| Summary: | <i>γ</i>-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. <i>Saccharomyces cerevisiae</i> 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 <i>γ</i>-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, <i>γ</i>-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene <i>atg36</i> further improved <i>γ</i>-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted <i>γ</i>-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of <i>ERG9</i>, <i>γ</i>-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 <i>γ</i>-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in <i>S. cerevisiae</i>.
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