Two new (1 and 2) along with six known (3–8) dixiamycins were isolated from the culture broth of a cold-seep-derived actinomycete, Streptomyces olivaceus OUCLQ19-3. Structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations. In the antibacterial test, compounds 1–8 exhibited notable growth inhibitions against a panel of multi-drug-resistant (MDR) strains with MIC values of 0.78–6.25 μg/mL, among which 1, 2, and 5–7 were more potent than the positive control tetracycline.

Summary Methylation is envisioned as a promising way to rationally improve key pharmacokinetic characteristics of lead compounds. Although diverse tailoring enzymes are found to be clustered with cyclodipeptide synthases (CDPSs) to perform further modification reactions on the diketopiperazine (DKP) rings generating complex DKP-containing compounds, so far, a limited number of methyltransferases (MTs) co-occurring with CDPS have been experimentally characterized. Herein, we deciphered the methylation steps during drimentines (DMTs) biosynthesis with identification and characterization of DmtMT2-1 (from Streptomyces sp. NRRL F-5123) and DmtMT1 (from Streptomyces youssoufiensis OUC6819). DmtMT2-1 catalyzes N 4-methylation of both pre-DMTs and DMTs; conversely, DmtMT1 recognizes the DKP rings, functioning before the assembly of the terpene moiety. Notably, both MTs display broad substrate promiscuity. Their combinatorial expression with the dmt1 genes in different Streptomyces strains successfully generated eight unnatural DMT analogs. Our results enriched the MT tool-box, setting the stage for exploring the structural diversity of DKP derivatives for drug development.

Cold‐seeps are areas of the ocean floor in which hydrogen sulfide and methane are released into the open water. The cold‐seep microbes are an emerging source of novel bioactive natural products. Four new ansa‐ring opened linear ansamycin analogues, named olimycins E−H (1–4) were isolated from the cold‐seep‐derived Streptomyces olivaceus OUCLQ19‐3. The planar and stereochemical structures of the isolated compounds were elucidated based on extensive MS and NMR spectroscopic analyses together with ECD calculations.