Tao Hu
Key Laboratory of Horticulture Science for Southern Mountainous Regions，Ministry of Education，College of Horticulture and Landscape Architecture，Southwest University，Chongqing 400715，ChinaGuangtao Zhang
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaYiguang Zhu
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaSumei Li
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaHaibo Zhang
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaGaiyun Zhang
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaXiaohong Yang
Key Laboratory of Horticulture Science for Southern Mountainous Regions，Ministry of Education，College of Horticulture and Landscape Architecture，Southwest University，Chongqing 400715，ChinaJianhua Ju
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaChangsheng Zhang
2CAS Key Laboratory of Marine Bio-resource Sustainable Utilization，RNAM Center of Marine Microbiology，Guangdong Key Laboratory of Marine Materia Medica，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，ChinaSupported by the National Science Foundation of China (30870060，31125001)，by the Key Project of Chinese National Programs for Fundamental Research and Development (2010CB833805)，by the Funds of the Chinese Academy of Sciences for Key Topics in Innovation
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．
Tao Hu, Guangtao Zhang, Yiguang Zhu, Sumei Li, Haibo Zhang, Gaiyun Zhang, Xiaohong Yang, Jianhua Ju, Changsheng Zhang. Cloning，expression and characterization of the nucleotidylyltransferase gene -amiE in amicetin biosynthesis. [J]. Acta Microbiologica Sinica, 2012, 52(2): 214-220
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