Abstract Background To elucidate the biosynthetic pathways of ribostamycin and related antibiotics, cloning, expression and functional determination of certain genes out of the respective gene clusters have to be carried out. Methods and findings Analysis of the respective antibiotic biosynthetic gene clusters showed that the RibN protein had a remarkable difference in its primary structure relative to NeoN, ParN, and LivN homologous proteins. In this study, the ribC from the ribostamycin biosynthetic gene cluster of Streptomyces ribosidificus NRRL B-11466, and the parN gene from the paromomycin biosynthetic gene cluster of Streptomyces rimosus subsp. paromomycinus NRRL 2455 were amplified using PCR, cloned into the cloning plasmid pUCPU21 producing pURC and pURCPN recombinant plasmids, respectively. RibC protein was expressed in E. coli JM109(DE3) under the control of T7 promoter. The expressed RibC protein was analyzed and shown to produce the functional protein 2-deoxy-scyllo-inosose synthase which catalyses the formation 2-deoxy-scyllo-inosose from glucose-6-phosphate. Alignment of RibN and its homologous proteins revealed a stretch of nonconserved amino acid sequence was detected at the positions from 185 to 261 in the RibN amino acid sequence as determined by multiple amino acid alignment sequences. Both ribC and parN were cloned into pUWL201PW shuttle vector producing pUWRC and pUWPN, respectively. The resulted recombinant plasmids were transformed into S. ribosidificus for the purpose of gene duplication and studying their influence on ribostamycin production. Conclusion RibC was successfully cloned, heterologous expressed in E. coli. The produced protein was biochemically proved to be involved in the synthesis of 2-deoxy-scyllo-inosose from Glucose-6-p. RibN was proved to have endogenous frame-shift mutation which was proposed to be reason for formation of ribostamycin as an end product. RibN was cloned into pUWL201PW shuttle vector as a prerequisite step for its influence on ribostamycin production.