State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, NJ, 07103, USA.
Appl Biochem Biotechnol. 2020 Apr;190(4):1271-1288. doi: 10.1007/s12010-019-03138-3. Epub 2019 Nov 19.
Nucleoside triphosphates and deoxynucleoside triphosphates are important biochemical molecules. In this study, recombinant Escherichia coli that could display nucleotide kinases (INP-N-NMKases) and acetate kinase (INP-N-ACKase) on the cell surface were constructed by fusing an enzyme (NMKase/ACKase) to the N-terminus of ice nucleation protein (INP-N). By using intact recombinant bacteria cells as a catalyst coupled with an ACKase-catalyzed adenosine-5'-triphosphate (ATP) regeneration system, nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) could be synthesized efficiently. In a reaction system with 5 mmol/l substrate, the conversion rates of cytidine-5'-triphosphate (CTP) and deoxycytidine-5'-triphosphate (dCTP) were 96% and 93%, respectively, the conversion rate of ATP and deoxyadenosine-5'-triphosphate (dATP) was 96%, the conversion rate of deoxythymidine-5'-triphosphate (dTTP) was 91%, and the conversion rate of uridine-5'-triphosphate (UTP) was 80%. There was no obvious degradation. At 37 °C, the stability of the surface-displayed fusion protein, especially in the presence of the substrate, was significantly improved. Each whole cell could be reused more than 8 times.
核苷三磷酸和脱氧核苷三磷酸是重要的生化分子。在这项研究中，通过将酶（NMKase/ACKase）融合到冰核蛋白（INP-N）的 N 端，构建了能够在细胞表面展示核苷酸激酶（INP-N-NMKases）和乙酸激酶（INP-N-ACKase）的重组大肠杆菌。通过使用完整的重组细菌细胞作为催化剂，并结合 ACKase 催化的腺苷-5'-三磷酸（ATP）再生系统，可以有效地合成核苷三磷酸（NTPs）和脱氧核苷三磷酸（dNTPs）。在 5mmol/l 底物的反应系统中，胞苷-5'-三磷酸（CTP）和脱氧胞苷-5'-三磷酸（dCTP）的转化率分别为 96%和 93%，ATP 和脱氧腺苷-5'-三磷酸（dATP）的转化率为 96%，脱氧胸苷-5'-三磷酸（dTTP）的转化率为 91%，尿苷-5'-三磷酸（UTP）的转化率为 80%。没有明显的降解。在 37°C 下，表面展示融合蛋白的稳定性，特别是在存在底物的情况下，显著提高。每个完整的细胞可以重复使用超过 8 次。
