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

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