## 3 Discussion
coli_ when grown on minimal medium with glucose as sole carbon source [66]. We attempted to estimate G6P and F6P concentrations in cells of LJ110 and GL3 strains. Cells were first grown on MM fructose, then washed and transferred to MM glucose and incubated for 2 h. Under these conditions, cells of wt LJ110 grew on glucose, while GL3 cells did not. G6P and F6P were then determined enzymatically after extraction from cells by a boiling method. Concentrations of F6P and G6P stayed below the threshold values in LJ110 (<10 uM for sugar phosphates). Cells of GL3 showed intracellular concentrations of G6P at \(1.6\pm 0.6\) mM and F6P of \(1.1\pm 0.4\) mM (average values of two biological replicates, each with three technical replicates). While we are aware that our conditions differed from that of [66] and that the concentrations of the two hexose phosphates may be determined with more accuracy, the results obtained in our study nonetheless allow the statement that the intracellular F6P concentration in GL3 is far from saturating for FSAA while FSAA A129S would be at least closer to its optimal rate. Growth of GL3/pJF119/_saA_\({}^{A129S}\) on glucose was restored with a mean generation time of about 6 h (u of 0.12 h\({}^{-1}\)) whereas the wt strain LJ110 in our study showed a u of 0.59 h\({}^{-1}\). Therefore, the FSAA A129S reaction is not a simple stand-in for the PFK function in the GL3 strain. Several limitations in central metabolism can be envisioned: insufficient enzyme activities and efficiencies, lack of NADPH redox cofactors, inappropriate effector provision and unwanted side effects of a new metabolite, to name a few. Here we will discuss some of these issues and questions. 1. We have shown here that a bypass of the PFK block mutation is possible if a suitable enzyme activity of FSA is present in _E. coli_ cells. Cleavage of F6P into G3P and DHA with subsequent phosphorylation of DHA would lead to the same couple of triose phosphates as in EMP, and no extra energy expenditure is necessary (see Figure 4). Therefore, one might ask why did Nature not choose this pathway as a general alternative to EMP? The reason could be that DHA may not be funneled completely or fast enough into DHA. DHA is a short chain sugar which is well-known to readily react with proteins as it forms adducts with lysine and arginine residues [67] leading to browning; this effect is exploited for skin tanning [68], where DHA is an ingredient in many self-tanning compounds. We also have observed that during the course of incubation, only the cultures of the strains that produce DHA became yellow. Still, DHA can also lead to protein inactivation and it is known as a mutagen for _E. coli_ under certain circumstances [67]. Both consequences would be detrimental for cell metabolism and genetic stability. This might be the reason why an elaborate high affinity PTS for DHA phosphorylation (DHAKLM) is present in _E. coli_[5; 69; 58] whose main function arguably lies not so much in catabolism (yielding DHAP which can be catabolized in glycolysis or used as building block for glycerophospholipid formation) but rather in effective detoxification of an undesired metabolite. We found increased gene activity of the _dhaK_ gene in GL4 which we take for evidence that the DHAKLM system is indeed involved in further metabolism of DHA which stems from F6P cleavage. 2. FSAA A129S not only opens a bypass at the block between F6P and F1,6 BP (see Figure 4 and Figure S3). Through its action, G3P is formed which can be further metabolized in the lower glycolytic trunk to PEP and ultimately pyruvate. As well, G3P together with F6P serves as substrate for transketolase (TKT) from the PPP. TKT transfers two-carbon dihydroxyethyl units from F6P to G3P forming E4P and X5P; transaldolase then performs a DHA transfer reaction from F6P onto E4P yielding G3P and S7P [70; 71]. FSA could also add DHA on E4P to form S7P and be a sink for DHA [25]; thus, not all DHA is to be expected to go to glycerol (in strain GL7). 3.