Bioinformatics analysis. trans-AT PKSs containing b-methylation modules were identified using refs. 3 and 8. For comparative analysis of ACP domains, all PKS subunit sequences (with the exception of VirFG10) were retrieved from the Protein data base (http://www.ncbi.nlm.nih.gov/protein), and domain boundaries were established relative to the solved structures of Vir ACPs 5a and 5b (PDB IDs: 2MF4, 4CA3)10. Sequence alignments shown in figures were generated using the NPS@ web server (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html)35 and the figures created with ESPript36.
Materials and DNA manipulation. Biochemicals and media were purchased from VWR (glycerol, NaPi, NaCl, MgSO4), BD (tryptone, yeast extract), Thermo Fischer Scientific (Tris, EDTA), Euromedex (isopropyl β-D-1-thiogalactopyranoside; IPTG), and Sigma-Aldrich (betaine, imidazole, Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), starch), and Roquette (corn steep). L-proline-2,5,5-D3 and L-serine-2,3,3-D3 were purchased from CDN Isotopes. The enzymes for genetic manipulation were purchased from Thermo Fisher Scientific. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Clean‑up or NucleoSpin® Plasmid DNA kits (Macherey Nagel). Standard PCR reactions were performed with Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific); and reactions were carried out on a Mastercycler Pro (Eppendorf). DNA sequencing was carried out by Eurofins.
Strains and media. E. coli BL21(DE3) strains (Supplementary Table 2) were obtained from Novagen and were cultured in LB medium (yeast extract 10 g L-1, tryptone 5 g L-1, NaCl 10 g L-1, adjusted to pH 7.0 with NaOH) or on LB agar plates (LB medium supplemented with 20 g L-1 agar) at 37 °C. Streptomyces pristinaespiralis ATCC 25486 (DMSZ, Germany) and the derived mutants were sporulated on RP agar plates (20 g L-1 starch, 20 g L-1 soybean flour, 0.5 g L-1 valine, 0.5 g L-1 K2HPO4, 1 g L-1 MgSO4 × 7H2O, 2 g L-1 NaCl, 3 g L-1 CaCO3, 20 g L-1 agar in tap water) for 7 days at 30 °C. All strains were maintained in 20% (v/v) glycerol and stored at -80 °C. E. coli ET12567/pUZ8002 was used for conjugation and appropriate antibiotics were added to LB liquid and agar cultures at the following concentrations: ampicillin 100 mg L-1, kanamycin 50 mg L-1, apramycin 25 mg L-1, chloramphenicol 25 mg L-1 and nalidixic acid 25 mg L-1. For metabolite production by S. pristinaespiralis and its mutant (Supplementary Table 2), 20 µL of spores were used to inoculate 25 mL inoculum medium (10 g L-1 corn steep powder, 15 g L-1 saccharose, 10 g L-1 (NH4)2SO4, 1 g L-1 K2HPO4, 3 g L-1 NaCl, 0.2 g L-1 MgSO4 × 7H2O, 1.25 g L-1 CaCO3 in tap water, pH 6.9), followed by incubation at 30 °C and 180 rpm on rotary shaker for 72 h. Production medium (25 g L-1 soybean flour, 7.5 g L-1 starch, 22.5 g L-1 glucose, 3.5 g L-1 yeast extract, 0.5 g L-1 ZnSO4 × 7H2O, 6 g L-1 CaCO3 in tap water, pH 6.0) was inoculated with 2% of precultures, and incubated at 30 °C, 180 rpm on a rotary shaker for 96 h. To evaluate its effect, certain cultures were supplemented with 2% XAD-16 resin (Sigma-Aldrich). For feeding experiments, cultures were supplemented individually with L-proline-2,5,5-D3 or L-serine-2,3,3-D3, or a combination of L-proline-2,5,5-D3 and L-serine-2,3,3-D3, at 4, 24 and 48 h after incubation, in equal portions, to a final concentration of 3 mM.
