Efficient biosynthesis of morolic acid in Saccharomyces cerevisiae cell factories

Abstract:

[Objective] Morolic acid is derived from germanicol, has excellent anti-HIV and anti-inflammatory activities properties as oleanane-type triterpenoid, thus their potential applications in the pharmaceutical industry. In this study, synthetic biology was used to construct Saccharomyces cerevisiae cell factories for efficient biosynthesis of morolic acid. [Methods] Firstly, using CRISPR/Cas9 technology, three oxidosqualene cyclases (OSCs), namely MdOSC, PbOSC and RcOSC were integrated into S. cerevisiae to screen for high yield germanicol chassis cells. Then, to construct the engineered strain capable of morolic acid production, the cytochrome P450 oxidase (CYP716AL1) derived from Catharanthus roseus and the cytochrome P450 reductase derived from Jatropha curcas (JcCPR) were further integrated into S. cerevisiae. Moreover, to improve morolic acid biosynthesis, the CYP716AL1 was coexpressed with CPRs (AtCPR, LjCPR, GuCPR and MtCPR) derived from different plants. Finally, the mevalonate (MVA) pathway was modified by overexpressing the key pathway enzymes to improve morolic acid production. [Results] The engineered strain S201 obtained by integrating oxidosqualene cyclase MdOSC from Malus domestica yielded the highest amount of germanicol of 68.3 mg/L. The engineered strain S401 obtained by integrating CYP716AL1 and JcCPR in S201 achieved biosynthesis of morolic acid, and the yield reached 15.0 mg/L. Furthermore, the CYP716AL1 was coexpressed with AtCPR from Arabidopsis thaliana in S201, resulting in engineered strain S402, which yielded the highest amount of morolic acid of 24.3 mg/L. Finally, overexpression of key enzymes in the metabolic pathway of MVA in engineered strain S402, FPP synthase (ERG20) and squalene epoxidase (ERG1), resulted in a morolic acid yield of 34.1 mg/L (S6). [Conclusion] In this study, morolic acid cell factories were successfully constructed, which provides a theoretical and technical basis for the construction of high-yield oleanane-type triterpenoids cell factories.