Strains and culture conditions

Cloning was performed in Escherichia coli DH10B using standard recombinant DNA techniques. E. coli BL21(DE3) (Invitrogen) was used for protein expression. Both E. coli strains were grown in LB (10.0 g l–1 tryptone, 5.0 g l–1 yeast extract, 5.0 g l–1 NaCl, pH 7.0) at 37 °C. S. cerevisiae YZL141 (ref. 45) was used for the heterologous expression of ZTR_06220 (KUL85185) from T. verruculosus TS63-9 (ref. 21), MPH_02178 (EKG20455) from M. phaseolina MS6 (ref. 22) and Cgl13855 (NMDCN0000R73) from C. gloeosporioides ES026 (ref. 43) and grown in YPD medium (20 g l–1 tryptone, 10 g l–1 yeast extract, 20 g l–1 glucose, pH 7.2) at 30 °C. T. verruculosus TS63-9 was selected for functional verification of TvTS in vivo and grown in PDB medium (20 g l–1 potato extract, 20 g l–1 glucose, pH 6.5).

Phylogenetic analysis of TvTS and MpMS

To characterize the evolutionary relationships of TvTS, MpMS and CgCS, 56 characterized fungal chimeric class I TSs were selected and multiple-sequence alignment was performed using ClustalW (version 2.0.12). The Poisson correction model based on the maximum-likelihood method was used to infer the evolutionary history of these enzymes, and MEGA7 was used to conduct the evolutionary analysis46. Bootstrap values were obtained using 1,000 replications. The initial tree for the heuristic search was acquired automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model and then selecting the topology with a superior log-likelihood value. After eliminating all positions that contained gaps and missing data, 186 positions were left in the final dataset. The plant-derived sesterterpene synthase AtTPS25 (ref. 47) was selected as an outgroup.

Functional characterization of TrTS candidates in vivo

Synthetic genes encoding TvTS (ZTR_06220), MpMS (MPH_02178), CgCS and the nine remaining candidates (Supplementary Table 7), codon optimized for expression in yeast, were obtained from GenScript. After digestion with HindIII and ScaI, the genes were cloned into HindIII- and ScaI-digested pYJ117 plasmid20 to produce plasmids pXM139 (from ZTR_06220), pXM018 (from MPH_02178), pRC181 (from Cgl13855) and five additional plasmids. The plasmids were individually transformed into S. cerevisiae YZL141 to yield corresponding strains, which were grown in 5 ml YPD medium (2% glucose) at 30 °C overnight. From this starter culture, 5 ml was added to a 500-ml flask containing 200 ml YPD (2% glucose and 1% galactose), followed by growth at 30 °C with shaking at 220 r.p.m. for 3 d. Strains were collected, and the mycelium was collected and extracted with hexane/ethyl acetate (4:1). The organic layers were combined for gas chromatography mass spectrometry (GC–MS) analysis.

Fermentation of engineered S. cerevisiae strains and isolation of 1 and 3

For isolation of 1 and 3, S. cerevisiae XM139 and RC181 strains were scaled up in 2-litre shaker flasks containing 1 litre YPD (2% glucose and 1% galactose). Subsequently, the mycelium was collected and extracted with acetone three times. The acetone-extracted layer was distilled under reduced pressure to remove acetone and then partitioned between ethyl acetate and water to afford the ethyl acetate fraction (1.7 g for 1 and 1.56 g for 3). For 1, the residue was subjected to a silica gel column (80–100 mesh) and elution with petroleum ether/ethyl acetate (100:0, 99.8:0.2, 99:1, 98:2, 95:5, 0:100) to give fractions A–F. Following this, 1 was identified in fraction B by GC–MS detection and further purified by semi-preparative HPLC (Ultimate 3000 HPLC equipped with an XBridge Prep C18 column (Waters, 113; 10 × 250 mm, 5 μm)) to afford compound 1 (7.2 mg). For 3, the crude extract was dissolved in methanol/DMSO (10:1, vol/vol) and subjected to semi-preparative HPLC (column: Agilent ZORBAX BS-C18, 5 μm, 9.6 × 250 mm internal diameter; solvent: acetonitrile/H2O, 99:1; flow: 3 ml min–1; detector: 210, 230 nm) to yield compound 3 (30.5 mg, tR = 24.2 min).

