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Identifying producers of antibacterial compounds by screening for antibiotic resistance

Abstract

Microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges. We have designed a screen to exploit the self-protection mechanism of antibiotic producers to enrich microbial libraries for producers of selected antibiotic scaffolds. Using resistance as a discriminating criterion we increased the discovery rate of producers of both glycopeptide and ansamycin antibacterial compounds by several orders of magnitude in comparison with historical hit rates. Applying a phylogeny-based screening filter for biosynthetic genes enabled the binning of producers of distinct scaffolds and resulted in the discovery of a glycopeptide antibacterial compound, pekiskomycin, with an unusual peptide scaffold. This strategy provides a means to readily sample the chemical diversity available in microbes and offers an efficient strategy for rapid discovery of microbial natural products and their associated biosynthetic enzymes.

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Figure 1: Comparison of resistance-based GPA screening with other screening approaches and their rates of success.
Figure 2: The heptapeptide core of the glycopeptides varies in amino acid composition and intra-strand cross-links, creating scaffold diversity.
Figure 3: The abundance and diversity of GPA producers.
Figure 4: GPAs identified in the screen.
Figure 5: GPA biosynthetic clusters from the draft genomes of WAC strains.
Figure 6: Rifampin-assisted isolation of ansamycin producers.

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Acknowledgements

We are grateful to X.D. Wang for isolation of actinomycetes, K. Koteva for sharing expertise in GPA purification, C. King for genome sequencing and C. Quinn, TA Instruments for help with ITC data. This research was funded by a Canadian Institutes of Health Research (CIHR) Grant MT-14981, Natural Sciences and Engineering Research Council Grant (237480) and by a Canada Research Chair in Antibiotic Biochemistry (G.D.W.).

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M.N.T. designed the experiments, isolated genomic DNA, designed PCR primers, standardized PCR conditions, and carried out BOX PCRs and other PCRs, performed isolation and purification of GPAs, performed in silico genome analysis, determined MIC values and wrote the manuscript. W.W. purified the GPAs, carried out NMR experiments and their analysis, and elucidated the structures. P.S. designed and carried out experiments for the resistance-based discovery of ansamycins. N.W. performed genome assemblies, created phylogenies and bioinformatic analyses, and submitted sequences. A.M.K. designed primers and isolated genomic DNA. R.M. isolated genomic DNA, performed 16S rDNA PCRs and fingerprinting PCRs. G.D.W. developed the concept, designed the experiments and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Gerard D Wright.

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The authors declare no competing financial interests.

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Supplementary Figures 1–21 and Supplementary Tables 1–9 (PDF 4376 kb)

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Thaker, M., Wang, W., Spanogiannopoulos, P. et al. Identifying producers of antibacterial compounds by screening for antibiotic resistance. Nat Biotechnol 31, 922–927 (2013). https://doi.org/10.1038/nbt.2685

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