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 ΔthrAΔpsuTΔargFΔpepA 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 ΔthrAΔpsuTΔargFΔpepAΔudkΔudpΔppnp 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.