A survey of Burkholderiales secondary metabolites using library-scale stable isotope labeling reveals selective production of methyl-esterified 3-hydroxybutyrate oligomers

Large-scale untargeted bacterial metabolomics studies are often challenging to interpret, particularly for natural product discovery. Complex raw mass spectrometry data contains media components, biotransformation products, workup contaminants, and in-source fragments, making it difficult to resolve true biosynthesized bacterial products from background features. Incorporating stable isotope labels into untargeted workflows enables the selective detection of actively biosynthesized compounds, drastically reducing candidate mass spectrometry features to highlight novel or robustly expressed metabolites. Recently, our lab introduced IsoAnalyst, a parallel stable isotope labeling platform that links secondary metabolites to biosynthetic gene clusters by determining the incorporation rates of a panel of isotopically labeled building blocks. In this study we applied the IsoAnalyst protocol to a library of 114 Burkholderiales strains with the goals of profiling the specialized metabolite chemistry of this bacterial order and connecting detected natural products to their cognate BGCs based on their labeling profiles. While our strain-matching protocol, which evaluates correlations between detected metabolites and BiG-SCAPE-defined gene cluster families, did not yield confident BGC assignments, it successfully highlighted widely distributed metabolites with distinct labeling profiles. We targeted a set of these molecules for isolation, revealing two classes of compounds derived from primary biomolecule metabolism that are implicated in cellular stress responses. The first class of molecules were identified as lysophosphatidyl ethanolamine analogues while the second was characterized via NMR spectroscopy and mass spectrometry as methyl-esterified 3-hydroxybutyrate oligomers (HB6-OMe, HB7-OMe, and HB8-OMe), representing higher-degree polymer units than previously reported. One-sentence summary Large-scale isotope labelling of a panel of 114 Burkholderiales strains reveals the secondary metabolite diversity of this microbial order, leading to the discovery of novel butyric acid polymers.