Rate-Limiting Enzyme Design in Propionaldehyde Oxidation-Driven Dual-Cofactor Regeneration System

Abstract

In vitro biomanufacturing offers an efficient platform for green synthesis; however, simultaneous in situ regeneration of NADPH and ATP remains challenging. Herein, a three-enzyme cascade driven by propionaldehyde oxidation is constructed for dual-cofactor regeneration, comprising an engineered NADP+-preferring aldehyde dehydrogenase (PduP), phosphotransacetylase (Pta), and propionate kinase (TdcD). Kinetic analysis identifies PduP as the primary thermosensitive bottleneck (half-life: 30 min at 37 °C). MD simulations and ancestral sequence reconstruction are combined to yield the R223G mutant, which retains 93.5% residual activity after incubation at 37 °C for 30 min, and a 4.3-fold longer thermal half-life. Stacking a second substitution, P327L, on R223G yields the double mutant M2, which shows a 3.15-fold higher kcat/Km but a 40.5% lower thermal half-life than R223G. Incorporation of R223G extends the catalytic window, increasing ATP conversion from 27.0% to 58.3% (2.16-fold). In a 2.00 mM scaled-up system, NADPH and ATP regeneration rates reach 2.12 and 1.15 mM/h, respectively. Analysis reveals substrate inhibition by excess NADP+ and product inhibition by accumulated NADPH and propionate. Future integration of a cathodic electrocatalytic module to construct an “electro-enzyme” network may overcome these limitations, providing a reference for the rational design of multienzyme cascade systems.