## 5 Conclusions  
The disruption of the balance of energy production and utilization in the PTS\({}^{-}\) strain of _E. coli_ PB12 by plasmid expression of the _atpAGD_ operon encoding the soluble portion of ATP synthase F\({}_{1}\).ATPase(PB12 AGD\({}^{+}\) mutant) resulted in a reduction of the [ATP]/[ADP] ratio and the intracellular level of cAMP. The mutant grows slowly in glucose, but showed a higher \(q_{GL}\) and lower biomass yield than the strain PB12. Higher relative transcription of _galP_ and _pgi_ genes is associated with these traits, suggesting an increased carbon flux through glycolysis and the Entner-Duodoroff pathway. The upregulation of the acetate transport and genes involved in acetate synthesis and utilization, with a lower acetate concentration detected in supernatant cultures, supports the coutilization of acetate and glucose to increase the proposed carbon flux in this strain in response to the reduction in the [ATP]/[ADP] ratio.  
Upregulation of most genes encoding the TCA cycle and the glyoxylate shunt indicates that the AGD\({}^{+}\) mutant employs these pathways for energy and the reduction in power supply growth in glucose. The upregulation of several respiratory genes indicates increased respiratory capabilities, coupled possibly with the increased availability of electron donor compounds from the TCA cycle, resulting in an increased respiratory capability of 240% in the _apt_ mutant than in the PB12. The upregulation of genes involved in stress response in the _apt_ mutant, including _soxR_ and _soxS_ (superoxide response regulon), probably in response to a higher oxygen consumption observed in this mutant, makes sense with the increased respiratory capability. The upregulation of _spoT_ (\(\sim\)357X in the _apt_ mutant) confirms the severe stress condition resultant in response to the severe energetic disruption imposed in this mutant.  
Finally, the reduction in the intracellular concentration of cAMP in the _atp_ mutant resulted in a reduced number of upregulated genes than in PB12, showing a downregulation of the expression of 15 genes regulated by cAMP-CRP compared to the PB12 strain, suggesting that the mutant has a very robust genetic background despite the severe disruption in its energetic level. Our results provide valuable information to understand the capacity of this evolved mutant PB12 to adapt to a drastic energetic perturbation.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/biotech13020010/s1](https://www.mdpi.com/article/10.3390/biotech13020010/s1)]([https://www.mdpi.com/article/10.3390/biotech13020010/s1](https://www.mdpi.com/article/10.3390/biotech13020010/s1)), Table S1: Primer set used for RT-qPCR assays.  
Conceptualization, S.S., A.E. and F.B.; methodology, S.S., R.d.A. and N.F.; writing--original draft preparation, S.S., A.E., O.E.C.-R. and F.B.; writing--review and editing, A.E. and F.B.; funding acquisition, F.B. All authors have read and agreed to the published version of the manuscript.  
This research was funded by PAPIIT UNAM, grant IN209421.  
Not applicable.  
Data Availability Statement: Data are contained within the article.  
We thank [PERSON] and [PERSON] for their technical support, [PERSON], [PERSON], and [PERSON] for synthesizing oligonucleotides IBT-UNAM (Mexico), and [PERSON], Escuela de Ingenieria en Alimentos, Biotecnologia y Agronomia (ESIABA), Tecnologico de Monterrey, Queretaro, Mexico, for the proposed manuscript structure.  
The authors declare no conflicts of interest.