Engineering Saccharomyces cerevisiae for Hyperproduction of β-Amyrin by Mitigating the Inhibition Effect of Squalene on β-Amyrin Synthase

The study aims to enhance β-amyrin production in Saccharomyces cerevisiae by peroxisome compartmentalization. First, overaccumulated squalene was determined as a key limiting factor for the production of β-amyrin since it could inhibit the activity of β-amyrin synthase GgbAs1. Second, to mitigate the inhibition effect, the enhanced squalene synthesis pathway was compartmentalized into peroxisomes to insulate overaccumulated squalene from GgbAs1, and thus the specific titer of β-amyrin reached 57.8 mg/g dry cell weight (DCW), which was 2.6-fold higher than that of the cytosol engineering strain. Third, by combining peroxisome compartmentalization with the “push-pull-restrain” strategy (ERG1 and GgbAs1 overexpression and ERG7 weakening), the production of β-amyrin was further increased to 81.0 mg/g DCW (347.0 mg/L). Finally, through fed-batch fermentation in a 5 L fermenter, the titer of β-amyrin reached 2.6 g/L, which is the highest reported to date. The study provides a new perspective to engineering yeasts as a platform for triterpene production.