## 2 Results
### Transient Formation of Dihydroxyacetone from Glucose  
While the in vivo presence of FSAA A129S (as observed by SDS-PAGE and enzyme activity in heat-treated extracts) and the restoration of growth in GL3 nicely fit with the assumption of a bypass  
Figure 3: Growth behavior of GL3 (\(\Circinus\)), GL3/pJF119 EH (\(\triangledown\)), GL3/pJF119_fsaA_ (**i**), and GL3/pJF119_fsaA_A129S (**a**). Shake flask cultures (37 \({}^{\circ}\)C) on MM with glucose as sole C source (100 uM IPTG, ampicillin 100 ug/mL if appropriate). Note that data for GL3 are taken from Figure 2 for comparison. Mean values from two independent biological replicates are presented. Precultures (overnight) on MM fructose + 100 uM IPTG were washed and inoculated to a starting OD\({}_{600}\) of approx. 0.05. For details of growth measurements see Section 4.  
reaction at the metabolic block at F6P (see Figure 4), further evidence for the in vivo functionality of this enzyme is necessary. Cleavage of F6P should liberate DHA and G3P in stoichiometric amounts. G3P could be directly further metabolized in the lower trunk of glycolysis to provide energy and reducing equivalents as well as PEP for glucose uptake via the PTS; furthermore, through action of transketolase on F6P and G3P the PPP could be supplied. The fate of DHA liberated intracellularly in _E. coli_ would be less clear as DHA is not considered as usual intermediate of central metabolic pathways [55]. Therefore, we looked for the appearance of DHA in culture supernatants of GL3/pJF119/saA\({}^{\text{A1295}}\) during growth on glucose (\(\approx\)30 mM initial concentration). Glucose consumption and DHA appearance was measured by HPLC, results are shown in Figure 5. As can be seen, with the onset of fast glucose consumption (after about 12 h of incubation) DHA appeared in the supernatant and reached a maximal concentration of approx. 12 (\(\pm\) 4) mM after 60 h of incubation. Thereafter, DHA successively disappeared from the medium and was no longer detected at 96 h, while glucose was already completely consumed at about 80 h.  
This observation could be explained by DHA reuptake from the medium and metabolism, putatively by the PEP-dependent DHAKLM system [55; 56; 57; 58]. We take the appearance of DHA (in millimolar concentrations) as further evidence that the activity of FSAA A1295 is indeed necessary to restore growth of the triple mutant GL3 on glucose. Our efforts to improve the formation of DHA are shown below. Figure 4 summarizes the putative bypass pathway at F6P which allows growth on glucose even if PFK is missing. Figure 4: Schematic of a glucose bypass metabolism in the strains GL3/pJF/saA\({}^{\text{A1295}}\) and GL4. DHA, dihydroxyacetone; _dhaKLM_, genes for dihydroxyacetone kinase; DHAP, dihydroxyacetone phosphate; EII\({}^{\text{Glc}}\), glucose transporter; F1,6 BP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; FRT, FLP recognition target; _faA_, fructose 6-phosphate aldolase gene; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; _pfkA_, _pfkB_, phosphofructokinase genes; _pgi_, phosphoglucose isomerase gene; P\({}_{\text{tac}}\), _tac_ promoter; _zwf_, glucose 6-phosphate dehydrogenase gene. A Single Chromosomal Copy of fsaA\({}^{\text{A1295}}\) Is Sufficient for Growth of the Triple Mutant  
As the mutant gene _fsaA\({}^{\text{A1295}}\)_ had been expressed from a multicopy plasmid vector [51], we wanted to know whether a single copy of the mutant gene in the chromosome of GL3 would suffice to allow growth on glucose. Therefore, by insertion of the _fsaA\({}^{\text{A1295}}\)_ gene under the control of P\({}_{\text{tac}}\) at the _lacZ_ locus of GL3, strain GL4 was constructed; this strain is plasmid-free and, thus, allows study in the absence of antibiotic selection. GL4 grew indeed on MM glucose though with a lower growth rate (\(\upmu\) approx. 0.05 h\({}^{-1}\)) than GL3/pJF119_fsaA\({}^{\text{A1295}}\)_ as can be seen in Figure 6. During growth, only minor DHA concentrations were detected (0.5 mM after 30 h of incubation; see Table 4).