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

Abstract:

Abstract:［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 ucleotidylytransferase 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 AmiEcatalyzedreactions 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．