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A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18‐/14‐membered sesterterpenoids including five new ones and two 14‐membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3‐ and 2.5‐fold, respectively, more sesterterpenoids than the wild‐type enzyme were engineered. Both 18‐ and 14‐membered ring sesterterpenoids displayed significant inhibitory activity on the IL‐2 and IFN‐γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well‐trodden territory of plant terpenoid biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: Eupatorium adenophorum is a malignant invasive plant possessing extraordinary defense potency, but its chemical weaponry and formation mechanism have not yet been extensively investigated.We identified six cadinene sesquiterpenes, including two volatiles (amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol) and four nonvolatiles (9‐oxo‐10,11‐dehydroageraphorone, muurol‐4‐en‐3,8‐dione, 9‐oxo‐ageraphorone and 9β‐hydroxy‐ageraphorone), as the major constitutive and inducible chemicals of E. adenophorum. All cadinenes showed potent antifeedant activity against a generalist insect Spodoptera exigua, indicating that they have significant defensive roles.We cloned and functionally characterized a sesquiterpene synthase from E. adenophorum (EaTPS1), catalyzing the conversion of farnesyl diphosphate to amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol, which were purified from engineered Escherichia coli and identified by extensive nuclear magnetic resonance (NMR) spectroscopy. EaTPS1 was highly expressed in the aboveground organs, which was congruent with the dominant distribution of cadinenes, suggesting that EaTPS1 is likely involved in cadinene biosynthesis. Mechanical wounding and methyl jasmonate negatively regulated EaTPS1 expression but caused the release of amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol. Nicotiana benthamiana transiently expressing EaTPS1 also produced amorpha‐4,7(11)‐diene and (–)‐amorph‐4‐en‐7‐ol, and showed enhanced defense function.The findings presented here uncover the role and formation of the chemical defense mechanism of E. adenophorum – which probably contributes to the invasive success of this plant – and provide a tool for manipulating the biosynthesis of biologically active cadinene natural products. [ABSTRACT FROM AUTHOR]

Cedrol is an extremely versatile sesquiterpene alcohol that was approved by the Food and Drug Administration of the United States as a flavoring agent or adjuvant and has been commonly used as a flavoring ingredient in cosmetics, foods and medicine. Furthermore, cedrol possesses a wide range of pharmacological properties including sedative, anti-inflammatory and cytotoxic activities. Commercial production of cedrol relies on fractional distillation of cedar wood oils, followed by recrystallization, and little has been reported about its biosynthesis and aspects of synthetic biology. Here, we report the cloning and functional characterization of a cedrol synthase gene (Lc-CedS) from the transcriptome of the glandular trichomes of a woody Lamiaceae plant Leucosceptrum canum. The recombinant Lc-CedS protein catalyzed the in vitro conversion of farnesyl diphosphate into the single product cedrol, suggesting that Lc-CedS is a high-fidelity terpene synthase. Co-expression of Lc-CedS , a farnesyl diphosphate synthase gene and seven genes of the mevalonate (MVA) pathway responsible for converting acetyl-CoA into farnesyl diphosphate in Escherichia coli afforded 363 μg/L cedrol as the sole product under shaking flask conditions. Transient expression of Lc-CedS in Nicotiana benthamiana also resulted in a single product cedrol with a production level of 3.6 μg/g fresh weight. The sole production of cedrol by introducing of Lc-CedS in engineered E. coli and N. benthamiana suggests now alternative production systems using synthetic biology approaches that would better address sufficient supply of cedrol. A sesquiterpene cyclase for sole production of cedrol using synthetic biology approaches. Image 1 • A high-fidelity cedrol synthase was cloned and identified from Leucosceptrum canum. • Escherichia coli harboring Lc-CedS produced 363 μg/L cedrol as a single product. • Nicotiana benthamiana expressing Lc-CedS produced cedrol with a yield of 3.6 μg/g FW. [ABSTRACT FROM AUTHOR]