Engineering a self-organized enzyme cascade for enhanced cell-free biosynthesis of 3-hydroxypropionic acid

The bio-based platform chemical 3-hydroxypropionic acid (3-HP) has attracted considerable attention as a precursor for the production of various value-added chemicals, motivating extensive efforts to biosynthesize it using engineered microbial cell factories. Among the known pathways, the cascade of glycerol dehydratase (GDHt) and α-ketoglutaric semialdehyde dehydrogenase (KGSADH) offers high theoretical yield and strong industrial potential. However, the implementation of this pathway as a freely diffusing two-enzyme system faces two major limitations. The reactive intermediate 3-hydroxypropionaldehyde (3-HPA) is cytotoxic, and diffusion of this intermediate into the surrounding bulk phase leads to intermediate loss and reduced catalytic coupling between the two Enzymes. Here, we report a cell-free biosynthetic platform for 3-HP production from glycerol featuring a designer two-enzyme complex composed of GDHt and KGSADH. Each enzyme was rationally fused to complementary Cys-engineered α-helical domains that enable selective heterodimer formation, bringing the two Enzymes into close proximity and minimizing diffusion of the labile intermediate into the bulk solution. Under optimized conditions, the self-organized enzyme cascade achieved more than a 17-fold increase in 3-HP yield and a twofold increase in productivity relative to the free enzyme mixture. These findings demonstrate how precise molecular engineering of enzyme organization can substantially enhance the efficiency of cascade biocatalysis and provide a promising strategy for cell-free production of 3-HP.