Gene cloning and site-directed mutagenesis. All protein-encoding constructs were amplified directly from Streptomyces virginiae genomic DNA using forward and reverse primers incorporating BamHI and HindIII restriction sites, respectively (Supplementary Table 1), and were ligated into the sites of vector pBG-102 for VirE and VirD and its mutant (VirD E128Q) or pLM-302 for VirC. Vector pBG-102 codes for a His6-SUMO tag and pLM-302 codes for a His6-maltose binding protein (MBP) tag (Centre for Structural Biology, Vanderbilt University). Following cleavage of the tags, the proteins incorporated a non-native N-terminal Gly-Pro-Gly-Ser sequence. The sequences of all constructs were verified by DNA sequencing prior to protein expression studies. Site-directed mutations were introduced into ACP5a and VirD by PCR using mutagenic oligonucleotides (Supplementary Table 1) and Phusion High-Fidelity polymerase, followed by digestion of the parental DNA by 1 μL of DpnI Fast digest (Thermo Fischer Scientific). The presence of the correct mutations was confirmed by sequencing.
Expression and purification of recombinant proteins ACP domains, VirC, VirC quadruple mutant (C114A/Q334A/R335A/R338A), VirD, VirD E128Q and VirE. All constructs were transformed into E. coli BL21(DE3) cells and grown at 37 °C in LB medium supplemented with 50 mg mL-1 kanamycin to an A600 of 0.8, and then IPTG added to a final concentration of 0.5 mM. Following incubation at 20 °C for 18 h, the cells were harvested by centrifugation at 3000g for 30 min at 4 °C, and cell pellets stored immediately at ─80 °C. Vir ACP5a and ACP5b purification was performed as described previously10, and all APC5a mutants, ACP5a─ACP5b didomain, ACP6 and ACP7 purified using the same method. In the case of all proteins of the b-methylation cassette, the cell pellets were suspended in His-buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 10 % glycerol for VirC and the VirC quadruple mutant, or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 10 % glycerol (VirD, VirD E128Q and VirE)) containing 8 U mL-1 of Benzonase (Merck) and 5 mM MgSO4. Cells were lysed by sonication and clarified by centrifugation (35,000 g for 40 min). Cell extracts were loaded onto a 5 ml HisTrap column (Cytiva) and washed with resuspension buffer supplemented with 20 mM imidazole. The supernatant was loaded onto a HisTrap 5 mL column equilibrated with His-buffer using an Akta Pure system (Cytiva). The proteins were eluted using a linear gradient of 0–50% His-elution buffer (50 mM NaPi pH 7.5, 250 mM NaCl, 300 mM imidazole for VirC and the VirC quadruple mutant or 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 300 mM imidazole (VirD, VirD E128Q and VirE)) over ten column volumes.
All His6-tagged constructs were then incubated with His-tagged human rhinovirus 3C protease (1 µM) for 12─16 h at 4 °C to cleave the affinity/solubility tags. The constructs were then separated from the remaining His-tagged proteins by loading onto a HisTrap 5 mL column, followed by elution in resuspension buffer containing 20 mM imidazole. VirD, VirD E128Q and VirE were subsequently injected onto a Q-sepharose column (trimethylammonium on 6% agarose) equilibrated in buffer (20 mM Tris-HCl pH 8.5, 20 mM NaCl, 10 % glycerol). All proteins were then eluted using an NaCl gradient (100 mM─1 M) at 5 mL min−1. Eluted fractions found to contain protein of the correct molecular weight as judged by SDS-PAGE analysis were pooled, concentrated using an Amicon Ultracel-10 (Merck Millipore) by centrifugation at 4000g, and loaded onto a Superdex 200 16/60 (Cytiva) equilibrated with 20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol (VirD, VirD E128Q and VirE) or a Superdex 75 16/60 column (Cytiva) (VirC and the VirC quadruple mutant). Following a concentration step, the purity of the recombinant proteins was determined by SDS-PAGE (Extended Data Fig. 1), and their concentrations were determined by NanoDrop (or Qubit for ACP6) (Thermo Scientific), with extinction coefficients calculated using the ExPASy ProtParam tool37.
