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The sesquiterpene β-bisabolene possessing R and S configurations is commonly found in plant essential oils with antimicrobial and antioxidant activities. Here, we report the cloning and functional characterization of a (R)-β-bisabolene synthase gene (CcTPS2) from a Lamiaceae medicinal plant Colquhounia coccinea var. mollis. The biochemical function of CcTPS2 catalyzing the cyclization of farnesyl diphosphate to form a single product (R)-β-bisabolene was characterized through an engineered Escherichia coli producing diverse polyprenyl diphosphate precursors and in vitro enzyme assay, indicating that CcTPS2 was a high-fidelity (R)-β-bisabolene synthase. The production of (R)-β-bisabolene in an engineered E. coli strain harboring the exogenous mevalonate pathway, farnesyl diphosphate synthase and CcTPS1 genes was 17 mg/L under shaking flask conditions. Ultimately, 120 mg of purified (R)-β-bisabolene was obtained from the engineered E. coli, and its structure was elucidated by detailed spectroscopic analyses (including 1D and 2D NMR, and specific rotation). Four chimeric enzymes were constructed through domain swapping, which altered the product outcome, indicating the region important for substrate and product specificity. In addition, (R)-β-bisabolene exhibited anti-adipogenic activity in the model organism Caenorhabditis elegans and antibacterial activity selectively against Gram-positive bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Terpenoids are the largest class of natural products with complex structures and extensive bioactivities; their scaffolds are generated by diverse terpenoid synthases (TPSs) from a limited number of isoprenoid diphosphate precursors. Promiscuous TPSs play important roles in the evolution of terpenoid chemodiversity, but they remain largely unappreciated. Here, an extremely promiscuous terpenoid synthase (CcTPS1) of the TPS-b subfamily was cloned and functionally characterized from a leaf-specific transcriptome of the Lamiaceae plant Colquhounia coccinea var. mollis . CcTPS1 is the first sester-/di-/sesqui-/mono-TPS identified from the plant kingdom, accepting C 25 /C 20 /C 15 /C 10 diphosphate substrates to generate a panel of sester-/di-/sesqui-/mono-terpenoids. Engineered Escherichia coli expressing CcTPS1 produced three previously unreported terpenoids (two sesterterpenoids and a diterpenoid) with rare cyclohexane-containing skeletons, along with four sesquiterpenoids and one monoterpenoid. Their structures were elucidated by extensive nuclear magnetic resonance spectroscopy. Nicotiana benthamiana transiently expressing CcTPS1 also produced the diterpenoid and sesquiterpenoids, demonstrating the enzyme's promiscuity in planta . Its highly leaf-specific expression pattern combined with detectable terpenoid products in leaves of C . coccinea var. mollis and N. benthamiana expressing CcTPS1 suggested that CcTPS1 was mainly responsible for diterpenoid and sesquiterpenoid biosynthesis in plants. CcTPS1 expression and the terpenoid products could be induced by methyl jasmonate, suggesting their possible role in plant-environment interaction. CcTPS1 was localized to the cytosol and may differ from mono-TPSs in subcellular compartmentalization and substrate tolerance. These findings will greatly aid our understanding of plant TPS evolution and terpenoid chemodiversity; they also highlight the enormous potential of transcriptome mining and heterologous expression for the exploration of unique enzymes and natural products hidden in plants., (© 2021 The Author(s).)

Eurysoloids A ( 1 ) and B ( 2 ), two novel diastereomeric sesterterpenoids possessing a pentacyclic 5/6/5/10/5 framework with an unusual macrocyclic ether system, were isolated from Eurysolen gracilis Prain. Their structures were unambiguously determined by spectroscopic, single-crystal X-ray diffraction and DP4+ analyses. A plausible biosynthetic pathway for compounds 1 and 2 was proposed. Both compounds exhibited immunosuppressive activity via inhibiting the production of cytokine IFN-γ of T cells, and compound 2 inhibited adipogenesis in 3T3-L1 adipocytes.

