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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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