Degree Type

Dissertation

Date of Award

2019

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

Major

Chemical Engineering

First Advisor

Thomas J. Mansell

Abstract

Since the turn of the millennium, we have witnessed the emergence of synthetic biology as a field which set the stage for transforming biotechnology and medicine. The tools developed have enabled us to apply general engineering approaches to manipulate biological systems towards achieving desired functions. In this work, we harness the programmability of microbes to understand the role of prebiotic oligosaccharides. Prebiotics are important glycans that shape the human gut microbiota from as early as, shortly after birth. Human milk oligosaccharides (HMOs), found in breast milk, are naturally occurring chemicals that selectively promote growth or activity in a microbial community. Given the benefits of prebiotics, especially HMOs, there has been a lot of interest in replicating their function by synthesizing them. Numerous approaches have been developed for their large-scale synthesis, however, the structural analysis of glycans is challenging and is under steadily increasing demand. The range of analytical techniques currently accessible to analyze glycans is limited by a lack of suitable high-throughput techniques, relying mostly on chromatographic methods. Biological systems have enormous potential to solve many engineering problems, and synthetic biology provides an attractive approach to harness this potential. In this work, I first describe two platforms for the development of high-throughput, linkage-specific screening of glycans. The first is based on a genetically engineered whole-cell biosensor, which produces a response to lactose. I created a library of specific glycosidases that can trim complex HMOs down to lactose and trigger a response. The second platform is based on an enzymatic, paper-based assay for determination of the type of fucosylation and sialylation in glycans. The paper device was developed with immobilized enzymes that was coupled to a colorimetric, redox assay for rapid screening. This part of the work provides new techniques to enable a streamlined synthesis process for HMOs with a significant reduction in analysis time of new producer strains. The second platform paves the way for development of a glyco-barcode assay for rapid analysis of key glycosylation patterns not only in biosynthesis platforms but also for diagnosis or monitoring disease states in different biomarkers. The other focus of my thesis is directed towards manipulating engineered microbial communities. Targeted biochemical modulation of microbiota can utilize prebiotics that are designed to modulate microbial function or growth. Here, I demonstrated engineered bacterial strains that, owing to selective utilization of HMOs, enabled growth-based selection in mixed cultures. The dynamic regulation of growth and protein production in mixed culture models is also demonstrated. We also illustrate chemical synthesis of bio-inspired novel inducer molecules for orthogonal control of protein expression The first chapter of the dissertation introduces the concept of prebiotics and highlights some key advantages and their mechanisms of action. Chapter two covers genetically encoded whole cell biosensors that enable high-throughput, linkage-specific detection of HMOs and enable dynamic regulation of growth and protein expression in mixed populations. The third and fourth chapters look at paper-based sensing, keeping rapid, simple, low-cost real-time detection, in mind. Chapters four and five describe approaches for microbiome manipulations, a shift towards understanding and engineering community-level interactions in mixed populations. Finally, chapter six introduces chemically synthesized novel inducer molecules that were inspired by biological mimics. With this work, the goal was to contribute towards expanding the glycobiology toolbox and help elucidate the staggering complexity of glycans. The promises of the ability to engineer microbial populations are immense in that we are living in symbiosis with bacteria, and their interactions play roles that heavily impact human health. Taken together, these projects demonstrate a variety of approaches for glycan analysis and utilization, taking inspiration from the denizens of the human gut microbiome and suggests multiple avenues forward towards clinical and health impact.

Copyright Owner

Fatima Enam

Language

en

File Format

application/pdf

File Size

204 pages

Available for download on Tuesday, October 27, 2020

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