Interrogating central carbon metabolism in Escherichia coli via the mapping of flux responses to gene knockouts and adaptive evolution

Long, Christopher P.

Interrogating central carbon metabolism in Escherichia coli via the mapping of flux responses to gene knockouts and adaptive evolution

Author(s)	Long, Christopher P.
Date Accessioned	2018-11-14T14:41:23Z
Date Available	2018-11-14T14:41:23Z
Publication Date	2018
SWORD Update	2018-07-27T13:03:29Z
Abstract	Biological systems have enormous potential for chemical conversion, including otherwise inaccessible syntheses and utilizing renewable feedstocks. Designing production strains through metabolic engineering requires detailed systems knowledge, particularly in the mapping of genotype (i.e., where manipulations are most typically made) to the metabolic phenotype (the output, e.g., overproduction of a chemical product). In this work, advances were made in metabolic characterization methods, and these methods were then applied to map metabolic responses to gene knockouts and adaptive laboratory evolution. Specifically, novel methods for measuring biomass composition, useful new measurements for 13C metabolic flux analysis (13C-MFA), and strategies for optimal tracer design were developed. These optimized methods were used to comprehensively assess metabolic responses to 45 E. coli gene knockout strains of enzymes in central carbon metabolism. Analysis of flux rewiring in these strains revealed bottlenecks and areas of flexibility in metabolism, a novel reversibility of Enzyme I of the PTS system and a glucose secretion phenotype. These results constitute a significant new resource for systems biology, particularly for metabolic modeling where they will be directly applied to the development of ensemble kinetic models. ☐ Genetic and metabolic responses to adaptive laboratory evolution in two strains were also characterized, providing new insights into the processes of microbial adaptation and fitness enhancement. Growth recovery (of up to 3.6-fold) in an E. coli knockout strain of a core glycolytic enzyme was enabled by a unique set of mutations which alleviated rate limiting steps in metabolism. In evolved E. coli wild-type strains, growth rate enhancements of 50% did not correspond to intracellular flux rewiring, indicating broad and proportional regulatory change. Mutations in both experiments also suggest critical roles for global regulators in adaptation. Finally, the metabolism of Vibrio natriegens, a very fast-growing and potential next-generation host organism, was elucidated by 13C-MFA. This provides an important baseline of knowledge to facilitate modeling and engineering of this organism. Further investigation into the mechanisms of fast-growth, both natural and evolved, will enable the development of hosts with superior productivity and economic potential.	en_US
Advisor	Antoniewicz, Maciek R.
Degree	Ph.D.
Department	University of Delaware, Department of Chemical and Biomolecular Engineering
DOI	https://doi.org/10.58088/rrx3-v238
Unique Identifier	1065523258
URL	http://udspace.udel.edu/handle/19716/23900
Language	en
Publisher	University of Delaware	en_US
URI	https://search.proquest.com/docview/2090033130?accountid=10457
Keywords	Biological sciences	en_US
Keywords	Applied sciences	en_US
Keywords	Adaptive evolution	en_US
Keywords	Escherichia coli	en_US
Keywords	Flux	en_US
Keywords	Gene knockouts	en_US
Keywords	Metabolic modeling	en_US
Keywords	Metabolism	en_US
Title	Interrogating central carbon metabolism in Escherichia coli via the mapping of flux responses to gene knockouts and adaptive evolution	en_US
Type	Thesis	en_US