Strain improvement Part II, Generation of mutants producing high level of primary metabolites.

This presentation, describes about various mutants that can be generated by carrying out process of mutation, so that high yielding mutants can be obtained that can be used for industrial production of primary metabolite.

1. Mentor: Ms. Renu NK Jaisinghani Prepared By : Ms . Shifa Siddiqui Assistant Professor Ms. Akshta Desai Department of Microbiology Smt. CHM College STRAIN IMPROVEMENT Part II Generation of mutants producing high level of primary metabolites

2. Primary Metabolite A primary metabolite is the one which is directly involved in normal growth, development of an organism. It is produced in only that quantity as required by the organisms. If excess of primary metabolite is required to be produced from organism, we need to mutate it in such a manner that it will become free of FBI/FBR and produce it in the desired concentration.

3. Types of mutants A] Mutants with altered cell permeability. B] Auxotrophic mutant. C] Analogue resistant mutant. D] Revertant mutant.

4. A] Mutants with altered cell permeability Biosynthesis of glutamate by C. glutamicum . Heavy lines indicate the route to glutamate and the light lines indicate the route in the regeneration of oxaloacetate via the glyoxylate cycle.

5. A] Mutants with altered cell permeability • Such mutants have leaky cell membrane, therefore end product synthesized within the cell are constantly secreted outside keeping concentration of end product very low in the cytoplasm. • End product at such low concentration cannot exert FBI/FBR and metabolic pathway become free of regulation to give very high yield of product. E.g. Corynebacterium glutamicum used for production of glutamic acid. The metabolic pathway leading to synthesis of glutamic acid. • These mutants lack α-ketoglutarate (α-KG)dehydrogenase that converts α-KGA to succinic acid in TCA cycle. • Thus a metabolic block results in the accumulation of large concentration of α-KGA which is then converted to glutamic acid. • To complete TCA cycle OAA is generated by the activity of glyoxylate pathway .

6. A] Mutants with altered cell permeability Corynebacterium glutamicum auxotrophic mutant(biotin - ) is used for production of glutamic acid. Mutant when grown in sub-optimal concentration of Biotin, leads to formation of leaky cell membrane. Cell membrane are made of phospholipids and biotin serves as precursor for synthesis of fatty acids. If fatty acids are not synthesized in enough amount, then lipids won’t be formed in enough amount,thus the membrane formed would be leaky.

7. Initial steps in synthesis of Fatty acids

8. B] Auxotrophic mutant • In these mutants key enzyme, catalysing conversion of second last intermediate product to final end product is not synthesized or synthesized in inactive form. Thus metabolic pathway and production becomes free of regulation by the end product. Instead of end product there is accumulation of intermediate product at very high concentration in the cell. E.g. Corynebacterium glutamicum auxotrophic for lysin is used in lysin production. • Auxotrophic mutant of Corynebacterium glutamicum ( auxotrophic for methionine, threonine, isoleucine) lacking homoserine dehydrogenase can be obtained to get higher yield of lysine since all aspartyl semialdehyde synthesized will get converted solely to Diaminopimellic acid.

9. The control of the aspartate family of amino acids in C. glutamicum .

10. B] Auxotrophic mutant a) Aspartyl kinase the 1 st enzyme of pathway is controlled by lysine and threonine together through FBI. b) Homoserine dehydrogenase is controlled by threonine through FBI and Methionine through FBR. Similarly auxotrophic mutant of Corynebacterium glutamicum , auxotrophic for lysin is free of FBI by end product lysin and such mutant if grown on medium with limited lysin with accumulation of Diaminopimellic acid in large quantity. It can be converted to lysin using another organism or protrophic Corynebacterium glutamicum . Dual fermentation is carried out to get high yield

11. C] Analogue Resistant Mutant : These mutants do not recognize the presence of inhibitory or repressing levels of the end product or enzyme produced by mutant is altered at regulatory site so that it fails to interact with inhibitor. Thus mutant becomes free of regulation and can give excess of end product.

12. C] Analogue Resistant Mutant : Definition of analogue : Analogue is a compound which is very similar in structure to another compound but toxic in nature. Analogue of amino acid, nucleotide are mostly growth inhibitory may be because : When used in biosynthetic pathway of macromolecule results into production of defective, inactive cellular component which may interfere with its biosynthesis.

13. C] Analogue Resistant Mutant : From biosynthesis pathway it is clear that lysine production is regulated by concerted FBI( feedback inhibition) by lysine & threonine by inactivating key enzyme aspartyl kinase . Mutant of B. flavum , resistant to analogue of lysine ie . ( 2amino ethyl cysteine )gives very high yield, since aspartyl kinase the key enzyme is more sensitive to high concentration of lysin. It is mainly because site of mutation in resistant mutant is ‘aspartyl kinase ‘which is also site of control by an end product. By mutation aspartyl kinase changes without loss of activity and becomes insensitive to end product exerting FBI.

14. C] Analogue Resistant Mutant : Eg. Product Analogue Organism 1) Phenylalanine β – thiethylamine E coli 2) Methionine Ethionine Candida utilis 3) Methionine Norleucine E coli 4) Arginine 2-thiazolealanine Coryn glutamicum B flavum 5)Lysine 2- aminoethyl cysteine B flavum

15. D] Revertant Mutant : Definition of Revertant Mutant: Isolated auxotrophic mutants may get altered in recognition of control factors and thus become free of regulation and give high yield of desirable product , these are called revertant.

16. D] Revertant Mutant : Let us take an hypothetical example A B C D E F P Let ’A’ be the substrate getting converted to product ‘P’ through series of intermediate products B, C, D&E .Let ’a’ be the key enzyme controlled by high concentration of product ‘P’ i.e. FBI regulation.

17. D] Revertant Mutant : A mutant not producing enzyme “a” of pathway is auxotrophic mutant requiring product ‘P’ for growth. If this auxotroph is exposed to mutagenic agent for 2 nd mutation to occur in locus concerned with production of enzyme a, then Revertant Mutant may be obtained, having ability to synthesize active enzyme “a” & thus revertant becomes prototrophic in nature. Many times, the enzyme “ a”produced by revertant is functionally same as enzyme “a” produced by wild type prototroph but structurally vary from it. Thus enzyme “a” of revertant is not susceptible to control by product ‘P’ & becomes free of regulation to synthesize excess of product.

18. Reference Stanbury, P.F., Whitaker, A. Principles of Fermentation Technology 2E.

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