Sacred lotus is widely used in the agricultural, nutraceutical, and pharmaceutical industries. Terpenes are not only crucial components of sacred lotus essential oil, but also serve as signaling molecules involved in plant–environment interactions. However, the biosynthesis of terpenes in sacred lotus has not yet been reported. Thus, gene-directed heterologous mining and combinatorial biosynthesis methods were used in this study to systematically characterize the function of terpene synthase genes in the sacred lotus. As a result, two monoterpene, 11 sesquiterpene, and three diterpene products were synthesized, and a highly efficient γ-eudesmol synthase was discovered. In addition, a mechanistic study revealed that N314 is the key amino acid responsible for the secondary cyclization that produces γ-eudesmol. In vitro assays demonstrated that γ-eudesmol exhibited substantial insecticidal and antimicrobial activities. Furthermore, de novo biosynthesis of γ-eudesmol was achieved in a yeast chassis through a series of metabolic engineering strategies, reaching a titer of 801.66 mg/L in a shake flask, the highest yield reported to date. The present study uncovered the biosynthesis of terpenes in sacred lotus, as well as successfully synthesized the bioactive compound γ-eudesmol by synthetic biology. This comprehensive strategy can be readily adapted for investigation and the production of other valuable plant-derived natural products.

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