General. Specific bacterial strains and plasmids used in this study were summarized in Table S1, PCR primers were listed in Table S2. General enzymes, chemicals, Kits, media, and molecular biological reagents were from standard commercial sources. Bioinformatics analysis, DNA isolation, manipulation, construction of gene replacement and complementation mutants were preformed following the standard methods.
Large Scale Fermentation, Isolation of Metabolites from S. aureus suzhoueusis SP-371 Mutant Strains. S. aureus suzhoueusis TG6004 were grown in TSB (tryptic soy broth) (3%) at 30 °C for 24 h as a seed culture, then 5 mL of seeding culture suspension was transferred into a 500-mL flask containing 100 mL fermentation broth (1% soy bean, 1% peanut meal, 5% glucose, 2% corn starch, 0.6% NH4NO3, 0.3% NaCl, 0.6% CaCO3, pH = 7.2) and the flask was cultured at 30 ℃and 220 rpm for an additional 5 d.
Hydrolysis of fermentation broth. The fermentation broth of ΔtjhO5::2R (TG6027) was dissolved by suitable amount of methanol with 0.25 M hydrochloric acid at 25 ℃ stirring for 1–3 h.
Construction of double knockout mutant Δ tjhO5 / B3. The plasmid TG6018 was introduced from E. coli S17-1 into S. aureus suzhoueusis SP-371 and apramycin-resistant clones were screened at 30 ℃. After picking candidate exconjugants to obtain single-crossover mutants in TSB (tryptic soy broth, 3%) liquid medium with apramycin at 37 ℃, the single-crossover mutants were grown in TSB without antibiotics. The double knockout mutants S. aureus suzhoueusis ΔtjhO5/tjhB3 (S. aureus suzhoueusis TG6032) were confirmed by PCR using TjhB3-yz-f/r as primers (Table S2).
Enzymatic assays and metabolite analysis. High performance liquid chromatography (HPLC) analysis was conducted on Thermo Scientific Dionex Ultimate 3000 (Thermo Fisher Scientific Inc., USA) with a reverse-phase Alltima C18 column (5 µm, 4.6×250 mm). Semi-preparative HPLC was performed on a Shimadzu LC-20-AT system using an YMC-Pack ODS column (YMC, 250×10 mm, 5 µm). HPLC electrospray ionization MS (HPLC-ESI-MS) was performed on the Thermo Fisher LTQ Fleet ESI-MS spectrometer (Thermo Fisher Scientific Inc., USA). High-resolution ESI-MS analysis was conducted on the 6230B Accurate Mass TOF LC/MS System (Agilent Technologies Inc., USA).
HPLC analysis was carried out on a HPLC was performed using a reverse-phase Alltima C18 column (5 µm, 4.6×250 mm) with UV detection at 270 nm. The column was equilibrated with 76% solvents A (H2O and 0.1% HCOOH) and 24% B (MeCN and 0.1% HCOOH) and developed with the following program: T = 0 min, 24% B; T = 24 min, 60% B; T = 29 min, 80% B; T = 30 min, 95% B; T = 33 min, 95% B; T = 35 min, 24% B, with a flow rate of 1 mL/min.
Protein Expression and purification. The TjhO5 gene was amplified by PCR from genome DNA using the primers shown in Table S2. The purified PCR products were ligated to pMD19-T and confirmed by sequencing. The code of TjhD4 was optimized and this gene was synthesized and cloned into pUC57 by Genewiz. The NdeI/HindIII fragment was cloned into the same sites of pET28a to yield plasmid pTG6032 (TjhO5) and pTG6033 (TjhD4), in which enzyme will be overproduced as a C-terminal 8x His-tagged fusion protein. For TjhO5 and TjhD4 expression and purificatin, plasmid pTG6032 and pTG6033 was transformed respectively into BL21(DE3) competent Escherichia coli cells which were grown at 37 ℃ in 800 mL LB with 50 µg/mL kanamycin to an OD600 of 0.6–0.8. Then the cells were induced with 0.1 mM IPTG for 20 hr at 16 oC. The cells were harvested by centrifugation (4,000 rpm, 8 min) and resuspended in 35 mL of Tris buffer (50 mM Tris, pH 8.0, 100 mM NaCl) and lysed by ultrasonication on ice. The lysates were clarified by centrifugation (30 min, 16,500 rpm), and the His-tagged fusion proteins were purified with nickel-affinity chromatography though a linear gradient of 25–500 mM imidazole in the lysis buffer. The fractions were collected in a buffer containing 50 mM Tris, pH 8.0, and 50 mM NaCl. Proteins were analysed by SDS-PAGE gels. Finally, their concentrations were estimated from the absorbance at 280 nm with their corresponding absorption coefficients.
Enzymatic assay.
1) The enzymatic assay of TjhO5 using 15 was carried out at 30 ℃ in a 50 µl aliquot containing 50 mM Tris-HCl (pH 8.0), 10 µM TjhO5, 1 mM NADPH, 0.2 mM 15. After 30 min, the assay was quenched by 50 µl MeOH.
2) The enzymatic assay of TjhD4 using 16 was carried out at 30 ℃ in a 50 µl aliquot containing 50 mM Tris-HCl (pH 8.0), 5 µM TjhD4, 0.5 mM NADH, 0.1 mM 16. After 10 min, the assay was quenched by 50 µl MeOH.
