Enhanced Production of Santalols by Engineering the Cytochrome P450 Enzyme to the Peroxisomal Surface in Yeast

Cytochrome P450 enzymes are crucial for producing complex plant natural products (PNPs), but their expression and activity in heterologous systems are often limited. This study explores subcellular engineering to improve PNPs production by synthesizing (Z)-santalols─high-value components of sandalwood oil─in GRAS (Generally Recognized As Safe) Saccharomyces cerevisiae. By directing the mevalonate pathway and sandalwood oil biosynthesis enzymes to peroxisomes using the ePTS1 peptide, we increased total santalene and (Z)-santalol titer by 5.1-fold, reaching 0.9 g/L, with a conversion rate of 31.0% based on santalenes. Enhanced targeting the crucial cytochrome P450 enzyme SaCYP736A167–46tATR1 to the peroxisomal surface via Pex15 fusion increased (Z)-santalols titer to 0.45 g/L, with a conversion rate of 36.2%. We then increased the number of peroxisomes, further improving the conversion rate to 60.6%. Ultimately, fed-batch fermentation achieved a (Z)-santalols titer of 10.4 g/L, the highest reported to date, demonstrating the potential of engineered yeast peroxisome for PNPs synthesis.