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 FaNES1G498Q 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.
Keywords: Yarrowia lipolytica; pathway compartmentation; peroxisomal acetyl-CoA pool; protein engineering; trans-nerolidol.