PhD Final Defense – Yoonjoo Seo

Engineering Microbial Cell Surface-displayed Protein Platforms for Environmental Sensing, Enzyme Discovery, and Waste Biodegradation

Advisor: Professor Na Wei

Abstract

Microbial cell surface display has emerged as a promising platform for deploying functional proteins to address critical challenges in environmental engineering. Moreover, advances in synthetic biology have continuously expanded the repertoire of natural and engineered protein sequence, providing a robust and extensive toolkit for developing innovative applications of cell surface-displayed proteins. Representative examples of proteins related to environmental applications include antibodies with high specificity and binding affinity for pollutants, as well as enzymes with superior catalytic activity for pollutant degradation. However, intrinsic biological characteristics of microbial cells, such as limited permeability to target molecules or poor tolerance to the elevated temperatures required for optimal enzyme activity, remain critical bottlenecks that limit the applicability and performance of this platform. In this dissertation, novel and robust microbial cell surface display systems were developed and applied to three environmental biotechnology applications, each of which has long been considered challenging tasks either for conventional whole-cell microbial systems or purified protein-based approaches. 

First, yeast surface-displayed quenchbody was created as a novel whole-cell biosensor for one-step detection of influenza A(H1N1) virus. We used Saccharomyces cerevisiae as a VHH-antibody surface display platform where antigen-antibody interaction can occur extracellularly. Further, we introduced quench body engineering to surface-displayed VHH-antibodies for efficient signal transduction and detection. The biosensor quantitatively detected H1N1 hemagglutinin (0.5–16 µg/mL) and H1N1 virus particles (2.4×10⁴–1.5×10⁷ PFU/mL), exhibiting high selectivity and storage stability. Second, a whole-cell–based screening platform for identifying efficient plastic-degrading enzymes was demonstrated using a biofilm-integrated nanofiber display (BIND) system. Using this approach, a library of 120 machine learning–predicted polycaprolactone (PCL)-degrading enzyme candidates was rapidly evaluated. Notably, the platform achieved an approximately 80% success rate in enzyme expression, substantially reducing the risk of overlooking promising candidates due to challenges associated with protein purification. Subsequent purification and biochemical characterization of the top five enzymes identified by the BIND screening led to the discovery of four promising candidates exhibiting superior PCL-degrading activities compared to current benchmarks, demonstrating the effectiveness of the BIND platform for rapid early-stage screening of plastic-degrading enzymes. Lastly, a yeast cell surface-displayed Humicola insolens cutinase (HiC) was developed as a novel whole-cell biocatalyst for polyethylene terephthalate (PET) depolymerization. The engineered biocatalyst produced approximately 6 mM of total PET degradation products after 14 days at 40 °C, achieving depolymerization efficiency comparable to that of the commercial HiC enzyme. To further improve the applicability of the platform under elevated-temperature conditions, the sterol composition of the host yeast cell membrane was engineered through genetic and chemical approaches to enhance the thermostability of the surface-displayed enzyme.

Overall, this research established three complementary strategies to advance microbial cell surface-displayed protein platforms and demonstrated their potential to address three critical environmental challenges. Owing to the modular nature of cell surface display systems, these strategies provide a versatile framework for developing plug-and-play platforms that can be readily adapted to diverse enzymes and target applications, expanding the utility of engineered microbial systems for environmental monitoring, waste management, and sustainable biotechnology.