Engineering microorganisms for synthesizing value-added products

Lee, Jaewon

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

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

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

Copyright and License Information

Copyright 2019 Jaewon Lee

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