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

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.

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.

The N-(His)6-tag AmiE was expressed in E. coli in soluble form and was successfully purified via Ni2+ 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.

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.