Supplementary Figure 11: High-resolution LC–MS and LC–MS/MS analysis of WAC6273 Δorf17 fermentation broths.
(A) High-resolution LC–MS of WAC6273 Δorf17 showing the bamicetin (top) and amicetin (bottom) m/z values. (B) MS/MS of b/amicetin family of compounds. Both families of compounds show fragments with an additional m/z of 56, suggesting that the fermentation broth contains b/amicetin type molecules with replacements of the α-methylserine at the ortho position of p-aminobenzoic acid for other amino acids in these novel derivatives, and pointing towards promiscuity in the biosynthetic enzymes responsible for α-methylserine incorporation21. LC–MS/MS results are representative of two independent analyses. (C) Molecular network of ions in a WAC6273 Δorf17 fermentation broth. Edges and nodes are colored as in Supplementary Fig. 6, with each node representing the relative abundance of each mass in wild-type WAC6273 (red) or streptothricin knockout Δorf15 pCRISPR-Cas9 (blue). The network nodes show parent ions that are related as determined by similar fragmentation patterns present in the MS/MS spectra. The inset lists pairs of parent ions that have similar fragmentation patterns as b/amicetin. Asterisks indicate masses of the known compounds streptcytosine A, amicetin and bamicetin, suggesting that all other masses represent unknown amicetin derivatives. Pie charts are drawn based on one biological replicate but are representative of data collected from three biological replicates.
