Bacterial strains, plasmids, and chemicals

Strains, plasmids, and PCR primers used in this study are listed in Supplementary Information. PCR primers were obtained from Sigma-Aldrich. Q5 high-fidelity DNA polymerase, restriction endonucleases, and T4 DNA ligase were purchased from NEB and used by following the protocols provided by the manufacturers. DNA gel extraction and plasmid preparation kits were purchased from Omega Bio-Tek. DNA sequencing was conducted by Eton Bioscience. The REDIRECT Technology kit for PCR-targeting homologous recombination was provided by The John Innes Center (Norwich, UK)35. pOJ260 was used as a shuttle vector for gene homologous recombination36. E. coli ET12567/pUZ8002 was used as the host for intergeneric conjugations37. pUWL201PWT, which is a derivative of pUWL201PW38 containing an oriT sequence that was cloned into its PstI site, was used as the shuttle vector for gene complementations, biotransformation, and heterologous production of PtmU4 in Streptomyces. Cosmid libraries were screened by PCR using OneTaq 2× Master Mix with GC buffer (NEB). For Southern analysis, digoxigenin labeling of DNA probes, hybridization, and detection were performed according to the protocols provided by the manufacturer (Roche Diagnostics Corp.). S. platensis CB0073915, CB0076515, MA73279, and MA733910, and their pathway-specific negative regulator ptmR1 inactivation mutants, SB1202615, SB1202914, SB1202715, SB1200118, and SB1260016 were reported previously. S. albus J107439, S. lividans K4-11440, S. avermitilis SUKA2241, and S. coelicolor M114642 were used as model Streptomyces hosts for small-molecule biotransformation and protein production. Other common chemicals, biochemical, and media components were purchased from standard commercial sources.

In-frame deletion of ptmA3 in SB12029 to afford SB12039

To construct the plasmid for in-frame deletion of ptmA3, two 3-kb fragments of the genes upstream and downstream of ptmA3 were amplified from cosmid pBS12037, a cosmid containing a partial ptm gene cluster15, with the primers 739A3up_F, 739A3up_R, 739A3down_F, and 739A3down_R. Both fragments were cloned into the HindIII and EcoRI sites of pOJ260 to obtain pBS12075. pBS12075 was transformed into E. coli ET12567/pUZ8002 and introduced into S. platensis SB12029 by intergeneric conjugation35. After several rounds of passaging the exconjugants, double crossovers via homologous recombination were selected by the apramycin-sensitive phenotype. The genotype of the in-frame deletion mutant SB12039 was verified by PCR analysis and Southern analysis.

Inactivation of ptmU4 in SB12029 to afford SB12040

The ptmU4 gene was replaced with the aac(3)IV+oriT resistance cassette from pIJ773 using λRED-mediated PCR-targeting mutagenesis35 in E. coli BW25113/pIJ790 harboring pBS12037, a cosmid containing a partial ptm gene cluster15. The genotype of the resultant ΔptmU4 mutant cosmid, pBS12074, was confirmed by PCR analysis using primers 739U4ID_F and 739U4ID_R. pBS12074 was transformed into the nonmethylating E. coli ET12567/pUZ8002 and introduced into S. platensis SB12029 by intergeneric conjugation. Single crossovers of ΔptmR1/ΔptmU4 were selected by screening for apramycin resistance on ISP4 medium. After another round of passaging the single-crossover exconjugants in solid ISP4 medium, the ΔptmR1/ΔptmU4 mutant SB12040, a result of double-crossover homologous recombination, was selected for by screening for an apramycin-resistant and kanamycin-sensitive phenotype. The genotype of SB12040 was confirmed by PCR and Southern analysis.

Inactivation of ptmS1, ptmS2, and ptmS4 and disruption of ptmS3 was performed using the protocol described above for the ΔptmR1/ΔptmU4 mutant SB12040. Each genotype was verified by PCR analysis and Southern analysis.

Heterologous production of 5-SH in model Streptomyces hosts

pUWL201PWT was used as an E. coli–Streptomyces expression shuttle vector to construct a 5-SH production system in model Streptomyces hosts. The candidate thioacid cassette genes, ptmA3 and ptmU4, were amplified by PCR using the primers 739U4pUW_F and 739U4pUW_R, and 739A3pUW_F and 739A3pUW_R from pBS12037 and individually cloned into pET-44b(+). ptmU4 was cloned into the NdeI and PstI sites and ptmA3, placed downstream of ptmU4, was cloned into the PstI and HindIII sites to yield pBS12084. The constructed fragment of ptmU4–ptmA3 was cut from pBS12084 at the NdeI and HindIII sites and cloned into pUWL201PWT at the same sites to construct pBS12085.

