Hygromycin B is an aminoglycoside antibiotic with a structurally distinctive orthoester linkage. Despite its long history of use in industry and in the laboratory， its biosynthesis remains poorly understood. We show here， by in-frame gene deletion in vivo and detailed enzyme characterization in vitro， that formation of the unique orthoester moiety is catalyzed by the α-ketoglutarate- and non-heme iron-dependent oxygenase HygX. In addition， we identify HygF as a glycosyltransferase adding UDP-hexose to 2-deoxystreptamine， HygM as a methyltransferase responsible for N-3 methylation， and HygK as an epimerase. These experimental results and bioinformatic analyses allow a detailed pathway for hygromycin B biosynthesis to be proposed， including the key oxidative cyclization reactions.