Summary
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.
Subject areas: Biosynthesis, Mycology
Graphical abstract
Highlights
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Bergamotene derivatives with unusual (2R,6R) configurations from marine fungus
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(+)-α-trans-bergamotene synthase NsBERS as a gateway to identify LsBERS and BcBOS
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The endo-anti cyclization of left-handed helix FPP involving (6R)-bisabolyl cation
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Volatiles elicited significant EAG responses suggesting their biocontrol potential
Biosynthesis; Mycology
Introduction
Terpenoids are derived from C5 isoprene units and possess intriguing structural diversity (Christianson, 2008). More than 80,000 terpenoids have been identified to date (Christianson, 2017). Sesquiterpenoids represent the largest subgroup of terpenoids which have varied applications in pharmaceutical (e.g. artemisinin), biofuels (e.g. farnesene and bisabolene), flavor (e.g. carotenoids), and fragrance (e.g. nerolidol) industries (Miller and Allemann, 2012; Peralta-Yahya et al., 2011). As the well-known constituents of plant essential oils, bergamotenes are a type of bicyclic sesquiterpenes. Some bioactive bergamotene lactones such as massarinolin A (Oh et al., 1999), expansolides C and D (Ying et al., 2017), and purpurolides A and B (Wang et al., 2018) have been isolated from fungi. In the last few decades, bergamotene-type sesquiterpenes having backbones with (2S,6S,7R/S) absolute configuration derived from (6S)-bisabolyl cation have been described and important examples include (−)-α-trans/cis-bergamotene and β-trans/cis-bergamotene (Cane et al., 1990b; Coates et al., 1988; Gibson and Erman, 1969). In this study, we isolated two new (+)-α-trans-bergamotene derivatives 1 and 2 from marine fungus Nectria sp. HLS206. These compounds possess unusual (2R,6R) absolute configurations, which challenged the concept that bergamotene skeleton was exclusively cyclized from (6S)-bisabolyl cation (Li et al., 2019; Sallaud et al., 2009).
In contrast to the increasing number of terpenoids being reported, the characterization of terpene synthases (TPSs) is yet to be explored comprehensively. Nevertheless, in the post-genomic era, the blossoming of sequencing technology and bioinformatics provides the opportunity for the identification of fungal TPSs. The active site contour of TPS serves as a template for catalysis, ensuring that the substrate adopts proper conformation yielding the correct product by complex cascade reaction (Christianson, 2017; Tantillo, 2011). Known fungal sesquiterpene synthases (STSs) mainly belong to class I TPSs which catalyze an ionization-dependent cyclization of farnesyl diphosphate (FPP) and are characterized by signature metal binding motifs DDXXD and NSE which are located on helix D and helix H, respectively (Christianson, 2017).
So far, several plant-derived promiscuous bergamotene synthases belonging to soluble class I TPSs have been identified (Jones et al., 2011; Landmann et al., 2007; Sallaud et al., 2009). However, the only identified fungal bergamotene synthase Fma-TC (β-trans-bergamotene synthase) best known for its significant role in the biosynthesis of fumagillin belongs to the UbiA superfamily (Lin et al., 2013). To the best of our knowledge, no fungal (−)-α-trans-bergamotene synthase has been characterized to date, let alone (+)-α-trans-bergamotene synthase. To get more insight into this kind of enzyme and to understand its enzymatic mechanism, we sequenced the whole genome of Nectria sp. HLS206. By genome mining and heterologous expression strategies, we found and characterized the first (+)-α-trans-bergamotene synthase NsBERS (NecD). Upon sequencing and phylogenetic analysis, we identified another (+)-α-trans-bergamotene synthase LsBERS (GenBank: TVY81921.1) and BcBOS (GenBank: XP_001546971.2) that produced (3R)-nerolidol and (+)-α-bisabolol with unusual configurations. We proposed that the enzymatic mechanism involved endo-anti cyclization of a left-handed helix FPP, which was supported by molecular docking.
Results
Isolation and structure elucidation of new bergamotene derivatives necbergamotenoic acids A and B
Two new (+)-α-trans-bergamotene derivatives with unusual (2R,6R) configurations named necbergamotenoic acid A (1) and necbergamotenoic acid B (2, its ionized form 2′) were isolated from marine Nectria sp. HLS206 (Figure 1A), and their structures were elucidated by a combination of NMR (Figure 1B) and X-ray diffraction analysis (Figure 1C).
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.
Necbergamotenoic acid A (1), colorless crystals with +51.5 (c 0.2, CH3OH), had the molecular formula C15H20O4 based on HRESIMS data (m/z 287.1246 [M + Na]+, calcd for C15H20O4Na, 287.1254, Figure S8), indicating six degrees of unsaturation. Analysis of the 1H, 13C and heteronuclear single quantum coherence (HSQC) NMR data (Table 1, Figures S10–S12) revealed the presence of two methyl groups, four methylene carbons, four methine carbons (which included two olefinic methine carbons), and five quaternary carbons (which included two disubstituted olefinic carbons and two carboxyl carbons). These results indicated that compound 1 might be a sesquiterpene containing two rings with two double bonds and two carboxylic acid groups. The heteronuclear multiple bond correlations (HMBCs) from H-4 to C-2, C-6 and C-15, from 15-CH3 to C-2 and C-4, from H-1a/1b to C-3 and C-5, from H-1a to C-14, from H-2 to C-4 and C-15 (Figure S13), suggested that compound 1 was α-bergamotane sesquiterpene with bicyclo[3.1.1]heptane ring (Oh et al., 1999), in which 14-CH3 was oxygenated into carboxylic acid. Besides, HMBCs from 13-CH3 to C-10, C-11 and C-12, from H-10 to C-8, C-12 and C-13, from H-9 to C-7 and C-11, from H-8 to C-2, C-6, C-10 and C-14, indicated the presence of α,β-unsaturated carboxylic acid group in the side chain (Figures 1B and S13). The double bond between C-10 and C-11 was assigned as E geometry based on the strong rotating frame overhauser effect spectroscopy (ROESY) correlation between H-9 and 13-CH3 (Figure S15). The homonuclear correlation spectroscopy (COSY) spectrum (Figures 1B and S14) revealed two spin systems (-4CH5CH26CH1CH22CH- and -8CH29CH210CH-), which further elucidated the planar structure of 1 as shown in Figure 1A. The single crystals of 1 were generated, and X-ray crystallography was performed subsequently using Gu Kα radiation, which confirmed the structure and determined the absolute configuration of 1 (2R,6R,7R) (Figure 1C, Table S1). To our knowledge, this is the first time that a natural bergamotene derivative in this configuration has been identified.