Expression of labelled protein samples for structural biology. Seleniated wild type VirD was produced in M9 minimal medium (50 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, 20 mM NH4Cl, adjusted to pH 7.2 with NaOH) for SAD/MAD phasing. Autoclaved M9 medium was supplemented with 50 mg L−1 of thiamine and riboflavin, 4 g L−1 glucose, 100 μM CaCl2, 2 mM MgSO4, 40 mg L−1 selenomethionine, and 40 mg L−1 of the 19 amino acids, based on the methionine biosynthesis inhibition method38. 13C,15N-enriched Vir ACP5a, ACP6 and ACP7 were produced in M9 medium supplemented with 15NH4Cl (0.5 g L-1) and 13C-glucose (2.0 g L-1), as the only sources of nitrogen and carbon. The labelled proteins were purified to homogeneity as described above.
Svp-catalysed modification of ACP domains and verification by HPLC-MS. Following size exclusion chromatography, apo-ACPs (1 mM) were incubated in buffer (20 mM Tris-HCl pH 8.5) with 5 mM (acyl-)CoASH, 40 mM PPTase Svp14, 10 mM MgCl2 and 50 mM TCEP for 22 h at 20 °C. The ACPs were then purified using a Superdex 75 16/60 column (Cytiva) equilibrated in 20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP. Quantitative modification was verified for all of the ACPs by HPLC-MS (Extended Data Fig. 2) using either a Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer, or an LTQXL mass spectrometer, both equipped with an in-line photodiode array detector (PDA) and an atmospheric pressure ionization interface operating in electrospray mode (ESI). All samples were diluted with Milli-Q water to a concentration of 50 µM and injected onto an Alltima™ C18 column (2.1 × 150 mm, 5 µm particle size). Analysis was carried out with Milli-Q water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B), using the elution profile: 0−15 min, linear gradient from 10−98% solvent B; 15−20 min, constant 98% solvent B; 20.1−26 min, constant 10% solvent B. In the case of the LTQXL, MS scans were performed in ESI+ in the mass range m/z = 100−2000, at 3 K resolution, with MS parameters as follows: spray voltage, 5 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 20, 5 and 5 arbitrary units min-1; capillary temperature, 350 °C; capillary voltage, 7 V; tube lens, split lens and front lens voltages 180 V, ─22 V and ─11.75 V, respectively. MS data acquisition was carried out using the Xcalibur v. 2.1 software (Thermo Scientific). For the Orbitrap, MS scans were performed in heated ESI positive ion mode (HESI+) in the mass range m/z = 150−2000, at 7.5 K or 60 K resolution (full width of the peak at its half maximum, fwhm, at m/z = 200) with MS parameters as follows: spray voltage, 4 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 30, 5 and 5 arbitrary units min-1; vaporiser and ion transfer tube temperatures were both set at 300 °C; maximum injection time, 50 ms; AGC target: 100000; normalised AGC target: 25%; microscans, 10; RF-lens, 35%; data type, profile. Mass spectrometer calibration was performed using the Pierce FlexMix calibration solution (Thermo Scientific). MS data acquisition was carried out using the Xcalibur v. 4.3 software (Thermo Scientific). For data obtained at low resolution (3 or 7.5 K), only the major isotopic peak was detected, while analysis at high resolution (60K) afforded the full isotopic spectrum (Extended Data Fig. 2).
