University of Illinois Urbana-Champaign

Engineering microorganisms for synthesizing value-added products

Lee, Jaewon

Content Files
LEE-DISSERTATION-2019.pdf

Permalink

https://hdl.handle.net/2142/106481

Description

Title
Engineering microorganisms for synthesizing value-added products
Author(s)
Lee, Jaewon
Issue Date
2019-12-04
Director of Research (if dissertation) or Advisor (if thesis)
Jin, Yong-Su
Doctoral Committee Chair(s)
Miller, Michael J
Committee Member(s)
  • Donovan, Sharon M
  • Rao, Christopher V
Department of Study
Food Science & Human Nutrition
Discipline
Food Science & Human Nutrition
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2020-03-02T22:38:56Z
Keyword(s)
Value-added products, microorganisms
Abstract
The overall goal of my thesis research is to produce value-added products using engineered microorganisms. Recent developments in metabolic engineering have allowed us to improve endogenous metabolic pathways or introduce heterologous metabolic pathways into microorganisms so that the engineered microorganisms have desired properties and phenotypes. As a result, value-added products that can only be synthesized by chemical processes can be produced in a more economical and sustainable way through biological processes using engineered microorganisms. Escherichia coli and Saccharomyces cerevisiae served as a biotechnological production organisms as well as a prokaryotic and eukaryotic model system in my thesis research, respectively. Since both strains are model strains, tremendous metabolic engineering tools and fermentation process techniques have already been developed and applied, but there are still more improvements that must be made to reach the titer and productivity of a target product for industrial scale production. The first goal of my thesis study was to overcome the drawbacks of high-level expression of rate-limiting enzymes for improving target products production. Increasing the expression level of the rate limiting enzyme via overexpression of the gene negatively affects the viability of host strain, making it difficult to produce a target product in a sustainable way. Instead of increasing the copy number using a high-copy plasmid or improving the transcription level using a strong promoter, I simply deleted two genes without affecting host strain’s viability and obtained the improved titer and productivity of a target product (2’-Fucosyllactose) in engineered E. coli. The second goal of my thesis study was to enhance the production of a target product (2’-fucosyllactose) in engineered S. cerevisiae by reducing by-product (ethanol) production. The second objective was based on three approaches. First, the primary carbon source was changed from glucose to xylose to minimize ethanol production as a by-product and maximize a target product production. Second, all heterologous enzyme needed for 2’-Fucosyllactose was chromosomally integrated an expressed by using CRISPR-Cas9 based genetic modification. Third, the heterologous gene was additionally integrated into chromosome to increase the enzymes activities expressed on chromosome. The third goal of my thesis study was to effectively resolve without glycerol formation as a by-product the redox imbalance caused by the reduction or elimination of ethanol production. To produce other target products from glucose than ethanol, ethanol producing genes (PDC: pyruvate decarboxylase, ADH: acetaldehyde dehydrogenase) should be mitigated in S. cerevisiae. Due to the redox imbalance, S. cerevisiae exhibited low growth rate and glucose consumption rate, resulting in a failure to reach the productivity of a target product suitable for industrial scale production. Therefore, by introducing an alternative metabolic pathway that can efficiently oxidize cytosolic NADH, we aimed to improve the productivity of a target product without producing glycerol as a by-product.
Graduation Semester
2019-12
Type of Resource
text
Permalink
http://hdl.handle.net/2142/106481
Copyright and License Information
Copyright 2019 Jaewon Lee

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Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Food Science and Human Nutrition