Table 1.
The 1H NMR (500 MHz) and13C NMR (125 MHz) data of necbergamotenoic acid A (1), necbergamotenoic acid B (2) and its ionized form (2′)
No | 1a |
2b |
2′b |
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δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | δH mult. (J in Hz) | δC | |
1a | 1.28, d (8.0) | 29.7 | 1.23, d (8.5) | 31.9 | 1.21, d (8.0) | 32.0 |
1b | 2.28, dd (8.0, 6.0) | 2.38, dd (8.5, 6.0) | 2.36, m | |||
2 | 2.26, d (6.0) | 43.5 | 2.30, m | 44.0 | 2.30–2.32c, m | 43.8 |
3 | 146.1 | 145.0 | 145.0 | |||
4 | 5.15, brs | 116.5 | 5.27, d (1.5) | 117.8 | 5.26, brs | 117.9 |
5a | 2.61, m | 31.7 | 2.20, m | 31.9 | 2.20, m | 32.0 |
5b | 2.24, m | 2.23, m | ||||
6 | 2.57, m | 39.3 | 2.36, m | 41.0 | 2.30–2.32c, m | 41.2 |
7 | 52.9 | 45.0 | 45.0 | |||
8a | 2.15, brt (6.0) | 33.4 | 1.95, dd (15.0, 3.5) | 42.2 | 1.95, dd (15.0, 4.0) | 42.8 |
8b | 2.05, dd (15.0, 9.5) | 1.98, dd (15.0, 8.5) | ||||
9 | 2.37, m | 24.9 | 4.70, ddd (9.5, 8.5, 3.5) | 67.1 | 4.63, ddd (9.5, 8.5, 4.0) | 67.3 |
10 | 6.81, t (6.0) | 144.9 | 6.74, dq (8.5, 1.0) | 145.9 | 6.53, dq (9.5, 1.0) | 141.1 |
11 | 128.0 | 128.3 | 133.7 | |||
12 | 173.7 | 171.6 | 177.6 | |||
13 | 1.78, s | 12.2 | 1.88, d (1.0) | 12.8 | 1.87, d (1.0) | 14.1 |
14a | 181.6 | 3.72, d (10.5) | 63.7 | 3.71, d (10.5) | 63.9 | |
14b | 3.37, d (10.5) | 3.30, d (10.5) | ||||
15 | 1.71, d (1.0) | 22.6 | 1.73, d (1.5) | 22.8 | 1.73, d (1.5) | 22.8 |
Recorded in CDCl3.
Recorded in CD3OD.
Overlapped.
Necbergamotenoic acid B (2) was obtained as yellowish gum with [α]20 D +100.9 (c 0.2, CH3OH), and its molecular formula was established as C15H22O4 on the basis of HRESIMS at m/z 289.1405 [M + Na]+ (calcd for C15H22O4Na, 289.1410, Figure S17), requiring five degrees of unsaturation. Compared with the 1H NMR and 13C NMR spectra of compound 1, one oxygenated methine signal at δH 4.70, δC 67.1, two oxygenated methylene signals at δH 3.72, 3.37, δC 63.7 appeared and one carboxyl carbon signal at δC 181.6 was missing (Table 1, Figures S19–S21), which indicated the presence of two hydroxyl groups in the α-bergamotenoic acid. Further analysis of COSY and HMBC spectra determined the hydroxyl groups were assigned to C-9 and C-14 (Figures 1B, S22 and S23). The relative stereochemistry of the skeleton of compound 2 was determined as (10E)-α-trans-bergamotenoic acid based on ROESY correlations (Figures S24) between H-14a/H-4 and H-9/13-CH3, which was the same as 1. Since the qualified crystals of 2 were not obtained and any useful information could not be acquired by Mosher’s method, we deduced the absolute configuration of the skeleton of 2 as 2R,6R,7R based on the biosynthetic pathway, but the absolute configuration of C-9 is still waiting for the determination (Figure 1A).
The ionized form of necbergamotenoic acid B (2′) was also isolated as a yellowish gum. Its HRESIMS spectrum showed the positive ion peak at m/z 289.1403 (calcd for C15H22O4Na, 289.1410, Figure S26), which was the same as that of 2. In the IR spectrum of 2′ (Figure S27), the carbonyl group had two bands situated at 1573 and 1426 cm−1 that are typical of ionized groups (RCOO−) (Max and Chapados, 2004). On basis of 1D NMR (1H and 13C NMR, Table 1, Figures S28, S29 and 2D NMR (HSQC, HMBC, COSY and ROESY, Figures S30–S33) spectra, the structure of 2′ was confirmed and all the1H and 13C NMR chemical shifts were assigned as Table 1. The carbon signals of C-11 and C-12 for α,β-unsaturated carboxylic acid group in ionized form (2′) shifted downfield about 5 ppm, which was consistent with the results reported in the literature (Cistola et al., 1982).
The sesquiterpenome of Nectria sp. HLS206
To explore how α-trans-bergamotene was synthesized in fungi, specifically (+)-α-trans-bergamotene, the whole genome of Nectria sp. HLS206 was sequenced and submitted to anti-SMASH 4.0 (Blin et al., 2017) for prediction of terpene biosynthetic gene clusters (BGCs). Excluding prenyltransferases and squalene synthases, only two candidate STSs (Nec04 and Nec26) were predicted. However, no oxidative tailoring enzyme was found adjacent to them. Using local BLAST with over 50 fungal STSs probes, another candidate sesquiterpene synthase NecD (scaffold 23) was mined although it was targeted by only a few probes with low identities/positives (∼25%/45%). The gene annotation of upstream and downstream of necD revealed three cytochrome P450 monooxygenases (NecB, NecC and NecE), which could be responsible for the oxidation from (+)-α-trans-bergamotene to 1 and 2 (Figure 2A, Table S4). Furthermore, necA encodes a p53 inducible quinone oxidoreductase PIG3 involving cellular responses to oxidative stress (Porte et al., 2009) which seemed to be a self-resistance enzyme (SRE). As a result, we hypothesized that NecD may be responsible for the biosynthesis of (+)-α-trans-bergamotene skeleton.
