[Show abstract][Hide abstract] ABSTRACT: A gene inactivation study was performed on gntE, a member of the gentamicin biosynthetic gene cluster in Micromonospora echinospora. Computer-aided homology analysis predicts a methyltransferase-related cobalamin-binding domain and a radical S-adenosylmethionine domain in GntE. It is also found that there is no gntE homolog within other aminoglycoside biosynthetic gene clusters. Inactivation of gntE was achieved in both M. echinospora ATCC 15835 and a gentamicin high-producer GMC106. High-performance liquid chromatographic analysis, coupled with mass spectrometry, revealed that gntE mutants accumulated gentamicin A2 and its derivative with a methyl group installed on the glucoamine moiety. This result substantiated that GntE participates in the first step of pseudotrisaccharide modifications in gentamicin biosynthesis, though the catalytic nature of this unusual oxidoreductase/methyltransferase candidate is not resolved. The present gene inactivation study also demonstrates that targeted genetic engineering can be applied to produce specific gentamicin structures and potentially new gentamicin derivatives in M. echinospora.
Biochemical and Biophysical Research Communications 09/2008; 372(4):730-4. DOI:10.1016/j.bbrc.2008.05.133 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The biosynthetic gene cluster for bluensomycin, a member of the aminoglycoside family of antibiotics, was isolated and characterized from the bluensomycin producing strain, Streptomyces bluensis ATCC27420. PCR primers were designed specifically to amplify a segment of the dTDP–glucose synthase gene based on its conserved sequences among several actinomycete strains. By screening a cosmid library using amplified PCR fragments, a 30-kb DNA fragment was isolated. Sequence analysis identified 15 open reading frames (ORFs), eight of which had previously been identified by Piepersberg et al. But seven are novel to this study. We demonstrated that one of these ORFs, blmA, confers resistance against the antibiotic dihydrostreptomycin, and another, blmD, encodes a dTDP–glucose synthase. These findings suggest that the isolated gene cluster is very likely to be responsible for the biosynthesis of bluensomycin.