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 <i>Rhodotorula toruloides</i> toward patchoulol production. First, the patchoulol biosynthesis baseline was constructed by employing a chimeric enzyme of the <i>Pogostemon cablin</i> 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 <i>R. toruloides</i> in producing patchoulol, and should shed light on the microbial synthesis of other sesquiterpenes.