Sustainable bioproduction of triterpenoid sapogenins and meroterpenoids in a metabolically engineered medicinal mushroom†

Plant-derived oleanolic and ursolic acids are sought-after triterpenoid sapogenins used in modern curative and preventive medicines. Several plant species have been overexploited for triterpenoid sapogenin extraction. In this study, we reconfigured the metabolic fingerprints of Ganoderma lucidum and produced oleanolic and ursolic acids, ganoderic acids, and meroterpenoids. Oleanolic and ursolic acids were first synthesized in the medicinal mushroom by expressing amyrin-synthases and beta-amyrin 28-monooxygenase from plants. The production of sapogenin precursors (2,3-oxidosqualene) and ganoderic acid was enhanced by reconstructing the mushroom terpenoid biosynthetic pathway using a new terpenoid gene cluster recovered from the mycelium. Overexpression of the VeA–VelB velvet and LaeA proteins upregulated secondary metabolism and stimulated the hyperproduction of a renoprotective meroterpenoid. The VeA–VelB velvet and LaeA protein variants developed a radically distinctive yellow phenotype that has not yet been reported in any of the mushroom mycelial variants. CRISPR-AsCpf1-based lanosterol synthase editing repressed the competing ganoderic acid pathway and further enhanced 2,3-oxidosqualene accumulation and triterpenoid sapogenin biosynthesis. The oleanolic and ursolic acid titer reached 1.478 g L⁻¹ and 0.87 g L⁻¹, respectively, when the fermentation conditions were optimized in a 5 L lab bioreactor. This study presents fascinating metabolic engineering strategies that remodel Ganoderma's metabolic route and produce oleanolic acid, ursolic acid, ganoderic acids, and meroterpenoids. These new strains could replace wild plant species as a green source of triterpenoid sapogenins.