Abstract
Significant advances in enzyme discovery, protein and reaction engineering have transformed biocatalysis into a viable technology for the industrial scale manufacturing of chemicals. Multi-enzyme catalysis has emerged as a new frontier for the synthesis of complex chemicals. However, the in vitro operation of multiple enzymes simultaneously in one vessel poses challenges that require new strategies for increasing the operational performance of enzymatic cascade reactions. Chief among those strategies is enzyme co-immobilization. This review will explore how advances in synthetic biology and protein engineering have led to bioinspired co-localization strategies for the scaffolding and compartmentalization of enzymes. Emphasis will be placed on genetically encoded co-localization mechanisms as platforms for future autonomously self-organizing biocatalytic systems. Such genetically programmable systems could be produced by cell factories or emerging cell-free systems. Challenges and opportunities towards self-assembling, multifunctional biocatalytic materials will be discussed.
Highlights
We suggested that new nanoscale enzyme co-localization strategies, including genetically engineered scaffolding and confinement systems, will be important for increasing reaction rates and efficiencies of longer enzyme cascades
We concluded that the spatial organization of multi-enzyme systems until recently had long been ignored by the field in industrial biocatalysis due to prioritizing the optimization of enzyme properties [27]
Rapid progress in synthetic biology, de novo protein design and cell-free approaches have created a robust foundation for the design of genetically engineered enzyme assemblies for integration and upscaling in industrial processes
Summary
Nucleic acid and protein or peptide-based systems can be genetically encoded and are suitable for programming the bottom–up design of enzyme assemblies. More control over biocatalyst organization along with the ability to incorporate additional functionalities is possible by immobilizing enzymes on selfassembling protein or nucleic acid scaffolds This can be achieved either by direct genetic fusion of the scaffold building blocks to the enzymes or fusion of cognate interaction tags to enzymes and scaffold building blocks. A more versatile strategy is the use of interaction tags fused to scaffold building blocks as they will less likely interfere with scaffold formation while creating a highly flexible platform for enzyme immobilization. With such a scaffolding system, the organization of enzymes can be controlled by the spacing, orientation, types, and linkers of the displayed tags. Emphasis will be placed on in vitro applications with a few exceptions to discuss a particular aspect of a system or where in vitro examples are currently lacking
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