Cloning, expression and knock-out of ribE gene involved in the biosynthesis of Ribostamycin in Streptomyces ribosidificus NRRL B-11466

Abstract

Streptmyces ribosidificus NRRL B-11466 is a producer of ribostamycin, a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic (2DOS-ACAGA). Analysis of the ribostamycin and the related 2DOS-ACAGAs such as, paromamycin, lividomycin, butirosin, neomycin, kanamycin, gentamicin and tobramycin biosynthetic gene clusters showed a conserved ribE gene (1.023 kb) and its homologous. The ribE gene or its homologous, were anticipated to encode 2-deoxy-scyllo-inosamine 1-dehydrogenase required for the biosynthesis of 2DOS, the basic aglycone moiety in all 2DOS-ACAGAs. In order to investigate and prove the biochemistry of regarded gene product, cloning and expression of the ribE gene was carried out. The ribE was amplified via PCR from the chromosomal DNA of Streptomyces ribosidificus NRRL B-11466 using appropriate primers. The PCR product was cloned into cloning pUCPU21 and expression pET16b vectors. Heterologous expressions of RibE protein was performed under the control of the T7 promotor in Escherichia coli JM109 (DE3). The RibE protein (35.42 kDa) was obtained in a soluble His-tagged form as determined by SDS-PAGE and Western blot assay. The ribE- knock-out mutant was created and showed no antibiotic production as compared to the wild-type. The knock-out mutant reproduced ribostamycin after plamid-mediated RibE expression confirming the involvement of RibE protein in the biosynthesis of ribostamycin.