In silico evaluation of a complex multi-enzymatic system using one-pot and modular approaches: Application to the high-yield production of hydrogen from a synthetic metabolic pathway

Multi-enzymatic processes are becoming increasingly attractive for the production of chemicals and pharmaceuticals at an industrial level due to a drastic reduction on the down-stream steps that leads to a better exploitation of resources. One-pot processes (all the enzymes placed in the same reactor) are the most commonly used approach although they present a number of limitations. In this work we explore the use of modular processes (enzymes distributed in different reactors) as an alternative to overcome the limitations of one-pot processes. An in silico evaluation of different process alternatives was performed, taking as an example a multi-enzymatic system for high-yield production of hydrogen from biomass catalyzed by a synthetic metabolic pathway composed of 13 enzymes. A modular process where an hyperthermophilic hydrogenase is kept at higher temperature than the rest of the enzymes lead to a 2-fold increase in productivity, whereas a separate reactor that consumes an inhibitor of other enzymes lead to an 8-fold increase in productivity, compared to the one-pot operation. A multi-objective optimization of the one-pot process for the maximization of productivity and yield was also performed using a genetic algorithm. Productivities of up to 355 mmol/L/h was expected to be achieved without compromising the high yield of the process (11.3 mol of H2 produced per mol substrate) under the conditions studied.

► A in silico analysis of a complex synthetic metabolic pathway (13 enzymes) was done.
► Simulation of different reactor configurations allowed identifying bottle-necks.
► A multi-objective genetic algorithm was applied to maximize productivity and yield.
► These in silico tools can be extended to other complex multi-enzymatic systems.