[Objective]The aim of this study is to clone and express the nucleotidylytransferase encoding gene-amiE from the biosynthetic gene cluster of amicetin,a disaccharide nucleoside antibiotic,and to characterize AmiE in vitro.[Methods]The amiE,encoding a nucleotidylytransferase of 257 amino acid,was PCR amplified and cloned into pET28a,resulting in the plasmid pCSG4001,which was transformed into E.coli BL21(DE3) for expressing N-(His)6-tag AmiE.The recombinant AmiE was purified by affinity chromatography via AKTA Purifier 10 system.The AmiE-catalyzed reactions were performed using TTP(or UTP) and glucose-1-phosphate as substrates.The enzyme assays were analyzed by HPLC;the substrate flexibility of AmiE was probed with three unnatural sugars-1-phosphate,including galactose-1-phosphate,galactosamine-1-phosphate and mannos-1-phosphate.[Results]The N-(His)6-tag AmiE was expressed in E.coli in soluble form and was successfully purified via Ni+2 mediated affinity chromatography;in vitro biochemical experiments showed that AmiE could convert glucose-1-phosphate into TDP-glucose(or UDP-glucose) in the presence of TTP(or UTP).However,galactose-1-phosphate,galactosamine-1-phosphate and mannos-1-phosphate were not substrates of AmiE.[Conclusion]The amiE was successfully cloned and expressed in E.coli,and the purified AmiE was biochemically confirmed to be a nucleotylyltransferase in amicetin biosynthesis pathway.

Cloning,expression and characterization of the nucleotidylyltransferase gene-amiE in amicetin biosynthesis

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