Abstract As a kind of pyrimidine nucleoside, cytidine has been widely recognized as an important precursor for the production of some antiviral and antitumor drugs. In this study, the systematic construction of metabolically engineered Escherichia coli strain for cytidine over-production was investigated, where the regulation of downstream pathway from uridine monophosphate (UMP) toward cytidine was focused on. Firstly, the engineered strain CR016 obtained by deleting the cytidine catabolism-related genes in E. coli MG1655 showed a significantly slower degradation rate of cytidine. Further modifications were carried out to amplify the metabolic flux from UMP to cytidine. The resulted strain CR023(pTrc03) began to accumulate 352.59 mg/L of cytidine in shake flask culture for 36 h. After optimizing the supply of three precursors, the final engineered strain CR023(pTrc03/pBAD021) produced 704.19 mg/L of cytidine with a yield of 0.15 g/g glucose, upon the supplementation with 2 mM glutamate. A fed-batch fermentation was then performed in a 5-L reactor to increase cytidine titer by 11.13-fold to 7.84 g/L in 48 h. The multistep metabolic engineering strategies presented here enabled a stepwise increase in the production of cytidine.

中文翻译：

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代谢工程大肠杆菌的多步构建以增强胞苷生物合成

摘要 作为一种嘧啶核苷，胞苷被广泛认为是生产一些抗病毒和抗肿瘤药物的重要前体。在这项研究中，研究了代谢工程大肠杆菌菌株的系统构建以防止胞苷过量产生，其中重点关注从尿苷单磷酸 (UMP) 到胞苷的下游途径的调节。首先，通过删除大肠杆菌MG1655中胞苷分解代谢相关基因获得的工程菌株CR016显示出明显较慢的胞苷降解速率。进行了进一步的修改以放大从 UMP 到胞苷的代谢通量。所得菌株 CR023(pTrc03) 在摇瓶培养 36 小时内开始积累 352.59 mg/L 胞苷。在优化了三种前驱体的供给后，最终的工程菌株 CR023(pTrc03/pBAD021) 在补充 2 mM 谷氨酸盐后产生 704.19 mg/L 胞苷，葡萄糖产量为 0.15 g/g。然后在 5-L 反应器中进行补料分批发酵，以在 48 小时内将胞苷滴度提高 11.13 倍至 7.84 g/L。这里介绍的多步代谢工程策略使胞苷产量逐步增加。