Xiaomei Li1, Yanying Yu1, Jiaqing Tang1, Bingxue Gong1, Wenjing Li1, Tingting Chen1, Xianxuan Zhou2. 1. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China. 2. School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, China. zhoux@hfut.edu.cn.
Abstract
OBJECTIVES: Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. RESULTS: The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. CONCLUSIONS: These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.
OBJECTIVES: Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. RESULTS: The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. CONCLUSIONS: These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.
Authors: Jinghua Chen; Fikri Y Avci; Eva M Muñoz; Lynda M McDowell; Miao Chen; Lars C Pedersen; Lijuan Zhang; Robert J Linhardt; Jian Liu Journal: J Biol Chem Date: 2005-10-31 Impact factor: 5.157