The three ADHBA biosynthetic genes, ptmB1, ptmB2, and ptmB3, were amplified as a single fragment by PCR using the primers 739B1B3pUW_F and 739B1B3pUW_R from pBS12037, which was subsequently cloned into the HindIII and EcoRI sites of pBS12085. The resulting construct, pBS12086, possessed ptmU4-ptmA3-ptmB1-ptmB2-ptmB3 (Supplementary Figure 7). pBS12086 was transformed into E. coli ET12567/pUZ8002 and introduced into four Streptomyces model strains (S. albus J1074, S. lividans K4-114, S. avermitilis SUKA22, and S. coelicolor M1146) by intergeneric conjugation. Clones containing pBS12086 were selected with thiostrepton.

PTM fermentation medium, supplemented with thiostrepton, was used for the production of 5-SH in the model Streptomyces hosts. After fermentation for 2 days at 28 °C, the fermentation broth was directly used for LC-MS analysis.

Gene cloning

The ptmA3, ptmU4, ptmS2GG, ptmS3GG, and ptmS4 genes from S. platensis CB00739 were amplified by PCR from genomic DNA with Q5 DNA polymerase (NEB). The PCR product was purified, treated with T4 polymerase, and cloned into pBS308043 according to ligation-independent procedures to afford pBS12087 (harboring ptmA3), pBS12088 (harboring ptmU4), pBS12089 (harboring ptmS4), pBS12090 (harboring ptmS2GG, amino acid residues 1–90), and pBS12091 (harboring ptmS3GG, amino acid residues 1–91). The E. coli fabF gene containing a site-directed mutation resulting in FabF C163Q was cloned into pBS308043 as described above, resulting in pBS12096. pUWL201PWT was used as an E. coli–Streptomyces expression shuttle vector and protein expression of PtmU4 in Streptomyces. The full-length ptmU4 gene together with an N-terminal His 6 -tag sequence was amplified by PCR from pBS12088 using the 739StrU4_F and 739StrU4_R primers. Thus, ptmU4 was cloned into the NdeI and HindIII sites of pUWL201PWT affording pBS12092. For site-directed mutagenesis of ptmU4, the ptmU4 gene from pBS12092 was amplified in two steps by primer extension44 using the 739StrU4_F and 739Stru4_R primers with internal primers containing the desired mutation. The mutant ptmU4 genes were then cloned into pUWL201PWT as described above yielding pBS12093–pBS12095.

Gene expression and protein production and purification

PtmA3, PtmS2GG, PtmS3GG, and PtmS4 were produced in E. coli. For enzyme activity assays, the plasmid harboring each gene was transformed into E. coli BL21(DE3) (Life Technologies) and grown in 1 L of lysogeny broth (LB) at 37 °C with shaking at 250 rpm until an OD 600 of 0.6 was reached. The culture was cooled to 4 °C, gene expression was induced with the addition of 0.25 mM isopropyl β-d-1-thiogalactopyranoside, and the cells were grown around 18 h at 18 °C with shaking. After harvesting the cells by centrifugation at 4000 g for 15 min at 4 °C, the pellet was resuspended in lysis buffer (50 mM Tris, pH 8.0, containing 300 mM NaCl and 10 mM imidazole), lysed by sonication, and centrifuged at 15,000 g for 30 min at 4 °C. The supernatant was purified by nickel-affinity chromatography using an ÄKTAxpress system (GE Healthcare Life Sciences) equipped with a HisTrap column. The resultant protein with an N-terminal His 6 -tag was desalted using a HiPrep desalting column (GE Healthcare Biosciences) and concentrated using an Amicon Ultra-15 concentrator (Millipore) in 50 mM Tris, pH 7.8, containing 100 mM NaCl, 50 mM KCl, and 5% glycerol. Protein concentrations were determined from the absorbance at 280 nm using a molar absorptivity constant of each protein. Individual aliquots of each protein were stored at –80 °C until use.

