Integrated protein and metabolic engineering enable high-yield biosynthesis of resveratrol derivatives in Yarrowia lipolytica

Piceatannol and pterostilbene, two resveratrol derivatives with superior pharmacological properties, hold significant promise for therapeutic and nutraceutical applications. Despite significant progress in their biosynthesis from resveratrol, microbial de novo biosynthesis of these compounds remains underexplored. Here, we engineered Yarrowia lipolytica to achieve de novo production of piceatannol and pterostilbene from glucose via integrated protein and metabolic engineering. First, a high-yielding resveratrol-producing Y. lipolytica strain was constructed by optimizing heterologous pathway and overexpressing key enzyme, achieving 526.0 mg/L resveratrol. Then, the introduction of HpaBC monooxygenase from Pseudomonas aeruginosa produced 273.1 mg/L piceatannol, while expression of caffeic acid O-methyltransferase (COMT) from Arabidopsis thaliana yielded 61.8 mg/L pterostilbene. Subsequently, the piceatannol production increased by 25.3 % (342.1 mg/L) through HpaBC genes duplication and cofactor optimization. On the other hand, directed evolution of COMT generated a superior mutant (H164Q/T308N/V311I/A160V/H321L/N322F), which was combined with multi-copy chromosomal integration, increased the production of pterostilbene by 190.3 % to 179.4 mg/L. Overall, this work establishes an efficient de novo biosynthesis platform for piceatannol and pterostilbene in Y. lipolytica, delivering the highest microbial titers reported to date and demonstrating the potential of this yeast for scalable production of bioactive stilbenes.