Genetically Encodable Scaffolds for Optimizing Enzyme Function

Abstract

Enzyme engineering is an indispensable tool in the field of synthetic biology, where enzymes are challenged to carry out novel or improved functions. Achieving these goals sometimes goes beyond modifying the primary sequence of the enzyme itself. The use of protein or nucleic acid scaffolds to enhance enzyme properties has been reported for applications such as microbial production of chemicals, biosensor development and bioremediation. Key advantages of using these assemblies include optimizing reaction conditions, improving metabolic flux and increasing enzyme stability. This review summarizes recent trends in utilizing genetically encodable scaffolds, developed in line with synthetic biology methodologies, to complement the purposeful deployment of enzymes. Current molecular tools for constructing these synthetic enzyme-scaffold systems are also highlighted.

Structure of a Minimal α-Carboxysome-Derived Shell and Its Utility in Enzyme Stabilization

“…Protein shells have been garnering attention as platforms for conferring stability on enzymes against physical insults, such as heating or freezing, or chemical insults, such as the presence of organic co-solvents or nonphysiological pH. , Enzyme confinement often reduces their conformational flexibility, which sometimes confers stability against structural changes that lead to denaturation. , Currently, homomeric protein shells are more established for hosting enzymes, attributable to their relative ease of assembly and particle size homogeneity, which improves predictability and tractability during engineering. ,− Due to their heteromeric composition, minimal BMC-derived shells represent emerging scaffolds for hosting enzymes, as these shells can provide more avenues for purposeful modifications, while their generally homogeneous particle size still confers predictability to facilitate engineering. ,,,, However, minimal BMC-derived shells have yet to be explored for hosting heterologous enzymes. , This encouraged us to investigate if the Cso-shell could host and stabilize enzymes. Empty Cso-shells (Cso-P A H A ) were first tested for their stability against heat shock, freezing, presence of methanol co-solvent, and environments with pH from 2 to 13.…”

“…Protein shells have been garnering attention as platforms for conferring stability on enzymes against physical insults, such as heating or freezing, or chemical insults, such as the presence of organic co-solvents or nonphysiological pH. 47,48 Enzyme confinement often reduces their conformational flexibility, which sometimes confers stability against structural changes that lead to denaturation. 49,50 Currently, homomeric protein shells are more established for hosting enzymes, attributable to their relative ease of assembly and particle size homogeneity, which improves predictability and tractability during engineering.…”

“…56,67 Among known protein shells, a distinctive feature of the Csoshell is its ability to stabilize encapsulated enzymes under basic environments. 48 The utility of the Cso-shell can be further explored in remediation of alkaline industrial effluent. 68 Enzymes that are currently used for wastewater treatment, such as laccases, peroxidases, and nitrilases, often suffer from diminished activities in high-pH environments.…”

“…We have shown that the Cso-shell is able to host and stabilize enzymes with markedly different molecular sizes and reaction chemistries against common denaturing factors . Stabilization of APEX2 and β-galactosidase may expand their usefulness in cellular imaging and food processing applications, respectively. , Among known protein shells, a distinctive feature of the Cso-shell is its ability to stabilize encapsulated enzymes under basic environments . The utility of the Cso-shell can be further explored in remediation of alkaline industrial effluent .…”

Mechanistic Aspects for the Modulation of Enzyme Reactions on the DNA Scaffold

“…The enzyme scaffolded by DNA structures often displays enhanced activity and stability over its free form; however, the actual mechanisms for the higher catalytic ability are still under debate [ 68 ]. As studied previously, Glettenberg et al [ 69 ] covalently conjugated peroxidase to different DNA oligonucleotides (ODN).…”