An isotopic labeling approach linking natural products with biosynthetic gene clusters

Abstract

Major advances in genome sequencing and large-scale biosynthetic gene cluster (BGC) analysis have prompted an age of natural product discovery driven by genome mining. Still, connecting molecules to their cognate BGCs is a substantial bottleneck for this approach. We have developed a mass spectrometry-based parallel stable isotope labeling platform, termed IsoAnalyst, which assists in associating metabolite stable isotope labeling patterns with BGC structure prediction in order to connect natural products to their corresponding BGCs. Here we show that IsoAnalyst can quickly associate both known metabolites and unknown analytes with BGCs to elucidate the complex chemical phenotypes of these biosynthetic systems. We validate this approach for a range of compound classes, using both the type strain Saccharopolyspora erythraea and an environmentally isolated Micromonospora sp. We further demonstrated the utility of this tool with the discovery of lobosamide D, a new and structurally unique member of the family of lobosamide macrolactams.

Metabolomics and Genomics Enable the Discovery of a New Class of Nonribosomal Peptidic Metallophores from a Marine Micromonospora

“…The highly unusual structural features required an array of spectroscopic approaches to establish its structure. An isotopic enrichment strategy 30 using uniformly labeled 13 C glucose and 15 NH 4 Cl in fermentations figured prominently in these efforts and is described in the Supporting Information (SI). Acquisition of 13 C− 13 C gCOSY and 15 N HMBC spectra and detailed analysis of the correlations [details in SI, Section 4.2] supported the structure of 1 (Figure 4A).…”

Integrated Metabolomic–Genomic Workflows Accelerate Microbial Natural Product Discovery

“… 61 Recently developed by Linington and colleagues, the IsoAnalyst platform uses isotopic labeling of biosynthetic precursors in parallel feeding experiments paired with MS to link BGCs to their natural products. 62 The platform utilizes biosynthetic relatedness based on the isotopic patterns rather than deriving structural information on fragments from the tandem mass spectral data. Validated with erythromycin and its analogues, IsoAnalyst was also used to discover a new lobosamide analogue and a new desferrioxiamine compound from Micromonospora sp.…”

“…Validated with erythromycin and its analogues, IsoAnalyst was also used to discover a new lobosamide analogue and a new desferrioxiamine compound from Micromonospora sp. 62 …”

“…The 28.7 kbp BGC for cryptomaldamide was subsequently heterologously expressed in a genetically tractable host, Anabaena PCC, giving further significance to this method of natural product discovery . Recently developed by Linington and colleagues, the IsoAnalyst platform uses isotopic labeling of biosynthetic precursors in parallel feeding experiments paired with MS to link BGCs to their natural products . The platform utilizes biosynthetic relatedness based on the isotopic patterns rather than deriving structural information on fragments from the tandem mass spectral data.…”

“…The platform utilizes biosynthetic relatedness based on the isotopic patterns rather than deriving structural information on fragments from the tandem mass spectral data. Validated with erythromycin and its analogues, IsoAnalyst was also used to discover a new lobosamide analogue and a new desferrioxiamine compound from Micromonospora sp …”