Tryptophan fluorescence quenching. All tryptophan fluorescence spectroscopy experiments were performed on a SAFAS Fluorescence Xenius Spectrophotometer (SAFAS, France) in a 2 mL quartz cuvette. The excitation wavelength was fixed at 295 nm and emission spectra were collected between 300−400 nm with a slit width of 2 nm. The temperature was maintained at 25 °C by an external thermostatic water circulator. To measure protein-ligand interactions, recombinant VirC, VirD, VirD E128A mutant and VirE at 5 mM were allowed to equilibrate in TE buffer (20 mM Tris-HCl pH 8.5, 2 mM EDTA) for 10 min under constant stirring, before being titrated with ligand solutions. The proteins were analysed against increasing concentrations of ligand (0─150 mM), depending on the specific ligand used. Data from two independent experiments were analysed using nonlinear regression, with application of the one site-specific binding model (F = Fmax*X / (Kd + X), where X is the ligand concentration, F is the fluorescence intensity, Fmax is the maximum specific binding and Kd is the equilibrium binding constant) using SciDAVis v2.3.0.
Circular dichroism measurements. Circular dichroism measurements were performed on a Chirascan CD (Applied Photophysics) in 100 mM NaPi, 150 mM NaF pH 8.0. Data were collected at 0.5 nm intervals in the wavelength range of 180─260 nm at 20 °C, using a temperature-controlled chamber. 30 mL of 100 mM ACP5a, ACP5a E6761A/L6764N and VirD were analysed in a 0.01 cm cuvette, while 100 mL of 100 mM VirD E128Q was analysed in a 0.1 cm cuvette. Each spectrum represents the average of three scans, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone. Spectrum deconvolution was carried out using the CDNN2.1 software39 (Extended Data Fig. 1).
Small-angle X-ray scattering (SAXS) data collection. SAXS data were acquired on the SWING beamline at the Synchrotron SOLEIL (France). The frames were recorded using an Eiger 4M detector at an energy of 12 keV. The distance between the sample and the detector was set to 2000 mm for VirD, VirE, holo-ACP5b−VirC and holo-ACP5b−VirE complexes, leading to scattering vectors q ranging from 0.0005−0.5 Å−1. The scattering vector is defined as 4p/l sinq, where 2q is the scattering angle. The protein samples were injected using the online automatic sample changer into a pre-equilibrated HPLC-coupled size-exclusion chromatography column (Bio-SEC 100 Å, Agilent), at a temperature of 15 °C.
The eluted fractions were delivered using an online purification system developed on the SWING beamline40. After equilibrating the column in the protein buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol), the buffer background was recorded (100 successive frames of 0.75 s). A 50 mL aliquot of the protein sample (at 5 mg mL-1) was then injected, and complete data sets were collected. The protein concentration downstream of the elution column was followed via the absorbance at 280 nm with an in situ spectrophotometer. In lieu of analysing several protein concentrations within a standard range (e.g., 0.1−10 mg mL−1), the coupling of data collection to a gel filtration column allows analysis of multiple concentrations of protein within a single experiment, as many distinct positions within the elution peak are sampled during the course of the measurement (typically 50−100 frames are acquired)40.
Following on from this, the dedicated in-house application FOXTROT was used to perform data reduction to absolute units, frame averaging, and solvent subtraction. Each acquisition frame of the experiment yielded a scattering spectrum, which was then analysed by FOXTROT to produce an Rg (radius of gyration) as well as an I(0) value (the I(0) depends on the protein concentration at that position in the elution peak, as described by the Guinier law (approximation I(q) = I(0) exp(−q2Rg2/3) for qRg < 1.3). Notably, observing a constant Rg for a significant proportion of the concentrations present in the gel filtration peaks showed that the measurements were concentration-independent, and thus that they were effectively carried out under conditions of infinite dilution.
Finally, all the frames exhibiting identical Rg as a function of I(0) were corrected for buffer signal and averaged. This step ensured that the data reflected only the signal arising from the protein structure and not from intermolecular interactions. The distance distribution function P(r) and the maximum particle diameter Dmax were then calculated by Fourier inversion of the scattering intensity I(q) using GNOM41. The SAXS data are presented in Supplementary Table 3.
