## 3 Discussion
The block of PFK in glycolysis leads to accumulation of phosphorylated sugars (G6P, F6P) from PTS substrates and causes the so-called sugar phosphate stress. This elicits activation of SgrS [59; 72; 73]. _SgrS_ sRNA interacts with _ptsG_ mRNA to reduce translation of new PtsG (enzyme IIBC\({}^{\text{Glc}}\)) molecules [74] whereas already existing PtsG molecules are still active as protein turnover is slow [75]; _SgrS_ sRNA also activates synthesis of a sugar phosphatase (YigL) which dephosphorylates G6P to help with glucose homeostasis [75]. This could lead to less G6P and, in turn, to less F6P in the cells. Moreover, the gene product of _sgrS_, when expressed ectopically, is a small polypeptide SgrT which inhibits PtsG activity in vivo [76; 77]. Thus, although FSAA A129S removes F6P, the uptake rate of glucose might be lower in GL3 than in LJ110 and contribute to the slower growth on glucose. 4. The missing glucose 6-phosphate dehydrogenase function (\(\Delta\)_zwf_) in strain GL3 and the resulting lack of a subsequent 6-phosphogluconate dehydrogenase step pose another problem as the NADPH supply by these two enzymes is not restored by introduction of FSAA A129S in the bypass pathway. Cells of GL3/pJF119/s\(aA^{A1295}\) have thus to rely either on transhydrogenase or isocitrate dehydrogenase as NADPH sources [45] for anabolism. This could also contribute to the observed slower growth rates. 5. Why does the triple mutant GL3 grow slower on C sources other than glucose? Fructose is in part transported via the enzyme II for D-mannose to yield F6P [14] and thus could end up at the same blockade as with glucose. Xylose and other C sources which are initially routed through the PPP, also deliver F6P which could then accumulate and/or be interchanged with G6P. Both would not contribute to the EMP. In comparison to GL3, in GL3/pJF119/s\(aA^{A1295}\) and strain GL7 growth on xylose and other C sources was only partially restored while being clearly slower than in the wild type, LJ110 (data not shown). This warrants further investigations. Another problem in the bypass pathway lies in the provision or recycling of PEP. As DHAKLM depends on PEP for the phosphorylation step (such as in strain GL3/pJF119 _fsaA_A1295 or in GL4), 2 moles of PEP are necessary for activation of glucose (via the glucose PTS) and of DHA, while only 2 PEP are formed during the catabolism of glucose through the EMP (see Figure S3). Thus, in theory, no net PEP would remain to fulfill its many roles as substrate of pyruvate kinases (PYKA and PYKF, for ATP production), PEP carboxylase (PPC, anaplerotic reaction) [78], DAHP-synthases and EPSP synthase (in aromatic biosynthesis), or MurA (cell wall biosynthesis). The cell would thus have to rely on PEP recycling to come up with its various anabolic and anaplerotic needs. PEP could be recycled from pyruvate by a PEP synthetase (PPS), from pyruvate via malate (malic enzyme, pyruvate + HCO\({}^{3-}\) + NAD(P)H\({}_{2}\) > malate + NAD(P)\({}^{+}\)) in conjunction with malate dehydrogenase and PEP carboxykinase (PCK; oxaloacetate + ATP > PEP + ADP). In any way, this would afford ATP. An additional problem which may contribute to slow growth of cells which have the bypass pathway is the low concentration of F1,6 BP. This molecule is not only an intermediate of the EMP, it also plays a role as a \"sensing metabolite\" in _E. coli_[66; 79] as it activates enzymes of the lower glycolytic trunk (PYKF) and in anaplerosis (PPC), and thereby contributes to faster glycolytic metabolism. As the FSA-based bypass does not directly lead to F1,6 BP, however, the enhancement of PYKF and PPC might be less, which could explain the slower growth of GL3-derived strains on compounds via F6P. We do not know how much F1,6 BP is formed from the gluconeogenic aldol addition condensation of G3P + DHAP. Having shown that a plasmid-encoded _fsaA_A1295 restored growth on glucose, we went on to integrate a copy of the mutant gene under control of an inducible P\({}_{\text{tac}}\) promoter in the chromosome of GL3 to come up with GL4.