Zhang Xiangjun, Niu Pilian, Liu Lu, Ye Tong, Ding Wei, Wei Xiaobo, Zhu Tengteng, Li Zongqian, Fang Haitian, Liu Huiyan
School of Life Science, Ningxia University, Yinchuan, Ningxia 750021, China.
School of Food Science and Engineering, Ningxia Key Laboratory for Food Microbial-Applications Technology and Safety Control, Ningxia University, Yinchuan, Ningxia 750021, China.
J Agric Food Chem. 2025 May 28;73(21):12776-12786. doi: 10.1021/acs.jafc.5c01391. Epub 2025 May 13.
As a pyrimidine nucleoside, cytidine is widely used in the medicine and food fields. Therefore, it is important to construct a microbial cell factory for efficient and sustainable cytidine production. Here, we perform modular metabolic engineering modifications on 168 to achieve efficient synthesis of cytidine. First, the cytidine titer reached 0.88 g/L by blocking cytidine degradation and enhancing the cytidine synthesis pathway. Next, the central carbon metabolism was modulated by knocking down CcpA, but the cytidine titer decreased instead. Transcriptome analysis revealed that differential genes were mainly enriched in PTS, glycolysis, TCA cycle, PP pathway, pyrimidine metabolism, aspartate metabolism, and glutamate metabolism. Then, by enhancing the l-aspartate and glutamine synthesis pathways, the cytidine titer was increased to 3.83 g/L. By strengthening the PP pathway to increase PRPP synthesis, the cytidine titer was further increased to 7.03 g/L. Finally, the cytidine titer reached 31.41 g/L by fed-batch fermentation in a 5 L fermenter, which was 4.47-fold that of shake flask fermentation. Overall, the efficient production of cytidine was accomplished through modular metabolic engineering, opening new pathways for the production of cytidine and other nucleosides.
作为一种嘧啶核苷，胞苷在医药和食品领域有着广泛应用。因此，构建一个用于高效可持续生产胞苷的微生物细胞工厂具有重要意义。在此，我们对168进行模块化代谢工程改造以实现胞苷的高效合成。首先，通过阻断胞苷降解并增强胞苷合成途径，胞苷产量达到0.88 g/L。接下来，通过敲低CcpA来调节中心碳代谢，但胞苷产量反而下降。转录组分析表明，差异基因主要富集在磷酸转移酶系统（PTS）、糖酵解、三羧酸循环（TCA）、磷酸戊糖途径（PP）、嘧啶代谢、天冬氨酸代谢和谷氨酸代谢中。然后，通过增强L-天冬氨酸和谷氨酰胺合成途径，胞苷产量提高到3.83 g/L。通过强化磷酸戊糖途径以增加5-磷酸核糖-1-焦磷酸（PRPP）合成，胞苷产量进一步提高到7.03 g/L。最后，在5 L发酵罐中通过补料分批发酵，胞苷产量达到31.41 g/L，是摇瓶发酵的4.47倍。总体而言，通过模块化代谢工程实现了胞苷的高效生产，为胞苷及其他核苷的生产开辟了新途径。
