13C-Metabolic flux analysis uncovers rewiring in Escherichia coli ΔmetA metabolism and secretion of amino acids in response to methionine limitation.

In this study, we investigated how methionine availability influences the phenotype and metabolism of the methionine-auxotrophic E. coli ΔmetA strain. To identify metabolic and physiological changes, the strain was cultured under two conditions: i) a methionine excess condition, where 1 mM of methionine was added to the medium; and ii) a methionine limited condition, where the medium did not contain any methionine, but instead methionine was fed slowly to maintain methionine concentration below 1 μM. To quantify metabolic fluxes, isotopic tracers [1,2-13C]glucose and [1,6-13C]glucose were applied and 13C-metabolic flux analysis (13C-MFA) was performed. To obtain acceptable fits of the labeling data the metabolic network model had to be updated. First, secretion reactions for four amino acids, i.e. glutamate, threonine, lysine and glycine, were added, which were found to accumulate in the medium under methionine limitation, both under growth and non-growth conditions. Second, a reaction was added to one-carbon metabolism that explicitly captures the incorporation of labeled methyl carbon derived from glucose into methionine. This cycle has thus far been overlooked in 13C-MFA models, however, our results suggest that taking the methylation of methionine into consideration is critical for successfully implementation of 13C-MFA. Under methionine limitation, we observed: 1) reduced oxidative pentose phosphate pathway flux; 2) significant increase in the TCA cycle flux and anaplerotic flux into the TCA cycle; 3) activation of a normally dormant pathway from threonine to glycine; 4) secretion of glutamate, threonine, lysine and glycine; and 5) recycling of 15% of intracellular methionine through the methylation cycle. In contrast, when the ΔmetA strain was grown in the presence of excess methionine, all of the unique features of this strain were masked and the flux phenotype simply reflected wild-type E. coli phenotype with only minor flux changes directly related to the gene knockout itself. We also performed co-culture experiments using ΔmetA and ten different auxotrophic E. coli strains, and cultured the same strains on spent medium from ΔmetA culture. We found that the growth behavior of the co-cultures matched with the growth behavior observed for the auxotrophic strains grown on ΔmetA's spent medium. Taken together, this study presents a robust, high-resolution approach to dissect nutrient-limited metabolism and provides novel insights that can advance our understanding of syntrophic interactions in microbial communities.