| Streptomyces tenebrarius H6 produces three kinds of antibiotics:ampramycin, carbamoyltobramycin and some carbamoylkanamycinB.To study the gene function of tacB from the biosynthetic gene cluster of tobramycin,a fragment including the intact tacB was amplified,and then the SalI-SalI fragment internal to tacB was released and self-ligated.The disruption vector pHZ132-â–³tacB was constructed and transferred into Streptomyces tenebrarius H6 by conjugation.Single-crossover exconjugants were obtained using erythromycin as a selective marker,and then erythromycin-sensitive strains were selected after sporulation in the absence of erythromycin.The double-crossover disruption mutant named S.tenebrarius tacB~- was confirmed by PCR and sequencing,and a new component in the fermentation broth of S.tenebrarius tacB~- was detected by further analysis.The new antibiotic produced by the disruption strain was isolated and purified with ino-exchange method,and identified as 3'-deoxy-carbamoylkanamycin C by MS and NMR analysis.In order to exclude the possibility of polar effects in the disruption mutant,restoration of the intact tacB into S.tenebrarius tacB~- was carried out.The complementary plasmid pSET-tacB was constructed,in which the expression of the intact tacB gene was under the control of the mercury resistant promoter(PmerR).pSET-tacB was transferred into the disruption mutant S.tenebrarius tacB~- by conjugation,and erythromycin-resistant exconjugants were selected.HPLC-ELSD analysis confirmed that carbamoyltobramycin production was partially restored in the complementary strain S.tenebrarius tacB~--tacB.The structural difference of 3'-deoxy-carbamoylkanamycin C and carbamoyltobramycin is that the former has a 6'-hydroxyl group while the latter has a 6'-amino group.Blast analysis proposed the function of TacB as a dehydrogenase,this paper further determined TacB as 6'-dehydrogenase,and which will contribute to clarify the complete biosynthetic pathway of tobramycin.
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