Improved pinene production in a recombinant yeast by fusion linker optimization and chaperon coexpression

Pinene is an active natural monoterpene from plants and has important applications in flavorings, fragrances,and pesticides. Especially, pinene dimers are regarded as renewable fuels with high density. However, the microbial pinene production was limited by the low activity pinene synthase. In this study, the pinene synthase activity was improved by fusion linker optimization and chaperon coexpression. To construct the pinene pathway in Saccharomyces cerevisiae, YPL062W gene was deleted to increase the MVA pathway precursor acetyl-CoA. Truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMG1), isopentenyl-diphosphate isomerase (IDI1), and farnesyl diphosphate synthase mutant (ERG20F96W−N127W) were then integrated to improve the GPP pool. Pinene synthase tPt1 was expressed in the constructed engineered yeast, and the titer of pinene reached 0.166 mg/L. GPP is the direct precursor of pinene, ERG20ww and tPt1 were fused by different linkers and orders to improve the accessibility of GPP. Pinene titer reached 9.94 mg/L by fusion these proteins in the order of ERG20ww and tPt1 and with a flexible linker (G)8. After that, several chaperons were coexpressed and the chaperon Sil1p improved the pinene titer to 10.2 mg/L with a yield of 1.63 mg/L·OD600. The results presented here provide novel information on the applications of protein fusion and protein chaperons in microbial pinene production.