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Agarwood is an aromatic resin produced by Aquilaria sinensis and other plants of the Thymelaeaceae family in response to external injury. It is valued both medicinally and as a fragrance material. Sesquiterpenoids are key components responsible for the characteristic aroma and bioactivity of agarwood, but their biosynthetic pathway remains incompletely elucidated. In this study, three sesquiterpene synthase genes AsTPS2, AsTPS10, and AsTPS41, were cloned and identified from methyl jasmonate (MeJA)-induced callus of A. sinensis. Recombinant proteins were obtained by expression and purification in Escherichia coli. In vitro enzymatic assays revealed that AsTPS2 and AsTPS10 exhibit similar functions: Both catalyze farnesyl diphosphate (FPP) to produce α-humulene and β-caryophyllene, and also convert geranyl diphosphate (GPP) into acyclic monoterpenes (geraniol and nerol) as well as monocyclic monoterpenes (α-terpineol and isopulegol). AsTPS41 catalyzes FPP to form β-elemene, α-guaiene, and δ-guaiene, while also acting on GPP to generate geraniol, nerol, and α-terpineol. Protein modeling and docking of AsTPS2 and AsTPS10 using AlphaFold3 and AutoDock Vina indicated that, despite sharing 83.5% sequence identity, residues lining their active cavities are completely identical, supporting their overlapping catalytic activities. Further site-directed mutagenesis was performed on key amino acids lining the active cavity of AsTPS2. The single-site mutant Y411A generated two new products, β-elemene and shyobunol, while retaining the functionality of AsTPS2. Building upon Y411A, the double-site mutant G410A-Y411A produced four additional new products: α-Elemol, nerolidol, trans-farnesol, and pogostol. In the single-site mutant C449A, the proportion of β-caryophyllene increased from 7.2% in wild-type AsTPS2 to 55.1%. Based on these findings, a Saccharomyces cerevisiae expression system was established for AsTPS2, its mutant AsTPS2C449A, and AsTPS41. Following isolation and structural analysis by nuclear magnetic resonance (NMR), the products α-humulene, β-caryophyllene, and δ-guaiene were identified. These results provide insights into the molecular mechanisms underlying agarwood formation and lay a foundation for further research on enzymes involved in the post-modification steps of agarwood sesquiterpenoid biosynthesis.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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