Enhancing Trans-Nerolidol Productivity in Yarrowia lipolytica by Improving Precursor Supply and Optimizing Nerolidol Synthase Activity

The low enzymatic capability of terpene synthases and the limited availability of precursors often hinder the productivity of terpenes in microbial hosts. Herein, a systematic approach combining protein engineering and pathway compartmentation was exploited in Yarrowia lipolytica for the high-efficient production of trans-nerolidol, a sesquiterpene with various commercial applications. Through the single-gene overexpression, the reaction catalyzed by nerolidol synthase (FaNES1) was identified as another rate-limiting step. An optimized FaNES1^G498Q was then designed by rational protein engineering using homology modeling and docking studies. Additionally, further improvement of trans-nerolidol production was observed as enhancing the expression of an endogenous carnitine acetyltransferase (CAT2) putatively responsible for acetyl-CoA shuttling between peroxisome and cytosol. To harness the peroxisomal acetyl-CoA pool, a parallel peroxisomal pathway starting with acetyl-CoA to trans-nerolidol was engineered. Finally, the highest reported titer of 11.1 g/L trans-nerolidol in the Y. lipolytica platform was achieved in 5 L fed-batch fermentation with the carbon restriction approach.