Fermentation, extraction, and sample preparation54 wild type strains (A1090, A1123, A11345, A1137, A1301, A1532, A1636, A2056, A2278, A2705, A2957, A30639, A33995, A34001, A34053, A40707, A40926, A4217, A44034, A5252, A53961, A58051, A5858, A61715, A6562, A80510, A8274, A8567, ATCC 23862, ATCC 31975, T10, T108, T118, T1195, T12, T1236, T1312, T1415, T1416, T1425, T1628, T168, T175, T265, T271, T298, T302, T343, T354, T36, T39, T467, T4680, T676) and their 459 edited mutants were received from the Agency for Science, Research and Technology (A*STAR)’s Natural Organism Library17. They were cultured on ISP2 plates [malt extract 10 g/L, Bacto yeast extract 4 g/L, glucose 4 g/L, Bacto agar 20 g/L] at 28 °C for 5 days. Three agar plugs of 5 mm diameter from the culture plate were then used to inoculate into 250 mL Erlenmeyer flasks each containing 50 mL SV2 seed media [glucose 15 g/L, glycerol 15 g/L, soya peptone 15 g/L, calcium carbonate 1 g/L, pH 7.0] and incubated for 4 days at 28 °C, with shaking at 200 rpm. A volume of 2.5 mL of the homogenized seed cultures were then inoculated into 250 mL Erlenmeyer flasks each containing 50 mL fermentation medium (Table 2). Marine actinomycetes strains were fermented in the same media with addition of 40 g/L sea salt, these media are annotated with the “M” prefix (i.e., MCA02LB instead of CA02LB). All cultures were fermented at 28 °C for 9 days shaking at 200 rpm with 50 mm throw. At the end of the incubation periods, cultures were freeze dried. A total of 2,138 fermentation samples were prepared. The lyophilized samples were extracted overnight (16 h) with methanol (14 mL) with shaking at 150 rpm. The extracted methanolic mixture was passed through cellulose filter paper (Whatman Grade 4, 1004-185) and the filtrate concentrated on a rotary evaporator, 0.1 mg of the dried methanol extract was then submitted for LC-MS/MS analysis.Table 2 Media compositions employed for the fermentation of 54 wild type and 459 mutant strains.Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data acquisitionFermentation extract samples were analysed on an Agilent 1290 Infinity LC System coupled to an Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer. 5 µL of extract was injected onto a Waters Acquity UPLC BEH C18 column, 2.1 × 50 mm, 1.7 µm. Mobile phases were water (A) and acetonitrile (B), both with 0.1% formic acid. The analysis was performed at flow rate of 0.5 mL/min, under gradient elution of 2% B to 100% B in 8 min. LC-MS/MS data was acquired in positive electrospray ionization (ESI) mode MS1 was acquired between m/z 100–2500 at a scan rate of 3 spectra/sec while MS/MS was acquired between m/z 100–2000 at a scan rate of 4 spectra/sec. For MS/MS fragmentation, a ramped collision energy method was employed, whereby the collision energy was determined according to the following formula:$${collision\; energy}\left({eV}\right)=\frac{({precursor\; mz}\times 5)}{100}+2.5$$The typical QTOF operating parameters were as follows: sheath gas nitrogen, 12 L/min at 325 °C; drying gas nitrogen flow, 12 L/min at 350 °C; nebulizer pressure, 50 psi; nozzle voltage, 1.5 kV; capillary voltage, 4 kV. Lock masses in positive ion mode: purine ion at m/z 121.0509 and HP-0921 ion at m/z 922.0098.Molecular networkingMSConvert v3.0.22198-0867718 from Proteowizard28 was used for initial processing of raw liquid chromatography-tandem mass spectrometry (LC-MS/MS) data into an open-source file format (.mzML). All tandem mass spectra (MS/MS) signals with intensity values below 1000 signal intensity were removed as background correction. Classical molecular networking was performed on resulting MS/MS spectra using the online workflow from the GNPS website (http://gnps.ucsd.edu). All peaks in a +/−17 Da around the precursor ion mass were deleted to remove residual precursor ions, and peaks not in the top 6 most intense peaks in a +/−50 Da window were filtered out. The precursor ion mass tolerance was set to 0.02 Da and the MS/MS fragment ion tolerance was set to 0.02 Da. Nearly identical MS/MS spectra with precursor ion m/z within the mass tolerance are combined into a single representative spectrum via the MS-Cluster algorithm29 and annotated as individual metabolites. Representative spectra created from a minimum number of 2 MS/MS spectra were considered for molecular networking. A network was then created where edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks. Further, edges between two nodes were kept in the network if and only if each of the nodes appeared in each other’s respective top 10 most similar nodes. Finally, the maximum size of a molecular family was set to unlimited. The spectra in the network were then searched against GNPS’ spectral libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 6 matched peaks.