3) The enzymatic assay of TjhO5 and TjhD4 using 15 was carried out at 30 ℃ in a 50 µl aliquot containing 50 mM Tris-HCl (pH 8.0), 5 µM TjhD4, 10 µM TjhO5, 0.5 mM NADPH, 0.2 mM 15. Under the condition of 0.4 mM 15, the specific concentrations in the experiments of changing ratio of TjhO5 and TjhD4: (1) 15 µM TjhD4 and 5 µM TjhO5; (2) 10 µM TjhD4 and 10 µM TjhO5; (3) 5 µM TjhD4 and 15 µM TjhO5. After 1 h, these assays were quenched by 50 µl MeOH. 4) The enzymatic assay of TjhO5 using 4 was carried out at 30 ℃ in a 50 µl aliquot containing 50 mM Tris-HCl (pH 8.0), 5 µM TjhO5, 1 mM NADPH, 0.1 mM 4. After 30 min, this assay was quenched by 50 µl MeOH.
4) In the enzymatic assay of TjhO5/TaGDH or BmGDH, 50 mM Tris-HCl (pH 8.0), 1 mM NADP+, 10 mM D-[1-2H]-glucose, 10 µM BmGDH or TaGDH was incubated in 37 ℃ for 30 min, and then after making the reaction cool down to 30 ℃, added TjhO5 and 15 to 5 µM and 0.1 mM respectively.
5) In the enzymatic assay of TjhD4/TaGDH or BmGDH, 50 mM Tris-HCl (pH 8.0), 1 mM NADP+, 10 mM D-[1-2H]-glucose, 10 µM BmGDH or TaGDH was incubated in 37 ℃ for 30 min, and then after making the reaction cool down to 30 ℃, added TjhD4 and 16 to 2.5 µM and 0.025 mM respectively.
6) In the enzymatic assay of TjhO5 with H218O. 15 was incubated in 50 µL 97% H218O for 30 min in 37 ℃. Then after making the reaction cool down to 30 ℃, TjhO5, NADPH and Tris-HCl Buffer were incubated in 30 ℃ for 30 min. The enzymatic reactions were extracted by equal amount of CH2Cl2, centrifuged at 12,000 g for 5 min. The organic phase was evaporated and added 5 µL DMSO and 100 µL H2O for overnight.
Large scale preparation of enzymatic products.
1) 16: A 10 mL reaction contain 50 mM Tris-HCl (pH 8.0), 10 µM TjhO5, 2 mM NADPH, 0.2 mM 15. The solution was extracted by CH2Cl2. The crude extracts were purified by semi-preparative HPLC with isocratic elution by acetonitrile and water using a flow rate of 3 mL/min (50% acetonitrile).
2) 2H-16: 500 µL x100 reaction contain 50 mM Tris-HCl (pH 8.0), 2 mM NADP+, 20 mM D-[1-2H]-glucose, 20 µM TaGDH incubated in 37 ℃ for 1 h, and then after making the reaction cool down to 30 ℃, added TjhO5 and 15 to 10 µM and 0.25 mM respectively. The solution was extracted by CH2Cl2. The crude extracts were purified by semi-preparative HPLC with isocratic elution by acetonitrile and water using a flow rate of 3 mL/min (50% acetonitrile).
3) 2H-4, 2H-8, and 2H-17: 500 µL x200 reaction contain 50 mM Tris-HCl (pH 8.0), 2 mM NADP+, 20 mM D-[1-2H]-glucose, 20 µM BmGDH incubated in 37 ℃ for 1 h, and then after making the reaction cool down to 30 ℃, added TjhO5, TjhD4 and 15 to 5 µM, 10 µM and 0.25 mM respectively. The solution was extracted by ethyl acetate. The crude extracts were purified by semi-preparative HPLC with isocratic elution by acetonitrile and water using a flow rate of 3 mL/min (30% acetonitrile).
Analytical Data.
Compound 15. yellow solid; HR-ESI-MS (-) found m/z 383.0781 [M-H]− (calcd [M-H]− for
C20H15O8, 383.0772); NMR data see Supplementary Table 3 and Supplementary Fig. 19–23.
Compound 16. yellow solid; HR-ESI-MS (-) found m/z 369.0990 [M-H]− (calcd [M-H]− for
C20H17O7, 369.0980); NMR data see Supplementary Table 4 and Supplementary Fig. 24–29.
Compound 17. yellow solid; HR-ESI-MS (-) found m/z 371.1145 [M-H]− (calcd [M-H]− for
C20H19O7, 371.1136); NMR data see Supplementary Table 5 and Supplementary Fig. 30–35.
Compound 4. yellow solid; HR-ESI-MS (-) found m/z 369.0989 [M-H]− (calcd [M-H]− for
C20H17O7, 369.0980); NMR data see Supplementary Table 6 and Supplementary Fig. 36–41.
Compound 2H-16. yellow solid; HR-ESI-MS (-) found m/z 371.1112 [M-H]− (calcd [M-H]− for C20H152H2O9, 371.1105); NMR data see Supplementary Fig. 42.
Compound 2H-4. yellow solid; HR-ESI-MS (-) found m/z 370.1051 [M-H]− (calcd [M-H]− for
C20H162H2O7, 370.1043); NMR data see Supplementary Fig. 43–44.
Compound 2H-8. yellow solid; HR-ESI-MS (-) found m/z 372.1208 [M-H]− (calcd [M-H]− for
C20H182H2O9, 372.1199); NMR data see Supplementary Fig. 45–46.
Compound 2H-17. yellow solid; HR-ESI-MS (-) found m/z 372.1210 [M-H]− (calcd [M-H]− for C20H152H2O9, 372.1199); NMR data see Supplementary Fig. 47–48.