Comprehensive study of metabolic flux rewiring in E. coli knockout strains

Christopher P. Long, Maciek R. Antoniewicz
Department of Chemical and Biomolecular Engineering, University of Delaware
150 Academy Street, Colburn Laboratory, Newark, DE 19716

Abstract

Cellular metabolic and regulatory systems are of fundamental interest to systems biology and
metabolic engineering, but incomplete understanding of their complex properties remains an obstacle to progress in those fields. An established method for obtaining information on network structure, regulation, and dynamics is to study the system following a perturbation such as a genetic knockout. The Keio collection of all viable Escherichia coli single-gene knockouts is now facilitating a systematic investigation of the regulation and metabolism of E. coli. Of all omics measurements available to observe the perturbed phenotype, the metabolic flux profile (the fluxome) provides the most direct representation and the most relevant to metabolic engineering. Recent advances in 13C-metabolic flux analysis (13C-MFA) are permitting highly precise and accurate flux measurements for investigating cellular systems and guiding metabolic engineering efforts.
In this project, the core metabolism of E. coli is studied by measuring metabolic fluxes of all single gene knockout mutants associated with central carbon metabolism (glycolysis, pentose phosphate pathway, TCA cycle, and anaplerotic reactions). First, a parallel labeling experimental design was optimized to improve (by about 10-fold) flux estimation precision in the E. coli network model. Our optimized experiment design is based on performing parallel experiments with [1,2-13C]glucose, [1,6-13C]glucose and [4,5,6-13C]glucose tracers, coupled with mass spectrometry measurement of isotopic labeling and 13C-MFA. In addition to these isotopic labeling measurements, we also measure changes in biomass composition and cellular yields for each Keio strain. These measurements constitute a thorough assessment of the metabolic states
of each knockout. This information is then used to learn about possible novel or unannotated reactions in central carbon metabolism. The metabolic fluxes are also compared to predictions made by commonly used constraint-based models including FBA, MOMA, and ROOM.
In this presentation, we will present our first rigorous assessment of wild-type E. coli and two knockout strains (pgi-KO and zwf-KO). These two enzymes catalyze the two sides of the branch point between glycolysis and the pentose phosphate pathway: pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate dehydrogenase). We will also present ongoing work on the knockout strains of the pentose phosphate pathway and glycolysis. When completed, this project will provide the first comprehensive assessment of the regulation of E. coli central carbon metabolism; it will allow detailed analysis of the accuracy of constraint-based models such as FBA, MOMA, and ROOM to predict metabolic fluxes in perturbed systems, which has been difficult to date because of limitations in existing knockout flux data; and finally, it will provide valuable information for new model development, for example, in training and fitting parameters in mechanistic (i.e. kinetic) metabolic models and regulatory models.