Artificial Multienzyme Scaffolds: Pursuing in Vitro Substrate Channeling with an Overview of Current Progress

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 ...