Designing a Novel Multienzyme Cascade with a Coenzyme Self-Recycling System for the Synthesis of 2-Phenylethyl-β-<scp>d</scp>-glucopyranoside from Phenylalanine

Phenylethanoid glycosides (PhGs) are glycoconjugates composed of phenylethyl alcohols and glycoside moieties, exhibiting diverse bioactivities. 2-Phenylethyl-β-d-glucopyranoside (2PE-Glc) is the simplest form of PhGs. We successfully designed and implemented a novel multienzyme cascade system with cofactor self-recycling capabilities for the efficient synthesis of 2PE-Glc from l-phenylalanine (L-Phe). First, different phenylalanine dehydrogenases involved in the cascade system were compared, and <i>Ls</i>PheDH_{V33A/A206D/L283F} was selected for the deamination reaction. Subsequently, the rate-limiting step was determined to be phenylacetaldehyde reduction, and <i>Pm</i>ADH was screened out and evolved by consensus design, leading to a mutant <i>Pm</i>ADH_I32V, which exhibited 1.74 times the activity of the wild type. Finally, five enzymes were coexpressed in <i>E. coli</i> BL21 (DE3) to construct a multienzyme cascade system, and the reaction conditions of conversion of l-Phe to 2PE-Glc catalyzed by the multienzyme cascade system were optimized. Under the optimal reaction conditions, the yield of 2PE-Glc reached 27.62 mM (7.85 g/L) in a fed-batch reaction. This work not only provides an effective strategy for the biosynthesis of 2PE-Glc but also demonstrates the potential of the multienzyme cascade system in the synthesis of biologically active complex glycoconjugates.