Chemical investigation on the root of Phlomoides betonicoides led to the isolation of six undescribed diterpenoid glycosides, phlomoidesides A-F, along with two known ones using various chromatographic techniques. The structures of these compounds were determined by extensive spectroscopic analyses (including 1D, 2D-NMR and HRMS), single crystal X-ray diffraction, and calculated 13 C NMR. The glycoside modifications of phlomoidesides A-F are rare in natural products, and a plausible biosynthetic pathway for these unusual glycosides was proposed. Phlomoidesides A, D, F, and phlomisosides V, Ш were cytotoxic against three human tumor cell lines, NCI-H1975, HepG2 and MCF-7, with IC 50 values ranging from 7.5 to 75.7 μM. Phlomoideside B only showed weak cytotoxicity against NCI-H1975, with IC 50 of 53.0- μM., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Plant glandular trichomes (GTs) are adaptive epidermal structures that synthesize and accumulate diverse specialized metabolites well-known as defense chemicals against biotic attacks, but their roles against abiotic challenges including UV radiation and cold climates remain largely unexplored. Colquhounia vestita Wall is a Chinese-Himalayan Lamiaceae plant with dense peltate and capitate GTs on its leaf and stem surfaces under a scanning electron microscope. Three diterpenoid acids, including a clerodane 5-epi-hardwickiic acid and two labdanes polyalthic acid and E-communic acid, were identified from the peltate GTs of C. vestita through laser microdissection coupled with UPLC-MS/MS. Under UV radiation and cold stresses, the major GT component polyalthic acid increased the biomass of Arabidopsis thaliana seedlings and decreased their malondialdehyde content. Furthermore, polyalthic acid promoted photosynthetic efficiency and the expression of genes encoding peroxidative enzymes under UV radiation, and stimulated Ca 2+ elevation and the expression of calmodulin binding transcription activator gene CAMTA3 and two downstream cold-responsive genes CBF3 and RD29A under cold stress. Therefore, polyalthic acid in GTs is likely to endow the plant with enhanced tolerance to UV radiation and cold stresses, which extends the current understanding of the function of GT compounds in plant adaptation to abiotic environments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Leaves of Leucosceptrum canum harbor abundant toxic aromatic abietanoids, and they are rarely attacked by insect herbivores, except for the larvae of Nacna malachitis . The excrements of the insect that fed on L. canum leaves were investigated, leading to the isolation and identification of two unprecedented 11,12- seco -abietane diterpene polyenes: nacnabietanins A ( 1 ) and B ( 2 ). This discovery heralds a unique detoxification mechanism of plant aromatic abietanoids by insects through enzymatic cleavage of stable benzene rings into more easily degraded polyenes.

Obesity that is highly associated with numerous metabolic diseases has become a global health issue nowdays. Plant sesterterpenoids are an important group of natural products with great potential; thus, their bioactivities deserve extensive exploration. RNA-seq analysis indicated that leucosceptroid B, a sesterterpenoid previously discovered from the glandular trichomes of Leucosceptrum canum, significantly regulated the expression of 10 genes involved in lipid metabolism in Caenorhabditis elegans. Furthermore, leucosceptroid B was found to reduce fat storage, and downregulate the expression of two stearoyl-CoA desaturase (SCD) genes fat-6 and fat-7, and a fatty acid elongase gene elo-2 in wild-type C. elegans. In addition, leucosceptroid B significantly decreased fat accumulation in both fat-6 and fat-7 mutant worms but did not affect the fat storage of fat-6; fat-7 double mutant. These findings indicated that leucosceptroid B reduced fat storage depending on the downregulated expression of fat-6, fat-7 and elo-2 and thereby inhibiting the biosynthesis of the corresponding unsaturated fatty acid. These findings provide new insights into the development and utilization of plant sesterterpenoids as potential antilipemic agents., (Copyright © 2019 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)