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.
The Aspergillus nidulans host system has been proved to be capable of heterologous terpene production. To verify our above-mentioned hypothesis, we cloned nec04, nec26 and necD genes from HLS206 gDNA and conducted heterologous expression in A. nidulans for functional analysis. The organic layers covering on the fermentation were analyzed by GC-MS. In comparison with the mock transformant, the sole product of AN-necD at 9.9 min (3) and the main product of AN-nec26 at 12.9 min (4) were identified as bergamotene and trichoacorenol by NIST library respectively, while no new product was found in AN-nec04 (Figure 2B).
Compounds 3 and 4 were purified from AN-necD and AN-nec26 fermentation, respectively, and their structures were determined using NMR spectroscopy. The NMR data of 4 (Table S3, Figures S38 and S39) were consistent with reported data of trichoacorenol and [α]20 D −6 (c 0.1, CHCl3) of 4 was consistent with previous study [α]20 D −5.2 (c 0.12, CHCl3) (Brock and Dickschat, 2011). Thus 4 was assigned as (−)-trichoacorenol and Nec26 was defined as NsTAS. The cyclization mechanism of (−)-trichoacorenol has been elaborated in a recent study (Rinkel and Dickschat, 2020).
By comparing the NMR data (Table S2, Figures S35 and S36) with the reported data (Sy and Brown, 1997), the relative structure of 3 was identified as α-trans-bergamotene consistent with that of compound 1. Furthermore, the specific rotation of 3 was positive and opposite to that of (−)-α-trans-bergamotene (Chapuis et al., 1998), implying that the absolute configuration of 3 was 2R,6R,7S (Figure 2B). The positive cotton effect at 209 nm of 3 was contrary to that of (−)-S-α-pinene in the CD spectrum (Figure S37) (Mason and Schnepp, 1973), further proving that the absolute configuration of 3 was consistent with that of 1. To the best of our knowledge, this is the first report of (+)-α-trans-bergamotene skeleton.
NsBERS converts (2E,6E)-FPP to (+)-α-trans-bergamotene
In comparison with the only known fungal bergamotene synthase Fma-TC with transmembrane helices, the NecD (hereby named NsBERS) was a 435-amino-acid protein containing the aspartate-rich motif (DDXXD/E), NSE motif and R, RY sensors (Figure S5), which are usually conserved in class I terpene synthases. By fusion with N-terminal MBP-His6 tag, the soluble fusion protein (91 kDa) was acquired by affinity chromatography (Figure S1). Incubation of NsBERS with FPP and Mg2+ resulted in the formation of sole product 3 (Figure S2). Additionally, Mn2+ and Co2+ can also be used as the cofactors of NsBERS and the optimal concentration of divalent metal ion was tested (Figure S3). The enzyme activity was insensitive to the concentration of Mg2+ (at the range from 0.005 mM to 500 mM). The total amount of product reached a maximum at concentrations of 0.2 mM Mn2+ and 1 mM Co2+, while higher concentrations led to activity loss. Substrate promiscuity assay showed that NsBERS cannot accept GPP and GGPP as substrates (Figure S4).
Phylogenetic analysis of fungal STSs and the NsBERS-like subclade
The exhaustive work by Schmidt-Dannert’s group has demonstrated the correlation between clade and cyclization mechanism in basidiomycetes STSs (clade I and clade IV) (Figure 3) (Agger et al., 2009; Wawrzyn et al., 2012), and has been seminal in guiding the development of in silico approaches for the directed discovery of new STSs with a specific cyclization mechanism (Quin et al., 2014; Zhang et al., 2020). However, the dispersive and unsystematic identifications reported till date do not allow cyclization mechanism or product type prediction rules to be set in place for ascomycetes STSs.
Figure 3.
Phylogenetic analysis of fungal sesquiterpene synthases
Fma-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.
As the first identified (+)-α-trans-bergamotene synthase, NsBERS shares the same initial 1,6 cyclization mechanism as NsTAS in clade IV (Figure 3). However, the clade IV conserved in 1,6 cyclization is predominantly constituted by basidiomycetes STSs than ascomycetes STSs and genes in clade IV seem to be involved in horizontal gene transfer from basidiomycetes to ascomycetes. NsBERS is away from this clade and located in clade III in which FgFS (Bian et al., 2018), FfAAS (Brock et al., 2013) and Hyp3 (Shaw et al., 2015) also exhibited 1,6 cyclization but shared only ∼26% identity with NsBERS (Figure S5). This phenomenon signified that there must exist STSs which are more phylogenetically related to NsBERS, therefore NsBERS may have the potential to facilitate the discovery of relevant fungal STSs. We performed a homology search in NCBI and got a set of uncharacterized NsBERS homologues belonging to the genera of Botrytis, Bipolaris, Metarhizium, Lachnellula, and so on separately (Table S6). After redundancy removal, the sequences with over 45% identity with NsBERS were chosen to analyze the phylogenetic relation, and a new subclade was formed (Figure 3). We hypothesized that this NsBERS-like subclade may well possess a similar cyclization mechanism and form similar products.
NsBERS serves as a gateway to identify LsBERS and BcBOS
To verify our hypothesis, TVY81921.1 from Lachnellula suecica (Giroux and Bilodeau, 2020) and XP_001546971.2 described as Bcstc4 from Botrytis cinerea B05.10 (Van Kan et al., 2017) (Figure 3, Table S6) were chosen for fast identification in Saccharomyces cerevisiae. As the key enzymes in mevalonate (MVA) pathway, HMGR, and FPP synthase (ERG20) were overexpressed to produce sufficient FPP for the production of sesquiterpenoids (Figure 4A). The GC-MS analysis of the organic layers covering the fermentation broth revealed that the product retention time of TVY81921.1 was consistent with that of NsBERS, while XP_001546971.2 mainly generated trans-nerolidol (47%), α-bisabolol (52%) and a minor product (Figure 4B).