PtmU4 was produced in S. avermitilis SUKA22 for enzyme activity assays. pBS12092 was transformed into E. coli ET12567/pUZ8002 and introduced into S. avermitilis SUKA22 by intergeneric conjugation. Positive colonies were selected using thiostrepton and named S. avermitilis SB12307. Fresh spores of SB12307 were inoculated into TSB seed medium supplemented with thiostrepton and cultured for 2 days. Three liters of TSB medium was inoculated with 5% (v/v) seed culture supplemented with thiostrepton and incubated at 28 °C and 250 rpm for 2 days. After harvesting the cells by centrifugation at 3750 g for 30 min at 4 °C, the pellet was resuspended in lysis buffer (50 mM Tris, pH 8.0, containing 300 mM NaCl and 10 mM imidazole) and 1 mg mL−1 lysozyme and 1.5 tablets of Protease Inhibitor Cocktail (Roche) were added. After incubation on ice for 2 h, the pellet was lysed by sonication, and centrifuged at 15,000 g for 30 min at 4 °C. The supernatant containing PtmU4 was purified in three steps using an ÄKTA FPLC system (GE Healthcare Biosciences): (a) nickel-affinity chromatography equipped with a HisTrap HP, 5 mL column (GE Healthcare Life Sciences), which was first washed with 300 mL Wash buffer (50 mM Tris, pH 8.0, containing 300 mM NaCl and 20 mM imidazole) and then eluted with 100 mL 50% Elution buffer (50 mM Tris, pH 8.0, containing 100 mM NaCl and 500 mM imidazole); (b) anion exchange chromatography equipped with a HiTrap Q HP, 5 mL column (GE Healthcare Life Sciences) using a gradient increasing the concentration of sodium chloride from 0 to 1 M in 50 mM Tris, pH 8.0; (c) size-exclusion chromatography equipped with a Superdex 200 16/600 column (GE Healthcare Life Sciences) using a buffer of 50 mM Tris, pH 7.8, containing 100 mM NaCl, 50 mM KCl, and 5% glycerol. The resultant protein with an N-terminal His 6 -tag was concentrated using an Amicon Ultra-15 concentrator (Millipore). Protein concentrations were determined from the absorbance at 280 nm using a molar absorptivity constant (ε 280 = 103,280 M−1 cm−1). Individual aliquots of PtmU4 were stored at –80 °C until use. Each of the PtmU4 site-directed mutants was produced and purified as described above.

Enzymatic activity of PtmA3

Preliminary incubations were performed in 50 mM phosphate, pH 7.6, containing 1 mM ATP, 1 mM CoA, 5 mM MgCl 2 , 1 mM 5, and 2 μM PtmA3 in a total volume of 50 μL. After incubation at 30 °C for 10 min, 50 μL of CH 3 OH were added to quench the reaction. The reaction mixture was then centrifuged and 2 μL of the supernatant were injected and analyzed by LC-MS. Substrate and product were detected by monitoring 260 nm with a photodiode array detector. The reactions conditions for PtmA3 were optimized by monitoring 5-CoA production using the HPLC method with a flow rate of 0.8 mL min−1 and a 6 min solvent gradient from 2.5–20% CH 3 CN in 10 mM ammonium acetate. Buffers (Tris and phosphate) and different concentrations of ATP, CoA, and MgCl 2 were all tested for improved PtmA3 activity. The optimized reaction conditions were determined to be 50 mM Tris, pH 8.0, containing 2.5 mM ATP, 2.5 mM CoA, and 5 mM MgCl 2 , and used for the kinetic studies of PtmA3.

Kinetic studies of PtmA3

All kinetics assays were performed in the optimized reaction conditions with varying concentration of aryl acids (5–10) in a total volume of 50 μL. Each reaction was incubated at 30 °C for 10 min and boiled 1 min to quench the reaction. After centrifugation, the reaction mixtures were analyzed by HPLC as described above, but using different solvent gradients (5 and 8, 2.5–20% CH 3 CN in 8 min; 6, 7, 9, and 10, 2.5–30% CH 3 CN in 8 min) and the integrated area under curve (AUC) at 260 nm was calculated. A standard curve of 5-CoA–10-CoA was used to convert AUC into the amount of product formed. Each kinetic assay was performed in triplicate.

