Substrate Channeling on Bienzyme Assembly Significantly Enhances Cascade Biocatalysis of Ene Reductase and Aldehyde Dehydrogenase at High Substrate Concentrations

Abstract Multienzyme cascades enable efficient synthesis of value-added chemicals by coupling sequential biotransformations. In the ene reductase (OYE)–aldehyde dehydrogenase (ALDH) cascade for chiral α-substituted carboxylic acids, accumulation of the saturated aldehyde intermediate inhibits OYE and thereby limits cascade performance, particularly at high substrate concentrations. Here, complementary RIDD/RIAD (DD/AD) peptides were used to organize OYE and ALDH into an activity-matched 2:1 assembly. Linker optimization identified the DD–(EAAAK)4–OYE fusion (DD–EK4OYE), which retained 86.4% of free OYE activity, whereas the AD–ALDH fusion (ADALDH) retained full ALDH activity. The assembly achieved complete conversion of 5 mM substrate within 8 h, reducing the reaction time by 1/3 relative to the free enzyme system and by 2/3 relative to the activity-matched free system. The assembly exhibited 43–87% higher product yields than the free-enzyme system across the 3–25 mM substrate range after 0.5 h. Moreover, the assembly modestly improved the stability of the rate-limiting OYE and shortened the cascade lag time from 2.7 to 0.7 min. Molecular dynamics simulations showed that DD bound the aldehyde intermediate at multiple exposed surface regions, forming an intermediate–enrichment interface that functioned as a surface-mediated substrate channeling, thereby leading to enhanced transfer to and catalysis by ALDH. These results demonstrate that peptide-mediated assembly could work as an effective substrate-channeling approach to promoting cascade biocatalysis by alleviating intermediate inhibition at high substrate concentrations.