Plasmid construction for in vitro and labelling experiments

For expression of recombinant TvTS and MpMS in E. coli, codon-optimized synthetic genes were obtained from GenScript. The synthetic gene for MpMS was digested with NdeI and EcoRI and cloned into pET28a to produce pRC088. The sequences encoding the PT and TC domains of TvTS were amplified separately by PCR with Phusion DNA polymerase using primer pairs P1/P2 and P3/P4 (Supplementary Table 2). The amplified nucleotide sequence for the PT domain was digested with NdeI and EcoRI and cloned into pET28a to produce plasmid pRC009, and the amplified nucleotide sequence for the TC domain was digested with HindIII and XhoI and cloned into pET21a to generate plasmid pRC041. The sequence encoding the TC domain-inactivated MpMS D114A/N115A variant was amplified from pRC088 using primer pairs P5/P6 and P7/P8 (Supplementary Table 2) and then assembled by overlap extension PCR and cloned into NdeI- and EcoRI-digested pET28a to produce pRC088-D114A/N115A. Correct gene insertion was verified by sequencing, and plasmids were used to transform E. coli BL21(DE3) competent cells using a calcium-based protocol.

Protein expression and purification for in vitro assays

For gene expression, a fresh LB culture of E. coli BL21(DE3) transformants (containing 100 mg l–1 ampicillin for pRC041 (TvTS-TC) and 50 mg l–1 kanamycin for pRC009 (TvTS-PT), pRC088 (MpMS) and pRC088-D114A/N115A) was inoculated from a glycerol stock and grown overnight. The precultures were used to inoculate the desired volume of LB (1 ml per litre) amended with the appropriate antibiotic. Cultures were grown at 37 °C with shaking until an OD600 of 0.6–0.8 was reached. The cultures were cooled to 16 °C, and isopropyl β-d-1-thiogalactopyranoside (0.1 mM) was added to induce expression. Proteins were expressed overnight (~20 h) at 16 °C with shaking. Cells were collected by centrifugation (8,000g, 5 min). The supernatant was discarded, and the cell pellet was resuspended in buffer A (50 mM Tris-HCl, 300 mM NaCl, 4 mM β-mercaptoethanol, pH 7.6; 10 ml per litre of culture). Cells were lysed by sonication on ice (5 × 30 s). The cellular debris was removed by centrifugation (14,710g, 2 × 7 min), and the supernatant was subjected to Ni-NTA affinity chromatography (Protino Ni-NTA, Macherey-Nagel) through a syringe filter. The resin was washed with buffer A (20 ml per litre of culture), followed by elution of the His6-tagged proteins using elution buffer (buffer A + 300 mM imidazole; 10 ml per litre of culture). The proteins were concentrated using centrifugal filters (10-kDa cut-off; 5,000g, 4 °C; Amicon Ultra-15 (Millipore) or Vivaspin 20 (Sartorius)) and diluted with incubation buffer (50 mM Na2HPO4, 10% glycerol, 2 mM MgCl2). Enzyme concentrations were determined by Bradford assay and adjusted to 20 μΜ. Incubation experiments were carried out at 30 °C overnight using combinations of enzymes and substrates as listed in Supplementary Table 5. For experiments with unlabelled substrates, hexane (650 μl) was used for extraction, whereas for experiments with labelled substrates extraction was performed using C6D6 (650 μl). Samples were directly analysed by GC–MS and/or NMR spectroscopy.

