High-level De novo Biosynthesis of Plant-Derived Pinosylvin in Yarrowia lipolytica via Metabolic Engineering and Surfactant-Mediated Fermentation.
Pinosylvin, a naturally occurring antimicrobial and pharmaceutical stilbene found in pine heartwood, faces challenges in sustainable production due to the environmentally burdensome nature of its extraction from plant sources. To overcome this limitation, the oleaginous yeast Yarrowia lipolytica, which possesses a high intracellular malonyl-CoA supply, was selected as the host for pinosylvin biosynthesis. Initially, the pinosylvin synthetic pathway was constructed in Y. lipolytica by introducing three heterologous enzymes: phenylalanine ammonia lyase (PAL), 4-coumaryl-CoA ligase (4CL) and stilbene synthase (STS). Through the implementation of a multi-copy strategy to enhance the expression of key enzymes, flux strengthening through the shikimate pathway to redirect carbon metabolism, and modification of the lipid synthesis pathway to boost precursor supply, a pinosylvin titer of 202.88mg/L was achieved. Additionally, the cytotoxic effect of pinosylvin was identified, and a surfactant‑assisted fermentation strategy effectively alleviated such cytotoxicity by promoting the extracellular secretion of pinosylvin. Finally, by optimizing the carbon source concentration, the pinosylvin titer reached 774.65mg/L, representing the highest level reported to date in shake flask culture. This work highlights the exceptional potential of Y. lipolytica as a cell factory for high-level pinosylvin production and presents an effective strategy for achieving high-level biosynthesis of cytotoxic natural products through engineered secretion.