Simultaneous synthesis of resveratrol by dual-compartment metabolic engineering in Saccharomyces cerevisiae

Resveratrol, a plant-derived polyphenolic compound, is widely used in medicine, health care, and nutrition. Synthetic biologyenables the microbial synthesis of resveratrol as apromising alternative to traditional plant extraction. However, an insufficient precursor supply remains a critical bottleneck limiting the overall yield. Here, for the first time, a dual-compartment engineering strategy was developed to simultaneously synthesize resveratrol in the peroxisomes and cytoplasm of Saccharomyces cerevisiae and to increase resveratrol synthesis in the presence of a sufficient supply of precursors. Heterologous tyrosine ammonia-lyase, 4-coumarate-CoA ligase, stilbene synthase and endogenous acetyl-CoA carboxylase were targeted to peroxisomes via a C-terminal signaling peptide, establishing a resveratrol synthesis pathway within this compartment. Three heterologous genes were increased to 11 copies using an efficient multilocus integration method, thereby improving the peroxisomal synthetic pathway and enhancing the resveratrol titer by 200 %. Systematic organelle engineering was further applied to efficiently increase the availability of malonyl-CoA, acetyl-CoA, and fatty acid at nodes, resulting in a 161 % increase in the resveratrol titer. Combining the cytoplasmic and peroxisomal pathways to synthesize resveratrol simultaneously in both compartments enabled the titer to 552.16 mg/L in 5-L bioreactors. ¹³C metabolic flux analysis revealed that the resveratrol accumulation of the dual-compartment strain approximated the sum of that of the two single-compartment strains. Notably, the peroxisome was rewired to become an organelle factory by redirecting acetyl-CoA into the organelle compartment, establishing an important and feasible site for resveratrol synthesis. This work established a novel and generalizable dual-compartment engineering paradigm for biochemical synthesis.