Reaction-kinetic model-guided biocatalyst engineering for dual-enzyme catalyzed bioreaction system

Current research on dual-enzyme-catalyzed bioreaction systems has focused on single-enzyme engineering and enzyme cascade development. However, few studies have investigated the bioreaction kinetic model, which creates a gap between the biocatalyst and reaction systems. To fill this gap, we developed a reaction-kinetic model-guided biocatalyst engineering strategy, including the iterative cycle of “kinetic model construction, limiting factor analysis, and biocatalyst engineering”. In the proof-of-concept experiment, a dual-enzyme-catalyzed bioreaction of the atorvastatin precursor (3R,5R)-2 was selected and the iterative cycle was performed in two rounds. First, a double-substrate-ordered Bi–Bi kinetic model was constructed and validated. Thereafter, the limiting factors were analyzed using the constructed model and further confirmed experimentally. The limiting factor was glucose dehydrogenase total activity in the first round, whereas it changed to aldo–keto reductase total activity in the second round. Under this guidance, the bioreaction system was improved through enzyme selection, protein engineering, promoter optimization, and optimization of the expression conditions. Finally, the coupling efficiency of the two enzymes increased, resulting in high space–time yields (>1 kg/L d-1) of the atorvastatin precursor in both 250 mL flasks and 50 L bioreactors.