Silent biosynthetic gene clusters represent a potentially rich source of new bioactive compounds. We report the discovery, characterization, and biosynthesis of a novel doubly glycosylated 24-membered polyene macrolactam from a silent biosynthetic gene cluster in Streptomyces roseosporus by using the CRISPR-Cas9 gene cluster activation strategy. Structural characterization of this polyketide, named auroramycin, revealed a rare isobutyrylmalonyl extender unit and a unique pair of amino sugars. Relative and absolute stereochemistry were determined by using a combination of spectroscopic analyses, chemical derivatization, and computational analysis. The activated gene cluster for auroramycin production was also verified by transcriptional analyses and gene deletions. Finally, auroramycin exhibited potent anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity towards clinical drug-resistant isolates.

The first systematic study of the thermal rearrangement/fragmentation of 5,5‐disubstituted 1,4,2‐oxathiazoles into isothiocyanates is reported. Structure–activity relationships reveal that the choice of substituent at the 5‐position of the 1,4,2‐oxathiazoles is the predominant factor to influence the ease of fragmentation.

Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid.We showed that Streptococcus thermophilus CRISPR1 Cas9 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology-directed repairmediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production, and diversification of natural products.These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.