Studies on squalene biosynthesis and the standardization of its extraction methodology fromSaccharomyces cerevisiae

Aims The present work focuses on studies on squalene improvement in the S. cerevisiae and development of squalene extraction procedure based on mechanical disruption of cells. Methods and Results In this study, a homogenization-based extraction method was developed and was compared to five conventional methods of squalene extraction. Squalene recovered from this novel procedure gave 3.5– fold, 10-fold, 16-fold and 8.1-fold higher yield than standard procedures viz., saponification with 60% KOH, acidic saponification, saponification with 18% KOH and glass beads method, respectively. Furthermore, this procedure has been evaluated on laboratory S. cerevisiae strains such as BY4742 and CEN.PK2-1C (native), deletion strains ( ERG6 and ERG11 ) and tHMGl overexpressed S. cerevisiae strains. When sonication method of cell lysis was replaced with homogenization it was found that the yields were significantly higher and reached a value of 9 mg/g DCW in case of BY4742. In addition, Squalene yield in ergosterol mutant strains has been analyzed and was found to be 1.8-fold and 3.4-fold higher in ERG6 and ERG11 deletion strains, respectively, than BY4742. Squalene was also found to be higher at the optimized temperature of 30°C and pH 6.0. Furthermore, tolerance of S. cerevisiae to external squalene at various concentration has been carried and found that the organism was tolerant up to 25 g/L of squalene. Conclusions Homogenization based mechanical disruption was observed to yield higher squalene and the SEM analysis corroborates these findings. The synergistic effect of ERG11 downregulation and tHMG1 over-expression has led to significant increase in squalene yield. Significance and Impact of the Study Sonication and homogenization has been used as a cell-disruption method for the first time in squalene extraction and squalene yield from homogenization method of cell lysis in BY4742 has been found to be 9 mg/g DCW which is the highest reported so far in a wild-type strain.