Clorobiocin is an aminocoumarin antibiotic containing a pyrrole-2-carboxyl moiety, attached through an ester bond to a deoxysugar. The pyrrole moiety is important for the binding of the antibiotic to its biological target, gyrase. The complete biosynthetic gene cluster for clorobiocin has been cloned and sequenced from the natural producer, Streptomyces roseochromogenes DS 12.976. In this study, the genes cloN1 and cloN7 were deleted separately from a cosmid containing the complete clorobiocin cluster. The modified cosmids were introduced into the genome of the heterologous host Streptomyces coelicolor M512 by using the integration functions of the PhiC31 phage. While a heterologous producer strain harbouring the intact clorobiocin biosynthetic gene cluster accumulated clorobiocin, the cloN1- and cloN7-defective integration mutants accumulated a clorobiocin derivative that lacked the pyrrole-2-carboxyl moiety, while also producing free pyrrole-2-carboxylic acid. The structures of these metabolites were confirmed by NMR and MS analysis. These results showed that CloN1 and CloN7, together with the previously investigated CloN2, are involved in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin. A possible mechanism for the role of these three proteins in the acyl-transfer process is suggested.

Aminocoumarin antibiotics are highly potent inhibitors of bacterial gyrase and represent a class of antibiotics that are very suitable for the generation of new compounds by metabolic engineering. In this study, the putative methyltransferase gene cloP in the biosynthetic gene cluster of clorobiocin was inactivated. Expression of the modified gene cluster in the heterologous host Streptomyces coelicolor M512 gave three new aminocoumarin antibiotics. The structures of the new compounds were elucidated by MS and 1H NMR, and their antibacterial activities were determined. All three compounds lacked clorobiocin's methyl group at 4-OH of the deoxysugar moiety, noviose. They differed from each other in the position of the 5-methylpyrrole-2-carbonyl group, which was found to be attached to either 2-OH, 3-OH or 4-OH of noviose. Attachment at 4-OH resulted in the highest antibacterial activity. This is the first time that an aminocoumarin antibiotic acylated at 4-OH in noviose has been detected.

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1 H-NMR and mass spectroscopy and their antibacterial activity was assayed.

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New Aminocoumarin Antibiotics from a cloQ-Defective Mutant of the Clorobiocin Producer Streptomyces roseochromogenes DS12.976. -The three new antibiotics (I) only differ in the position of the pyrrole ring. -(FREITAG, A.; GALM, U.; LI, S.-M.; HEIDE*, L.; J. Antibiot. 57 (2004) 3, 205-209; Pharm. Inst., Eberhard-Karls-Univ. Tuebingen, D-72076 Tuebingen, Germany; Eng.) -C. Oppel 36-209