## 3 Discussion  
In this work, by deletion of the genes for PFKA and PFKB (_pfkA, pfkB_) as well as of _zwf_ (glucose 6-phosphate dehydrogenase), a glucose-negative mutant of _E. coli_ K-12 was created. Very recently, other groups have reported the construction of such triple-negative strains, however, they were interested in different features and did not report the glucose phenotype [16; 17; 18]. Thus, we present here the broader characterization of a \(\Delta zwf\Delta p\)_fkB_\(\Delta p\)_fkA_ triple deletion strain (such as GL3) on various C sources. GL3 was found to be unable to grow on C sources which are taken up by the PEP-dependent sugar:phosphotransferase system (PTS) such as glucose, \(N\)-acetylglucosamine, mannose, mannitol, and others. These substrates have in common that they enter the EMP at F6P or G6P [6]. Although glucose in _E. coli_ might be oxidized via a PQQ-dependent glucose dehydrogenase to gluconic acid and thereby could circumvent the coerced genetic blocks, this oxidation apparently did not occur in GL3, most likely because PQQ as cofactor cannot be formed by _E. coli_ cells [61; 62]. As well, GL3 showed severe impairments when growing on D-galactose, glucose 6-phosphate, glycerol, pentose sugars, or other C sources which have to pass through F6P. Degradation of sugars which enter glycolysis at the stage of DHAP such as L-fucose or L-rhannose [63], however, was little affected in GL3 (data not shown); growth of GL3 on D-fructose was still possible, presumably because it can enter the EMP at the stage of F1,6 BP [14]. There is ample evidence for microbial cells being able to cope with genetic perturbations of central metabolic pathways (including blocks in the EMP) by activation of inherent metabolic pathways [64; 65]. In line with several genome scale metabolic models for _E. coli_ which already had incorporated FSA as a constituent of central metabolism [32; 33; 34], we had expected that a block in PFK in _E. coli_ might be overcome by activation of the dormant genes _fsaA_ or _fsaB_ as these genes encode a F6P aldolase activity [20]. However, even after prolonged incubation with glucose as sole C source, we did not detect GL3 mutants which could grow again on glucose. We therefore focused on the question whether recombinant expression of the gene for FSAA on a plasmid might create a metabolic bypass in living _E. coli_ cells. In the triple deletion strain GL3, we compared the expression of the wild type gene (_fsaA_) with the allele encoding a variant enzyme (FSAA A129S) with a known enhanced catalytic efficiency  
Figure 7: Maximal glycerol concentrations formed by GL7 during growth on different C-sources. Hexoses (D-glucose, D-galactose) were set at \(\approx\)28 mM initial concentrations, pentoses (L-arabinose, D-xylose) at \(\approx\)33 mM. Actual starting concentrations were determined by HPLC. Mean values of two independent biological replicates are given. for F6P as substrate. As a result, a glycolytic bypass leading to growth on glucose was only detected in GL3 with the plasmid-borne gene variant. What differentiates the mutant _fsaA_ gene from the wt _fsaA_ gene? Apparently, the difference lies in the improved catalytic efficiency for F6P which is known for FSAA A129S [24; 27; 35]. FSAA has a rather high \(K_{\text{M}}\) value for F6P in the range of 7-19 mM as determined by various researchers (however not under the same conditions) ([24; 27; 35], and [PERSON], unpublished results). FSAA A129S however, showed an improved affinity for F6P (range from 1.5 to 4 mM) as well as an improved activity with this compound as substrate leading altogether to an approximate 20-fold improved catalytic efficiency in vitro ([24; 27; 35], and [PERSON], unpublished results). If the affinity of the two FSA enzymes for F6P is important, knowledge on the intracellular F6P concentrations in _E. coli_ could be helpful. The [PERSON] group determined intracellular concentrations for F6P of about 2.5 mM and for G6P in the range of 8 mM for _E.