strain improvement and preservation

strain development , preservation , rDNA technology , different types of strain improvement , crypreservation

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’.

8. 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.

9. 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 .

10. 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.

11. 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.

12. 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

13. 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.

14. 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%.

15. 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

16. 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

17. 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.

18. 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

19. • 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

20. 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.

21. 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).