Enzymatic activity of PtmU4

The reaction was first incubated in 50 mM phosphate, pH 7.4, containing 500 μM ATP, 2 mM MgCl 2 , 2 mM Na 2 S 2 O 3 , 100 μM PtmS2GG or 100 μM PtmS3GG and 40 μM PtmS4 at 30 °C for 30 min. Then, 100 μM 5-CoA and 5 μM PtmU4 was added in a total volume of 50 μL. After incubation at 30 °C for another 30 min, the reaction was quenched by boiling for 1 min. The reaction mixture was then centrifuged and 20 μL of the supernatant were injected and analyzed by HPLC. Each sample was run on an Agilent 1260 HPLC system equipped with an Agilent Poroshell 120 EC-C18 column (50 mm×4.6 mm, 2.7 μm) using a 6 min solvent gradient (0.8 mL min−1) of 2.5–20% CH 3 CN in 10 mM ammonium acetate. Substrate and product were detected by monitoring 260 nm with a photodiode array detector.

When potassium hydrosulfide (KSH) was used to replace the native sulfur donor (sulfur-carrier protein), the reaction was performed in 50 mM phosphate, pH 7.4, containing 5 mM KSH, 200 μM 5-CoA, and 5 μM PtmU4 in a total volume of 50 μL. After incubation at 30 °C for 10 min, the reaction was quenched by boiling for 1 min. The reaction mixture was then centrifuged and 10 μL of the supernatant were injected and analyzed by HPLC as described above. The relative activities of all PtmU4 mutants were determined using a 5-CoA concentration of 500 μM. Due to slower turnovers of all mutants, enzyme concentration and incubation time were increased to 10 μM and 1 h, respectively, to facilitate product detection. The reaction mixture was then centrifuged and 10 μL of the supernatant were injected and analyzed by HPLC as described above. Substrate promiscuity assays of PtmU4 were determined using 500 μM of different CoA substrates and 0.5 μM PtmU4. The aryl thioacid peaks were collected and analyzed by LC-MS using either positive or negative mode (Supplementary Fig. 48).

The one-pot reaction to synthesize 5-SH from 5 using a combination of PtmA3 and PtmU4 was performed using 5 (100 μM), CoA (50 μM), ATP (1 mM), Mg2+ (4 mM), KSH (2 mM), PtmA3 (10 μM), and PtmU4 (5 μM) in phosphate buffer (50 mM, pH 7.4) at 30 °C.

Antibiotic binding using surface plasmon resonance

Experiments were performed on a Biacore X100 (GE Healthcare) instrument at 25 °C and data were analyzed using Biacore X100 evaluation software. HBS-P+buffer (0.1 M HEPES, 1.5 M NaCl, 0.5% v/v surfactant P20, pH 7.4) containing 0.1% dimethyl sulfoxide (DMSO) was used as the running buffer. Cells 1 and 2 were used as the reference and experimental surface, respectively. FabF C163Q was diluted to 0.15 μM in HBS-P+buffer containing 0.1% DMSO and immobilized on an NTA sensor chip (GE Healthcare) at a flow rate of 30 μL min−1. For kinetic analysis, antibiotics (1–4) were prepared by two-fold serial dilutions with HBS-P+buffer containing 0.1% DMSO (0.078–10 μM) and injected over both surfaces at a flow rate of 30 μL min−1. A 120-s association phase was followed by a 350-s dissociation phase. Signals from the reference surface and buffer blank injections were subtracted and the corrected results were globally fit to a 1:1 binding model. The association rate constant (k a ) and dissocation rate constant (k d ) were used to determine the equilibrium dissociation constant (K d ) in units of M.

Computational details

The crystal structure of the E. coli FabF C163Q–PTM complex9 (PDB 2GFX) was used to extract the ADHBA moiety of PTM and the side chains of H303 and H340, or H303, H340, and Q163. For calculations with ADHBSH, one oxygen in the carboxylic acid group of ADHBA was replaced with a sulfur atom. Quantum mechanical DFT calculations were performed using Gaussian 0945. Each of the geometry optimizations were performed at the M06-2X/6-311+G(d,p) level of theory with the SMD implicit solvation model to account for the solvation effects of water and the interior of protein (ε = 4).

Data availability

Proteins from S. platensis CB00739 have been deposited to protein database of the National Center for Biotechnology Information (NCBI), under accession code AIW55578 for PtmA3, AIW55577 for PtmU4, AVR47602 for PtmS1, AVR47603 for PtmS2, AVR47604 for PtmS3, AVR47605 for PtmS4, AVR47606 for SpSCP1, and AVR47607 for SpSCP2. All other relevant data that support the findings of this study are available in the manuscript and the Supplementary Information.