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.
Further NMR analysis (Table S2) and the specific rotation indicated that the product of TVY81921.1 was (+)-α-trans-bergamotene ([α]20 D +20 (c 0.06, CHCl3)). Compound 5 (Table S3, Figures S40 and S41) was (3R,6E)-nerolidol ([α]20 D −16 (c 0.1, CHCl3)), and its specific rotation was consistent with [α]23D −12.5 (c 0.022, CHCl3) (Cane et al., 1990a). Compound 6 (Table S3, Figures S42 and S43) was (+)-α-bisabolol ([α]20 D +51.7 (c 0.06, CHCl3)), and the NMR data of which were different from those of epi-α-bisabolol (Chen et al., 2002) and its specific rotation was sufficiently close to the reported [α]22D +51.3 (c 0.06, CHCl3) (Bian et al., 2018; Gnther et al., 1993). Thus, TVY81921.1 as the second (+)-α-trans-bergamotene synthase was named as LsBERS, and XP_001546971.2 was designated as BcBOS which is the first identified fungal (+)-α-bisabolol synthase. Although (3R)-nerolidol and (+)-α-bisabolol were deemed to occur rarely as a constituent of natural products (Laurini et al., 2020), some related STSs derived from plants have been reported recently. For instance, the reports of maize TPS1 producing ∼29% (3R)-nerolidol (Schnee et al., 2002) and several (+)-α-bisabolol synthases from the genus Artemisia (Muangphrom et al., 2016, 2019) indicated the lack of comprehensive studies in the domain of sesquiterpene enantiomers.
LsBERS exhibited the same properties as NsBERS, and the in vitro assay of the 92 kDa soluble fusion protein also contributed to the formation of 3 (Figure S3). However, the ratio of BcBOS products was influenced by many factors such as the type and concentration of metal cofactor (Lopez-Gallego et al., 2010). The higher ratio of linear nerolidol can be detected in vitro when Mg2+ is used as the cofactor. Substitution of Mg2+ with Co2+ shifted the product profile to a higher (+)-α-bisabolol ratio, and higher concentration of Co2+ can contribute to higher ratio of cyclization product, implying that metal coordination interactions may play a significant role in governing the substrate binding conformation prior to the initiation of cyclization cascade (Christianson, 2017).
The proposed mechanism of (+)-α-trans-bergamotene
At this point in the study, NsBERS, LsBERS and BcBOS, which produced sesquiterpene backbones with unusual absolute configurations were characterized. Apparently, the C6 of 3 and 6 did not participate in further rearrangements and retained their configurations, that is, (+)-α-trans-bergamotene and (+)-α-bisabolol were both derived from (6R)-bisabolyl cation. These enzymes can be good models for studying the formation of (6R)-bisabolyl cation and pursing the difference with (6S)-bisabolyl cation. A general stereochemical model for bicyclic monoterpene proposed by Croteau et al. (Croteau, 1987) has been applied to elucidate the conformation control of (+)-bornyl diphosphate synthase (Whittington et al., 2002), 1,8-cineole synthase (Kampranis et al., 2007), (±)-α-pinene synthases (Croteau and Satterwhite, 1989) and (±)-limonene synthases (Kumar et al., 2017), respectively. A more recent computational chemistry study investigating the mechanism of santalenes produced by santalene synthase (SaSSy) has illustrated that the (6S)-bisabolyl cation is formed via endo-anti cyclization in the right-handed helix of FPP (Jindal and Sunoj, 2012) which is similar to those of (−)-α-pinene synthase and (−)-limonene synthase. Although (6R)-bisabolyl cation is possibly formed via the exo-anti cyclization of the right-handed helix of FPP, it is unfavorable for the subsequent C2–C7 bond formation. Therefore, we hypothesized that the (6R)-bisabolyl cation formed adopts the mirror image via endo-anti cyclization in the left-handed helix of FPP during the cyclization of (+)-α-trans-bergamotene (Figure 5).
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.
To verify the hypothesis, homology modeling of NsBERS was carried out by adopting AaTPS (PDB: 6LCD) as the template (He et al., 2020). The crystal structure of AaTPS only bound two Mg2+ and the DDXXD motif on the helix D was apparently away from the pyrophosphate (PPi), implying incomplete closure of the active site. Although this may lead to deviation in the position of PPi, the results of the molecular docking of (2E,6E)-FPP displayed almost consistent left-handed endo-anti conformation (Figure 5A). The alternative suitable substrate (3R)-NPP and the product (+)-α-trans-bergamotene also exactly resembled the predicted mechanism (Figures 5B and 5C). The bent contour of active cavity must play an important role in controlling the pre-folding state of the substrate, while sulfur-cation interaction (dative S → cation bonded state) (Dixit et al., 2017) may anchor the cation intermediate in a reactive conformation. Furthermore, LaBERS from Lavandula angustifolia (mainly producing (−)-α-trans-bergamotene with a ratio of 74%) was chosen to illustrate the symmetrical state (Figures 5D–5F), due to the fungus-derived (−)-α-trans-bergamotene synthase was still not identified. The stabilization effect of W263 and M280 on cation supported the right-handed conformation, but the vertical shape of active cavity cannot guarantee the formation of C2–C7 bond to its maximum extent, which led to formation of linear sesquiterpene byproducts.
