Artificial Multienzyme Scaffolds: Pursuing <i>in Vitro</i> Substrate Channeling with an Overview of Current Progress

Abstract

Artificial multienzyme scaffolds are being developed for in vitro cascaded biocatalytic activity and, in particular, accessing substrate channeling. This review covers progress in this field over the last 5 years with a specific focus on the scaffold materials themselves and the benefits they can provide for assembling multienzyme cascades in vitro. These benefits include improving biocatalytic efficiency, bypassing potential cellular toxicity, directed catalysis, modularity, incorporating enzymes from different prokaryotic and eukaryotic sources, and potentially the ability to create de novo designer cascades. We begin with an overview of the strongest impetus currently driving the rapid development of this field, namely, biomanufacturing and cell-free synthetic biology. We then discuss in detail pertinent mechanisms responsible for the benefits of artificial multienzyme scaffolds. In particular, we focus on substrate channeling, including the evolving debate about what leads to substrate channeling in artificial systemsproximity, confinement, or both and whether sequential enzyme order is really needed. How different scaffold materials/chemistries can in turn affect enzyme activity is also discussed. The bulk of the review then details progress in the development of different biotic (e.g., cells) and abiotic (e.g., nanoparticles) scaffolding materials and is divided up by class and subtype as needed. Within each material class of scaffolds, attention is given to their inherent chemical diversity, how they are engineered, how they allow for enzymatic attachment, their ease of use, their benefits (e.g., inherent three-dimensional architecture) and liabilities where appropriate, and other relevant issues. For each scaffolding material, a detailed overview of current progress is provided using examples of multienzyme cascades and data/schematics reproduced from the literature. Special attention is also given to the use of DNA scaffolds, as they can potentially provide the most versatile designer three-dimensional scaffold architectures. Finally, a short perspective on how this rapidly moving field will evolve in the near and long terms is provided.