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Actinobacteria, which are one of the largest bacterial phyla and comprise between 13 and 30% of the soil microbiota, are the main source of antibiotic classes in clinical use 1 . During screens for antimicrobials, as many as 50% of actinomycete strains are discarded because they produce a known antibiotic (Supplementary Fig. 1) (ref. 2 ). Despite each strain likely having the capacity to produce many compounds, strains are abandoned because the already characterized antibiotic could interfere with screening for, or purification of, newly discovered compounds 3 . We applied CRISPR-Cas9 genome engineering to knockout genes encoding two of the most frequently rediscovered antibiotics, streptothricin or streptomycin, in 11 actinomycete strains. We report that this simple approach led to production of different antibiotics that were otherwise masked. We were able to rapidly discover rare and previously unknown variants of antibiotics including thiolactomycin, amicetin, phenanthroviridin and 5-chloro-3-formylindole. This strategy could be applied to existing strain collections to realize their biosynthetic potential.

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.