The identification of a lobophorin biosynthetic gene cluster from the deep-sea derived Streptomyces sp. SCSIO 01127 reveals a paradigm of three glycosyltransferases (GTs) LobG1鈥揕obG3 being responsible for appending four sugars. Characterization of five differentially glycosylated metabolites from three GT gene-inactivation mutants allowed the assignment of GT functions and the implication of LobG3 as an iterative GT to attach two digitoxoses.