Publication Date

2023

Document Type

Dissertation/Thesis

First Advisor

Zhou, Shengde

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Biological Sciences

Abstract

Glucosamine (GlcN) and GlcN-based supplements, such as N-acetyl-glucosamine (GlcNAc), are commonly used by osteoarthritis patients to relieve pain and maintain the health of bone joints. However, the current production methods for GlcN-based products are not eco-friendly and pose risks to individuals with shrimp allergies. Microbial cell-based systems have been explored to address these issues as an eco-friendly alternative for GlcN and GlcNAc production. This study focused on developing an Escherichia coli (E. coli) strain for GlcNAc fermentation. E. coli has an endogenous pathway for peptidoglycan biosynthesis involving GlcNAc-1-P. However, altering this pathway could affect cell growth and integrity. Therefore, two key genes, glmS (glucosamine-6-P synthase) from E. coli and GNA1 (N-aceytlglucosamine-6-P N-acetyltransferase) from Saccharomyces cerevisiae, were cloned and assembled into an artificial operon (glmS-GNA1) for co-expression of an alternative GlcNAc pathway in E. coli. Efficient expression of the constructed artificial operon was confirmed through qPCR analysis, which showed a 15- and 114-fold increase in glmS and GNA1 mRNA expression levels, respectively. In combination with other chromosomal genes, the co-expression of the glmS-GNA1 operon established a novel pathway for GlcNAc production in the bacterium, which yielded an approximate concentration of 221 milligrams per liter of GlcNAc in the fermentation media.The study also involved metabolic engineering in developing a strain (E. coli AP520) specifically for GlcNAc production. This involved blocking carbon competing pathways and deleting GlcNAc uptake transporter genes to prevent a GlcNAc futile cycle. A salicylate inducible regulatory switch and an inducible artificial operon were also constructed to enable controlled cell growth and GlcNAc production. The final GlcNAc-producing strain, AP520, was developed by transforming the engineered expression vector cassette into the E. coli strain. Shaking flask fermentation with AP520 resulted in the production of 6.2 g/L of GlcNAc. These findings validated the concept of strain design and the engineering approach for constructing a functional GlcNAc fermentation pathway in E. coli. However, further improvements are necessary to enhance cell growth and increase product yield.

Extent

74 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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Microbiology Commons

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