Multi-Enzymatic Cascades In Vitro

The combination of enzymatic reactions in a simultaneous or sequential fashion by designing artificial synthetic cascades allows for the synthesis of complex compounds from simple precursors. Such multi-catalytic cascade reactions not only bear a great potential to minimize downstream processing steps but can also lead to a drastic reduction of the produced waste. With the growing toolbox of biocatalysts, alternative routes employing enzymatic transformations towards manifold and diverse target molecules become accessible. In vitro cascade reactions open up new possibilities for efficient regeneration of the required cofactors such as nicotinamide cofactors or nucleoside triphosphates. They are represented by a vast array of two-enzyme cascades that have been designed by coupling the activity of a cofactor regenerating enzyme to the product generating enzyme. However, the implementation of cascade reactions requires careful consideration, particularly with respect to whether the pathway is constructed concurrently or sequentially. In this regard, this chapter describes how biocatalytic cascades are classified, and how such cascade reactions can be employed in order to solve synthetic problems. Recent developments in the area of dynamic kinetic resolution or cofactor regeneration and showcases are presented. We also highlight the factors that influence the design and implementation of purely enzymatic cascades in one-pot or multi-step pathways in an industrial setting.