重组大肠杆菌全细胞催化制备假尿苷

Whole-cell catalytic production of pseudouridine by recombinant Escherichia coli

假尿苷是非编码RNA中含量最多的修饰核苷,在生物和医药领域用途广泛。现有的假尿苷制备方法普遍存在步骤繁琐、生产效率低、生产成本高等缺点。本研究在大肠杆菌中设计了新颖的酶级联反应路线,并实现了全细胞催化尿苷制备假尿苷。首先,质粒过表达内源的假尿苷-5-磷酸糖苷酶、核糖激酶和核糖核苷水解酶,构建出尿苷到假尿苷的代谢途径,实现了假尿苷的积累;然后,筛选内源的高活性核糖核苷水解酶,促进了尿苷的水解,为假尿苷的合成提供了更多前体;接着,对底物与产物的转运途径进行改造,提升假尿苷产量的同时避免了副产物尿苷的积累。最终获得的重组菌株Ψ-7在24 h内可以全细胞催化30 g/L尿苷产生27.24 g/L假尿苷,转化率为90.8%,生产效率为1.135 g/(L·h),假尿苷产量和生产效率为现有酶催化法报道的最高值。

Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields.However,current methods for pseudouridine production suffer from drawbacks such as complex procedures,low efficiency and high costs.This study presents a novel enzymatic cascade reaction route in Escherichia coli,enabling the whole-cell catalytic synthesis of pseudouridine from uridine.Initially,a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase,ribokinase,and ribonucleoside hydrolase,resulting in the accumulation of pseudouridine.Subsequently,highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis.Furthermore,modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine.The resulting recombinant strainΨ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h,achieving a conversion rate of 90.8%and a production efficiency of 1.135 g/(L·h).These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.