Engineering Encapsulin Nanocontainers for Biocatalysis Applications Public

Abstract

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.

Table of Contents

Chapter 1: Introduction 1

1.1 – Overview of Proteinaceous Micro-and Nanocontainers Used in Biotechnology 2

1.1.1 – Bacterial Microcompartments 2

1.1.2 – Microbial Encapsulins 8

1.1.3 – Virus-like Particles 13

1.1.4 – Lumazine Synthase 16

1.1.5 – Ferritins 19

1.1.6 – Synthetic Protein Cage Assemblies 24

1.2 – Therapeutic Applications of Protein Containers 30

1.2.1 – Polyvalent Vaccine Scaffolds 30

1.2.2 – Nanoscale Delivery Vehicles and Imaging Agents 32

1.3 – Protein Containers as Nanoscale Catalytic Scaffolds 37

1.3.1 – Enzymatic Catalysis 37

1.3.2 – Non-enzymatic Catalysis 40

1.3.3 – Dual Chemo-biocatalytic Cascades 43

1.4 – Novel Biomaterials from Nanocontainer-derived Supramolecular Assembly 44

1.4.1 – Applications and Advantages of Supramolecular Assembly 44

1.4.2 – Two-dimensional Assemblies 45

1.4.3 – Three-dimensional Assemblies 46

1.5 – Aims and Scope of the Dissertation 48

1.6 – References 50

Chapter 2: Functionalization of Encapsulin Surfaces via Genetically Embedded Peptide-binding Domains 86

2.1 – Introduction 87

2.2 – Results and Discussion 90

2.2.1 – External Display of Non-covalent Peptide Binding Domains 90

2.2.2 – Covalent Display of Functional DHFR Enzymes 98

2.3 – Conclusion 109

2.4 – Materials and Methods 112

2.4.1 – Reagents and Materials 112

2.4.2 – Molecular Cloning 113

2.4.3 – Protein Overexpression and Purification 116

2.4.4 – Dynamic Light Scattering Analysis 120

2.4.5 – In vitro SpyCatcher/SpyTag Conjugation Reactions 121

2.4.6 – Fluorescein Methotrexate Titrations 122

2.4.7 – Michaelis-Menten Kinetics Analyses 122

2.4.8 – Thermal Stability Assays 123

2.4.9 – Transmission Electron Microscopy Imaging 123

2.5 – References 124

Chapter 3: Development of a Bi-enzymatic Synthetic Metabolon Using Encapsulin-based Nanoreactors 133

3.1 – Introduction 134

3.2 – Results and Discussion 136

3.3 – Conclusion 148

3.4 – Materials and Methods 150

3.4.1 – Reagents and Materials 150

3.4.2 – Molecular Cloning 151

3.4.3 – Protein Overexpression and Purification 152

3.4.4 – LC/MS Analyses 155

3.5 – References 156

Chapter 4: User-triggered Assembly of Encapsulin Nanocontainers for the in vitro Loading of Non-native Cargo Under Mild Conditions 165

4.1 – Introduction 166

4.2 – Results and Discussion 170

4.2.1 – TmE Critical Assembly Concentration Determination 170

4.2.2 – CPA Proof-of-Principle 172

4.2.3 – Optimization of CPA Method Using TEV Protease 178

4.2.4 – Attempted Non-native Cargo Loading via CPA 181

4.3 – Conclusion 185

4.4 – Materials and Methods 189

4.4.1 – Reagents and Materials 189

4.4.2 – Molecular Cloning 190

4.4.3 – Protein Overexpression and Purification 191

4.4.4 – In vitro Loading via GdnHCl-based Denaturation 196

4.4.5 – Encapsulin CACapp Determination 197

4.4.6 – Size Exclusion Chromatography Analyses 198

4.4.7 – Proteolytic Cleavage Assays 199

4.4.8 – Transmission Electron Microscopy Imaging 199

4.5 – References 200

Chapter 5: General Conclusions and Final Thoughts 209

5.1 – General Conclusion 210

5.2 – Nanocontainer-based Superstructure Assemblies 211

5.3 – Synthetic Metabolons Using Nanocontainer Scaffolds 214

5.4 – In vitro Cargo Loading Strategies 217

5.5 – References 221

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Analytical Chemistry, Biochemistry Nanotechnology
Mot-clé	Synthetic biology Protein Engineering Biocatalysis Dihydrofolate Reductase Encapsulin LigM
Committee Chair / Thesis Advisor	Lutz, Stefan, Emory University
Committee Members	Conticello, Vincent, Emory University Salaita, Khalid, Emory University

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