Directed self-assembly strategy of a DET complex based on the application of artificial electron channeling

Custom exogenous ferredoxin (Fdx) created a new conduit for electrons to travel from the reductase to the oxygenase, which increased the 4-androstene-3,17-dione (AD) conversion rate. Here, the specific amino acid residues of the multienzyme complex involved in direct electron transfer (DET) pathways were further analyzed. Some unique residues from the reductase or ferredoxin domains were found to be involved in the DET pathways. The electrostatic interactions in mutant MT2 (Y110R in KshA, T85D in DmFdx2) were strengthened using an iterative saturation mutagenesis (ISM) strategy. Importantly, Fdx was first proposed to be used as a scaffold to modify the solid support, guiding the immobilization and self-assembly of the multi-enzyme complex. After modifying rate-limiting residues by substrate channel simulation, mutant MT3 (Y110R/Y226M in KshA, T85D in DmFdx2) was further introduced into the multienzyme immobilization system. By coupling the NADH coenzyme regeneration system with self-assembly directed immobilization, the MT3 complex showed a 155.47% increase in the conversion rate of AD. The scaffold-protein-modified self-assembly directed immobilization strategy offers new methods for catalyst recycling for efficient steroid conversion in industry. Custom exogenous ferredoxin (Fdx) created a new conduit for electrons to travel from the reductase to the oxygenase, which increased the 4-androstene-3,17-dione (AD) conversion rate.