Li Zonglin, Zhong Yahui, Qing Zhoulei, Li Zhimin
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130 Meilong Road, Shanghai, 200237, China.
Bioresour Bioprocess. 2024 Jan 4;11(1):6. doi: 10.1186/s40643-023-00724-6.
Cytidine triphosphate (CTP), as a substance involved in the metabolism of phospholipids, proteins and nucleic acids, has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline. In this study, we established an in vitro six-enzyme cascade system to generate CTP. To avoid thermodynamic bottlenecks, we employed a circuitous and two-stage reaction strategy. Using cytidine as the key substrate, the final product CTP is obtained via the deamination and uridine phosphorylation pathways, relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate. Several extremophilic microbial-derived deaminases were screened and characterized, and a suitable cytidine deaminase was selected to match the first-stage reaction conditions. In addition, directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition, along with a 1.7-fold increase in activity over the wild type. After optimizing the reaction conditions, we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM, and the yield of CTP exceeded 82% within 10.0 h.
三磷酸胞苷（CTP）作为一种参与磷脂、蛋白质和核酸代谢的物质，具有确切的药理作用，是合成胞磷胆碱等药物的直接前体。在本研究中，我们建立了一个体外六酶级联系统来生成CTP。为避免热力学瓶颈，我们采用了迂回的两阶段反应策略。以胞苷作为关键底物，通过脱氨和尿苷磷酸化途径，依靠三磷酸胞苷合酶的不可逆反应催化三磷酸尿苷的胺化反应，最终获得产物CTP。筛选并表征了几种嗜极微生物来源的脱氨酶，选择了合适的胞苷脱氨酶以匹配第一阶段反应条件。此外，对该途径中的限速酶CTP合酶进行定向进化改造，获得了一个成功解除产物反馈抑制的变体，其活性比野生型提高了1.7倍。优化反应条件后，我们最终在初始胞苷浓度为20 mM的条件下进行催化反应，10.0 h内CTP的产率超过82%。
