Fig. 1: Chemical structures of Ψ as well as related modified nucleosides and the designer artificial biosynthetic strategies for Ψ production in vitro and in E. coli.

a Chemical structures of Ψ and other functional modified nucleosides reported in mRNA². b The Ψ biosynthetic route utilizing an EcPsuG-PhoA cascade with uracil and ribose-5P as substrates. EcPsuG, ΨMP glycosidase; PhoA, phosphatase. c The Ψ biosynthetic route employing an AgmA-EcPsuG-PhoA cascade with uracil and AMP as substrates. AgmA, AMP phosphoribohydrolase. d A four-enzyme cascade promotes the conversion of CMP to Ψ. This cascade involves CMP hydrolase SgGouE, deaminase CodA, EcPsuG, and YjjG. e A three-enzyme cascade with a UMP nucleosidase NmYgdH engaged enables the efficient transformation of UMP to Ψ. The enzymes for the full-optimized pathway are highlighted by green color, and the ones marked by black color are superficially utilized for in vitro synthesis of Ψ. f Diagrammatic sketch of artificial Ψ biosynthetic pathway and the schemes for the engineering of E. coli cell factory. In (d) and (e), the phosphatase PhoA was utilized for the dephosphorylation of ΨMP for related assays in the initial stage.