Enzymatic Synthesis of Nucleoside Triphosphates and Deoxynucleoside Triphosphates by Surface-Displayed Kinases
- 57 Downloads
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.
KeywordsNucleoside triphosphates Deoxynucleoside triphosphates Nucleotide kinase Acetate kinase Ice nucleation protein Surface display ATP regeneration
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- 1.Li, Y., Li, X. M., Zhang, H., Xu, R. F., & Tao, G. C. (2008). Effect of cytidine triphosphate and sodium ferulate on nerve conduction velocity in painful diabetic neuropathy. Journal of the Fourth Military Medical University, 22, 2075–2077.Google Scholar
- 2.Fang, L., Wang, J., & Zhao, Z. H. (2007). Curative effect of cytidine triphosphate via intramuscular injection in 30 patients with diabetic peripheral neuropathy. Journal of the Fourth Military Medical University, 28, 2275–2277.Google Scholar
- 3.Li, X. M., Li, Y., Zhao, K. Y., & Sun, L. H. (2005). Effect of cytidine triphosphate on nerve conduction velocity in patients with diabetic peripheral neuropathy. Chinese Journal of Clinical Rehabilitation, 9, 152–153.Google Scholar
- 5.Bennett, W. D., Zeman, K. L., Foy, C., Shaffer, C. L., Johnson, F. L., Regnis, J. A., Sannuti, A., & Johnson, J. (2001). Effect of aerosolized uridine 5′-triphosphate on mucociliary clearance in mild chronic bronchitis. American Journal of Respiratory and Critical Care Medicine, 164(2), 302–306.CrossRefGoogle Scholar
- 7.Wihlborg, A. K., Balogh, J., Wang, L., Borna, C., Dou, Y., Joshi, B. V., Lazarowski, E., Jacobson, K. A., Arner, A., & Erlinge, D. (2006). Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction. Circulation Research, 98(7), 970–976.CrossRefGoogle Scholar
- 14.Yano, T., Yasohara, Y., Kashihara, M., Tachiki, T., Hidehiko, K., & Tatsurokuro, T. (1989). Production of deoxyribonucleoside triphosphates through coupled fermentation with energy transfer. Agricultural and Biological Chemistry, 53, 1935–1940.Google Scholar
- 15.You, Y., Ding, Q. B., & Ou, L. (2007). Biosynthesis of nucleosides triphosphate by immobilized beer yeast cells. Industrial Microbiology., 37, 31–35.Google Scholar
- 20.Becker, S., Hobenreich, H., Vogel, A., Knorr, J., Wilhelm, S., Rosenau, F., Jaeger, K. E., Reetz, M. T., & Kolmar, H. (2008). Single-cell high-throughput screening to identify enantioselective hydrolytic enzymes. Angewandte Chemie (International Ed. in English), 47(27), 5085–5088.CrossRefGoogle Scholar
- 24.Maruthamuthu, M. K., Selvamani, V., Nadarajan, S. P., Yun, H., Oh, Y. K., Eom, G. T., & Hong, S. H. (2018). Manganese and cobalt recovery by surface display of metal binding peptide on various loops of OmpC in Escherichia coli. Journal of Industrial Microbiology & Biotechnology, 45(1), 31–41.CrossRefGoogle Scholar
- 26.van Bloois, E., Winter, R. T., Janssen, D. B., & Fraaije, M. W. (2009). Export of functional streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis. Applied Microbiology and Biotechnology, 83(4), 679–687.CrossRefGoogle Scholar
- 28.Bao, S., Yu, S., Guo, X., Zhang, F., Sun, Y., Tan, L., Duan, Y., Lu, F., Qiu, X., & Ding, C. (2015). Construction of a cell-surface display system based on the N-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins. Journal of Applied Microbiology, 119(1), 236–244.CrossRefGoogle Scholar
- 33.Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.Google Scholar