## 3 Results
### Metabolite Profiling
We have evaluated this by estimating correlation coefficients based on the [PERSON]'s correlation, which identify uncorrelated patterns when comparing metabolite profiles along the three culturing conditions and between the two _E. coli_ cultures. Figure 2 represents those metabolite profiles that showed significant uncorrelated patterns among cultures and the estimated pairwise [PERSON]'s correlation coefficients. As illustrated in Figure 2, only one metabolite (succinate, _succ_) was found to have negatively correlated profiles, which means that the intracellular levels of this metabolite followed an opposite pattern in both _E. coli_ strains. However, six other metabolites showed poorly correlated patterns that are essentially caused by discrepancies at lower dilution rates (_i.e._, dilution rates of 0.1 and 0.05 h\({}^{-1}\)). Most of these uncorrelated profiles are associated with fatty acids, denoting that the coordination of fatty acids biosynthetic activities is somehow affected by the _rel_A gene mutation. Figure 1: Venn diagram showing the list of the intracellular metabolites that were significantly changed (p-value \(<\) 0.01) according to either factors: A (_i.e._, _E. coli_ strain) or B (_i.e._, dilution rate). To understand how these specific metabolic alterations are related to changes in biochemical activities, metabolite profiles were translated into metabolic pathway activities. Two enrichment analyses were performed: the Metabolite Biological Role (MBRole) a web-server tool that uses biological and chemical annotations from different databases to highlight the biological role of metabolomics data; and Pathway Activity Profiling (PAPi), an algorithm that uses the metabolite profiles and KEGG database to compare the activities of metabolic pathways between different experimental conditions. While MBRole highlights metabolic activities that are over-represented in the metabolomics data, PAPi used the quantification of metabolite levels to determine pathways activity measured by the Activity Score (AS). In both analyses, pathways like \"Aminoacyl-tRNA biosynthesis,\" \"ABC transporters,\" \"Citrate cycle (TCA cycle),\" \"Alanine, aspartate and glutamate metabolism\" and \"Fatty acid biosynthesis\" were highlighted (see Tables S3 and S4). However, PAPi showed that, particularly at the dilution rate of 0.1 h\({}^{-1}\), pathways such as \"Aminoacyl-tRNA biosynthesis,\" \"ABC transporters,\" \"Nicotinamide and nicotinamide metabolism,\" \"Sphingolipid metabolism\" and \"Sulfur metabolism\" presented higher activity scores in the _E. coli_ W3110 culture, whereas pathways of \"Biosynthesis of unsaturated fatty acids\" and \"Alanine, aspartate and glutamate metabolism\" showed lower activity scores. Clearly, metabolic pathway activities involving amino and fatty acids seem to be the most affected by the _rel_A gene mutation in these experiments. Figure 2: Metabolic patterns of the W3110 (represented by full diamonds and dashed lines) and \(\Delta\)_rel_A (represented by open circles and red lines) _E. coli_ cultures that presented low pairwise correlation coefficients (_r_ < 0.6). The error bars shown in the line graphs represent the relative standard deviation among the 3–4 sample replicates. Only metabolites that presented significant changes according to the Mack-Skillings test for the strain factor (factor A) were considered in this analysis. To illustrate these differences, metabolite profiles were also represented in the _E. coli_ metabolic map that includes these major metabolic pathways (Figure 3). **Figure 3.** Representation of metabolic profiles on the metabolic map of _E. coli_. White and grey boxes represent metabolites that were respectively detected and undetected by the GC-MS analysis. Black boxes refer to metabolites that were found to change significantly according to the Mack-Skillings test. Each plot next to metabolite black boxes displays the corresponding metabolite profiles from each _E. coli_ culture (dashed lines represent the metabolic profile of _E.