiting enzyme of the
biosynthetic pathway.
(2) blocking the competing metabolic pathways.
(3) heterologous gene expression.
(4) enzyme engineering.
Eg: It has been used to overproduce the amino acid isoleucine in
Corynebacterium glutamicum and ethanol by E.coli.
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23. CONCLUSION:
A number of genetic methods are available to improve fermentation
product yield and other strain characteristics.
Recombinant DNA methods have been especially useful in the
production of primary metabolites such as amino acids, but are also
finding increasing use in strain development programs for antibiotics.
The current strain improvement strategies have already contributed to
creating more efficient and safer enzymes production strains. The task
of both discovering and improving the new strain of interest ones have
become more and more challenging.
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