Metabolic and transport engineering enable efficient de novo biosynthesis of patchoulol in Saccharomyces cerevisiae

Patchoulol is a valuable sesquiterpene for fragrance and cosmetics, but plant extraction is slow and resource-intensive. Here, we combined enzyme, pathway and transporter engineering to build a yeast chassis for high-titer patchoulol production. Semi-rational engineering of patchoulol synthase (PTS) increased catalytic efficiency; together with multi-copy PTS expression, the shake-flask titer rose from 0.40 ± 0.05 mg/L (PT01) to 258.72 ± 27.35 mg/L. Strengthening glycolysis and the mevalonate pathway and down-regulating ERG9 redirected flux to FPP. Guided by docking and transcript analysis, we identified PDR12 as an effective efflux pump; inducible overexpression increased the titer to 372.80 ± 14.92 mg/L. In a 5-L two-stage fed-batch with a dodecane overlay, the best strain reached 2.86 g/L (2856.25 mg/L). This work provides a general framework to couple metabolic with transporter engineering for hydrophobic terpenoids.