Metabolic Flux Rewiring and Physiology in E. coli Upper Central Carbon Metabolism Knockout Strains

Metabolic and physiological responses to genetic perturbations are of fundamental interest to systems biology and metabolic engineering. They reveal new information on the network structure, for example, uncovering previously “hidden” reactions, as well as metabolic regulation and kinetics. The accurate prediction of metabolic fluxes following a genetic perturbation, critically important for rational metabolic engineering, has been a persistent challenge due to the complexity of cellular systems. There has been much work done to develop in silico predictive models of metabolism, most notably the constraint-based models. Progress in this area has been limited by a lack of high-quality, reproducible experimental metabolic flux (fluxomic) data of genetically perturbed strains. In this work, we leverage recent advances in ¹³C-metabolic flux analysis (¹³C-MFA) and the availability of all viable Escherichia colisingle gene knockouts from the Keio collection to perform a comprehensive physiological and fluxomic study of mutants associated with central carbon metabolism. The results will provide both novel biological insights and a significant new resource for advancing metabolic modeling and strain design tools.

The core metabolism of E. coli is studied by measuring metabolic fluxes and biomass composition for all single gene knockout mutants associated with central carbon metabolism (glucose transport, glycolysis, pentose phosphate pathway, ED pathway, TCA cycle, and anaplerotic reactions) during aerobic, exponential growth on glucose. Fluxes are estimated based on an optimized parallel experimental design using [1,2-¹³C]glucose and [1,6-¹³C]glucose tracers, coupled with mass spectrometry measurement of metabolite isotopic labeling and ¹³C-MFA. Complimentary biomass composition measurements were carried out using a novel GC-MS based method for the quantification of amino acids, RNA, fatty acids, and glycogen. These measurements, along with growth and yield observations, constitute a thorough assessment of the metabolic states of each knockout.

Here we will present the described rigorous assessments of the wild-type E. coli and 23 knockout strains which comprise the complete “upper half” of central carbon metabolism: the key glucose transporters, the entirety of the pentose phosphate and ED pathways, and the upper portion of the EMP pathway (glycolysis). Global correlations and trends involving biomass composition and fluxes are reported. The fluxomic data is leveraged to develop new insights into specific kinetic and regulatory responses, as well as more general stress responses. Particularly notable are cases in which the knockout strains exhibit unusual pathway usage, and/or previously unannotated or “hidden” reactions. All fluxes are compared with predictions made by common constraint-based models such as FBA, MOMA, ROOM, and RELATCH. Ongoing efforts in this project will aim to extend this analysis to lower central carbon metabolism, and to apply the flux data to new model development efforts such as the ensemble modeling approach.