Productive Amyrin Synthases for Efficient α-Amyrin Synthesis in Engineered Saccharomyces cerevisiae

alpha-Amyrin is a plant-derived pentacyclic triterpenoid, with a lot of important physiological and pharmacological activities. The formation of alpha-amyrin from (3S)-2,3-oxidosqualene is catalyzed by alpha-amyrin synthase (alpha-AS), a member of the oxidosqualene cyclase (OSC) protein family. However, alpha-amyrin is not yet commercially developed due to its extremely low productivity in plants. The engineered Saccharomyces cerevisiae with efficient alpha-amyrin production pathway could be used as an alternative and sustainable solution to produce alpha-amyrin from renewable raw materials. To efficiently improve alpha-amyrin production in S. cerevisiae, we identified two alpha-ASs, EjAS and MdOSC1 from Eriobotrya japonica and Malus x domestica, respectively, through strict bioinformatics screening criteria and phylogenetic analysis. The specific activities of purified EjAS and MdOSC1 were 0.0032 and 0.0293 mu mol/min/mg respectively. EjAS produced alpha-amyrin and beta-amyrin at a ratio of 17:3, MdOSC1 produced alpha-amyrin, beta-amyrin and lupeol at a ratio of 86:13:1, indicating MdOSC1 had significantly higher specific activity and higher ratio of alpha-amyrin than EjAS. Furthermore, MdOSC1 was introduced into S. cerevisiae combining with the increased supply of (3S)-2,3-oxidosqualene to achieve the encouraging alpha-amyrin production, and the titer of alpha-amyrin achieved 11.97 +/- 0.61 mg/L, 5.8 folds of the maximum production reported.