Lantibiotics are ribosomally synthesized oligopeptide antibiotics that contain lanthionine bridges derived by the posttranslational modification of amino acid residues. Here, we describe the cinnamycin biosynthetic gene cluster (cin) from Streptomyces cinnamoneus cinnamoneus DSM 40005, the first, to our knowledge, lantibiotic gene cluster from a high G؉C bacterium to be cloned and sequenced. The cin cluster contains many genes not found in lantibiotic clusters from low G؉C Gram-positive bacteria, including a Streptomyces antibiotic regulatory protein regulatory gene, and lacks others found in such clusters, such as a LanT-type transporter and a LanP-type protease. Transfer of the cin cluster to Streptomyces lividans resulted in heterologous production of cinnamycin. Furthermore, modification of the cinnamycin structural gene (cinA) led to production of two naturally occurring lantibiotics, duramycin and duramycin B, closely resembling cinnamycin, whereas attempts to make a more widely diverged derivative, duramycin C, failed to generate biologically active material. These results provide a basis for future attempts to construct extensive libraries of cinnamycin variants.C innamycin (Fig. 1) is a peptide antibiotic produced by several Streptomyces strains, including Streptomyces cinnamoneus cinnamoneus DSM 40005. It is assumed to be made by posttranslational modification and processing of a larger ribosomally synthesized peptide. Modifications include the formation of lanthionine bridges, which are thio-ether bridges made when cysteine residues react with dehydroalanine or dehydrobutyrine moieties that have in turn been formed by dehydration of serine or threonine residues, respectively. The possession of lanthionine bridges defines cinnamycin as a lantibiotic. The lantibiotics studied so far are made as prepeptides, each consisting of a leader peptide and a propeptide. Modification of amino acid residues in the propeptide occurs before cleavage of the leader sequence, which releases the mature lantibiotic. In addition to lanthionine residues, lantibiotics may also contain other posttranslationally modified amino acid residues. Thus, cinnamycin contains a -hydroxy-aspartate residue and a lysino-alanine bridge (apparently formed in a similar manner to a lanthionine bridge but with a lysine residue replacing cysteine; Fig. 1). Lantibiotic synthesis and classification have been reviewed (1).There are two major subclasses of lantibiotics. Type A lantibiotics have been studied intensively and have a tertiary structure that is relatively flexible and rod-like. In contrast, the less-studied type B lantibiotics have a relatively globular and inflexible tertiary structure (2). Whereas type A lantibiotics form pores in the cell membrane, type B lantibiotics may inhibit enzymes involved in cell-wall biosynthesis (3). Cinnamycin is closely related to type B lantibiotics duramycin, duramycin B, duramycin C, and ancovenin. These compounds are all derived from 19-aa propeptides and have lanthionine residues in similar position...