In vitro enzyme assays for TvTS-PT and MpMS D114A/N115A and detection of HexPP

Reactions were carried out using substrate (IPP and DMAPP, 100 μM each), 2 mM Mg2+, 10% glycerol and 10 μM enzyme (TvTS-PT or MpMS-PT D114A/N115A) in Tris-HCl buffer (200 μl; 50 mM, pH 7.6) at 30 °C overnight. The resulting HexPP was extracted with acetonitrile and analysed by liquid chromatography mass spectrometry (LC–MS). For high-resolution MS analysis of HexPP, an LTQ Orbitrap Elite instrument coupled to a Thermo Scientific Ultimate 3000 RSLC HPLC system and an ACE UltraCore 2.5 SuperC18 (2.1 × 100 mm) column was used for compound separation at 35 °C. The mobile phase (pH 9.5) containing 5 mM ammonium bicarbonate in water as solvent A and acetonitrile as solvent B was set to a flow rate of 0.2 ml min–1. The gradient programme was as follows: 98–10% solvent A (0–10 min), 10–0% solvent A (10–15 min), 0% solvent A (15–17 min), 0–98% solvent A (17–18 min) and 98% solvent A (18–22 min). EI was used in negative mode for detection. The ion source parameters were as follows: sheath gas, 40 arb; auxiliary gas, 5 arb; spray voltage, 3.1 kV; capillary temperature, 270 °C; S-lens RF level, 65 kV; auxiliary gas heater temperature, 250 °C. Full-scan MS mode with a resolution of 60,000 was used for qualitative analysis.

Site-directed mutagenesis and in vitro analysis of enzyme variants

Site-directed mutagenesis for the construction of MpMS and TvTS enzyme variants was performed with the PCR-based QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol, using Phusion DNA polymerase and the mutational primers listed in Supplementary Table 2. Plasmids pRC088 (containing the full-length gene for MpMS) and pMBP139 (containing the full-length gene for TvTS) were used as template. All mutants were verified by gene sequencing. Reactions for TvTS, MpMS and the variants were carried out using substrate (DMAPP and IPP, 100 μM and 500 μM, respectively), 2 mM Mg2+, 5% glycerol and 10 μM enzyme in Tris-HCl buffer (200 μl; 50 mM, pH 7.5) at 30 °C overnight. The resulting product was extracted with ethyl acetate and analysed by GC–MS. All experiments were performed in three biological replicates.

Isolation of macrophomene (2) from incubation with MpMS E104Y

MpMS E104Y was expressed in E. coli and purified by Ni-NTA affinity chromatography. The pooled enzyme fractions were concentrated, further purified by size-exclusion chromatography and concentrated again. Incubation was performed using GPP (30 mg, 0.08 mM) and IPP (100 mg, 0.34 mM) dissolved in NH4HCO3 buffer (25 mM, 20 ml), which was added to the enzyme solution in incubation buffer (100 ml) over a period of 1 h using a syringe pump. The mixture was incubated overnight and extracted with n-hexane (150 ml) three times. The organic layers were dried with MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography on SiO2 (pentane) to yield macrophomene as a colourless oil.

Construction of expression plasmids for protein crystallization

To increase the solubility of protein in E. coli, the coding sequence for TvTS was amplified using primer pair P21/P22 from the codon-optimized synthetic gene and ligated into NdeI- and HindIII-digested pET28-MBP-TEV, using the In-Fusion HD Cloning kit (TaKaRa), yielding plasmid pMBP139. For expression of protein for crystallization, the coding sequence for TvTS-TC was amplified from pRC041 using primer pair P23/P24. The amplified sequence was cloned into pET-SUMO, which was itself amplified with primer pair P25/P26, using the In-Fusion HD Cloning kit, resulting in plasmid pSUMO041.

Protein expression and purification for crystallization

Protein expression was performed with E. coli BL21(DE3) harbouring pSUMO041 using the same protocol as described above. For crystallization, after Ni-NTA purification, SUMO–TvTS-TC was dialysed against 2 × 1 litre of Tris-HCl buffer (pH 8.0) containing 5% (vol/vol) glycerol and 300 mM NaCl. After dialysis, SUMO–TvTS-TC was treated with SUMO protease Ulp1403-621 (ref. 48; prepared as previously described, 0.87 μM) in the presence of dithiothreitol (DTT, 1 mM) at 4 °C overnight. The protein solution was loaded onto a column filled with Ni-NTA resin. The His6–SUMO fragment and protease were then captured by the Ni-NTA resin, leaving TvTS-TC in the flow-through, and the remaining protein on the column was eluted with Tris-HCl buffer (pH 8.0) containing 5% (vol/vol) glycerol, 10 mM imidazole and 300 mM NaCl. The collected protein solution was incubated with 10 mM EDTA (pH 8.0) at 4 °C for 1 h. To obtain a protein preparation of high purity, further purification was performed using anion-exchange chromatography on a Resource Q column (Cytiva) by linearly increasing the salt concentration from 0 M NaCl to 1 M NaCl in buffer (50 mM Tris-HCl (pH 8.0), 1 mM DTT, 5% glycerol) over 20 column volumes. The desired protein was collected and then purified to homogeneity by size-exclusion chromatography on a HiLoad 16/600 Superdex 200 pg column (Cytiva) and eluted with a solution containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT and 5% glycerol. The resulting eluate was concentrated to 10 mg ml–1, using an Amicon Ultra-4 filter (molecular weight cut-off of 30 kDa) at 4 °C. The purity of the proteins was monitored by SDS–PAGE, and protein concentrations were determined with a SimpliNano microvolume spectrophotometer.

