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.

中文翻译：

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大肠杆菌从葡萄糖和尿嘧啶高效生产假尿苷的系统工程

在这项研究中，我们提出了一种利用工程大肠杆菌在体内有效地从葡萄糖和尿嘧啶产生假尿苷（Ψ）的生物学方法。通过宿主菌株和核心酶筛选，获得了过表达Ψ单磷酸（ΨMP）糖苷酶和ΨMP磷酸酶的大肠杆菌MG1655，其中Ψ浓度最高。然后，研究了 RBS 序列的优化、细胞中核糖 5-磷酸供应的增强以及膜转运蛋白 UraA 的过度表达。最后，在 5 L 发酵罐中分批补料发酵 ψ，48 小时内产量可达 27.5 g/L，尿嘧啶产率为 89.2 mol%，葡萄糖产率为 25.6 mol%，均为迄今为止最高。另外，发酵液经结晶后可纯化出纯度高达99.8%的Ψ产品。这项工作提供了一种高效且环保的方案，使工业规模的 Ψ 生物生产成为可能。