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Engineering Escherichia coli to Exploit Alternative Glycolytic Pathways upon Complete Loss of the Upper Embden-Meyerhof-Parnas Pathway

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Title
Engineering Escherichia coli to Exploit Alternative Glycolytic Pathways upon Complete Loss of the Upper Embden-Meyerhof-Parnas Pathway
Other Titles
대체 해당 경로가 활성화된 엠브덴-마이어호프-파르나스 경로 비활성화 대장균의 제작
Author
Kim, Ye Eun
Advisor
Lee, Sung Kuk
Issue Date
2019-08
Publisher
Graduate School of UNIST
Abstract
The glycolytic pathway has a central role in metabolism by supplying precursor, energy and redox molecules for supporting well being of microorganism at natural habitat. Advent of synthetic biology, however, facilitates proliferation of microorganisms beyo nd their original niche. The native glycolytic pathway often contains thermodynamic bottleneck and stoichiometry limiting their wide range application. Adopting alternative glycolytic structure bypassing such metabolic hurdles enables to expand and diversi fy traits of a microbial strain. Here, we completely inactivate the Embden-Meyerhof-Parnas (EMP) pathway in Escherichia coli which functions as major glycolytic route. In order to surpass sub optimality of latent alternative glycolytic routes in glucose ca tabolism, E . coli strain without the functional EMP pathway was subjected to adaptive evolution with glucose as sole carbon source. The evolved mutant exhibiting 6.6 fold higher growth rate was isolated. Based on whole genome sequencing data, key genotype changes were identified from genotype phenotype relationship. Disabilities of glucose uptake and glucose catabolism sole via the Pentose phosphate (PP) pathway were identified as primary metabolic bottlenecks upon complete loss of the upper EMP pathway. Th ese can be resolved by simultaneous enabling of glucose uptake and activating latent the Entner Doudoroff (ED) pathway for glucose catabolism. By rebalancing two alternative glycolytic pathways, the ED and PP pathway, tuning profile of carbon, energy and r edox equivalents yield from glucose was demonstrated. Combining the rebalancing strategy to adaptive evolution, 3-hydroxypropionic acid (3-HP) production titer was increased up to 3.2 fold compared to the unevolved strain
Description
Department of Chemical Engineering
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