Abstract：
In this study, we proposed a biological approach to efficiently produce pseudouridine (Ψ) from glucose and uracil in vivo using engineered Escherichia coli. By screening host strains and core enzymes, E. coli MG1655 overexpressing Ψ monophosphate (ΨMP) glycosidase and ΨMP phosphatase was obtained, which displayed the highest Ψ concentration. Then, optimization of the RBS sequences, enhancement of ribose 5-phosphate supply in the cells, and overexpression of the membrane transport protein UraA were investigated. Finally, fed-batch fermentation of Ψ in a 5 L fermentor can reach 27.5 g/L with a yield of 89.2 mol % toward uracil and 25.6 mol % toward glucose within 48 h, both of which are the highest to date. In addition, the Ψ product with a high purity of 99.8% can be purified from the fermentation broth after crystallization. This work provides an efficient and environmentally friendly protocol for allowing for the possibility of Ψ bioproduction on an industrial scale.
摘要：
在这项研究中,我们提出了一种使用工程大肠杆菌在体内有效地从葡萄糖和尿嘧啶中产生假尿苷（Φ）的生物学方法。通过筛选宿主菌株和核心酶，E.coliMG1655过表达的单磷酸（ΦMP）糖苷酶和ΦMP磷酸酶被获得，这显示出最高的Φ浓度。然后，RBS序列的优化，增强细胞中的核糖5-磷酸供应，并研究了膜转运蛋白UraA的过表达。最后,在5L发酵罐中补料分批发酵，可以达到27.5g/L，在48小时内，尿嘧啶的产量为89.2mol％，葡萄糖的产量为25.6mol％，两者都是迄今为止最高的。此外,可以从结晶后的发酵液中纯化出纯度为99.8%的产品。这项工作提供了一种有效且环保的协议，可以在工业规模上进行生物生产。
