Convergent biosynthetic transformations to a bacterial specialized metabolite

Abstract

Microbes produce specialized metabolites to thrive in their natural habitats. However, it is rare that a given specialized metabolite is biosynthesized via pathways with distinct intermediates and enzymes. Here, we show that the core assembly mechanism of the antibiotic indolmycin in marine gram-negative Pseudoalteromonas luteoviolacea is distinct from its counterpart in terrestrial gram-positive Streptomyces species, with a molecule that is a shunt product in the Streptomyces pathway employed as a biosynthetic substrate for a novel metal-independent N-demethylindolmycin synthase in the P. luteoviolacea pathway. To provide insight into this reaction, we solved the 1.5 Å resolution structure in complex with product and identified the active site residues. Guided by our biosynthetic insights, we then engineered the Streptomyces indolmycin producer for titer improvement. This study provides a paradigm for understanding how two unique routes to a microbial specialized metabolite can emerge from convergent biosynthetic transformations.

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Fig. 1: Indolmycin biosynthesis and gene clusters.
Fig. 2: In vitro analysis of indolmycin biosynthesis in P. luteoviolacea.
Fig. 3: Crystal structure of PluN2 in complex with 3.
Fig. 4: Proposed mechanism of PluN2 and engineered pathway to indolmycin.

Data availability

The structure of PluN2–3 has been deposited to the Protein Data Bank with accession number 6P29. All other data generated or analyzed in this study are available within the article and the Supplementary Information.

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Acknowledgements

We thank H. He for helpful discussion, S. Yao for metabolic analysis and Z. Zhou for tblastx analysis. This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (grant no. RGPIN-2016-03778), the Alfred P. Sloan Foundation (grant no. FG-20166503), the Canadian Institutes of Health Research (grant nos. FDN-148381 and 201312MSH-322191-209186), the Michael Smith Foundation for Health Research (grant no. 16776), the National Natural Science Foundation of China (grant no. 31872625) and the Zhejiang Provincial Science Foundation for Distinguished Young Scholars (grant no. LR19C010001). Data collection for the crystallographic research in this paper was performed using beamline 08ID-1 at the Canadian Light Source.

Author information

Y.-L.D. designed the study, performed biochemical characterization and structural work, and wrote the manuscript. M.A.H. performed sequence-similarity network analysis and participated in structural work. G.Z. determined steady-state kinetic parameters and conducted pathway engineering. K.S.R. designed the study and wrote the manuscript.

Correspondence to Yi-Ling Du or Katherine S. Ryan.

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Supplementary Information

Supplementary Tables 1–2 and Supplementary Figs. 1–13

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