Advances and opportunities for the design of self-sufficient and spatially organized cell-free biocatalytic systems.

During the past decades, biocatalysis has made important contributions to chemical manufacturing by using both whole-cell and cell-free biotransformation reactions. More recently, multi-enzyme systems that can run step-wise reactions in one-pot with high selectivity are increasingly being developed. The use of multiple isolated enzymes to perform a series of reactions offers operational and process advantages over the use of living or resting cells, but such cell free processes need to be optimized to meet industrial productivity and titer requirements. Major advances have been made in enzyme discovery and engineering in order to access new activities and increase catalytic efficiency and stability. Yet, the efficient operation of multiple enzymatic reactions simultaneously requires new approaches for optimization. Inspired by the spatial organization of metabolic networks in cells, researchers have recently begun to exploit these mechanisms to increase the efficiency of multi-enzyme systems. This review highlights recent examples that adopt cellular enzyme co-localization mechanisms for multi-enzyme biocatalysis, which include enzyme attachment to preformed surfaces, enzyme clustering and enzyme encapsulation. Co-immobilization of multiple enzymes is achieved by merging tools from protein engineering and synthetic biology with approaches from material sciences.