Cell-free regeneration of ATP based on polyphosphate kinase 2 facilitates cytidine 5'-monophosphate production.

Autor: Teng F; Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China., Wang L; Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: wl8893@163.com., Hu M; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China., Tao Y; Chinese Academy of Sciences Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address: taoyong@im.ac.cn.
Jazyk: angličtina
Zdroj: Enzyme and microbial technology [Enzyme Microb Technol] 2023 Apr; Vol. 165, pp. 110211. Date of Electronic Publication: 2023 Feb 11.
DOI: 10.1016/j.enzmictec.2023.110211
Abstrakt: Cytidine 5'-monophosphate (5'-CMP), a key intermediate for the production of nucleotide derivatives, has been extensively used in food, agriculture, and medicine industries. Compared to RNA degradation and chemical synthesis, the biosynthesis of 5'-CMP has attracted wide attention due to its relatively low cost and eco-friendliness. In this study, we developed a cell-free regeneration of ATP based on polyphosphate kinase 2 (PPK2) to manufacture 5'-CMP from cytidine (CR). McPPK2 from Meiothermus cerbereus exhibited high specific activity (128.5 U/mg) and was used to accomplish ATP regeneration. McPPK2 and LhUCK (a uridine-cytidine kinase from Lactobacillus helveticus) were combined to convert CR to 5'-CMP. Further, the degradation of CR was inhibited by knocking out cdd from the Escherichia coli genome to enhance 5'-CMP production. Finally, the cell-free system based on ATP regeneration maximized the titer of 5'-CMP up to 143.5 mM. The wider applicability of this cell-free system was demonstrated in the synthesis of deoxycytidine 5'-monophosphate (5'-dCMP) from deoxycytidine (dCR) by incorporating McPPK2 and BsdCK (a deoxycytidine kinase from Bacillus subtilis). This study suggests that the cell-free regeneration of ATP based on PPK2 has the advantage of great flexibility for producing 5'-(d)CMP and other (deoxy)nucleotides.
Competing Interests: Conflict of Interest The authors confirm that there is no conflict of interest regarding this article.
(Copyright © 2023 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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