Artificial Multienzyme Scaffolds: Pursuing in Vitro 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 systemsproximity, 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.

Quantum Dots and Gold Nanoparticles as Scaffolds for Enzymatic Enhancement: Recent Advances and the Influence of Nanoparticle Size

“…Note that NPs are defined as a particle between 1 and 100 nm in diameter [15,16]. The interested reader is directed to excellent reviews on other scaffolds for enzyme immobilization [4,[12][13][14]. Herein, we first describe the benefits of enzyme immobilization and potential mechanisms underlying these advantages, followed by factors affecting these benefits-in particular, construct size.…”

“…Enzymes can be immobilized either as single copies or as multiple enzymes which are part of a multienzyme cascade [4,[17][18][19][20][21][22]. Benefits of immobilizing single enzymes can include: (1) increased stability, (2) increased activity, (3) closeness and orientation to substrate, and (4) increased recoverability and reuse; whereas, the benefits of immobilizing multiple enzymes can include these plus: (5) increased (temporary) reaction rates, (6) bypassed intermediate toxicity, (7) bypassed offtarget pathways/directed catalysis, (8) reaction order, and (9) modularity ( Figure 1, Table 1) [4,10,[23][24][25][26][27][28][29][30]. Reaction order Dictating order of reaction by choosing which enzymes to place in proximity 9 Modularity Modular tags allow interchangeability of enzymes to change cascade (and product) or make de novo cascades…”

“…Enzymatic catalysis is exquisite in its ability to enhance specific reaction rates by orders of magnitude using precisely selected substrates from within a milieu of other compounds [1]. This ability is highlighted in the thousands of "one-pot" reactions that are not only biocatalyzed simultaneously yet specifically in the metabolic network of a cell, but can occur in water at ambient pressure and temperature and can produce regio-and stereo-selective products [2][3][4][5][6][7]. It is no wonder that enzymes have been studied and engineered for a range of applications, including biodegradation/bioremediation of toxic compounds, chemical biosynthesis, and detection using biosensors, among others [2,6,8,9].…”

“…This is evidenced by nature's use, and researchers' engineered exploitation, of metabolons (e.g., clustered TCA cycle enzymes) and scaffolds (e.g., cellulosomes of scaffoldin and cellulases) [3,10,11]. A range of both biotic and abiotic soluble scaffolds have been used to immobilize enzymes, including: cellular surfaces, peptides, proteins, DNA, polymers, metal-organic frameworks, and nanoparticles (NPs), among others [4,[12][13][14]. NPs notably present unique features for enzyme immobilization, and two in particular-quantum dots (QDs) and gold-nanoparticles (AuNPs)-will be the subject of this review.…”

DNA Microsystems for Biodiagnosis

“…Notwithstanding, the diffusion of enzyme-labeled DNA sequences (i.e., GOx and HRP) to the origami rectangular is also important for the reaction to happen [155]. In theory, the enzymatic activity here is expected to occur faster compared to that activity without the presence of the DNA nanostructure due to the dimensional reduction and the distance controllability [156,157]. By exploiting the 3D DNA nanostructures, the encapsulation of GOx and HRP into DNA origami modules coated with neutravidins (NTVs) and biotin demonstrates another dimension and versatility of DNA nanotechnology which effectively enhances the specific binding among targeted enzymes in a reactor-like system (Figure 7b).…”