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Unusual (2R,6R)-bicyclo[3.1.1]heptane ring construction in fungal α-trans-bergamotene biosynthesis.
Wen YH
,
Chen TJ
,
Jiang LY
,
Li L
,
Guo M
,
Peng Y
,
Chen JJ
,
Pei F
,
Yang JL
,
Wang RS
,
Gong T
,
Zhu P
.
Abstract
Bergamotenes are bicyclo[3.1.1]heptane sesquiterpenes found abundantly in plants and fungi. Known bergamotene derivatives all possess (2S,6S)-bergamotene backbone. In this study, two (+)-α-trans-bergamotene derivatives (1 and 2) with unusual (2R,6R) configuration were isolated and elucidated from marine fungus Nectria sp. HLS206. The first (+)-α-trans-bergamotene synthase NsBERS was characterized using genome mining and heterologous expression-based strategies. Based on homology search, we characterized another (+)-α-trans-bergamotene synthase LsBERS from Lachnellula suecica and an (+)-α-bisabolol synthase BcBOS from Botrytis cinerea. We proposed that the cyclization mechanism of (+)-α-trans-bergamotene involved endo-anti cyclization of left-handed helix farnesyl pyrophosphate by (6R)-bisabolyl cation, which was supported by molecular docking. The biosynthesis-based volatiles (3-6) produced by heterologous fungal expression systems elicited significant electroantennographic responses of Helicoverpa armigera and Spodoptera frugiperda, respectively, suggesting their potential in biocontrol of these pests. This work enriches diversity of sesquiterpenoids and fungal sesquiterpene synthases, providing insight into the enzymatic mechanism of formation of enantiomeric sesquiterpenes.
Figure 1. Structure elucidation of compounds 1, 2 and 2′ isolated from the marine fungus Nectria sp. HLS206(A–C) (A) Structures of compounds 1, 2 and 2′; (B) Key COSY, HMBC and ROESY correlations of 1 and 2; (C) X-Ray ORTEP drawing of 1.
Figure 2. The sesquiterpenome of Nectria sp. HLS206(A and B) (A) Schematic representation of the nec and lach gene clusters and their amino acid sequence identity (The genes necD and lachB encode NsBERS and LsBERS, respectively); (B) The mining and identification of STSs in Nectria sp. HLS206. The n-dodecane layers of AN-nec04, AN-necD and AN-nec26 expressed strains were analyzed by GC-MS. The asterisks represent minor products of AN-nec26 and the triangle represents dodecanol at 10.35 min.
Figure 3. Phylogenetic analysis of fungal sesquiterpene synthasesFma-TC of UbiA superfamily was chosen as the outgroup. Branches are labeled with triangles representing the percentage of 1000 bootstrap replicates. The initial cyclization mechanism catalyzed by identified enzymes are indicated in square brackets. Hyp3 (1,8-cineole synthase) from Hypoxylon sp. in clade III is the first identified fungal monoterpene synthase and its mutants N136S and N136A can yield sesquiterpene products. TVY81921.1 corresponds to LachB in Figure 2A.
Figure 4. Heterologous expression of NsBERS-like STSs (NsBERS, TVY81921.1 and XP_001546971.2) in S. cerevisiae BJ5464(A) Schematic representation of the engineered sesquiterpenoid biosynthetic pathway in S. cerevisiae. The key enzymes HMGR and ERG20 were overexpressed to produce sufficient FPP for the production of sesquiterpenoids.(B) The function characterization of TVY81921.1 and XP_001546971.2. The n-octane layers of S. cerevisiae engineered strains were analyzed by GC-MS. The asterisks represent minor product of XP_001546971.2.
Figure 5. The proposed left-handed endo-anti cyclization mechanism of NsBERS, LsBERS and BcBOS(A) (2E,6E)-FPP; (B) (3R)-NPP; and (C) (+)-α-trans-bergamotene were docked into NsBERS model, while (D) (2E,6E)-FPP; (E) (3S)-NPP; and (F) (−)-α-trans-bergamotene were docked into LaBERS model. The crystal structure of (4S)-limonene synthase complex (PDB: 2ONH) was used as the template for homology modeling of LaBERS.
Figure 6. The EAG responses (mV) of Helicoverpa armigera and Spodoptera frugiperda were elicited by (+)-α-trans-bergamotene (3), (−)-trichoacorenol (4), (3R,6E)-nerolidol (5) and (+)-α-bisabolol (6)The EAG responses are represented as mean ± SEM.
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