Crystallization and structure determination

Crystals of TvTS-TC were obtained after 1 d at 10 °C by using the sitting-drop vapor-diffusion method. Before crystallization, 125 μM of protein was incubated with 2 mM MgCl2 on ice for 30 min, and 0.5 μl of protein solution was then mixed with 0.5 μl of reservoir solution containing 0.1 M Tris-HCl (pH 8.5), 0.1 M MgCl2, 30% PEG 4000 and 0.2 M NDSB-211. Crystals of TvTS-TC in complex with 2,3-dihydro-HexPP were obtained by incubation of TvTS-TC crystals with 10 mM 2,3-dihydro-HexPP in the crystallization drop at 10 °C for 14 h. The crystals were transferred to cryoprotectant solution (reservoir solution with 25% (vol/vol) glycerol) and then flash cooled at –173 °C in a nitrogen gas stream. The X-ray diffraction datasets were collected at X06SA (Paul Scherrer Institut, Villigen, Switzerland) for the apo TvTS-TC structure and at BL-1A (Photon Factory, Tsukuba, Japan) for the structure of TvTS-TC in complex with 2,3-dihydro-HexPP, using a beam wavelength of 1.0 and 1.1 Å, respectively. The diffraction datasets for TvTS-TC were processed and scaled using the XDS package49 and Aimless50. The initial phase of the TvTS-TC structure was determined by molecular replacement, using PaFS (PDB, 5ER8) as the search model. Molecular replacement was performed with Phaser in PHENIX (version 1.19.2-4158-000)51,52. The initial phase was further calculated with AutoBuild in PHENIX52. The TvTS-TC structures were modified manually with Coot53 and refined with PHENIX.refine54. The cif parameters of the ligands for the energy minimization calculations were obtained by using the PRODRG server55. After soaking with 2,3-dihydro-HexPP, strong additional electron densities were observed close to the DDXXD motif and in the active site cavity (Supplementary Fig. 36a, b). Modelling of 2,3-dihydro-HexPP to the observed density was partly satisfactory, but some of the methyl groups along the isoprenoid chain stuck out (Supplementary Fig. 36c). For an alternative explanation, PEG used in the crystallization buffer was modelled to the density, but in this case large unassigned densities close to the DDXXD motif remained in the refined structure (Supplementary Fig. 36d). Thus, it is possible that the observed density originated from both 2,3-dihydro-HexPP and PEG with low occupancies. The final crystal data and intensity statistics are summarized in Extended Data Table 1. The Ramachandran statistics were as follows: 97.6% favoured and 2.4% allowed for apo TvTS-TC, 98.9% favoured and 1.1% allowed for TvTS-TC soaked with 2,3-dihydro-HexPP. Although the ligand was not assigned, the conformations of PEG and 2,3-dihydro-HexPP in the active site should be similarly defined by active site residues. Therefore, a docking model of 2,3-dihydro-HexPP based on the observed density was developed. All crystallographic figures were prepared with PyMOL (DeLano Scientific;

