Building and optimizing multi-enzyme in vitro cascade reactions

Biocatalysis is becoming increasingly attractive for the development of more efficient and cleaner chemical synthetic processes. The combination of multiple enzyme steps for cascade reactions allows for attractive one-pot processes with reduced operating costs. While the use of whole-cells have a number of advantages for these reactions, the competing needs of the cell and limited transport across the cell membrane can result in a low final product concentration. In contrast, the use of isolated enzymes allows reactions to be easily controlled, with the use of stable enzymes such as those from thermophiles offering economically competitive processes. For the construction of novel enzymatic cascade reactions, there is a need for well-defined modular enzyme building blocks that can be quickly assembled for new reactions. Carboxylic acid reductase (CARs) is a relatively undeveloped class of enzyme which meets a demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. A thorough biochemical characterization of four new CARs provides insight into the operating parameters of these enzymes, while the integration of a CAR into a seven enzyme in vitro cascade reaction demonstrates their potential for green chemistry. Mathematical modeling of the cascade allows for a detailed understanding of the reaction and gives opportunity for its optimization with respect to flux and cost. Our work highlights the virtue of thorough enzyme characterization, and of modeling reactions, to deliver new understanding and build robust pathways.

Author(s): Nicholas Harmer

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