Native and engineered clifednamide biosynthesis in multipleStreptomycesspp

Abstract

8Polycyclic tetramate macrolactam (PTM) natural products are produced by actinomycetes and 9 other bacteria. PTMs are often bioactive, and the simplicity of their biosynthetic clusters make 10 them attractive for bioengineering. Clifednamide-type PTMs from Streptomyces sp. JV178 contain 11 a distinctive ketone group, suggesting the existence of a novel PTM oxidizing enzyme. Here, we 12 report the new cytochrome P450 enzyme (CftA) is required for clifednamide production. Genome 13 mining was used to identify several new clifednamide producers, some having improved 14 clifednamide yields. Using a parallel synthetic biology approach, CftA isozymes were used to 15 engineer the ikarugamycin pathway of Streptomyces sp. NRRL F-2890 to yield clifednamides. 16Further, we observed that strong CftA expression leads to the production of a new PTM, 17clifednamide C. We demonstrate the utility of both genome mining and synthetic biology to rapidly 18 increase clifednamide production and identify a PTM tailoring enzyme for rational molecule

Native and Engineered Clifednamide Biosynthesis in Multiple Streptomyces spp.

“…The tandem oxidation of clifednamide A by CftA is analogous to the activity of the recently characterized IkaD 30 . A cytochrome P450 associated with some ikarugamycin biosynthetic clusters, IkaD primarily installs an epoxide across carbons 7 and 8.…”

“…Additionally, all four pHM11a constructs and two pDA1652 constructs resulted in a new peak having a characteristic PTM UV profile and m/z 509 (Figures 4, S4). MS/MS fragmentation produced a daughter ion with m/z of 139, a diagnostic daughter ion of PTM molecules lacking C25 hydroxylation such as clifednamide A, ikarugamycin, and capsimycin 30 . Furthermore, metabolic certified by peer review) is the author/funder.…”