Mining of terpene synthases in fennel (Foeniculum vulgare) and application of limonene and pinene synthase in engineered Escherichia coli

Foeniculum vulgare Mill. is an important medicinal and edible plant, whose volatile constituents are predominantly composed of monoterpenes and sesquiterpenes. However, studies on genes involved in terpenoid biosynthesis and their functional characterization in this species remain relatively limited. In this study, integrated metabolomic and transcriptomic analyses were conducted to systematically elucidate terpenoid metabolites and their associated biosynthetic genes in F. vulgare. A total of 53 terpenoid metabolites and 109 putative terpene biosynthetic genes were identified. Five candidate terpene synthase (TPS) genes were identified from full-length transcriptome sequencing and functionally characterized using an Escherichia coli heterologous expression system. FvTPS1 exhibited limonene synthase activity, whereas FvTPS2 primarily produced α-pinene together with minor amounts of β-pinene. Shake-flask fermentation experiments showed that the titer of limonene reached 295.75 ± 35.63 mg/L, while α-pinene production reached 254.01 ± 12.72 mg/L. Overall, this study provides a comprehensive overview of the terpenoid metabolome and transcriptome of F. vulgare, and reports for the first time the identification of limonene synthase and pinene synthase in this species. These findings offer valuable functional gene resources for the metabolic engineering and industrial production of high-value monoterpenes derived from F. vulgare.