strain improvement.pptx

Microbial strain improvement

1. STRAIN IMPROVEMENT Techniques

2. Introduction Why strain improvement? Natural isolates usually produce commercially important products in very low concentrations Increased yield may be achieved by optimizing the culture medium and growth conditions Productivity of the organism is controlled by genome. Therefore, the genome must be modified to increase the product yield Cultural requirements of the modified organism would be examined to provide suitable conditions to increase the yield. Strain improvement process involves continual genetic modifications of the culture followed by reappraisals of its culture requirements.

3. INTRODUCTION Strain- A Strain is a group of species with one/ 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 Strain K12 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 cultivation cost Shorter fermentation time.

5. Purpose of Strain Improvement Increase the productivities Regulating the activity of the enzymes Introducing new genetic properties into the organism by Recombinant DNA technology / Genetic engineering.

6. Approaches for Strain Improvement Natural variants Mutant Selection Recombination Rec o mbinant DNA T ec h nology

7. Natural variants Generally a microbial culture will undergo a vast number of divisions. There is a small probability of genetic change occurring each time a cell divides. The microbial culture becomes heterogenous. The heterogeneity of some cultures can present serious problems of yield degeneration because the variants are the inferior producers compared with the original culture. Therefore, periodically plate out the producing culture and screen a proportion of the progeny for productivity. Selection of natural variants may result in increased yields.

8. MUTA N T S E LECTION 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, etc. 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.”

9. 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 Ex: Streptomyces griseus- Streptomycin-Mannosidostreptomycin Ex: Streptomyces aurofaciens (S-604) – Produce 6-demethyl tetracycline in place of Tetracycline In contrast, most improvements in biochemical production have been due to the Stepwise accumulation of so called Minor genes. Ex: Pencillium chrysogenum – Strain E15-1 was obtained which yield 55% more penicillin than original strain

10. 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. First superior penicillin producing mutant , Penicillium chrysogenum X-1612,was isolated after X ray mutagenesis.

11. Isolation of mutants The following points highlight the four methods to detect and isolate mutants. The methods are: 1. Replica Plating Technique 2. Resistance Selection Method 3. Substrate Utilization Method 4. Carcinogenicity Test. 1. Replica Plating Technique : Lederberg and Lederberg (1952) have given replica plating technique. This technique is used to detect auxotrophic mutants which differentiates between mutants and wild type strains on the basis of ability to grow in the absence of an amino acid.

12. 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. Substrate Utilization Method: This method is employed in the selection of bacteria. Several bacteria 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. Carcinogenicity Test: to identify the environmental carcionogens that cause mutation and induce cancer in organisms. It’s based on detecting potential of carcinogens and testing for mutagenicity in bacteria.

13. Ames (1973) developed a method for deletion of mutagenicity of carcinogens which is commonly known as Ames test. It is widely used to detect the carcinogens. The Ames test is a mutational reversion assay in which several special strains of Salmonella typhimurium are employed. Each strain contains a different mutation in the operon of histidine biosynthesis.

14. The number of spontaneous reverants is low, whereas the number of reverants induced by the test mutagen is quite high. In order to estimate the relative mutagenicity of the mutagenic substance the visible colonies are counted and compared with control. The high number of colonies represents the greater mutagenicity. A mammalian liver extract is added to the above molten top agar before plating. The extract converts the carcinogens into electrophilic derivatives which will soon react with DNA molecule. The liver extract is added to this test, just to promote the transformation. The Ames test has now been used with thousands of substances and mixtures such as the industrial chemicals, food additives, pesticides, hair dyes and cosmetics.

15. RECOMBINATION Defined as formation of new gene combinations among those present in different strains. Recombination is used for both genetic analysis as well as strain improvement To generate new products Recombination may be based on:- Transformation Conjugation others like cross over and 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 antibiotic.

16. Protoplast fusion

17. RECOMBINATION 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 proteins Metabolic Engineering

18. 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 Metabolic Engineering :- When metabolic activities of an organism are modified by introducing into it transgenes, 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 .

19. Product Modification include the new enzymes which modifies the product of existing biosynthetic pathway e.g. Conversion of Cephalosporin C into 7- amino cephalosporanic acid by D-amino acid oxidase (in A. chrysogenum). Completely new metabolite formation include in which all the genes of a new pathway are transferred e.g. E.coli, transfer of 2 genes for polyhydroxybutyrate synthesis from Alcaligenes eutrophus. Enhance growth include enhanced substrate utilization. e.g. E.coli , glutamate dehydrogenase into M.methylotrophus carbon conversion increased from 4% to 7%

20. Novel genetic technologies Novel genetic tech. Metabolic e n gi n e e ring Genome shuffling

21. Metabolic engineering- The existing pathways are modified, or entirely new ones are 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. It has been used to over-produce the amino acid isoluecine in Corynebacterium glutamicum , & ethanol by E. coli and has been employed 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.

22. Genome Shuffling It is 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.

23. APPLICATIONS Large scale Production of vaccines, Enzymes, Interferon, growth factors, blood clotting factors. In the field of Microbiology to improve the microbe’s productivities or characteristics. Treatment of Genetic diseases like SCID by rDNA technology Production of medically useful biological products like insulin

24. CONCLU S ION These steps have been taken by firms in order to gap the bridge between basic knowledge and industrial application. The task of both 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 costs have come about mainly by using m utagenesis. In recent y ear s ,reco m bina n t DNA technolo g y has also been applied. The promise of the future is via extensive of new genetic techniques-Metabolic engineering and Genomic shuffling. The choice of approaches which should be taken will be driven by the economics of the biotechnological process and the genetic tools available for the strain of interest.

25. REFER E NCES http://www.yourarticlelibrary.com/micro-biology/strain- improvement ht t ps: / / w w w .resea r chgat e .net / .../226497441 _ Strai n _ imp r o vement ht t ps: / / w w w .j i c.ac.uk/... / Marine l li % 20Le c ture % 202%20pa rt%201.pdf http://technologyinscience.blogspot.in/2012/08/strain- improvement-importance-of-pure.html# http://www.cabri.org/guidelines/micro- organisms/M300.html A text book of Molecular Biology, Genetic Engineering and Industrial Biotechnology by B.D Singh

26. Presented to , Prof.Prasanna Srinivas Dept. of Microbiology RCASC