Identification of OxyE as an ancillary oxygenase during tetracycline biosynthesis

The double hydroxylation of 6-pretetramid to 4-keto-anhydrotetracycline is a key tailoring reaction during the biosynthesis of the broad-spectrum antibiotic tetracyclines. It has been shown previously by heterologous reconstitution that OxyL is a dioxygenase and is the only enzyme required to catalyze the insertion of oxygen atoms at the C-12a and C-4 positions. We report here that OxyE, a flavin adenine dinucleotide (FAD)-dependent hydroxylase homologue, is an ancillary mono-oxygenase for OxyL during oxytetracycline biosynthesis in Streptomyces rimosus. By using both gene disruption and heterologous reconstitution approaches, we demonstrated that OxyE plays a nonessential, but important role in oxytetracycline biosynthesis by serving as a more efficient C-4 hydroxylase. In addition, we demonstrated that partially oxidized biosynthetic intermediates can undergo various glycosylation modifications in S. rimosus. Our results indicate that the synergistic actions of OxyE and OxyL in the double hydroxylation step prevent accumulation of shunt products during oxytetracycline biosynthesis in S. rimosus.