Functional mining of transporters using synthetic selections

Abstract

Only 25% of bacterial membrane transporters have functional annotation owing to the difficulty of experimental study and of accurate prediction of their function. Here we report a sequence-independent method for high-throughput mining of novel transporters. The method is based on ligand-responsive biosensor systems that enable selective growth of cells only if they encode a ligand-specific importer. We developed such a synthetic selection system for thiamine pyrophosphate and mined soil and gut metagenomes for thiamine-uptake functions. We identified several members of a novel class of thiamine transporters, PnuT, which is widely distributed across multiple bacterial phyla. We demonstrate that with modular replacement of the biosensor, we could expand our method to xanthine and identify xanthine permeases from gut and soil metagenomes. Our results demonstrate how synthetic-biology approaches can effectively be deployed to functionally mine metagenomes and elucidate sequence–function relationships of small-molecule transport systems in bacteria.

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Figure 1: Synthetic selection system for thiamine uptake.
Figure 2: Functional metagenomic selection of thiamine transporters.
Figure 3: Phylogenetic and functional relationship of Pnu transporters.
Figure 4: Riboswitch-based xanthine alkaloid selection system.
Figure 5: Functional metagenomic selections identify xanthine uptake transporters.

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Acknowledgements

We thank Y. Yokobayashi (University of California, Davis, California, USA) for providing plasmid pLacthiM19tetA-gfpuv, J. Gallivan (Emory University, Atlanta, Georgia, USA) for providing plasmid pSKD314, D. Paiva (Technical University of Denmark, Kongens Lyngby, Denmark) for metagenomic libraries, R. Lavallee for technical HPLC support, and C. Munck for critical reading of the manuscript. This study was funded by the Novo Nordisk Foundation and the European Union Seventh Framework Programme (FP7-KBBE-2013-7 single stage) under grant agreement no. 613745, Promys. H.J.G. acknowledges additional financial support from Novozymes A/S.

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Contributions

H.J.G. and M.O.A.S. conceived the study. H.J.G. and S.D.P. developed the thiamine functional selection system, and H.J.G. and A.P.B. performed functional metagenomic selections for thiamine uptake. H.J.G., M.K. and L.S.G. developed the HPLC assay for measurement of thiamines. H.J.G. and A.P.B. cloned heterologous genes. H.J.G. and M.T.B. developed the xanthine alkaloid selection system, and H.J.G. performed functional metagenomic selections and analysis. H.J.G. and S.S. performed dose-response characterizations of selected xanthine importers. S.J.H. developed the LC-MS method for xanthine alkaloids and performed measurements of samples prepared by S.S. and H.J.G. H.J.G. wrote the manuscript with contributions from all other authors.

Corresponding author

Correspondence to Morten O A Sommer.

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Competing interests

H.J.G. and M.O.A.S. are named on a pending patent application relating to dual genetic selection systems (WO 2014/187829 A1).

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–4 and Supplementary Tables 1–10. (PDF 1926 kb)

Supplementary Table 11

Comparative genomics of thiamine biosynthesis, salvage and transport of 1752 complete bacterial genomes. (XLSX 166 kb)

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Genee, H., Bali, A., Petersen, S. et al. Functional mining of transporters using synthetic selections. Nat Chem Biol 12, 1015–1022 (2016). https://doi.org/10.1038/nchembio.2189

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