1200 series high

All samples taken from growth experiments for chemical analysis were centrifuged at 12,000 x g for 1 min at 4°C, and the supernatants were stored at -20°C until analysis. Analysis of the substrates and metabolites were performed using an Agilent Technologies (USA) 1200 Series high-performance liquid chromatography (HPLC) system with a Quaternary pump, an Autosampler and a 5-channel variable wavelength UV-Vis detector. The separation of compounds was achieved using an Agilent ZORBAX Eclipse XDB-C18 (4.6 x 250 mm i.d., 5 μm) analytical column and guard column (12.5 x 4.6 mm i.d.) of the same material (Agilent Technologies, USA). Elution was carried out at a flow rate of 0.80 ml min^-1 within 15 minutes with a mobile phase gradient as follows: 0 min B (methanol)/C (standard buffer of 0.1% formic acid and 1 mM ammonium acetate, pH ~2.8) 45/55, 9 min 75/25, 11 min 45/55, and 13 min 45/55. The separation of the mixtures (injection volume 20 μl) was carried out at room temperature and the UV-Vis detector was set at 274 nm. The combined standard uncertainties of the results were between 2.1% and 3.4%. The preparation of the sample, instrumental measurements (both samples and calibration standards) and integration of the peaks are the main contributions to the uncertainties [37 ].

An Agilent 1200 series high performance liquid chromatography (HPLC) system consisting of G2258A DLA dual loop auto-sampler and G1361A preparative pump were used for the further purification of CGEEA-F5 (0.42 g) using a X bridge C18 Prep column (5 μm, 19 mm × 100 mm) and monitored at 254 nm using a Waters G1315D DAD Diode Array Detector. Solvents (A: 0.1% v/v glacial acetic acid in water, B: 0.1% v/v glacial acetic acid in methanol) were used at a flow rate of 16 mL/min and a max pressure of 400 bar. A gradient of A and B solvent system (5% B for 5 min; 5–60% B over 25 min; and 100% B for 6 mins) was used. Twelve sub-fractions (CGEEA-F5-(1 to 12)) were collected and lyophilized to give: F1, 12 mg; F2, 2 mg; F3, 2 mg; F4, 2 mg; F5, 2 mg; F6, 4 mg; F7, 16 mg; F8, 21 mg; F9, 83 mg; F10, 223 mg; F11, 13 mg; and F12, 8 mg.

S. yeochonensis CN732 was initially cultivated in 50 mL of acidified ISP medium 2 (pH 5). After the strain was cultivated for 4 days on a rotary shaker at 170 rpm and 30 °C, 10 mL of the culture was transferred in 1 L of modified Bennet’s medium in a 2.8-L Fernbach flask. The entire culture (72 L) was extracted twice with ethyl acetate (150 L). The EtOAc extract was concentrated in vacuo to yield 10 g of dry material.
Optical rotations were measured using a JASCO P-1020 polarimeter. UV spectra were acquired on a Chirascan plus spectrometer of Applied Photophysics Ltd. Low-resolution electrospray ionization source mass spectra were acquired with an Agilent Technologies 6130 quadrupole mass spectrometer coupled to an Agilent Technologies 1200 series high-performance liquid chromatography (HPLC) instrument.