Synergistic engineering of monoterpenoid synthase and metabolic pathways to enhance α-terpineol production in Saccharomyces cerevisiae

Plant-derived monoterpenoid α-terpineol has gained significant attention due to its versatile applications in the food, cosmetic and pharmaceutical industries. Microbial biosynthesis offers an environmentally sustainable approach for its commercial-scale production. Here, we developed a combinatorial engineering strategy to enhance α-terpineol production in Saccharomyces cerevisiae . A truncated Vitis vinifera α-terpineol synthase (tVvTS) was optimized through alanine scanning, site-directed mutagenesis, and N-terminal truncation, yielding a variant with improved whole-cell production performance, tVvTS^M522K/13aa, which increased α-terpineol titer by 4.8-fold. Further enhancement of mevalonate pathway flux through enzyme fusion and mitochondrial compartmentalization increased the titer by 26.3-fold relative to the parental strain. Subsequent optimization of tolerance-associated genes expression, diploid construction, and fed-batch fermentation enabled the final strain WB11 to produce 81.78 mg/L α-terpineol in a 5-L bioreactor. This study provides an efficient combinatorial strategy for microbial production of natural terpene alcohols.