In vivo characterization of the dia biosynthetic gene cluster reveals diaporthinic acid as its main product

Description

Methods

HPLC-MS/MS analysis
LC-MS grade acetonitrile (ACN) and LC-MS grade formic acid were acquired from VWR chemicals (Radnor, PA, USA). Water (H2O) was purified in-house using a Barnstead™ Smart2Pure™ Water Purification System from Thermo Fisher Scientific (Waltham, MA, USA). Alternariol (CAS # 641-38-3) standard, Catalog # C5061 (Batch No. 1), was purchased from APExBIO (Houston, TX, USA).
The frozen culture media were thawed, vortexed, and 2 mL of each strain and quadruplicate were transferred into fresh Eppendorf tubes. These tubes were spun down for 2 minutes at 20,000 g at room temperature. From the supernatant, 10 μL were taken out and diluted 1:10 in 2 % ACN + 0.1 % FA. The injection volume was 1 μL.
For analysis of the NMR-confirmed diaporthinic acid standard, a stock solution of the compound in DMSO was diluted 1:10 in 2 % ACN + 0.1 % FA. The commercial alternariol standard (1 mg) was dissolved in 1 mL of 50% ACN (with two drops of NH4OH) and further diluted 1:100 in 2 % ACN + 0.1 % FA (c = 10 ng μL-1). The injection volume for both standards was 1 μL.
After dilution of the culture media and the reference standards, the samples were measured employing an untargeted metabolomics workflow in positive ionization mode on a Bruker timsTOF Pro equipped with a VIP-HESI source (Bruker Corporation, Billerica, MA, USA). The frontend was a Thermo Fisher Scientific Vanquish H UHPLC with a Waters Acquity BEH C18 column (150 mm × 1 mm ID, 1.7 μm; Waters Corporation, Milford, MA, USA). Mobile phase A was 0.1 % formic acid in water and solvent B acetonitrile containing 0.1 % formic acid. The following gradient was employed at 40 °C and a constant flow rate of 100 μL min-1: 0 min, 2 % B; 9 min, 98 %; 12 min, 98 % B; 12 min, 2 % B, followed by 4 min re-equilibration. The timsTOF Pro mass spectrometer was operated without trapped ion mobility spectrometry (TIMS off). For positive ionization mode, source capillary voltage was set to 4500 V and dry gas flow to 8 L min-1 at 230 °C. Sheath Gas Flow was set to 4.0 L min-1 at a temperature of 200 °C, with active exhaust being activated. Scan mode was set to Auto MS/MS with 12 Hz MS spectra rate and 16 Hz MS/MS spectra rate, resulting in a total cycle time of 0.5 s. Scan range was set from 20 to 800 m/z.
Data was acquired with Bruker Compass Hystar 6.3. Individual compounds were quantified on MS1 level employing the open-source application Skyline version 23.135,36 normalizing to the TIC, based on exact mass only.