Overproduction of Patchoulol in Metabolically Engineered Komagataella phaffii

Patchoulol, a plant-derived sesquiterpene compound, is widely used in perfumes, cosmetics, and pharmaceuticals. Microbial production provides a promising alternative approach for the efficient and sustainable production of patchoulol. However, there are no systematic engineering studies on Komagataella phaffii aimed at achieving high-yield patchoulol production. Herein, by fusion overexpression of FPP synthase and patchoulol synthase (ERG20LPTS), increasing the precursor supply, adjusting the copy number of ERG20LPTS and PTS, and combined with adding auxiliary carbon source and methanol concentration optimization, we constructed a high-yield patchoulol-producing strain P6H53, which produced 149.64 mg/L patchoulol in shake-flask fermentation with methanol as the substrate. In fed-batch fermentation, strain P6H53 achieved the highest production (2.47 g/L, 21.48 mg/g DCW, and 283.25 mg/L/d) to date in a 5 L fermenter. This study will lay a good foundation for the development of K. phaffii as a promising chassis microbial cell for the synthesis of patchoulol and other sesquiterpenes with methanol as the carbon source.