Electroantennographic (EAG) responses of H. armigera and S. frugiperda to compounds 3–6
A large amount of evidence indicated that microbial volatiles are eco-friendly and have important applications in agricultural practices for sustainable development (Kanchiswamy et al., 2015). For instance, nerolidol has insecticidal activity (Chan et al., 2016) and has been reported as an important pheromone secreted by queens of higher termites (Havlickova et al., 2019). Hence, the EAG responses of H. armigera and S. frugiperda to biosynthesis-based volatiles (+)-α-trans-bergamotene (3), (−)-trichoacorenol (4), (3R,6E)-nerolidol (5) and (+)-α-bisabolol (6) were recorded. The results revealed that all of these volatiles can induce EAG responses (Figure 6). Especially, 5 elicited the highest responses of both H. armigera and S. frugiperda. To H. armigera, the EAG response values of females to (+)-α-trans-bergamotene were significantly higher than those of males, which suggested that 3 can attract female moths to oviposit (Figure 6A).
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.
Discussion
Polycyclic terpenoids usually have multiple chiral centers, and the chiral terpene pool has long served as the building blocks for chemical synthesis of complex natural products (Brill et al., 2017). However, a lot of sesquiterpenoids were identified only based on comparison of standards and NIST library, which overlooked the stereochemistry. In fact, it is essential to emphasize the pertinence of a more comprehensive understanding of stereochemistry of sesquiterpenoids, which will not only facilitate understanding the biogenesis of naturally occurring enantiomers but also will provide insight into enzymatic mechanism of precise stereochemistry control. In the last decades, bergamotene derivatives isolated from plants and fungi have been known to possess the (2S,6S) ring configurations. In this study, the isolation and identification of necbergamotenoic acids A and B (1 and 2) from marine fungus Nectria sp. HLS206 with (2R,6R) configurations broke this notion and enriched the chemical diversity of the bergamotene skeleton.
To investigate the biosynthetic mechanism, the whole genome of Nectria sp. HLS206 was sequenced and detailed bioinformatics-based analyses were carried out. By genome mining and heterologous expression in A. nidulans, we characterized the first (+)-α-trans-bergamotene synthase NsBERS with high fidelity. Upon homology search and phylogenetic analysis, we identified another (+)-α-trans-bergamotene synthase LsBERS and the first fungal (+)-α-bisabolol synthase BcBOS which indicated that NsBERS is a representative of untouched land and has the potential to facilitate discovery of relevant fungal STSs. The unusual stereochemistry of compounds 3, 5 and 6 revealed that the structure elucidation of sesquiterpene enantiomers has been ignored to a degree. In addition, the disregard for NsBERS-like subclade proved that fungal STSs are still relatively unexplored and comprehensive studies designed to get a more in-depth understanding are pertinent. Furthermore, the phylogenetic analysis showed that fungal STSs initiating 1,6 cyclization reaction were apparently grouped into two clades (clade III and clade IV). Apart from metal ion binding motifs, these two clades have no sequence similarity and tend to be conserved in some residues respectively (Figure S6), which may contribute to differences in overall fold of protein structures. It also gives a hint that genes of STSs in clade IV may be involved in horizontal gene transfer from basidiomycetes to ascomycetes. Future studies involving these two clades may provide insight into the mechanism and evolution of fungal 1,6 cyclization STSs.
Based on the general stereochemical model for bicyclic monoterpene, it was inferred that the endo-anti cyclization in the left-handed helix of FPP formed the (+)-α-trans-bergamotene by (6R)-bisabolyl cation, which was further supported by our molecular docking results. The superposition of (±)-limonene synthases and substrate analogues complexes revealed that it is likely that the steric hindrance leads to the different conformations which yield the divergent absolute configurations subsequently (Kumar et al., 2017). As a result, the identification of fungal (−)-α-trans-bergamotene synthase will probably facilitate the mechanism with more details.
Known as the constituent of plant essential oil, (−)-α-trans-bergamotene was found to play a dual role in flowers and leaves of Nicotiana attenuata, solving the dilemma when pollinators are also herbivores (Zhou et al., 2017). The EAG results showed that (+)-α-trans-bergamotene (3) elicited a significantly higher response of females than males of H. armigera, which signified that 3 can potentially attract female moths to oviposit suggesting its potential in biocontrol of H. armigera. More behavioral orientation-based assays are underway.
Fungal SRE has been known to be insensitive to the inhibitors (compounds encoded by BGC) and enable the producing organism to survive, illustrating that the SRE in the BGC can serve as a predictive window to the bioactivity of the natural product (Yan et al., 2020). Some representative examples of SREs including the herbicide target AstD (Yan et al., 2018), MetAP-2 of fumagillin cluster (Lin et al., 2013), and HMGR of lovastatin cluster (Kennedy et al., 1999) have been well studied. Our further bioinformatic analysis showed that NecA is the second copy of PIG3 encoded in the genome and has 44% identity with the housekeeping copy PIG3 (scaffold 6) in HLS206, implying that NecA is a variant of housekeeping PIG3. Apart from the nec cluster, we observed that the PIG3 variants, NsBERS-like enzymes and cytochrome P450 monooxygenases co-localized in many phytopathogen BGCs (Figure S7), for instance, the lach cluster (GenBank: QGMK01000402.1) where LsBERS (TVY81921.1) is located (Figure 2A, Table S5). Based on this, we speculated that the encoded compounds of these BGCs serve as PIG3 inhibitors accelerating infection and plant cell death by regulating the host reactive oxygen species (Peng et al., 2010; Williams et al., 2011). In addition, knocking down PIG3 has been reported to increase the sensitivity of non-small cell lung cancer cells (NSCLC) to docetaxel by dysregulating the dynamics of microtubules (Li et al., 2017), signifying that the putative PIG3 inhibitor compounds 1 and 2 may be potential adjuvants of docetaxel for NSCLC. We will be committed to confirming these potential bioactivities and revealing the underlying mechanisms.
Limitations of the study
In this study, we deciphered the biosynthesis of unusual (2R,6R)-α-trans-bergamotene, and proposed the cyclization mechanism which was supported by molecular docking. However, the docking results did not present the completely symmetrical state because of the differences between plant-derived STSs and fungus-derived STSs in overall fold, especially in active cavity. In addition, in comparison with the protein crystal structure analysis, homology modeling and molecular docking have limitations and are not precise enough for some details. Therefore, it is hard to predict which residue was the determinant of absolute configuration. Thus, the crystalization and three-dimensional analysis of both two types of enantiomeric synthases and their enzyme-substrate complexes will facilitate the enzymatic mechanism deciphering with more details.
