Metabolic engineering and adaptive laboratory evolution of yeasts to achieve efficient microbial cell factory

Yook, Sangdo

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https://hdl.handle.net/2142/120570 Copy

Description

Title

Metabolic engineering and adaptive laboratory evolution of yeasts to achieve efficient microbial cell factory

Author(s)

Yook, Sangdo

Issue Date

2023-04-28

Director of Research (if dissertation) or Advisor (if thesis)

Jin, Yong-Su

Doctoral Committee Chair(s)

Miller, Michael J.

Committee Member(s)

• Rao, Christopher V.
• Stasiewicz, Matthew J.

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

Keyword(s)

• Metabolic engineering
• Adaptive laboratory evolution
• yeast
• Yarrowia lipolytica
• Saccharomyces cerevisiae
• Synthetic biology
• Value-added products

Abstract

The overall goal of this thesis research is to develop microbial cell factories using yeasts, particularly focusing on Yarrowia lipolytica, while also considering Saccharomyces cerevisiae by applying metabolic engineering and adaptive laboratory evolution (ALE). Until recently, metabolic engineering efforts for engineering yeast other than a model organism (S. cerevisiae) have been limited. However, with recent advancements in genetic engineering tools and sequencing technology, it is now feasible to manipulate non-conventional yeast to understand the relationship between genetic and phenotypic alterations. Despite these advancements, research on utilizing these tools to modify the phenotypes of yeasts, specifically Y. lipolytica, to generate value-added products remains relatively scarce. Consequently, this study emphasizes the use of advanced metabolic engineering tools, such as CRISPR-Cas9, Golden Gate assembly, and ALE, followed by whole-genome sequencing and reverse engineering, to produce lipids, modified fatty acids, styrylpyrones, and trehalose by yeast fermentation from abundant and inexpensive carbon sources such as cellulosic sugar (xylose). The first objective of this research is to incorporate a metabolic pathway of a non-edible sugar (xylose) derived from cellulosic biomass into a non-conventional yeast Y. lipolytica. To accomplish this, an oxidoreductase pathway was introduced into the wild-type Y. lipolytica strain (PO1f), and ALE was utilized to enhance xylose-utilizing phenotypes of resulting mutants. An engineered and evolved Y. lipolytica strain (XEV) can efficiently assimilate xylose to synthesize a native product, lipids. Whole-genome sequencing and copy number analysis were performed to identify genetic variations associated with the improved xylose assimilation phenotype of the XEV strain, and qPCR and CRISPR-mediated reverse engineering revealed that amplifications of xylose metabolic enzymes and a mutation in a cellular signaling protein might be responsible for enhanced xylose utilization. Ultimately, a Y. lipolytica strain capable of fermenting lignocellulosic hydrolysates as a substrate was constructed. The second objective of this research is to engineer Y. lipolytica capable of producing diverse value-added products from glucose and xylose. A cyclopropane fatty acid synthetic pathway was introduced into the XEV strain, enabling Y. lipolytica to produce cyclopropane fatty acids from xylose. The production of cyclopropane fatty acids was significantly enhanced by introducing S-adenosylmethionine synthetase (MetK) and supplementing methionine. To produce a medically significant styrylpyrones (Bisnoryangonin, 4-hydroxy-6-styryl-2-pyrone) from a mixture of glucose and cinnamic acid, an engineered 2-pyrone synthase (3APG) was introduced into Y. lipolytica. Integration of the 3APG gene at multiple locations in the genome and the reduction of the hydroxylation reaction of cinnamic acid substantially increased the synthesis of cinnamoyl-CoA from cinnamic acid, leading to enhanced styrylpyrone production. Lastly, the third objective of this study is to bolster the stress tolerance of S. cerevisiae through ALE and metabolic engineering. To achieve this, S. cerevisiae strains were subjected to ALE under ethanol and nitrogen-deficient conditions, resulting in the isolation of evolved strains with high trehalose and glycogen accumulations. The stress tolerance of these strains was assessed by measuring cell viability and fermentation efficiency after exposure to stress (freeze/thaw) conditions. Subsequent whole-genome sequencing and reverse engineering captured genetic variations that contributed to the enhanced tolerance of these strains under stress conditions.

Graduation Semester

2023-05

Type of Resource

Thesis

Copyright and License Information

Copyright @ 2023 Sangdo Yook

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