PhD Oral Exam - Trisha Ghosh, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Enzymes play a pivotal role in green chemistry as tools for biocatalysis, enabling a wide range of chemical transformations that may not be accessible through conventional synthetic approaches. Protein engineering strategies, such as directed evolution and rational design, rely on screening mutant libraries to enhance enzyme activity, specificity, and efficiency. Selection techniques and screening methods, including coupled enzyme assays and multi-enzyme pathways, are commonly used to evaluate functional variants and facilitate rapid assessment of enzyme performance. Cell-free platforms have accelerated protein and enzyme engineering by enabling rapid high throughput screening. Oxidoreductases are particularly valuable due to their ability to catalyze key electron transfer (redox) reactions, including asymmetric hydrogenation, oxygenation, hydroxylation, epoxidation, and BaeyerVilliger oxidation. Many of these enzymes depend on NAD(P)H as an electron donating co-substrate or NAD(P)+ as an electron acceptor. The ability to sensitively detect oxidoreductase activity is critical for identifying useful biocatalysts from nature and for engineering improved variants, particularly when native reaction rates are low. Herein, we report the development of a fluorescence-based coupled enzyme cascade system for detecting NAD(P)H-dependent oxidoreductase activity with orders of magnitude greater sensitivity than conventional absorbance-based methods. By recycling NAD(P)H from NAD(P)+, the coupled enzyme cascade triggers cleavage of a fluorogenically labeled probe, generating a strong fluorescent signal. This approach enables sensitive, continuous, and highthroughput detection of low levels of NAD(P)H/NAD(P)+-dependent oxidoreductase activity, providing an effective platform for screening and engineering redox enzymes in protein engineering and biocatalysis applications. We demonstrated that the coupled cascade approach can be extended beyond redox enzymes to non-redox enzymes as well.

Recognizing the need for selection strategies that directly link enzyme catalysis with gene amplification, this work also investigates an approach coupling NAD(P)H-dependent enzyme activity to DNA amplification. In this strategy, NAD(P)+ generated by target enzyme activity drives a sequential enzyme cascade that converts dNDPs into dNTPs, enabling polymerase chain reaction amplification. This provides a potential route for maintaining genotype–phenotype linkage in cell-free directed evolution platforms by selectively amplifying genes encoding active enzyme variants.

Overall, this work demonstrates the versatility of in vitro coupled enzyme cascades for developing sensitive fluorescence-based enzyme screens and lays the foundation for future cell-free selection platforms. Although mutant library screening remains beyond the scope of this thesis, the methods described here provide a framework for integrating enzymatic activity detection with highthroughput screening, microfluidic compartmentalization, and cell-free protein engineering.