## 1 Introduction  
Bacteria are often used as microbial cell factories for delivering functional biomolecules with industrial or pharmaceutical interest. As most of these bioprocesses are metabolically demanding, it is critical to understand the physiological behavior of these organisms and to characterize their metabolic capabilities. Many studies have demonstrated that, under stressful conditions, their metabolic activities are not growth-related, which results in lower biomass yields and productivity [1, 2]. Particularly, recombinant bioprocesses can be quite demanding for microbial cells due to the metabolic burden caused by the depletion of central metabolites like amino acids toward recombinant protein synthesis, which unbalances most central metabolic activities resulting in considerable productivity losses. Under low nutrient conditions, cells usually engage in a multitude of cellular responses that allow their survival until growth resumes. Typically, the coordination of these cellular responses involves the global regulator guanosine-3',5'-bis-pyrophosphate (ppGpp), a core molecule that primarily triggers the stringent response [3, 4, 5, 6]. Although the synthesis of ppGpp has been mainly associated with cellular responses to amino acid starvation, which in _E. coli_ are mainly initiated by the activation of the ribosome-associated enzyme encoded by the _rel_A gene catalyzing the conversion of cellular GDP into ppGpp [7], recent studies have indicated that this molecule also accumulates during carbon starvation [8, 9, 10]. A second ppGpp synthetase, _i.e._, the bifunctional enzyme SpoT that has both hydrolase and synthetase activities, has been described to be involved in ppGpp accumulation during carbon starvation [11, 12], but its activity was shown to be much weaker than the one of the RelA enzyme [13]. This suggests that RelA may be central in the response to carbon starvation. It was thus suggested that these two nutritional stress phenomena are strictly correlated, the exhaustion of carbon often resulting in a rapid decrease in amino acids availability, entangling the activity of both enzymes [8]. Therefore, it is expected that RelA, directly or indirectly, interferes in the cellular responses to carbon-limited conditions. These phenomena have been implicated in recombinant bioprocesses using _E. coli_ as an expression host [14]. It was found that ppGpp-deficient strains can maintain a metabolically productive state longer than the parent strains [15]. Thus, reducing the intracellular ppGpp levels seems to attenuate the pleiotropic effects on the metabolism, which is beneficial for the synthesis of foreign proteins. However, whether this is due to a less stress-responsive phenotype during recombinant production that eventually affects the metabolism, or to changes in the metabolic basis of this strain is still unclear. Despite the effects on the synthesis of foreign proteins, the impact of this regulator on the cellular metabolism of host strains needs to be characterized. To investigate the metabolic state of _E. coli_ cells and the role of the RelA enzyme (p)ppGpp synthetase in the _E. coli_ responses to nutrient-limited growth conditions, a metabolomics approach was applied in this study. The intracellular metabolite profiles measured by gas chromatography-mass spectrometry (GC-MS) were used to assess the main metabolic changes resulting from different steady state growth conditions. Aerobic chemostat cultivations were performed at different dilution rates that provided for constant nutrient-limiting conditions specific for a single nutrient (_i.e._, glucose) allowing steady state growth of cells (_i.e._, at steady state the specific growth rate of cells is equal to the dilution rate). At these conditions, transient growth effects and other stress-induced responses are avoided that could mask effects resulting specifically from nutrient limitation.