STAR★Methods
Key resources table
REAGENT or RESOURCE | SOURCE | IDENTIFIER |
---|---|---|
Strains | ||
Nectria sp. HLS206 | Lab stock | N/A |
Aspergillus nidulans | (Ohashi et al., 2017) | A1145 |
Saccharomyces cerevisiae | (Ohashi et al., 2017) | BJ5464 |
E. coli T1 | Transgen biotech | Cat# CD501-03 |
E. coli BL21(DE3) | Transgen biotech | Cat# CD601-03 |
Chemicals, peptides, and recombinant proteins | ||
GPP | Sigma-Aldrich | Cat# G6772 |
FPP | Sigma-Aldrich | Cat# F6892 |
GGPP | Sigma-Aldrich | Cat# G6025 |
Critical commercial assays | ||
Fungal RNA Kit | Omega Bio-tek | Cat# R6840-01 |
cDNA Synthesis Kit | Transgen biotech | Cat# AE311-02 |
Q5 high fidelity DNA polymerase | NEB | Cat# M0491L |
Zymoprep Yeast Miniprep Kit | Zymo Research | Cat# D2001 |
recombinase Exnase II | Vazyme Biotech | Cat# C112 |
Deposited data | ||
CIF of compound 1 | this paper | CCDC: 2098349 |
nec cluster necA | this paper | GenBank: MZ672110 |
nec cluster necB | this paper | GenBank: MZ672111 |
nec cluster necC | this paper | GenBank: MZ672112 |
nec cluster necD | this paper | GenBank: MZ672113 |
nec cluster necE | this paper | GenBank: MZ672114 |
nec26 | this paper | GenBank: MZ672115 |
Oligonucleotides | ||
For details of oligonucleotides generated and used, see Table S7 | this work | N/A |
Recombinant DNA | ||
For details of plasmids generated and used, see Table S8 | this work | N/A |
Software and algorithms | ||
MAFFT | (Katoh et al., 2002) | https://mafft.cbrc.jp/alignment/software/ |
MEGA7 | (Kumar et al., 2016) | https://www.megasoftware.net/ |
2ndFind | N/A | https://biosyn.nih.go.jp/2ndfind/ |
NCBI conserved domain database | (Lu et al., 2020) | https://www.ncbi.nlm.nih.gov/cdd |
SWISS-MODEL | (Waterhouse et al., 2018) | https://swissmodel.expasy.org/ |
AutoDock Vina | (Trott and Olson, 2010) | http://vina.scripps.edu/ |
Resource availability
Lead contact
Further requests for information should be addressed to Ping Zhu at zhuping@imm.ac.cn.
Materials availability
Requests for materials should be made via the lead contact.
Experimental model and subject details
Strain and culture conditions
Nectria sp. HLS206 isolated from the marine sponge Gelliodas carnosa (Liu et al., 2010) and deposited at our laboratory was cultured in rice medium for isolation compounds 1 and 2. After 3 days of culturing in YPD medium, the gDNA of Nectria sp. HLS206 was isolated by CTAB for next generation sequencing. E. coli T1 was used for plasmid construction and E. coli BL21(DE3) was used for protein expression. A. nidulans A1145 and S. cerevisiae BJ5464 were used for heterologous expression.
Method details
General experimental procedures
Optical rotations were recorded using a Rudolph Research Analytical automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Circular dichroism (CD) was measured on a JASCO J-815 CD spectro polarimeters (JASCO Corporation, Tokyo, Japan). IR spectra were acquired by KBr disk method on a Shimadzu FTIR-8400S spectrometer (Shimadzu Co. Ltd., Tokyo, Japan). 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-500 spectrometer (Bruker Biospin Group, Karlsruhe, Germany) with TMS as an internal standard. Mass spectra were measured on Thermo Scientific Q Exactive Focus Orbitrap mass spectrometer (Waltham, MA, USA) for HRESIMS. GC-MS analysis was conducted by GCMS-TQ8050 (Shimadzu Co. Ltd., Tokyo, Japan) using the Rtx-5MS column (30 m × 0.25 mm × 0.25 μm).
Semi-preparative HPLC was conducted on an Agilent C18 column (250 × 10 mm I.D., 5 μm) equipped with a LC-6AD pump and a Shimadzu SPD-20A UV-Vis detector (Shimadzu Co. Ltd., Japan). Column chromatography (CC) was performed with silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical plant, Qing dao, China), ODS-C18 (50 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (18–110 μm, GE healthcare, Fairfield, CT, USA). Chromatographic or analytical grade solvents were used for isolation and separation procedures.
Extraction and isolation
The EtOAc extract (28.2 g) of the rice solid cultures of fungus HLS206 (5 kg) was first separated using ODS column, and eluted successively with 20%, 30%, 40%, 50%, 60%, 80% and 100% CH3OH in H2O to produce eight fractions (Fr. 1–8). Fr.4 was subjected to Sephadex LH-20 column eluted by CH3OH to give 16 subfractions (Fr. 4.1–4.16). Compounds 2′ (7.1 mg, Rt 24 min) and 2 (40.8 mg, Rt 36 min) were obtained from Fr. 4.9 through semi-preparative HPLC eluted by 30% CH3CN-H2O. From Fr. 4.12, we obtained compound 1 (23.6 mg, Rt 21 min) by semi-preparative HPLC, eluted by gradient CH3CN-H2O (0 min, 20%-10 min, 40%-30 min, 80%).
For purification of compounds 3–6, the n-octane layers were concentrated using a rotary evaporator, and terpenoids were purified using semi-preparative HPLC with acetonitrile and water as the mobile phase (Bian et al., 2018). The GC-MS procedure was described as previous research (Bian et al., 2017).
