ABSTRACT
Protoplast fusion, a part of evolutionary engineering has a great potential for genetic analysis and strain improvement [3]. It breaks down the barriers to genetic exchange imposed by conventional mating systems. It can serve the purpose for developing a strain with mixed substrate fermentation abilities. Saccharomyces cerevisiae strains are suitable hexose fermenters but pentose fermentation is a setback for these strains. Although recombinant strains have been engineered but with limited success and certain constraints. Pichia stipitis strains are better pentose fermenters. Therefore, in this study, S. cerevisiae (LN) (~80% fermentation efficiency) and P. stipitis NCIM 3498 exhibiting xylose assimilation were taken. Firstly, selection markers were identified for the selection of fusants. S. cerevisiae LN was sensitive to hygromycin B (50 ppm) while P. stipitis NCIM 3498 was sensitive towards cycloheximide (5 ppm). Protoplast formation was optimized using glucanex enzyme (Sigma). 10 mg mL-1 enzyme concentration yielded highest protoplast frequency after 72 h. Fusion was carried out using 35% PEG and protoplasts incubated at 30 ºC for ~30 h. Fusants were observed under microscope after 16 h of incubation but they could not regenerate well in the regeneration medium.
