Dissertation

Protein Engineering of Encapsulin Nanocontainers for Biocatalysis Applications

Jenkins, Matthew C

2020

Overview

Nature has been shown to repeatedly employ proteinaceous containers spanning a broad range of length scales (i.e. approximately 10 to >2000 nm) to suit myriad biological functions including the propagation and infectious behavior of viruses, creating distinct microenvironments designed to facilitate specific metabolic processes, and the generation of metabolite storehouses for maintaining intracellular homeostasis. Such macromolecular cage assemblies are evolutionary marvels, forming highly symmetrical and monodisperse architectures in a hierarchical fashion from either singular or small subsets of structurally-related protein building blocks. In recognition of their vast diversity in terms of sizes, morphologies, physiochemical attributes, and dynamic functional behaviors, synthetic biologists have increasingly sought to repurpose naturally occurring protein containers for applications in a breadth of biotechnologically-relevant fields. Along these lines, this dissertation specifically focuses on the rational engineering of a recently discovered class of proteinaceous nanocontainers, referred to as encapsulins, in order to generate catalytically functional multienzyme nanoreactors.The first chapter provides general context for this dissertation by presenting a broad overview of select protein-based container structures found in nature, followed by several brief reviews of therapeutic, catalytic, and biomaterials applications for which these protein-based containers have been employed in recent decades. The second chapter describes efforts to rationally engineer the exterior surface of encapsulin nanocontainers derived from the hyperthermophilic bacterium Thermotoga maritima to present a series of solvent-exposed peptide interaction domains. T. maritima encapsulins (TmE) presenting external SpyCatcher covalent interaction domains were shown to capture up to 60 copies of an Escherichia coli dihydrofolate reductase (DHFR) variant enzyme both in vitro and in vivo. Surface-tethered DHFR enzymes maintained catalytic functionality with minimal deviations from their untethered Michaelis-Menten profiles. The third chapter expands upon the DHFR-decorated nanocontainers generated in chapter 2 to construct a bi-enzymatic nanoreactor metabolon in which the reduction of dihydrofolate by DHFR is used to fuel the demethylation of an aryl substrate by LigM, a tetrahydrofolate-dependent aryl-O-demethylase enzyme isolated from Sphingomonas paucimobilis SYK-6, which was encapsulated within the TmE lumen. The resulting bi-enzymatic nanoreactors were shown to be functional, though mutations previously used to enlarge the 5-fold symmetry pores natively distributed throughout the TmE shell were needed to facilitate efficient exchange of pathway metabolites between the interior and exterior spaces.The fourth chapter shifts focus to describe attempts to generate a novel in vitro cargo loading mechanism for TmE under benign solvent conditions by abrogating the native in vivo container assembly process using engineered steric obstructions. Recombinant fusion of a bulky protein domain to the lumen-oriented N-terminus of TmE was shown to prevent full container assembly, and subsequent proteolytic liberation of the TmE coat proteins resulted in rapid initiation of container assembly. However, cargo loading attempts in tandem with protease treatment proved unsuccessful, highlighting the need for refinement of the steric-based assembly design.The final chapter provides a general summary of the works presented in the preceding chapter. Additionally, several commentaries concerning the successes and failures of the nanocontainer engineering strategies employed in this dissertation are presented, along with general opinions pertaining to possible future directions within the field of nanocontainer engineering.

• Publisher: ProQuest Dissertations & Theses
• Subject: Analytical chemistry / Biochemistry / Nanotechnology
• ISBN: 9798691259166