SCREENING METHOD FOR HIGH YIELDING S-ADENOSYL-LMETHIONINE IN RECOMBINANT PICHIA PASTORIS STRAINS, BASED ON SELECTION PRESSURE AND GENE COPY NUMBERRAVI KANT HARIT, M. BALAMURALI AND SANKARANARAYANAN MEENAKSHISUNDARAM
In the present study we constructed a recombinant Pichia pastoris (P. pastoris) harboring the Saccharomyces cerevisiae (S.cerevisiae) SAM2 synthetase gene. Multiple copies of this gene were integrated into P. pastoris using constitutive intracellular expression vectors (PGAP). The recombinant P. pastoris clones were screened and selected on the basis of their high growth rate under the pressure of various concentrations of antibiotic Zeocin from 200μg/mL to 800μg/mL and the growth rate of these clones were correlated with number of copies of SAM2 gene integrated into the P. pastoris genome. Selected clones were hecked for the expression of the SAM2 gene and similarly for the SAM production. The SAM specific yield in recombinant P. pastoris transformants GAP1, GAP2 and GAP3 was 0.260, 0.220 and 0.350 respectively. Here-in this study GAP3 clone showed approximately 35% higher SAM yield as compared with GAP1 clone and 59.1% higher SAM than GAP2 clone. This is a novel approach for screening recombinant P. pastoris transformants for high SAM production on the basis of growth at higher concentrations of antibiotic zeocin and correlating SAM yield with integrated gene copy numbers.
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