## 4 Discussion
### Glycolysis, Gluconeogenic, Pentose Phosphate, and Entner-Doudoroff Pathways  
The glycolytic gene _psi_ encoding for glucose-6-P isomerase was upregulated in both PB12 mutants. The upregulation of _psi_ with _galP_ and _glk_ was proposed to contribute to the increased glycolytic flux observed in the PB12 mutant, which could result in a possibly higher pool of fructose-6-P in both PB12 mutants [11; 22]. Upregulation of _psi_ was associated with a reduced carbon flux from glycolysis to PPP and the Entner-Duodoroff pathways [22]. Other glycolytic genes upregulated in PB12 by at least 1.7-fold or higher were _fbaB_, _fbp_, _gpmA_, and _pykA_ (Table 3, Figure 2), encoding, respectively, to fructose-biphosphate aldolase (FbaB), fructose-1-6, biphosphatase 1 (Fbp), 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (GpmA), and pyruvate kinase 2 (PykA). Although these genes showed a decrease in their relative expression in the _atp_ mutant compared to PB12 (Table 3, Figure 2), they support an increased glycolytic flux in this mutant, as suggested by the increased _q\({}_{\text{Glc}}\)_ value (Table 2).  
The PPP genes observed upregulated in both PB12 mutants were _eda_, _rpiB_, _talA_, and _tktB_, encoding, respectively, to KHG/KDPG aldolase (Eda), allose-6-phosphate isomerase/ribose-5-phosphate isomerase B (RpiB), and the transketolase 2 (TktB). Nevertheless, _zwf_encoding the NADP+-dependent glucose-6-phosphate dehydrogenase catalyzing the reaction of glucose-6-P + NADP+ \(\rightarrow\) 6-P D-glucono-1,5-lactone + NADPH + H+ showed an increased relative transcription of 1.96 in PB12, but not in the _apt_ mutant (1.25) (Table 3, Figure 2). Despite the observed reduced relative expression of _zwf_ in the _apt_ mutant but associated with the observed upregulated genes of the PPP, both strains presented an increased carbon flux from glucose-6-P to the PPP.  
The expression of the _aptAGD_ results in a high turnover of ATP, which has been associated with a strong stimulation of the glycolytic flux resulting in a decrease in the growth rate of the cells [9]. In agreement with this observation, the decrement observed in growth in the _atp_ mutant (\(\mu\) = 0.24 h\({}^{-1}\)) may be associated with lower availability of ATP; the PB12 strains use the GalP to internalize glucose from the periplasm. In the glycolytic pathway, several reactions consume one ATP; phosphorylation of glucose from ATP by Glk (\(-\)1 ATP) and conversion of fructose-6-P to fructose-1,6-diP from ATP by PfkA/PfkB (\(-\)1 ATP), resulting in the consumption of 2 ATP (-2 ATP). Nevertheless, two reactions involve the synthesis of ATP from ADP: the conversion of 2[3-P-glycerol-P] to 2[3-P-glycerate] by Pgk (+2 ATP), and the conversion of 2[phosphoenolpyruvate] to 2[pyruvate] by PykF/PykA (+2 ATP). Additionally, as the gluconeogenic gene _ppsA_ (PpsA) was upregulated in both PB12 strains, it could be considered to be the conversion of at least one pyruvate molecule to phosphoenolpyruvate with 1 ATP. Then, the PTS- mutant PB12 showed the final balance of +1 ATP in the glycolytic pathway from glucose to PEP and the gluconeogenic reaction catalyzed by PpsA. Although the ATP balance in the PB12 AGD+ _atp_ mutant is the same as that in PB12, the higher ATPase (activity consuming) ATP and lower [ATP]/[ADP] ratio than PB12 indicates the lower growth in the _atp_ mutant than in PB12, but, as discussed above, the higher _qGlc_ showed by the _atp_ mutant indicates an increased carbon flux in response to the lower [ATP]/[ADP] ratio.