Production, purification, and analysis of polyenes

The polyenes were produced and analyzed using previously described methods^27,29. The production yield and purity >80% (NPP B1 peak area/areas of all polyene peaks at 405 nm) of the NPP B1 from the recombinant P. autotrophica strain were determined using HPLC analysis using the amphotericin B standard as a reference. Concentrations of NPP B1 were measured using an API2000 LC-MS/MS system (Applied Biosystems) equipped with TurboIon Spray source operated in the positive ion mode. Sample injection volume was 10 ul and separation was performed on an XTerra MS C18 column (2.1 × 50 mm, 3.5 um; Waters) maintained at 30 °C. The column was developed using the following linear gradient: from 0.1% formic acid/0% acetonitrile/100% deionized (DI) water to 0.1% formic acid/100% acetonitrile/0% DI water. The optimized electrospray ionization parameters were as follow: declustering potential (DP): 151 (NPP B1), 111 (amphotericin B) and 56 V (IS); collision energy (CE): 91 (NPP B1), 83 (amphotericin B) and 29 V (IS); focusing potential (FP): 170 (NPP B1), 280 (amphotericin B) and 360 V (IS); collision cell exit potential (CXP): 2 (NPP B1), 0 (amphotericin B) and 4 V (IS). Nebulizer gas (NEB), curtain gas (CUR) and collision gas (CAD) were set to 45, 10 and 6 psi, respectively. The monitoring ion were set as m/z 1127 → 105 for NPP B1, m/z 924 → 107 for amphotericin B and m/z 237 → 194 for the IS. The scan dwell time was 100 ms for each transition channel. All data were acquired and analyzed using the Analyst software (version 1.5.2, Applied Biosystems).