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Aminoglycosides are antibiotics used to treat many types of bacterial infections, particularly those caused by gram negative bacteria. Kanamycin which is isolated from Streptomyces kanamyceticus, is an aminoglycoside antibiotic, used to treat a wide variety of infections. Kanamycin contains kanosamine, a major part plays key factor in biological control of plant disease and highly inhibitory to plant-pathogenic oomycetes is glycosylated from UDP-kanosamine to 2-DOS. Here we tried to one pot enzymatic synthesis of UDP-kanosamine from the cheap substrate UMP as a starting precursor via UDP-glucose and UDP 3-keto glucose. UDP-kanosamine is synthesized in two steps. First, we synthesize UDP-glucose from UMP using ACK, TMK and GalU enzyme and second, from UDP-glucose to UDP-3-keto-glucose an intermediate for UDP-kanosamine. To synthesize UDP-kanosamine, two major enzymes KanC (UDP-glucose dehydrogenase) and KanD (aminotransferase) were cloned in pET32a+ and the soluble protein was expressed and enzymatic assay was carried out and analyzed.