1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. OC-SBT051-U01-05 Introduction Programmes and Courses  SEP – SBT051 – Unit 01

3. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.3 Credits  Academic Inputs by Arun Punaji More. M.Sc. (Microbiology) Experience: 11 Years arunmesh@hotmail.com

4. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of 40-60 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 40-60 minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

5. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives  After studying this module, you should be able to: Describe different techniques of strain improvement for fermentation processes. Explain the importance of strain improvement in fermentation technology.

6. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Introduction - 1  Microorganisms produce number of useful primary and secondary metabolites having pharmaceutical and therapeutic values.  However these useful metabolites are produced in very low concentration making the fermentation processes highly costlier.  To some extend, optimizing the cultural conditions help increase the productivity and yield, but after one limit, the productivity and yield of these metabolites are controlled by microorganisms’ genetic constituents.

7. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 Introduction - 2  Therefore, in order to increase the productivity and yield of the useful metabolites up to level that make fermentation processes economically feasible, organisms’ genomes must be modified.  The processes by which this is achieved by various techniques are collectively called as strain improvement.  Let us learn more about the strain improvement.

8. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 Strain Improvement  Strain improvement is carried out to fulfill following objectives: To increase productivity of a fermentation process. To increase yield of a fermentation process. To develop ability in microorganism to utilize cheapest available substrate. To develop ability in microorganism to excrete fewer byproducts so that the cost of downstream processing would be minimum.

9. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 Strategies for Strain Improvement-1  In case of improving strain to produce maximum primary metabolites, the cells metabolic energy is channelised to that biosynthetic pathway involved in producing the primary metabolite of our interest as follow:  Carbon flux is channelised to desired primary product for its highest yield by:-  Directing and optimizing the primary metabolite pathway flux to target product by removing rate limiting steps and by removing transcriptional and allosteric regulation.

10. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 Strategies for Strain Improvement-2 By modifying secondary metabolic pathways to enhance energy metabolism toward the product of our interest, and availability of required cofactors. By removing detrimental side products. For example, to improve synthesis of hydrocortisone by S. cerevisiae, recombinant strain of the organism was developed which expressed 13 assembled genes required for the synthesis of hydrocortisone, while side reactions by other endogenous genes were disrupted to increase carbon flux toward the target product i.e. hydrocortisone.

11. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Strategies for Strain Improvement-3 Recombinant strain is very prone to infection with bacteriophages. Strain improvement for such culture involve making this culture resistant to infection with bacteriophages. This can be achieved in two different ways:-  One by inducing mutation into the recombinant culture that will make the culture resistant to bacteriophage infections.  Second way is to introduce restriction/modification endonucleases system into this recombinant culture so that the viral DNAs entered into the host cells are degraded, preventing their infections.

12. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 Strategies for Strain Improvement-4  In recombinant strain many times, foreign proteins expressed as a result of recombinant techniques are usually degraded by lon proteases selectively.  This remarkably reduces overall productivity or fermentation process.  So the one approach to avoid this is to remove the gene responsible for lon protein synthesis by random mutagenesis techniques.

13. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Strategies for Strain Improvement-5 Strain improvement can be achieved by two approaches:- 1. Classical Strain Improvement Approach. Mutagenesis with UV rays and chemical mutagenic agents. 2. Genetic Recombination Technology  Conjugation  Transformation  Transduction  Protoplast Fusion Techniques 3. Genetic Engineering

14. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 Classical Strain Improvement Approach  Classical Strain Improvement Approach: This is empirical approach. Microorganisms to be improved for a desired phenotypic property are subjected to mutagenic treatment using mutagenic agents such as UV light, nitrosoguanidine (NTG). After exposing microorganism to a selected mutagenic agent,the survived microorganisms are cultured on a suitable nutrient specially designed to select only those microorganism which have come to possess the desired metabolic ability. This process is also called as screening of microorganisms having expressed desired metabolic ability useful for fermentation processes.

15. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 Genetic Recombination Technology - 1  Conjugation:- conjugation is method of recombination mediated by conjugative plasmid; in this process male bacterial cell (which possess conjugative plasmid) donate a part of DNA or whole DNA to the recipient cell. After the plasmid mediated transfer DNA fragment; recombination occurs between the foreign DNA fragment and the host DNA. The existent conjugation in bacteria of our interest can be utilize to transfer useful trait from one strain to another of the same spp.; or to pool the set of genetic traits into the single bacterial strain.

16. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 Genetic Recombination Technology - 2  Transformation:- Transformation is a method of genetic recombination naturally observed in some bacteria; in this process there is uptake of free DNA fragment of the same species of different species by a bacterial strain. After the uptake of free DNA fragment; recombination occurs between the foreign DNA fragment and the host DNA. The rate of recombination is dependent upon the homology that these two DNA fragments shared.

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 Genetic Recombination Technology –3  Transduction:- In transduction the transfer of DNA fragment take place through the agency of some viral vector having the ability to infect two different species of bacteria. After the transfer of DNA fragment and recombination events, new recombinant strain that arises come to possess some useful genetic trait. Screening of such useful recombinant strain is done and such strains are utilized as improved strain for fermentation.

18. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.18 Genetic Recombination Technology – 4  Protoplast Fusion Techniques :- Protoplasts are cells without cell walls. Protoplasts are prepared by treating cells with cells wall degrading enzymes in isotonic solution. Such protoplasts are forced to fused by chemical treatment using chemicals such as polyethylene glycol and p-fluorophenylalanine. In fused protoplasts, genetic recombination may occur giving rise to new recombinant protoplast which regenerate its cells wall. The new recombinant cells thus formed come to possess both its parents’ useful traits.

19. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.19 Genetic Recombination Technology – 5  Diagram shows Protoplast fusion techniques Parent cell protoplast Recombining DNAs Fused recombinant protoplast Recombinant cells with cell wall

20. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.20 Genetic Recombination Technology – 6  Protoplast fusion is successfully used to  improve strains of number of fungi.  produce improved strain of Penicillium chrysogenum, producing higher quantity of antibiotic than its parent strains. E.g. recombinant strain of cephalosporin producing C. acremonium was successfully produced by fusion of protoplasts of asporulating, slow growing strain with a sporulating, fast growing strain of C. acremonium. The resultant recombinant strain showed good sporulation, high growth rate and produced 40% more cephalosporin than their parent strains.

21. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 Genetic Engineering  In this techniques DNA enzymes such as endonucleases, DNA ligases and DNA polymerase are used to construct a recombinant DNA.  The recombinant DNA constructed is then transferred to a another bacterial cells through the agency of plasmid and are allowed to expressed in their new host. By genetic engineering we can incorporate the eukaryotic genes into bacterial DNA and are expressed in the bacteria producing the product of a eukaryotic cells from which the DNA fragment is transferred. Genetic engineering is sophisticated, tedious and time consuming techniques of strain improvement.

22. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 What we Learn ………….  The processes by which the productivity and yield of the useful metabolites is increased up to level that make fermentation processes economically feasible, organisms’ genomes are modified by various techniques are collectively called as strain improvement.  3 strategies for strain improvement are: Carbon flux is channelised to desired primary product By modifying secondary metabolic pathways By removing detrimental side products  Recombinant strain is very prone to infection with bacteriophages.  3 Approaches for Strain Improvement are: Classical Genetic Recombination Technology Genetic Engineering  Genetic engineering is sophisticated, tedious and time consuming techniques of strain improvement.

23. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.23 Critical Thinking Questions Which is the easiest technique of strain improvement?

24. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.24 Hints for Critical Thinking Questions The techniques with the least sophistication.

25. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.25 Study Tips  Book Title:Principles of Fermentation Technology. Author: P. F. Stanbury, A. Whitaker and S. J. Hall. Publication: Elsevier Science Ltd.  Book Title: Fermentation Microbiology and Biotechnology. Editors: E.M.T. El-Mansi, C.F.A. Bryce, A.L. Demain, A.R. Allman. Taylor and Francis group. Publication: Taylor and Francis group.

26. End of the Presentation Thank You !