Molecular weights and oligomeric structures in solution from SAXS data. Classically, molecular weights can be derived from SAXS data using the I(0) and the known protein concentration. However, this method was not appropriate in our case, as the delay between exiting the gel filtration column and the SAXS data acquisition may have altered the concentrations. We therefore determined the molecular weights of the constructs using Bayesian Interference in PRIMUS42. SAXS data were recorded on wild type VirE, as well as VirC and VirE complexed with holo-ACP5b. A model of a trimer of VirE was generated using ColabFold: AlphaFold219. On the basis of the structural homology between the VirE model and the solved VirD crystal structure (r.m.s.d. 3.38 calculated based on 200 Ca), we generated a model of the holo-ACP5b−VirE complex by superimposition on the crystal structure of holo-ACP5b−VirD using PyMOL43. The quality of the 3D modelling was determined using CRYSOL16 to compare the fit between the theoretical scattering curves from atomic coordinates with experimental scattering curves, and judged using the discrepancy c2, defined according to Konarev and colleagues17. SAXS data obtained on wild type VirC complexed with holo-ACP5b were directly compared with that calculated16 from the crystal structure of the acetyl-ACPD−CurD complex (PDB: 5KP6)13 using CRYSOL16.
Crystallisation and X-ray data collection. Se-VirD was purified and stored in buffer (20 mM Tris-HCl pH 8.5, 300 mM NaCl, 5% glycerol) at a final concentration of 5 mg mL−1. holo ACP5b was stored in buffer (20 mM Tris-HCl pH 8.5, 250 mM NaCl, 50 mM TCEP) at a final concentration of 20 mg mL−1. Prior to crystallization trials, sample homogeneity was checked by dynamic light scattering (DLS) using a Zetasizer NanoS (Malverne). Initial crystallisation hits were obtained using the Rigaku kit (Molecular Dimensions). The conditions consisted of 20% PEG 400, 20% PEG 800, 100 mM Tris-HCl, pH 7.5 for Se-VirD, while holo ACP5b−Se-VirD crystallised in 100 mM chloride calcium, 30% PEG 1500, 10% 2-propanol, 100 mM imidazole-HCl, pH 6.5.
Crystals grew in 10−15 days using the hanging drop method in Linbro® plates, with drops formed by mixing 2 mL of protein solution (ratio 1:4 for the holo-ACP5b−Se-VirD complex, 5 mg mL−1 Se-VirD) with 1 mL of crystallisation buffer. Crystals were then soaked in crystallisation buffer containing 30% ethylene glycol prior to freezing in liquid nitrogen. X-ray diffraction data on Se-VirD and the holo ACP5b−Se-VirD complex were collected at the SOLEIL synchrotron on the Proxima2 beamline. The crystals belong to the P41212 and H3 space groups, respectively (Supplementary Table 4). A complete MAD data set at four wavelengths was collected in order to solve the crystal structure of VirD. Data sets were indexed and integrated using XDS44 and scaled by using pointless and aimless (CCP4 package).
Structure determination and refinement. Initial phases were ultimately generated via SAD using the peak wavelength (λ = 0.979260 Å). Three high confidence Se sites were identified and refined by using the NCS using Phenix.autosol45,46. The figure of merit (FOM) from Phenix AutoSol is 0.32. Density modification and NCS were then used to improve the quality of the phases (FOM: 0.68 with a bias ratio of 1.36). The good quality of the electron density map allowed for building approximatively 80% of the backbone at 2.02 Å using Phenix.autobuild47. The final model of WT VirD was built using ARP/wARP48, followed by iterative cycles of manual rebuilding and refinement at 1.7 Å using COOT49 and REFMAC550. The structure of the holo-ACP5b−VirD complex was solved by molecular replacement using a monomer of VirD as search model with the program MOLREP in CCP451,52. The contrasted solution with final CC of 0.7252 and Tf/sig of 27.17, consists of 2 monomers of VirD in the asymmetric unit. The initial model was then refined by rigid body refinement at 3 Å followed by a restraint refinement at 2.1 Å resolution using REFMAC 5 CCP450. The excellent quality of the electron density maps allowed us to locate two extra electron density in the FoFc map corresponding to two ACP5b molecules in the asymmetric unit. The ACPs were then constructed manually in the electron density maps. Structure geometry was validated using the program MolProbity53. The structures of VirD and holo-ACP5b−VirD contain 99.26% and 97.91% of the residues in the allowed region of the Ramachandran plot respectively and contain no outliers (Supplementary Table 4). Figures were prepared using the program PyMOL43.
