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Title:Systematic and combinatorial approaches for metabolic engineering of optimal yeast strains to produce fuels and chemicals
Author(s):Quarterman, Joshua
Director of Research:Jin, Yong-Su
Doctoral Committee Chair(s):Miller, Michael
Doctoral Committee Member(s):Jin, Yong-Su; Rao, Chris; Zhao, Huimin
Department / Program:Food Science & Human Nutrition
Discipline:Food Science & Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):metabolic engineering
Saccharomyces cerevisiae
systems biology
flux balance analysis
evolutionary engineering
Abstract:The overall goal of my thesis study is to use a metabolic engineering approach for developing optimal yeast cell factories capable of efficiently fermenting various sugars abundant in renewable biomass. The research has broad applications for sustainable biological production of value-added fuels and chemicals. Initially, a systematic approach based on global reaction stoichiometry was applied to select gene knockout targets in Saccharomyces cerevisiae for enhancing bioethanol production from glucose with minimal impact on biomass yield. Due to the limited scope of stoichiometric models, genetic screening was combined with the model-based technique, and this led to identification of three knockout mutants (∆cox9, ∆qcr6, and ∆qcr9) with superlative characteristics for enhancing fermentation of glucose and other hexoses (sucrose, fructose, and mannose). Although deletion of a respiratory enzyme subunit (Cox9) identified by the model-based approach severely inhibited galactose metabolism, the deletion was a necessary intermediate step for the respiration-deficient yeast to reach efficient galactose fermentation rapidly through serial subculture in galactose media. The combination of systematic and combinatorial methods led to an optimal phenotype on galactose that could not have been achieved by either method independently and demonstrates a promising approach for directing adaptive evolution toward fermentative metabolism. To understand the genetic basis of the improved phenotype, genome sequencing was conducted and used to identify a loss of function mutation in a repressor of galactose metabolic genes (Gal80p); this mutation was found to act synergistically with inhibition of respiration for efficient galactose fermentation by S. cerevisiae. A similar ‘fermentative evolution’ approach, involving deletion of COX9 and serial subculture in xylose minimal media, was applied to improve ethanol fermentation by a xylose-fermenting yeast strain previously developed in our group (SR8). Genome sequencing and a yeast mating experiment led to identification of a frameshift mutation in a transcriptional regulatory complex subunit (Spt3p) for improving xylose fermentation in engineered respiration-deficient yeast. Ethanol production was significantly improved in the xylose-evolved mutant, but excessive xylitol production demonstrated a redox imbalance problem due to NAD+ shortage without respiration. Two separate strategies were effective for alleviating the redox imbalance problem and further improving ethanol fermentation from xylose in the respiration-deficient evolved strain: (i) expression of a NADH-consuming acetate reduction pathway, or (ii) expression of a mutant NADH-preferring xylose reductase. In summary, this metabolic engineering study utilizes model-based and evolutionary tools for development of yeast cell factories that can rapidly ferment sugars abundant in non-food plant biomass. Furthermore, we demonstrate a novel strategy for redirecting sugar metabolism toward the fermentation pathway by systematic deletion of a respiration-related gene and adaptive evolution in selective conditions. The work in this study advances knowledge of limiting factors for sugar fermentation under anaerobic conditions and describes metabolic engineering strategies for overcoming these limitations. Efficient sugar-fermenting strains that can function in the absence of oxygen (i.e. without respiration), such as the ones developed in this study, are desirable because they prohibit respiratory utilization of sugars and oxidative metabolism of alcohol or other fermentation products, both of which can reduce product yield. Thus, engineering and characterization of respiration-deficient yeast strains provides valuable knowledge and understanding that may have applications for industrial fermentation processes.
Issue Date:2015-01-21
Rights Information:Copyright 2014 Joshua Quarterman
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12

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