Abstract：
OBJECTIVE: 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.
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.
摘要：
目的：使用肝素作为起始多糖硫酸化，使用磺基转移酶生成完全精细的肝素，一种广泛使用的临床药物。然而,肝素和酶的制备被认为是繁琐的，因为这些材料必须在单独的发酵批次中制备。在这项研究中,一种普遍承认的益生菌，大肠杆菌菌株Nissle1917(EcN)，被设计为在细胞内表达磺基转移酶，同时,分泌肝素到培养基中。
结果：工程菌株EcN::T7M，携带编码T7RNA聚合酶的BL21（DE3）的λDE3区，在烧瓶中表达人N-脱乙酰酶/N-磺基转移酶-1（NDST-1）的磺基转移酶结构域（NST）和小鼠3-O-磺基转移酶-1（3-OST-1）的催化结构域。进行了携带表达NST的质粒的EcN::T7M的分批补料发酵，使NST的产量达到0.21g/L，乙酰肝素的产量达到0.85g/L，分别。此外，肝素被纯化，通过1H核磁共振（NMR）表征，并使用3'-磷酸腺苷-5'-磷酸硫酸盐（PAPS）作为磺基供体通过NST进行硫酸化。元素组成分析表明，肝素原的二糖重复序列中有80％以上是N-硫酸化的。
结论：这些结果表明EcN::T7M能够同时制备磺基转移酶和肝素原。EcN::T7M菌株也是表达由tac启动子和T7启动子驱动的外源蛋白的合适宿主。
结果：工程菌株EcN::T7M，携带编码T7RNA聚合酶的BL21（DE3）的λDE3区，在烧瓶中表达人N-脱乙酰酶/N-磺基转移酶-1（NDST-1）的磺基转移酶结构域（NST）和小鼠3-O-磺基转移酶-1（3-OST-1）的催化结构域。进行了携带表达NST的质粒的EcN::T7M的分批补料发酵，使NST的产量达到0.21g/L，乙酰肝素的产量达到0.85g/L，分别。此外，肝素被纯化，通过1H核磁共振（NMR）表征，并使用3'-磷酸腺苷-5'-磷酸硫酸盐（PAPS）作为磺基供体通过NST进行硫酸化。元素组成分析表明，肝素原的二糖重复序列中有80％以上是N-硫酸化的。
结论：这些结果表明EcN::T7M能够同时制备磺基转移酶和肝素原。EcN::T7M菌株也是表达由tac启动子和T7启动子驱动的外源蛋白的合适宿主。
