## 2 Results
### Expression of a Mutant Gene (fsaA\({}^{1295}\)) Restores Growth of GL3 on Glucose  
Recombinant FSAA is known to perform its eponymous reaction, F6P cleavage (retro-aldol reaction), in vitro [20]. We wanted to find out whether a plasmid-based expression of the _E. coli fsaA_ gene might open up a bypass reaction for glycolysis by cleavage of F6P to DHA and G3P in vivo. Therefore, the genes encoding the wild type FSAA or a mutant gene encoding the variant FSAA A129S, which has an improved catalytic efficiency towards F6P [24, 27, 35] were tested. These genes had been cloned earlier into the pJF119 EH expression plasmid which contains an ampicillin resistance marker for selection (see Table 3) and a _lacl_ gene for repression in the absence of inducer. Both recombinant _fsa_ genes are under the control of an IPTG-inducible P\({}_{\text{tac}}\) promoter [51, 52]. Both plasmids were introduced in GL3; as a control, the empty vector (pJF119 EH [51]) was used. Expression of _fsaA_ gene was examined in strain GL3 after growth on MM fructose and was verified by SDS-PAGE and enzyme analyses. As can be seen (Figure S1), cell-free extracts (cfe; soluble protein fraction) of GL3/pJF119_fsaA_ after growth on MM with 28 mM fructose and 100 u IPTG showed a prominent additional protein band at the size of approx. 24 kDa which is typical for FSAA [20]. This band was missing in cfe of wt strain LJ110. FSAA is known to be stable for a prolonged time at elevated temperatures (up to 75 \({}^{\circ}\)C) in vitro and retains most of its enzyme activity after such a temperature treatment [20]. Heat treatment at 75 \({}^{\circ}\)C for 20 min indeed led to an enrichment of the \(\approx\)24 kDa protein band in the supernatant of this strain (see Figure S1). FSAA enzyme activity (cleavage of F6P to DHA and G3P) of the heat-treated extracts of GL3/pJF119_fsaA_ was determined to be in the range of 0.7 \(\pm\) 0.0 U/mg of cell-free protein.  
\begin{table}
\begin{tabular}{c c c} \hline
\begin{tabular}{c} [PERSON] \\ (unpublished) \\ \end{tabular} \\ \hline \end{tabular}
\end{table}
Table 3: Plasmids used during this study. RBS, ribosome binding site; ts, temperature-sensitive replication. Amp\({}^{\text{R}}\), ampicillin resistance gene; Cm\({}^{\text{R}}\), chloramphenicolical resistance gene; Km\({}^{\text{R}}\), kanamycincis resistance gene; Spc\({}^{\text{R}}\), spc\({}^{\text{R}}\), spetinomycin resistance gene.  
Knowing that the plasmid-borne wild type gene _fsaA_ was sufficiently expressed and active in _E. coli_ strain GL3 (yielding soluble protein and active FSA enzyme in vitro), we expected that growth on MM glucose should be restored by expression of the recombinant gene copy. As can be seen from Figure 3, however, no growth on glucose occurred for 96 h with strains GL3 and GL3/pJF119 EH (controls), or with GL3/pJF119_fsaA_.  
From Figure 3 it also becomes evident that GL3 carrying the mutant gene _fsaA_A129S was able to grow on glucose and without a noticeable lag-phase. The doubling time was determined at about 6 h (359 \(\pm\) 52 min) corresponding to a growth rate (in the early exponential phase) of 0.12 \(\pm\) 0.01 h\({}^{-1}\).  
To the best of our knowledge, this is the first report that expression of an _fsa_ gene restores growth on glucose in a mutant background which presumably accumulates F6P by following the eponymous function of its encoded protein. FSAA A129S activity from heat-treated cfe (after growth in MM with glucose and 100 uM IPTG) was in the range of 4.9 \(\pm\) 1.7 U/mg of protein.  
The feature that plasmid-borne _fsaA_A129S confers a glucose-positive growth phenotype could also be seen on MM agar plates with glucose as sole carbon source (in the presence of IPTG). When cells of GL3 were transformed with plasmid DNA of pJF119_fsaA_A129S, about equal transformation efficiencies (colony forming units per ug of DNA) were found on LB with 100 ug/mL ampicillin agar plates compared to MM glucose + IPTG agar plates (data not shown).