Necbergamotenoic acid A (1): colorless crystals; [α]20 D +51.5 (c 0.2, CH3OH); CD (c 0.1(w/v)%, CH3OH) 218.5 (2.44) nm, Figure S16; IR (KBr) νmax 3039, 2985, 2963, 2933, 2660, 1697, 1674, 1640, 1427, 1289, 1255, 926, 802, 739, 708, 573 cm−1, Figure S9; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, Table 1; (+)-HR-ESI-MS m/z 287.1246 [M + Na]+ (calcd for C15H20O4Na 287.1254).
Necbergamotenoic acid B (2): yellowish gum; [α]20 D +100.9 (c 0.2, CH3OH); CD (c 0.4(w/v)%, CH3OH) 215.5 (5.46) nm, Figure S25; IR (KBr) νmax 3271, 2931, 2840, 2688, 1692, 1650, 1438, 1249, 1026, 791, 754, 662 cm−1, Figure S18; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, Table 1; (+)-HR-ESI-MS m/z 289.1405 [M + Na]+ (calcd for C15H22O4Na 289.1410 ).
Ionizedform of Necbergamotenoic acid B (2′): yellowish gum; IR (KBr) νmax 3271(w), 2971(w), 2924(w), 1573(s), 1426(s), 1042, 1014, 924,781, 649, 620 cm−1, Figure S27; 1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, Table 1; (+)-HR-ESI-MS m/z 289.1403 [M]+ (calcd for C15H22O4Na 289.1410).
(+)-α-trans-bergamotene (3): colorless oil; [α]20 D +22.5 (c 0.2, CHCl3); CD (c 0.1(w/v)%, n-hexane) 209 (0.70) nm, Figure S37; IR (KBr) νmax 3380, 2954, 2924, 2955, 1725, 1461, 1377, 1263, 1223, 1077, 1026, 973, 799, 740 cm−1, Figure S34; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, Table S2; GC-MS, Figure 2B.
(−)-trichoacorenol (4): colorless oil; [α]20 D −6 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): 5.46 (m, H-2), 4.27 (m, H-4), 0.83 (d, J = 6.5 Hz, CH3-12), 0.93 (d, J = 6.5 Hz, CH3-13), 0.82 (d, J = 6.5 Hz, CH3-14), 1.77 (brs, CH3-15); 13C NMR (CDCl3, 125 MHz) data, Table S3; GC-MS, Figure 2B.
(3R)-nerolidol (5): colorless oil; [α]20 D −16 (c 0.1, CHCl3); 1H NMR (CDCl3, 500 MHz): 5.92 (dd, J = 18.0, 11.0 Hz, H-2), 5.22 (dd, J = 18.0 Hz, H-1a), 5.07 (dd, J = 11.0 Hz, H-1b), 5.15 (t, J = 6.5 Hz, H-6), 5.09 (t, J = 6.5 Hz, H-10), 1.27 (d, CH3-12), 1.59 (s, CH3-13, CH3-15), 1.59 (d, CH3-14), 1.67 (brs, CH3-15); 13C NMR (CDCl3, 125 MHz) data, Table S3; GC-MS, Figure 4B.
(+)-α-bisabolol (6): colorless oil; [α]20 D +51.7 (c 0.06, CHCl3); 1H NMR (CDCl3, 500 MHz) 5.37 (m, H-2), 5.12 (t, J = 6.8 Hz, H-10), 1.62 (s, CH3-12), 1.68 (s, CH3-13), 1.10 (s, CH3-14), 1.64 (s, CH3-15); 13C NMR (CDCl3, 125 MHz) data, Table S3; GC-MS, Figure 4B.
In silico analysis
For phylogenetic analysis, the multiple sequence alignment was generated using MAFFT and phylogenetic tree was generated in MEGA7 using Neighbor-Joining method based on the poisson model. The parameter of Gamma Distributed was set as 3 and pairwise deletion was adopted in missing data treatment. The gene annotation results were generated by 2ndFind and NCBI conserved domain database. Homology modelling was conducted by SWISS-MODEL. The molecular docking was generated by AutoDock Vina and visualized using PyMOL.
Heterologous expression in A. nidulans A1145 and S. cerevisiae BJ5464
The plasmids pYTU-Nec04, pYTU-NecD and pYTU-Nec26 for A. nidulans expression were assembled by yeast homologous recombination (Ohashi et al., 2017). The genes nec04, necD and nec26 carrying ∼200 bp terminators were amplified from the gDNA of HLS206 using primers pYTU-Nec04-F/R, pYTU-NecD-F/R and pYTU-Nec26-F/R with flanking regions of pYTU (Table S7). The PCR products and Pac I digested pYTU were co-transformed into S. cerevisiae BJ5464. The plasmid was extracted from yeast using Zymoprep Yeast Miniprep Kit and transformed to E. coli T1 for sequencing. Preparation of the protoplast and transformation of A. nidulans A1145 were performed as previously described (Yan et al., 2019). The transformation mixture was spread on the selected GMM agar medium (10 g/L glucose, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4, 1 mL/L trace elements solution, 20 g/L agar) with 1.2 M sorbitol and supplementation of pyridoxine HCl (0.5 μg/mL) and riboflavin (2.5 μg/mL) (uracil and uridine dropout). After culturing at 37°C for 3 days, the transformants were transferred to fresh GMM plates and cultured for another 3 days. Then the transformants screening was conducted by PCR using primers U-F/R to confirm for the presence of corresponding plasmids. The correct transformants were incubated in 20 mL liquid CD-ST medium (20 g/L starch, 20 g/L tryptone, 6 g/L NaNO3, 0.52 g/L KCl, 0.52 g/L MgSO4·7H2O, 1.52 g/L KH2PO4, 1 mL/L trace elements solution) with supplementation of pyridoxine HCl and riboflavin overlaid with 2 mL n-dodacane at 25°C, 180 rpm and the organic layers were detected by GC-MS after 6 days.
