1260 infinity system

Samples were first centrifuged at 15,000×g for 10 min, and then the supernatants were diluted 10 times and determined by HPLC (Agilent Technologies 1260 Infinity system, Santa Clara, CA) equipped with a refractive index detector (RID) and a Rezex^TM ROA-Organic Acid H+ (8%) LC column (Phenomenex Inc., Torrance, CA). The column was eluted with 5 mM of sulfuric acid as a mobile phase at a flow rate of 0.6 mL/min and 50 °C. Agilent OpenLab ChemStation C.01.10 was used to collect the HPLC data. The pure EG, glycolate, glyoxylate, and succinate purchased from Thermo Fisher Scientific (Waltham, MA) were used to establish the calibration curve for EG, glycolate, glyoxylate, and succinate concentration calculation.

The wild-type S. koyangensis SCSIO 5802 and relevant gene-inactivated mutants were first grown on A1 medium agar [20 (link)] at 28° C for 4–5 days to achieve sporulation. A portion of mycelium and spores (1 cm²) for each strain was added to 250 mL flasks containing 50 mL of RA medium. Fermentations were then carried out at 28 °C on rotary shakers (200 rpm) for 8 days. After fermentation, each fermentation culture was washed with 100 mL butanone, and the butanone solvent was removed under reduced pressure to afford an oily residue. Residues were each dissolved into 1 mL MeOH and centrifuged at 13,000× g for 10 min; supernatants were subjected to HPLC–UV analyses, each of which was performed using an Agilent Technologies 1260 Infinity system using a Phenomenex ODS column (150 × 4.6 mm, 5 μm), eluting with a linear gradient of 5 to 65% solvent B (solvent B: CH₃CN + 0.1% trifluoroacetic acid (TFA); solvent A: H₂O + 0.1% TFA) over 20min, followed by 65% to 100% solvent B in 2 min, and then 100% solvent B for 5 min, at a flow rate of 1 mL/min. Since the UV absorption wavelengths of compounds 1–6 and compounds 7–9 are different, chromatograms employed UV detection at both 254 nm and 400 nm to ensure comprehensive detection of new compounds.

UV/Vis spectra were obtained on an Agilent (Agilent Technologies, USA) Cary 300 UV-visible spectrophotometer with a path length of 10 mm. ¹H and 2D- (gCOSY, gHSQCAD, gHMBCAD and NOESY) NMR spectral data were recorded on an Agilent 600 MHz NMR spectrometer equipped with a cold probe. ¹³C NMR spectral data were recorded at 100 MHz on an Agilent NMR spectrometer. Flash column chromatography was carried out on Lichroprep RP-18 (40–63 µm, Merck, USA). Routine HPLC analysis was performed on an Agilent 1260 Infinity system with a Phenomenex (USA) Luna C₁₈(2) (100 Å) 5 µm (4.6 × 150 mm) column and a Photo Diode Array (PDA) detector. The separation and purification of metabolites were performed using an Agilent Prepstar HPLC system with an Agilent Polaris C₁₈-A 5 µm (21.2 × 250 mm) column, a Phenomenex Luna C₁₈(2) or C₈(2) (100 Å) 10 µm (10.0 × 250 mm) column, and an Agilent Phenyl-Hexyl 5 µm (9.4 × 250 mm) column. Low-resolution electrospray ionization mass spectrometry (ESI-MS) data were measured on an Agilent 6120 Quadrupole LC/MS system with a Phenomenex Kinetex C₁₈ (100 Å) 5 µm (4.6 × 250 mm) column. High-resolution ESI-MS data were obtained using an Agilent iFunnel 6550 QTOF (quadrupole time-of-flight) MS instrument fitted with an electrospray ionization (ESI) source coupled to an Agilent 1290 Infinity HPLC system.