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| Amicetin, an antibacterial and antiviral agent, belongs to a group of disaccharide nucleoside antibiotics featuring an ¦Á -(1¡ú4)-glycoside bond in the disaccharide moiety. In this study, the amicetin biosynthetic gene cluster was cloned from Streptomyces vinaceus-drappus NRRL 2363 and localized on a 37 kb contiguous DNA region. Heterologous expression of the amicetin biosynthetic gene cluster in Streptomyces livdians TK64 resulted in the production of amicetin and its analogues, thereby confirming the identity of the ami -gene cluster. In silico sequence analysis revealed that 21 genes were putatively involved in amicetin biosynthesis, including 3 for regulation and transportation, 10 for disaccharide biosynthesis, and 8 for the formation of amicetin skeleton by the linkage of cytosine, para-benzoic acid (PABA), and the terminal (+)-¦Á -methylserine moieties. The inactivation of the benzoate-CoA ligase gene amiL and the N-acetyltransferase gene amiF, led to two mutants, which accumulated the same two compounds cytosamine and 4-acetamido-3- hydroxybenzoic acid. These data indicated that AmiF functioned as an amide synthethase to link cytosine and PABA. The inactivation of amiR, encoding an acyl CoA-acyl carrier protein transacylase, resulted in the production of plicacetin and norplicacetin, indicating AmiR to be responsible for attachment of the terminal methylserine moiety to form another amide bond. These findings implicated two alternative strategies for amide bond formation in amicetin biosynthesis. |
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