Purification of MpMS and cross-linking of the PT and TC domains

Protein expression was performed with E. coli BL21(DE3) harbouring pRC088 using the same protocol as described above. After Ni-NTA purification, MpMS protein solution was further purified using anion-exchange chromatography on a Resource Q column (Cytiva) by linearly increasing the salt concentration from 20 mM NaCl to 1 M NaCl in buffer (50 mM HEPES (pH 7.5) and 1 mM DTT) over 20 column volumes. The desired protein was collected and then purified by size-exclusion chromatography on a Superose 6 10/300 column (Cytiva) using buffers containing 20 mM HEPES (pH 7.5), 150 mM NaCl and 1 mM DTT. The resulting eluate was concentrated to 50 μM, using an Amicon Ultra-4 filter (molecular weight cut-off of 100 kDa) at 4 °C. The purity of the proteins was monitored by SDS–PAGE. To obtain the structure of full-length MpMS, cross-linking of the PT and TC domains via glutaraldehyde (25% in water; Nacalai Tesque) was performed. First, MpMS protein was purified by Ni-NTA and anion-exchange chromatography, as described above, to obtain pure protein. Subsequently, cross-linking was performed on ice by incubating 1 mg ml–1 MpMS protein with 0.06% glutaraldehyde for 10 min at a 100-μl scale. Reactions were quenched by adding 10 μl of 1.0 M Tris-HCl (pH 8.0). Reaction mixtures were pooled and further purified by size-exclusion chromatography on a Superose 6 10/300 column (Cytiva) to exclude aggregations. The resulting eluate was concentrated to 50 μM, using an Amicon Ultra-4 filter (molecular weight cut-off of 100 kDa) at 4 °C.

Cryo-EM sample preparation and data acquisition

For cryo-grid preparation, 3 μl of sample was applied to a holey carbon grid (Quantifoil, Cu, R1.2/1.3, 300 mesh). The grid was rendered hydrophilic by a 30-s glow discharge in air (11-mA current) with PIB-10 (Vacuum Device). The grid was blotted for 20 s (blot force of 0) at 18 °C and 100% humidity and then flash frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). For the MpMS-PT and MpMS cross-linking datasets, 1,888 and 1,529 movies were acquired, respectively, on a Talos Arctica (FEI) microscope operating at 200 kV in nanoprobe mode using EPU software for automated data collection. The movies were collected on a 4,000 × 4,000 grid using a Falcon 3EC direct electron detector (electron counting mode) at a nominal magnification of 120,000 (0.88 Å per pixel). Fifty movie fractions were recorded at an exposure of 1.00 electrons per Å2 per fraction, corresponding to a total exposure of 50 electrons per Å2. The defocus steps used were –1.0, –1.5, –2.0 and –2.5 μm. The movie fractions were aligned, dose weighted and averaged using RELION’s own implementation on 5 × 5 tiled fractions with a B-factor of 300. The non-weighted movie sums were used for contrast transfer function (CTF) estimation with Gctf 56. The dose-weighted sums were used for all subsequent steps of image processing. The subsequent processes of particle picking, two-dimensional classification, ab initio reconstruction, 3D classification, 3D refinement, CTF refinement and Bayesian polishing were performed using RELION-3.18 (ref. 57). For details of the cryo-EM data processing, see Supplementary Figs. 3740.

AlphaFold2 prediction and docking analysis

UCSF Chimera58 (version 1.12) and AutoDock Vina59 (version 1.1.2) were used to perform receptor and ligand preparation and molecular docking analysis. Before the docking procedure, the receptor structures predicted by AlphaFold2 and ligands were processed as follows. Metal ions were added to the TC domain binding site of receptors, referring to the homologous protein in PDB for the coordinates of the three Mg2+ ions. With the exception of Cgl13855 and FgMS, for which 5IMP and 5ER8 were used as the reference for metal ions, respectively, 6VYD was used as the reference for the other PTTCs. The receptor structures containing metal ions were then processed using the Chimera tool Dock Prep, in which hydrogen atoms and charges were added and other parameters were set as default. The energy-minimization molecule models included in Chimera were used to minimize the energy for the structures from the previous step. The ligand structures were drawn, charge was added and the structures were transformed to 3D conformations. The binding site of the TC domain was determined by referring to the crystal structures for homologous proteins. The grid box in the docking procedure was defined to include the metal ions, and corresponding residues appeared in the binding site of crystal structures for homologous proteins. Receptor and ligand options in AutoDock Vina were set as default. The number of binding modes, exhaustiveness of search and maximum energy difference (kcal mol–1) parameters were set as 9, 8 and 3, respectively.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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