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An isotopic labeling approach linking natural products with biosynthetic gene clusters

Nature Chemical Biology volume 18, pages 295–304 (2022)Cite this article

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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 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 demonstrate the utility of this tool with the discovery of lobosamide D, a new and structurally unique member of the family of lobosamide macrolactams.

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Fig. 1: Overview of the IsoAnalyst workflow.
Fig. 2: Overview of IsoAnalyst results for erythromycin A (1).
Fig. 3: SIL incorporation and structures for selected labeled ions detected in the S. erythraea metabolome.
Fig. 4: SIL incorporation and structures for selected labeled ions detected in the Micromonospora sp. metabolome.
Fig. 5: Comparison of lobosamide structures and biosynthesis.

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Data availability

Whole genome sequence (WGS) data for Micromonospora sp. RL09-050-HVF-A was uploaded to NCBI GenBank under the accession number JAGKQP000000000 and the BioProject ID PRJNA718589. The version described in this paper is version JAGKQP010000000. WGS data for Saccharopolyspora erythraea are available in NCBI with the accession code NC_009142. The processed mass spectrometry data, antiSMASH output for Micromonospora sp. RL09-050-HVF-A, and IsoAnalyst output files that support the findings of this study have been deposited in Zenodo [10.5281/zenodo.4711483]. The MIBiG database used in this study is available at https://mibig.secondarymetabolites.org/. Raw mass spectrometry data have been deposited to MassIVE under the accession numbers MSV000087824 (S. erythraea) and MSV00008723 (Micromonospora sp.). The structure of lobosamide D has been deposited to the Natural Products Atlas (www.npatlas.org). The NMR data for lobosamide D have been deposited to the Natural Products Magnetic Resonance Database (NP-MRD; www.np-mrd.org) under accession number NP0044012. MS/MS spectra for lobosamides C and D have been deposited to the Global Natural Products Social molecular networking database (GNPS; https://gnps.ucsd.edu) under accession numbers CCMSLIB00006685341 and CCMSLIB00006709936, respectively.

Code availability

The IsoAnalyst package is freely available via GitHub under an open access MIT software license (https://github.com/liningtonlab/isoanalyst).

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Acknowledgements

We thank E. Ye for assistance with NMR experiments, J. Shoults for fabricating 24-well plate holders, K. Kurita for discussion about label incorporation rates, and T. Clark for providing desferrioxamine compound standards. Funding was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery program (R.G.L.), the National Institutes of Health (AT008718 to R.G.L.) and the Netherlands eScience Center (NLeSC) Accelerating Scientific Discoveries Grant (ASDI.2017.030 to J.J.J.v.d.H. and M.H.M.).

Author information

Authors and Affiliations

  1. Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada

    Catherine S. McCaughey, Jeffrey A. van Santen & Roger G. Linington

  2. Bioinformatics Group, Wageningen University, Wageningen, the Netherlands

    Justin J. J. van der Hooft & Marnix H. Medema

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  1. Catherine S. McCaughey
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Contributions

C.S.M. and R.G.L. designed the study. C.S.M. performed all microbial cultures, isotopic labeling and mass spectrometric analyses. C.S.M. and J.A.v.S. wrote the code and code documentation. J.J.J.v.d.H. and M.H.M. identified the BGCs from the genome sequences and determined the predicted labeling for each BGC. C.S.M. isolated and identified all compounds. C.S.M. and R.G.L. wrote the manuscript with assistance from all authors. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Roger G. Linington.

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Competing interests

J.A.v.S. is a consultant for Unnatural Products Inc. M.H.M. is a member of the Scientific Advisory Board of Hexagon Bio and co-founder of Design Pharmaceuticals.

Additional information

Peer review information Nature Chemical Biology thanks Camila Crnkovic, Hosein Mohimani and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 TCA cycle labeling by [1-13C]acetate.

Red filled circles represent 13C derived from [1-13C]acetate following its direct transformation to acetyl-CoA. When a 13C labeled succinyl-CoA is converted to succinate, the position of the 13C label becomes ambiguous due to the symmetry of succinate, represented by open red circles, indicating that either position has an equal chance of being labeled. Labeled oxaloacetate re-enters the TCA cycle resulting in up to two 13C incorporation events in citrate, isocitrate, and a-ketoglutarate. Groups of amino acids that are derived from TCA cycle biosynthetic precursors are indicated. The interconversion of succinate and succinyl-CoA leads to the indirect incorporation of 13C into the C1 position of methylmalonyl-CoA as indicated by an open red circle while the direct labeling of the C4 position of methylmalonyl-CoA is lost to decarboxylation during the PKS condensation of methylmalonyl-CoA units.

