Highly Efficient Biosynthesis of γ-Bisabolene with a New Sesquiterpene Synthase AcTPS5 by Dual Cytoplasmic-Peroxisomal Engineering in Saccharomyces cerevisiae

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.