Engineering Glucose Enzymes Through Domain Insertion for Adaptive Bioelectronic Sensors

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dc.contributor.advisorAjo-Franklin, Carolineen_US
dc.contributor.advisorWarmflash, Aryehen_US
dc.creatorNgwadom, Chiagoziemen_US
dc.date.accessioned2025-01-16T19:32:38Zen_US
dc.date.created2024-12en_US
dc.date.issued2024-12-03en_US
dc.date.submittedDecember 2024en_US
dc.date.updated2025-01-16T19:32:38Zen_US
dc.description.abstractBiosensors are essential in diagnostics, monitoring, and therapeutics. A major example is the glucometer, which effectively utilizes glucose-oxidizing enzymes to generate accurate electrical signals that report blood sugar levels. This bioelectrochemical sensor’s affordability, manufacturability, and suitability for patient-side use make glucose enzymes highly appealing for broader sensing applications. Although existing studies have explored mutagenizing glucose redox enzymes to enhance their stability and activity, significant obstacles remain in repurposing these enzymes to detect other biomarkers. These challenges stem from an incomplete understanding of glucose enzyme design and the limited effectiveness of current protein engineering approaches. This thesis addresses these challenges by using pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH) as a robust platform for glucose-dependent oxidoreductase applications. Through comprehensive methods developed to identify structural elements crucial to its function, this work demonstrates the repurposing of PQQ-GDH to produce electrochemical output for non-glucose analytes. Additionally, a high-throughput screening system is introduced to accelerate the development of a broad range of bioelectronic sensors. In Chapter 2, we integrate small peptide sequences into PQQ-GDH to investigate the structure-sequence-function relationships at various structural levels. In Chapter 3, we engineer PQQ-GDH conformational switches to create electronic sensors capable of detecting cancer therapeutics in blood samples, pushing the boundaries of traditional glucose sensing. In Chapter 4, we establish a high-throughput selection system for glucose enzyme variants by manipulating glucose metabolism and NADPH regeneration in E. coli through targeted knockouts. Our research explores multiple strategies for functionalizing PQQ-GDH to enhance bioelectronic diagnostics. These findings provide critical insights into how the structure and sequence of PQQ-GDH influence its function—particularly at the active site and dimerization interface, which are essential for enzyme activity and stability. When integrated onto electrode interfaces, our functionalized PQQ-GDH variants demonstrate a significant electrical response to the cancer therapeutic 4-hydroxytamoxifen in blood. This advancement lays a solid foundation for real-time, point-of-care diagnostics in therapeutic monitoring. Additionally, our innovative growth complementation assay enriches enzyme variants in direct proportion to their activity levels, establishing a novel selection method for variants that exhibit superior performance. These contributions advance biosensing technologies and significantly expand the application scope of bioelectrochemical systems. We are paving the way for reliable point-of-care diagnostic devices and therapeutic monitoring platforms that promise to transform future healthcare solutionsen_US
dc.embargo.lift2025-06-01en_US
dc.embargo.terms2025-06-01en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.urihttps://hdl.handle.net/1911/118166en_US
dc.language.isoenen_US
dc.subjectSynthetic biologyen_US
dc.subjectlibrary generationen_US
dc.subjectnext-generation sequencingen_US
dc.subjectoxidoreductasesen_US
dc.subjectredox proteinsen_US
dc.subjecthigh-throughput screeningen_US
dc.subjectmetabolic engineeringen_US
dc.subjectbiosensorsen_US
dc.subjectprotein engineeringen_US
dc.subjectelectrochemistryen_US
dc.titleEngineering Glucose Enzymes Through Domain Insertion for Adaptive Bioelectronic Sensorsen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineBiochemistry and Cell Biologyen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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