Maximizing the Efficiency of Multi-enzyme Process byStoichiometry Optimization.

Multi-enzyme processes represent an important area of biocatalysis. Their efficiency can be enhanced by optimization of biocatalysts’ stoichiometry. Here we present a workflow for maximizing the efficiency of a three-enzyme system catalysing a five-step chemical conversion. Kinetic models of pathways featuring either wild-type or engineered enzymes were built and the enzyme stoichiometry of each pathway was optimized. Mathematical modelling and one-pot multi-enzyme laboratory experiments provided detailed insights into pathway dynamics, enabled the selection of suitable engineered enzyme and afforded high efficiency while minimizing biocatalyst loadings. The optimizing of stoichiometry in a pathway with engineered enzyme reduced the total biocatalyst load by an impressive 56 %. Our new workflow represents a broadly applicable strategy for optimizing multi-enzyme processes.