Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics The GenBank accession number for the sequence of cosmid K1F2 is AF329398.

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Abstract
The biosynthetic gene cluster of the aminocoumarin antibiotic clorobiocin was cloned by screening of a cosmid library of Streptomyces roseochromogenes DS 12.976 with two heterologous probes from the novobiocin biosynthetic gene cluster. Sequence analysis revealed 27 ORFs with striking similarity to the biosynthetic gene clusters of novobiocin and coumermycin A1. Inactivation of a putative aldolase gene, cloR, by in-frame deletion led to the abolishment of the production of clorobiocin. Feeding of the mutant with 3-dimethylallyl-4-hydroxybenzoic acid (Ring A of clorobiocin) restored clorobiocin production. Here, it is suggested that the formation of Ring A of clorobiocin may proceed via a retro-aldol reaction catalysed by CloR, i.e. by a mechanism different from the previously elucidated benzoic acid biosynthetic pathway in Streptomyces maritimus. A comparison of the gene clusters for clorobiocin, novobiocin and coumermycin A1 showed that the structural differences between the three antibiotics were reflected remarkably well by differences in the organization of their respective biosynthetic gene clusters.