Organizing Enzymes on Self‐Assembled Protein Cages for Cascade Reactions

Abstract

Cells use self-assembled biomaterials such as lipid membranes or proteinaceous shells to coordinate thousands of reactions that simultaneously take place within crowded spaces. However, mimicking such spatial organization for synthetic applications in engineered systems remains a challenge, resulting in inferior catalytic efficiency. In this work, we show that protein cages as an ideal scaffold to organize enzymes to enhance cascade reactions both in vitro and in living cells. We demonstrate that not only enzyme-enzyme distance but also the improved K m value contribute to the enhanced reaction rate of cascade reactions. Three sequential enzymes for lycopene biosynthesis have been co-localized on the exterior of the engineered protein cages in Escherichia coli, leading to an 8.5-fold increase of lycopene production by streamlining metabolic flux towards its biosynthesis. This versatile system offers a powerful tool to achieve enzyme spatial organization for broad applications in biocatalysis.

Tuning properties of biocatalysis using protein cage architectures

“…2,67 Simultaneously, the utility of protein cages is also growing in whole-cell catalysis and metabolic engineering. 35,[68][69][70] Therefore, translational outcomes of protein cage-associated biocatalysis can be expected in industry including pharmaceutical/ fine chemical manufacturing, biofuel/agricultural production, and environmental remediation.…”

“…73 A recent study showed the efficiency of a cascade reaction was enhanced by inter-enzyme proximity, which was induced by co-localization of the cascade enzymes on the exterior surface of a protein cage. 68 The molecular mechanism behind the increased efficiency, however, needs to be further investigated in the future. using biomimetic approaches.…”

“…73 A recent study showed the efficiency of a cascade reaction was enhanced by inter-enzyme proximity, which was induced by co-localization of the cascade enzymes on the exterior surface of a protein cage. 68 The molecular mechanism behind the increased efficiency, however, needs to be further investigated in the future. However, inter-enzyme proximity (which results in co-localization) has been used as an auxiliary means to induce substrate channeling together with other primary mechanisms that can control the diffusion of cascade intermediates, both naturally and synthetically (see Section 5.2 and 5.3).…”

Synthetic Multienzyme Assemblies for Natural Product Biosynthesis

“… [73] Likewise, Kang et al. showed a globular protein cage Mi3 from E. coli as a scaffold to organize three enzymes for lycopene biosynthesis, leading to an 8.5‐fold increase in cellular production [74] . Lim et al.…”

“…[73] Likewise, Kang et al showed a globular protein cage Mi3 from E. coli as a scaffold to organize three enzymes for lycopene biosynthesis, leading to an 8.5-fold increase in cellular production. [74] Lim et al also utilized a self-assembling filamentous protein, the γprefoldin (γ-PFD), to form the scaffold. The scaffold was engineered to display an array of peptide tags, through which enzymes can be assembled (Figure 2E).…”