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The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM40725,and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation.Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames,35 of which were defined to constitute the CHL gene cluster.