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Title:Strain engineering and biosensor development for efficient biofuel production by Saccharomyces cerevisiae
Author(s):Li, Sijin
Director of Research:Zhao, Huimin
Doctoral Committee Chair(s):Zhao, Huimin
Doctoral Committee Member(s):Rao, Christopher; Schroeder, Charles; Jin, Yong-su
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Saccharomyces cerevisiae
biofuel
biosensor
Abstract:Metabolic engineering of Saccharomyces cerevisiae is an attractive approach to enhance the production of cellulosic ethanol, fatty alcohols and other advanced biofuels. Production of cellulosic ethanol from lignocelluloses has attracted a lot of interest and significant improvement has been made to construct and optimize the recombinant S. cerevisiae strains capable of converting glucose or pentose sugars into ethanol. Unfortunately, pentose sugars, which constitute up to 30% of biomass hydrolysate, cannot be co-utilized simultaneously with glucose by recombinant S. cerevisiae strains. Great efforts have been made to improve the co-utilization efficiency of sugars derived from lignocellulose hydrolysates. A lot of research has been carried out to lower the effect of glucose repression that leads to inefficient pentose sugars utilization in the presence of glucose, but it remains challenging to overcome this issue by depletion of genes involved in transcriptional regulation or optimization of pentose sugar transportation and utilization. To overcome the glucose repression problem in S. cerevisiae, we designed a strategy to construct a S. cerevisiae strain capable of simultaneously utilizing cellobiose and xylose derived from lignocellulose. The high efficiency pathway containing a cellobiose transporter and a β-glucosidase enables fast cellobiose utilization and ethanol production, and glucose repression is avoided by the intracellular utilization of cellobiose. Distinguished from existing glucose derepression methods, glucose utilization is not impaired, while xylose utilization is improved because of the synergistic effects. To optimize the cellobiose utilization efficiency, the functional role of an important enzyme in glucose conversion, aldose 1-epimerase (AEP), was investigated. AEP is supposed to maintain the intracellular equilibrium of α-glucose and β-glucose when the spontaneous conversion between the two glucose anomers is not sufficient. However, the heterologous cellobiose utilization pathway results in excess β-glucose accumulation and lowers the rate of glucose glycolysis, which limits efficient utilization of cellobiose in engineered S. cerevisiae strains. We found three AEP candidates (Gal10, Yhr210c and Ynr071c) in S. cerevisiae and investigated their function in cellobiose utilization. Deletion of Gal10 led to complete loss of both AEP activity and cell growth on cellobiose, while complementation restored the AEP activity and cell growth. In addition, deletion of YHR210C or YNR071C resulted in improved cellobiose utilization. These results suggest that the intracellular mutarotation of β-glucose to α-glucose might be a rate controlling step and Gal10 plays a crucial role in cellobiose fermentation by engineered S. cerevisiae., The production of advanced biofuels, such as higher alcohols, fatty acid derived fuels, and hydrocarbons, is considered to be a better fuel alternative solution. Because their physiochemical properties are more compatible with the current gasoline-based infrastructure than ethanol. However, compared to current progress in ethanol production, a lot more efforts are needed to make these advanced biofuels commercially available. Recent efforts in advanced biofuels synthesis have been focused on the design, construction and optimization of pathways and strains, but detection becomes the bottleneck step that hinders high-throughput screening. Genetic biosensors convert chemical concentrations into detectable fluorescence signal via transcriptional regulation, and may serve as an important tool for screening and cell sorting. We have constructed a genomic sensor that correlates intracellular malonyl-CoA concentration to a fluorescence signal by transcriptional regulation. Malonyl-CoA is the building block for the biosynthesis of fatty acids, 3-hydroxypropionic acid, polyketides, and flavonoids, which can either be used directly or be used as a precursor for the production of biofuels and value-added chemicals. The sensor was combined with a genome wide mutant library in S. cerevisiae, and used to screen for mutants with higher productivity of malonyl-CoA, thus improving the downstream production of the reporter chemical, 3-hydroxypropionic acid. The constructed malonyl-CoA sensors can also be employed as control elements in order to modulate gene expression of biosynthetic pathways of important compounds that are of particular interest to the pharmaceutical and biofuel industries. The development of transcriptional-regulation based sensors relies on the discovery and identification of transcription factors and operators, which are usually heterologous to the platform microorganism. We explored a novel strategy to discover multiple sensors by transcriptional profiling. The strategy utilizes the native regulation mechanisms in S. cerevisiae, minimizes extrinsic manipulation and screens for multiple metabolite-responsive promoters with various transcription activities in a short time. A proof-of-concept sensor targeting acetyl-CoA was established and validated and the development of more sensors is in progress. This strategy provides an innovative approach for metabolite monitoring and pathway control. 
Issue Date:2014-07-09
Type:Thesis
URI:http://hdl.handle.net/123456789/3545
http://hdl.handle.net/2142/89263
Rights Information:Copyright 2014 Sijin Li
Date Available in IDEALS:2016-03-07
Date Deposited:2014-08


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