The RNA of A. nidulans recombinant AN-necD was obtained by using Fungal RNA Kit. The One-Step gDNA Removal and cDNA Synthesis Kit was used to synthesize the cDNA. For construction of plasmid pESC-URA-NsBERS, the intron-free necD was amplified with overhang primers pESC-URA-NsBERS-F/R from cDNA of AN-necD and cloned into pESC-URA (BamH I / Sal I). The genes of LsBERS and BcBOS were chemically synthesized by GeneScript (GenScript USA Inc., Piscataway, NJ, USA) and directly cloned into pESC-URA (BamH I / Sal I) yielding pESC-URA-LsBERS and pESC-URA-BcBOS. The pESC-LEU-tHMGR-ERG20 was co-transformed into S. cerevisiae BJ5464 with pESC-URA-NsBERS/LsBERS/BcBOS, respectively. The S. cerevisiae transformants were incubated in uracil-dropout medium and induced by galactose at 30°C, 220 rpm for 3 days. The n-octane organic layers overlaid the medium were detected by GC-MS.
Plasmid construction of E. coli overexpression
For construction of pET28a-MBP-NsBERS, the intron-free necD was amplified with overhang primers from cDNA using Q5 high fidelity DNA polymerase and cloned into modified pET28a (MBP His6 tagged) using recombinase Exnase II. For construction of pET28a-MBP-LsBERS, the DNA fragment lachB was amplified from pESC-URA-LsBERS and cloned into modified pET28a (MBP His6 tagged). For the construction of pET28a-BcBOS, the DNA fragment bcbos were ligated into BamH I / Not I digested pET28a.
Protein purification and in vitro assays
For protein purification, E. coli BL21 (DE3) harboring the expression plasmids were cultivated in 2 L LB medium supplemented with 50 mg/L kanamycin (KAN) at 37°C, 220 rpm until OD600 reached 0.4–0.6. After cooling to 16°C, 0.1 mM IPTG was added and the cultures were induced at 16°C, 150 rpm for 16 h. The cells were collected by centrifugation and resuspended in buffer A (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.6) and lysed by sonication on ice. The cellular debris were removed through centrifugation at 12,000 rpm, 4°C for 40 min. The protein was purified by AKTA system using Ni-NTA column (GE Healthcare). The purified protein was then concentrated and preserved in buffer C (50 mM Tris-HCl, 50 mM NaCl, 5% glycerol, pH 7.6) by desalting column (GE Healthcare) for in vitro assays.
Unless otherwise stated, the reactions were conducted using 5 μM purified proteins, 100 μM substrates (GPP, FPP or GGPP), and 10 mM Mg2+ in 50 μL of reaction buffer (50 mM Tris-HCl buffer, 10% glycerol, pH 7.6) covered 60 μL n-dodacane. The reaction mixtures were incubated at 30°C overnight. After centrifugation and dried with anhydrous sodium sulfate, the organic phase was analyzed by GC-MS.
EAG assays
The EAG assays were performed on both sexes of Helicoverpa armigera and Spodoptera frugiperda respectively according to previous reports (Guo et al., 2021). The antennae of 3-day-old virgin male and female moths were cut at the base of the flagellum. About 1 μg/μL stock solutions of individual compound was prepared with n-hexane. In each test, 10 μg of each compound was used for stimulation. Odor stimulation was controlled by a puff of purified air (0.2 s at 10 mL/s airflow) from a stimulus controller (CS-55, Syntech, Kirchzarten, Germany). EAG signals were recorded and monitored with an Intelligent Data Acquisition Controller (IDAC-4-USB, Syntech), then analyzed using Syntech EAG-software. For each compound, 4 or 5 antennae from different individual moths were tested. The EAG response values of each compound were calculated by subtracting that of the same antennae corresponding to a solvent blank of n-hexane.
Quantification and statistical analysis
In general, data were analyzed in Excel and figures are plotted in GraphPad Prism. Details of replicates and data analysis for each experiment can be found in the figure legends.
Acknowledgments
We thank Prof. Yi Tang from the University of California, Los Angeles, for providing Aspergillus nidulans heterologous expression host and the related plasmids. We acknowledge Prof. Guirong Wang from CAAS for his guidance in the insect pheromone assays. We also thank Prof. Meirong Jia from CAMS & PUMC for critical reading of the manuscript. We are grateful to the Department of Instrumental Analysis of our institute for measurement of the IR, NMR, and MS data. We acknowledge the State Key Laboratory of Natural and Biomimetic Drugs of Peking University School of Pharmaceutical Sciences for measurement of the X-Ray crystallography data. This work is supported by the National Key Research and Development Program of China (grant No. 2018YFA0901900), PUMC Graduate Innovation Fund Project (No. 2019-1007-04, China), PUMC Disciplinary Development of Synthetic Biology (201920100801, China), and CAMS Innovation Fund for Medical Sciences (No. CIFMS2021-I2M-1-029).
Author contributions
Y.H.W. designed and performed the experiments and drafted the manuscript. T.J.C., J.J.C., and F.P. helped for molecular docking. L.Y.J. and Y.P. provided assistance in the experiments. L.L. performed CD measurements. M.G. conducted EAG assays. R.S.W. took part in partial chemical work. J.L.Y. discussed the experimental results, commented on and proofread the manuscript. T.G. designed and performed the chemical work, drafted and revised the manuscript. P.Z. conceived, designed, and supervised the study and revised the manuscript. All authors approved the final manuscript.
Declaration of interests
The authors declare no competing interests.
Published: April 15, 2022
Footnotes
Supplemental information can be found online at https://doi.org/10.1016/j.isci.2022.104030.
Contributor Information
Ting Gong, Email: gongting@imm.ac.cn.
Ping Zhu, Email: zhuping@imm.ac.cn.
Supplemental information
Data and code availability
The crystallographic information file (CIF) of compound 1 was deposited into Cambridge Crystallographic Data Center with the deposition number 2098349. The nec cluster (necA-necE) were deposited into the GenBank with the accession number MZ672110~MZ672114. The nec26 was deposited into the GenBank under the accession number MZ672115.
A summary of analysis software and tools were provided in key resources table. This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The crystallographic information file (CIF) of compound 1 was deposited into Cambridge Crystallographic Data Center with the deposition number 2098349. The nec cluster (necA-necE) were deposited into the GenBank with the accession number MZ672110~MZ672114. The nec26 was deposited into the GenBank under the accession number MZ672115.
A summary of analysis software and tools were provided in key resources table. This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.