Protein NMR data acquisition. All ACPs proteins samples were buffer exchanged via gel filtration into 100 mM sodium phosphate (pH 6.0), 1 mM EDTA and 1 mM TCEP, concentrated to 1 mM, and then 350 μL of the samples (including 10% D2O) were loaded into 4 mm NMR tubes. All NMR data were recorded at 25 °C on a Bruker DRX600 spectrometer equipped with a cryogenic probe (Unité Mixte de Service (UMS) 2008 Ingénierie-Biologie-Santé en Lorraine (IBSLor)). Backbone and sequential resonance assignments were obtained by the combined use of 2D 15N−1H and 13C−1H HSQC spectra and 3D HNCA, HNCACB, CBCA(CO)NH, HNHA, HBHA(CO)NH, HN(CA)CO, and HNCO experiments. Assignments of aliphatic side chain resonances were based on 2D aromatic 13C−1H HSQC, (HB)CB(CGCDCE)HE, (HB)CB(CGCD)HD and 3D (H)CC(CO)NH, H(CC)(CO)NH, CCH−TOCSY, and HCCH-TOCSY experiments (reviewed in 54). To collect NOE-based distance restraints for the structure calculations, 3D 15N NOESY-HSQC and 13C NOESY-HSQC were recorded on uniformly 13C,15N enriched samples using a mixing time of 120 ms. NMR data were processed using Topspin 3.2 (Bruker) and were analysed using NMRFAM-SPARKY55.
Protein NMR structure calculations. Initial structures were generated using CYANA 3.98 software56. Starting from a set of manually-assigned NOEs, the standard CYANA protocol of seven iterative cycles of calculations was performed with NOE assignment by the embedded CANDID routine combined with torsion angle dynamics structure calculation57. In each cycle, 100 structures starting from random torsion angle values were calculated with 15,000 steps of torsion angle dynamics-driven simulated annealing. A total of 1822, 1208 and 1763 NOE-based distances, 110, 92 and 94 backbone angle restraints were used for structure calculation of the holo-ACP5a, holo-ACP6 and holo-ACP7 domains, respectively (Supplementary Table 5). The angle restraints were obtained from 13Cα, 13Cβ, 13C′, 15N, 1HN, and 1Hα chemical shifts using TALOS-N58 with an assigned minimum range of ±20°. 4¢-phosphopantetheine-serine was created as a serine modified residue within the CYANA library using 4¢-phosphopantetheine coordinates from the solution structure of holo-ACP PfACP from Plasmodium falciparum (PDB ID: 2FQ0)59.
The second stage consisted of the refinement of the 50 lowest CYANA target function conformers by restrained molecular dynamic (rMD) simulations in Amber 1460,61, following published protocols62. Phosphopantetheinyl serine library and force field parameters63 were used for AMBER minimisation. The final representative ensembles correspond to the 20 conformers from each calculation with the lowest restraint energy terms. The structures of holo-ACP5a, holo-ACP6 and holo-ACP7 contain 98.6%, 94.4% and 97.1% in the most favoured region and 1.4 %, 5.6% and 2.9% of the residues (non-glycine and non-proline) in the additional allowed region of the Ramachandran plot, respectively. PROCHECK statistics were calculated using PROCHECK-NMR64. The proportion of residues in the most favoured/additionally allowed/generously allowed/disallowed regions of the Ramachandran plot for the ACPs are as follows: holo-ACP5a (97.1/2.9/0/0); holo-ACP6 (94.3/5.7/0/0); holo-ACP7 (92.4/7.1/0.1/0.4).
