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A scalable platform to identify fungal secondary metabolites and their gene clusters

Nature Chemical Biology volume 13, pages 895–901 (2017)Cite this article

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Abstract

The genomes of filamentous fungi contain up to 90 biosynthetic gene clusters (BGCs) encoding diverse secondary metabolites—an enormous reservoir of untapped chemical potential. However, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS. Using fungal artificial chromosomes (FACs) and metabolomic scoring (MS), we screened 56 secondary metabolite BGCs from diverse fungal species for expression in Aspergillus nidulans. We discovered 15 new metabolites and assigned them with confidence to their BGCs. Using the FAC-MS platform, we extensively characterized a new macrolactone, valactamide A, and its hybrid nonribosomal peptide synthetase–polyketide synthase (NRPS–PKS). The ability to regularize access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

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Figure 1: The FAC-MS platform.
Figure 2: Analysis of benzomalvin A/D biosynthesis.
Figure 3: Analysis of three gene clusters on AtFAC9J20.
Figure 4: Structure and proposed biosynthesis of valactamide A (facms0017).

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Acknowledgements

This work was supported in part by the US National Institutes of Health (National Institute of Allergy and Infectious Diseases SBIR award in the form of grant R44AI094885 to C.C.W., J.W.B., and N.L.K.; grant R01AT009143 to N.L.K.; grant R01-AI065728 to N.P.K.; grant 5T32GM105538-04 to G.P.M.). NMR instrumentation and assistance was provided by the Integrated Molecular Structure Education and Research Center (IMSERC) at Northwestern University.

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  1. Kenneth D Clevenger, Jin Woo Bok and Rosa Ye: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Northwestern University, Evanston, Illinois, USA

    Kenneth D Clevenger, Galen P Miley, Maria H Verdan & Neil L Kelleher

  2. Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA

    Kenneth D Clevenger, Peng Gao, Paul M Thomas & Neil L Kelleher

  3. Department of Medical Microbiology and Immunology and Department of Bacteriology, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Jin Woo Bok, Thomas Velk, KaHoua Yang & Nancy P Keller

  4. Intact Genomics, Inc., St. Louis, Missouri, USA

    Rosa Ye, Cynthia Chen, Matthew Lamprecht, Md Nurul Islam & Chengcang C Wu

  5. Department of Molecular Biosciences, Northwestern University, Evanston, Illinois, USA

    Matthew T Robey, Paul M Thomas & Neil L Kelleher

  6. Center for Forest Mycology Research, Northern Research Station, US Forest Service, Madison, Wisconsin, USA

    Jonathan M Palmer

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Contributions

R.Y., C.C.W., M.L., and M.N.I. worked on the FAC library assembly, FAC end sequencing, FAC DNA preparation, and FAC engineering. C.C.W., J.M.P., C.C., and M.L. carried out fungal secondary metabolite gene cluster prediction, FAC bioinformatic analyses, and FAC next-generation sequencing assembly and annotation. J.W.B., T.V., and K.H.Y. performed the A. nidulans transformation with FACs and prepared samples for metabolite identification and structure determination. K.D.C. and P.G. carried out LC–mass spectrometry analyses. K.D.C. conducted analysis of LC–mass spectrometry data, including development of the analysis pipeline, invention of the FAC-Score, discovery of valactamide A, and bioinformatic analyses of the benzomalvin and valactamide gene clusters, under the supervision of P.M.T. and N.L.K. P.M.T. identified benzomalvin A. G.P.M. carried out total synthesis of benzomalvin A/D. M.H.V., G.P.M., and K.D.C. carried out purification and structural characterization of valactamide A. The paper was written by K.D.C. under the supervision of N.L.K. The Online Methods and supplementary material were prepared by K.D.C., N.P.K., C.C.W., J.W.B., R.Y., G.P.M., M.H.V., and M.T.R. All authors read and approved the final draft of the manuscript. C.C.W., N.P.K., and N.L.K. conceived of and supervised the project.

Corresponding authors

Correspondence to Chengcang C Wu, Nancy P Keller or Neil L Kelleher.

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Clevenger, K., Bok, J., Ye, R. et al. A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat Chem Biol 13, 895–901 (2017). https://doi.org/10.1038/nchembio.2408

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