Abstract
13C-Metabolic flux analysis (13C-MFA) traditionally assumes that kinetic isotope effects from isotopically labeled compounds do not appreciably alter cellular growth or metabolism, despite indications that some biochemical reactions can be non-negligibly impacted. Here, populations of Escherichia coli were adaptively evolved for ~1000 generations on uniformly labeled 13C-glucose, a commonly used isotope for 13C-MFA. Phenotypic characterization of these evolved strains revealed ~40% increases in growth rate, with no significant difference in fitness when grown on either labeled (13C) or unlabeled (12C) glucose. The evolved strains displayed decreased biomass yields, increased glucose and oxygen uptake, and increased acetate production, mimicking what is observed after adaptive evolution on unlabeled glucose. Furthermore, full genome re-sequencing revealed that the key genetic changes underlying these phenotypic alterations were essentially the same as those acquired during adaptive evolution on unlabeled glucose. Additionally, glucose competition experiments demonstrated that the wild-type exhibits no isotopic preference for unlabeled glucose, and the evolved strains have no preference for labeled glucose. Overall, the results of this study indicate that there are no significant differences between 12C and 13C-glucose as a carbon source for E. coli growth.
Highlights
Metabolic flux analysis (MFA) has become an invaluable technique with which to probe the metabolic flux states of an organism [1, 2]
Wild-type Escherichia coli K-12 MG1655 was used to establish six independent cultures that were serially propagated in M9 minimal medium with uniformly labeled 13C-glucose as the sole carbon source
An automated system was used [10] to perform this Adaptive Laboratory Evolution (ALE) experiment, continually tracking the cultures’ growth rates and passing them to fresh media flasks before depletion of the glucose could serve as a limiting factor to growth
Summary
Metabolic flux analysis (MFA) has become an invaluable technique with which to probe the metabolic flux states of an organism [1, 2]. Knowledge of these intracellular fluxes is frequently used in complement with metabolic engineering, where it can be utilized to guide the rational design of genetic manipulations necessary for chemical production [3]. MFA relies on the isotopic labeling of a “tracer” compound that is passed through the reaction network of a cell, and 13C is typically used as the stable isotope of choice [4]. Inherent to 13C-MFA experiments is the assumption that the labeled compound is not metabolized differently than the unlabeled form, PLOS ONE | DOI:10.1371/journal.pone.0151130. Inherent to 13C-MFA experiments is the assumption that the labeled compound is not metabolized differently than the unlabeled form, PLOS ONE | DOI:10.1371/journal.pone.0151130 March 10, 2016
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