Fig. 2: Rapid identification of known chemical scaffolds using high-resolution (HR) mass spectrometry and bioinformatics.

a Identification of enniatin from active fractions of WAC11175 through GNPS based on HRMS and MS/MS data. (i) The enniatin GNPS molecular network, with MS/MS fragment ion matches between the sample (blue) and reference enniatin B1 (black)(inset). (ii) HR-LCMS analysis for enniatins detected in WAC11175 active fractions, including structures and representative HRMS spectra of enniatin A₁ (iii), enniatin B (v), and enniatin B₁ (vii). Corresponding MS/MS spectra acquired at 20 eV and fragmentation analyses are shown for enniatin A₁ (iv), B (vi), and B₁ (viii). N–Me–Val, N–Me–Ile, and Hiv represent N-Methyl-L-valine, N-Methyl-L-isoleucine, and 2-hydroxyisovaleric acid, respectively. b Identification of the enniatin BGC in WAC11175 genome and depiction of its biosynthesis. The enniatin synthetase Esyn1 contains the following domains: C (condensation), A (adenylation), P (phosphopantetheine attachment site), and nMT (N-methyltransferase). L-valine and D-hydroxycarboxylic acid precursors are activated at the A domain, and the building blocks are transferred between modules via P-domains. The final condensation, cyclization, and product release are catalyzed by the C-domains. c Identification of surfactin from active fractions of WAC11084. (i) GNPS molecular network, constructed from HR-MS/MS data, showing MS/MS fragment ion matches (inset panel) between the identified entity (blue) and the reference surfactin C (black). MS/MS fragmentation patterns and structures are shown below: (ii) Surfactin A ([M + H]⁺, m/z 1008.6565) fragments into main ions at 667.4, 568.3, and 455.3; (iii) Surfactin B ([M + H]⁺, m/z 1022.6731) into 909.5, 681.4, and 582.4; (iv) Surfactin C ([M + H]⁺, m/z 1036.6907) into 923.6, 695.4, and 596.4. Diagnostic fragment ions corresponding to specific amino acid sequences were observed, including m/z 227.1750 [Leu + Leu + H]⁺, 229.1145 [Asp + Leu + H]⁺, and 441.2699 [Leu + Asp + Val + Leu + H]⁺. d Genetic organization of the surfactin BGC in WAC11084 and proposed biosynthesis. The surfactin synthetase complex comprises SrfA, SrfB, and SrfC, which assemble the heptapeptide core, while SrfD serves as a key regulatory component. Functional domains include C (condensation), A (adenylation), T (thiolation), E (epimerization), and TE (thioesterase), the latter mediating cyclization and product release.