De novo production of (−)-limonene, (−)-carveol and (−)-carvone via engineering Rhodotorula toruloides

De novo biosynthesis of (−)-limonene, (−)-carveol and (−)-carvone was achieved via rewiring the mevalonate pathway in the oleaginous yeast Rhodotorula toruloides . Strategies including enzyme selection, protein fusion, pathway optimization (MVA upstream enhancement, cofactor supply), and fermentation condition optimization were applied to improve the monoterpenoids production. Finally, the (−)-limonene titer of 1.6 g/L and (−)-carveol of 668.0 mg/L were achieved through fed-batch fermentation in a 3-L bioreactor. Furthermore, the feasibility of biosynthesis of (−)-carvone was also demonstrated. This study represents an example for de novo microbial production of (−)-limonene, (−)-carveol and (−)-carvone with the oleaginous red yeast R. toruloides , demonstrating its potential as a versatile chassis for monoterpenoid biosynthesis.