Systematic metabolic engineering of Escherichia coli for high-level production of pseudouridine via pathway optimization and precursor enhancement

Pseudouridine, the C5-ribose epimer of uridine with significant biological functions and clinical applications, was efficiently produced through systematic metabolic strategy of Escherichia coli in this study. Initial overexpression of pseudouridine-5-phosphate glycosylase gene psuG and alkaline phosphatase gene YjjG in E . coli pRSFDuet-1- YjjG - psuG yielded 0.43 g L −1 pseudouridine, which increased 8.56-fold with 5 g L −1 uridine supplementation. Subsequent deletion of thrA , psuT , argF , and pepA enhanced titer by 1.29-fold in E . coli Δ thrA Δ psuT Δ argF Δ pepA pRSFDuet-1- YjjG - psuG , while ribonucleoside hydrolase gene rihA overexpression boosted titer to 5.57 g L −1 . Further optimization through deleting the uridine kinase gene udk and overexpressing the ribokinase gene rbsK in strain E . coli Δ thrA Δ psuT Δ argF Δ pepA Δ udk Δ udp Δ ppnp pRSFDuet-1- YjjG - psuG pCDFDuet-1- rihA-rbsK achieved 6.23 g L −1 pseudouridine, increasing to 11.34 g L −1 with two-stage uridine feeding. Fed-batch fermentation in a 5-L bioreactor yielded a record 102.2 g L −1 pseudouridine. This work provides an efficient and scalable bioprocess for industrial pseudouridine manufacturing to meet the growing demands of mRNA-based applications.