De Novo Synthesis of Friedelin in Saccharomyces cerevisiae via Combination of Metabolic and Lipid Droplet Engineering

Friedelin, a pentacyclic triterpenoid, exhibits anti-inflammatory, anticancer, antibacterial, and neuroprotective properties. It also serves as a precursor to celastrol, a potential anticancer drug. Despite the potential of friedelin as a valuable triterpenoid, its low natural abundance in plants and high extraction costs, along with the challenges of chemical synthesis, highlight the need for more efficient and sustainable production methods. While synthetic biology and microbial cell factories, particularly Saccharomyces cerevisiae, have emerged as promising alternatives, further optimization of these systems is required to enhance friedelin biosynthesis. In this study, we aimed to construct a de novo biosynthetic pathway for friedelin in S. cerevisiae by expressing the friedelin synthase gene, TwOSC1. The results showed that friedelin production in the Z16 strain increased to 270 mg/L after enhancing the activity of key enzymes in the pathway, alleviating promoter inhibition, and reducing the metabolic flux of competing pathways in YPD medium. Further improvements in friedelin production were achieved via medium optimization and lipid droplet engineering, which enhanced cell density and mitigated product cytotoxicity. The friedelin titer of the Z28 strain reached a record level of 1500 mg/L in 250 mL shake-flask fermentation, representing a 30-fold improvement compared to that observed in the initial strain. This study establishes a foundation for the microbial production of friedelin and provides an important precursor for the biosynthesis of high-value compounds such as celastrol.