Multi-omics-guided discovery of CYP5035 enables heterologous production and engineering of pachymic acid in yeast.

Pachymic acid is a key bioactive triterpenoid of Poria cocos, a medicinal fungus widely used in traditional Chinese medicine, and is responsible for its anti-tumor and anti-inflammatory activities. However, its low natural abundance and reliance on pine wood-based cultivation limit large-scale production and therapeutic application. Methyl jasmonate (MeJA) is known to stimulate triterpenoid biosynthesis in plants and fungi. In this study, we treated liquid-cultured Poria cocos mycelia with MeJA and integrated transcriptomic and targeted metabolomic analyses to identify a cytochrome P450 gene, CYP5035, whose expression strongly correlates with pachymic acid accumulation. We then functionally validated CYP5035 by heterologous expression in Saccharomyces cerevisiae. Subsequent metabolic engineering of the yeast strain-through redox partner optimization and dynamic flux redirection-significantly enhanced pachymic acid production. Our work not only reveals the molecular basis of MeJA-induced triterpenoid synthesis but also establishes a sustainable microbial platform for the green and efficient production of pachymic acid.