Extended Data Fig. 2 IsoAnalyst MS data processing workflow.

(a) Required data pre-processing steps to generate input files for IsoAnalyst. Files (indicated as parallelograms) highlighted with a light gray box are required input files generated by third party tools that can perform the processes shown in dashed rectangles. We recommend MSconvert and MZmine for these processes however other programs may be used as long as the.csv input meets the requirements described in the software documentation available in the GitHub repository. The ground truth feature list of features aligned across samples is highlighted in a dark gray box and may be generated by the ‘Prep’ step of the IsoAnalyst program (highlighted in green) or by third party tools (for example, MZmine). (b) IsoAnalyst performs the following steps: all isotopologue peak information for every feature is first scraped from the.mzML data files in the ‘Scrape’ step (highlighted in blue). In the ‘Analyze’ step (highlighted in orange), the isotopologue ratios are compared for every feature in each SIL condition to determine the extent of labeling. Finally, a summary file is generated containing all of the SIL incorporation profiles for every feature that contains labeling in two or more conditions.

Extended Data Fig. 3 MS Data centroiding and isotopologue ratio plotting.

(a) Diagram of LC-MS data acquisition showing mass spectra collected at regular time intervals across a chromatographic peak. Each orange line represents a single mass spectrum, or scan, containing m/z vs. intensity values across the instrument’s range. Data are first centroided, or peak picked, to give a single data point corresponding to the intensity of every m/z value in a scan. (b) All centroided scan data for a given feature plotted together as single points. (c) All centroided scan data for the first two isotopologue peaks, M1 and M0, plotted by matching scans. The slope of the linear trend line in (c) is the isotopologue ratio used in the analysis step of the IsoAnalyst workflow.

Extended Data Fig. 4 SIL incorporation for erythrochelin (7).

(a) Substrates used in the biosynthesis of 7, and both the expected and observed labeling of 7. (b) Structures and mass spectra for the iron adduct (m/z 657.2064) and the protonated adduct (m/z 604.2950) of 7 in the [1-13C]acetate and [1-15N]glutamate conditions. [1-13C]Propionate and [methyl-13C] methionine conditions are not shown as 7 was not produced under the [1-13C] propionate condition and no SIL incorporation occurred under the [methyl-13C]methionine condition.

Extended Data Fig. 5 SIL incorporation in fragments of 7.

Mass spectra of fragments m/z 390.1990 (a), m/z 303.1666 (b), m/z 173.0932 (c) and m/z 131.0827 (d) under [1-13C]acetate and [1-15N]glutamate conditions indicate that the detected SIL incorporation is within the expected labeling maximums for the biosynthetic subunits derived from ornithine and acetyl-CoA.

Extended Data Fig. 6 IsoAnalyst profiles for desferrioxamine analogues.

(a) Diagram showing all labeled MS features detected in the metabolome of Micromonospora sp. Circles represent features with IsoAnalyst profiles resembling the desferrioxamines. Diamonds represent features with IsoAnalyst profiles resembling the lobosamides. (b) Structures and IsoAnalyst profiles for desferrioxamine compounds indicated by filled gray circles in (a).

Extended Data Fig. 7 IsoAnalyst profiles of microferrioxamines and their fragments.

Each box contained the expected and observed SIL incorporation for various ions associated with compounds 11, 12, and 13. Due to the substrate flexibility of the acyl transferase DesC39, the exact number of acetate-derived subunits cannot be accurately predicted.

Extended Data Fig. 8 IsoAnalyst profiles of desferrioxamines and their fragments.

(a) Structures with observed fragmentation indicated and mass spectra for [M + H]+ ions of 8 and 14. Expected (Exp.) and observed (Obs.) labeling for 8 and 14 in each condition is indicated in boxes. (b) Mass spectra of observed b-ion and y-ion fragments for 8 and 14. Expected and observed labeling for fragments indicated in boxes.

Extended Data Fig. 9 Lobosamide MS adducts.

Adducts and in-source fragment ions corresponding to compounds 15, 16, and 17, which had SIL incorporation detected by IsoAnalyst.

Supplementary information

Supplementary Information

Supplementary Figs. 1–37, Tables 1–5 and Notes 1–3.

Reporting Summary

Supplementary Table 6

Supplementary_Table_6_Saccharopolyspora.xlsx.

Supplementary Table 7

Supplementary_Table_7_Micromonospora.xlsx.

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McCaughey, C.S., van Santen, J.A., van der Hooft, J.J.J. et al. An isotopic labeling approach linking natural products with biosynthetic gene clusters. Nat Chem Biol 18, 295–304 (2022). https://doi.org/10.1038/s41589-021-00949-6

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