Engineering microorganisms for synthesizing value-added products

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Food Science & Human Nutrition

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2020

Author dc:creator

• Lee, Jaewon

Contributors dc:contributor

• Jin, Yong-Su
• Miller, Michael J
• Donovan, Sharon M
• Rao, Christopher V

• Value-added products, microorganisms

Statement dc:rights

• Copyright 2019 Jaewon Lee

Language dc:language

en

Handle dc:identifier

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

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/106481