Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox ...
Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.