Biosynthesis of patchoulol via metabolic engineering the oleaginous red yeast Rhodotorula toruloides

Patchoulol is a widely used sesquiterpenoid in perfumes, cosmetics, foods and pharmaceuticals. The plant-dependent production is suffering from limited growing area, long seasonal cycle, etc. Microbial production represents a sustainable alternative as it is featured with mild operating conditions and eco-friendliness. Herein, we engineered the oleaginous Rhodotorula toruloides toward patchoulol production. First, the patchoulol biosynthesis baseline was constructed by employing a chimeric enzyme of the Pogostemon cablin originated patchoulol synthase and the native FPPS. Second, the supply of essential intermediates was streamlined by redeploying the mevalonate (MVA) pathway while the recycling of NADPH was enhanced through over-expressing related enzymes. Third, the patchoulol production was further enhanced to 724.8 mg/L, 6.0 mg/L/h and 36.2 mg/g glucose by down-regulating the squalene biosynthesis and tuning the cultivation condition in shake flask. Finally, the production of patchoulol was increased to 1.31 g/L and 13.8 mg/g glucose in the minimal medium in a 3-L bioreactor. Our study demonstrated the potential of R. toruloides in producing patchoulol, and should shed light on the microbial synthesis of other sesquiterpenes.