Elucidation of the Late Steps during Hexacosalactone A Biosynthesis in Streptomyces samsunensis OUCT16-12.

Hexacosalactone A (1) is a polyene macrolide compound featuring a 2-amino-3-hydroxycyclopent-2-enone (C 5 N)-fumaryl moiety. While compound 1 has been proposed to be assembled via a type I modular polyketide synthase (PKS) system, most of the putative biosynthetic steps lack experimental evidence. In this study, we elucidated the post-PKS tailoring steps of compound 1 through in vivo gene inactivation and in vitro biochemical assays. We demonstrated that the amide synthetase HexB and O -methyltransferase HexF are responsible for the installations of the C 5 N moiety and the methyl group at 15-OH of compound 1, respectively; two new hexacosalactone analogs, named hexacosalactones B (4) and C (5), were purified and structurally characterized, followed by anti-multidrug resistance (anti-MDR) bacterial assays, revealing that the C 5 N ring and the methyl group are necessary for the antibacterial bioactivities. Through database mining of C 5 N-forming proteins HexABC, six uncharacterized biosynthetic gene clusters (BGCs), putatively encoding compounds with different types of backbones, were identified, providing potentials to discover novel bioactive compounds with C 5 N moiety. IMPORTANCE In this study, we elucidate the post-PKS tailoring steps during the biosynthesis of compound 1 and demonstrate that both C 5 N and 15- O Me groups are critical for the antibacterial activities of compound 1, paving the way for generation of hexacosalactone derivatives via synthetic biology strategy. In addition, mining of HexABC homologs from the GenBank database revealed their wide distribution across the bacterial world, facilitating the discovery of other bioactive natural products with C 5 N moiety.