Mining and modification of Oryza sativa-derived squalene epoxidase for improved β-amyrin production in Saccharomyces cerevisiae.

Abstract

β-Amyrin is a pentacyclic triterpenoid and has anti-viral, anti-bacterial and anti-inflammatory activities. The synthetic pathway of β-amyrin has been analyzed and its heterogeneous synthesis has been achieved in Saccharomyces cerevisiae. Squalene epoxidase (SQE) catalyzes the oxygenation of squalene to form 2,3-oxidosqualene and is rate-limiting in the synthetic pathways of β-amyrin. The endogenous SQE in S. cerevisiae is insufficient for high production of β-amyrin. Herein, eight squalene epoxidases derived from different plants were selected and characterized in S. cerevisiae for improved biosynthesis of β-amyrin. Among them, the squalene epoxidase from Oryza sativa (OsSQE52) showed the best performance compared to other plant-derived sources. Through protein remodeling, the mutant OsSQE52L256R, obtained based on modeling analysis, increased the titer of β-amyrin by 2.43-fold compared to that in the control strain with ERG1 overexpressed under the same conditions. Moreover, the expression of OsSQE52L256R was optimized with the improvement of precursor supply to further increase the production of β-amyrin. Finally, the constructed strains produced 66.97mg/L β-amyrin in the shake flask, which was 6.45-fold higher than the original strain. Our study provides alternative SQEs for efficient production of β-amyrin as well as other triterpenoids derived from 2,3-oxidosqualene.