Generation of S. pristinaespiralis pathway inactivation mutant. For construction of the pathway mutant, the pCRISPomyces-2 plasmid25 was used for CRISPR-Cas9-based genome editing. Spacer sequences (Supplementary Table 1) were chosen using the online CRISPy-web software65, and were generated by annealing two 24 nt oligonucleotides. Next, 1 kb homologous arms HAL and HAR were amplified by PCR, the pCRISPomyces-2 plasmid was linearised with the restriction enzyme XbaI (Thermo Fisher Scientific), and then assembly of the editing templates and the pCRISPomyces-2 plasmid was performed using the In-Fusion HD Cloning kit (Ozyme, France). Correct plasmid assembly was confirmed by diagnostic digestion and sequencing (Supplementary Fig. 5). Recombinant plasmids were introduced into E. coli 12567 (pUZ8002) by electroporation. Conjugation of plasmids into Streptomyces spores was performed using the protocol described elsewhere66. Following conjugation, clearance of the plasmid was accomplished by repeated high-temperature cultivation (37 °C) for 2–3 days, followed by replica plating on selective and nonselective plates to confirm restoration of apramycin sensitivity. Apramycin-sensitive colonies were then picked into liquid ISP2 medium (4 g L-1 yeast extract, 4 g L-1 dextrose, 10 g L-1 malt extract adjusted to pH 7.3 with NaOH) for genomic DNA isolation using the Wizard Genomic DNA Purification Kit (Promega). Genomic modifications were confirmed by PCR and sequencing of the modified regions (Supplementary Fig. 5).
Analysis by HPLC-MS of the S. pristinaespiralis pathway inactivation mutant. S. pristinaespiralis cultures were extracted twice with ethyl acetate (v/v). When present, XAD-16 resin was harvested by sieving, and also extracted twice with ethyl acetate (v/v). The solvent was removed by evaporation, the extracts resuspended in 1:1 ACN/water (v/v) and then the sample was passed through a 0.4 µm syringe filter. HPLC-MS analysis was performed in positive and/or negative electrospray mode (ESI+/−) on the Thermo Scientific Orbitrap ID-X Tribrid Mass Spectrometer using an Alltima™ C18 column (2.1 × 150mm, 5 µm particle size) at 25°C (flow rate, 0.2 mL min-1). Separation was carried out with Milli-Q water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B), using the following elution profile: 0−48 min, linear gradient 5−95% solvent B; 48−54 min, constant 95% solvent B; 54−60 min, constant 5% solvent B. Mass spectrometry operating parameters were as described previously. Metabolite yields (Extended Data Table 2) were estimated by generating a calibration curve using commercially-available virginiamycin M 1 (Sigma-Aldrich), over the concentration range of 0.00128−20 mg L-1 (10 mL of each sample was injected). This approach afforded a linear correlation between the quantity of metabolite and the respective integrated peak area in the extracted ion chromatogram (EIC) (the areas of the peaks corresponding to the parental ions [M+H]+ were used systematically) (Supplementary Fig. 4). For analysis of metabolite yields in extracts, following conversion of peak areas to titres, the results were divided by 200 to correct for the enrichment of the sample during preparation, as the extracts from 20 mL of culture were resuspended in 100 mL of solvent prior to HPLC-MS analysis (as with the standard, 10 mL of each sample was injected).
Data availability
Crystal structures of VirD and the holo-ACP5b−VirD complex have been deposited in the Protein Data Bank with their respective diffraction data under accession codes 8AHZ and 8AHQ, respectively. Coordinates and chemical shifts for the NMR structures of holo-ACP5a, holo-ACP6 and holo-ACP7 has been deposited in the Biological Magnetic Resonance Bank with accession codes 8A7Z, 8AIG, and 8ALL, respectively. SAXS and HPLC-MS data have been deposited in the data repository DOREL (Données de la Recherche Lorraines) [https://doi-org.insis.bib.cnrs.fr/10.12763/PEYXHP]. The remaining data supporting this study are included in the Supplementary Information. Source data are provided with this paper, and all biological materials are available from the authors upon request.
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