## 3 Discussion
GL4 grew slower on glucose (\(\upmu\) 0.05 h\({}^{-1}\)) apparently due to the lower gene dosage. Transiently, DHA appeared in cultures on glucose but was consumed again. GL4 reachednearly the same cell yield as LJ110 (Table 4). By deletion of the _dhaKLM_ genes, GL5 was constructed. This strain produced up to 3 mM of DHA but this still disappeared during further incubation. The fate of DHA here is less clear. It might be that DHA is first reduced to glycerol by GLDA and then phosphorylated by GLPK to glycerol 3-phosphate or it could be phosphorylated directly by GLPK which has a high activity but low affinity (\(K_{\text{M}}\) value of \(\approx\)0.5 mM) towards DHA [80]. Indeed, in strain GL6 which has the _glpK_ gene deleted, DHA accumulated up to 8.8 mM. As a new product, glycerol appeared as minor by-product (up to 1.9 mM). Most likely DHA was in part converted to glycerol by GLDA which is known to act preferentially on DHA [81]. As GLPK is missing now, glycerol is a dead end product. To our best knowledge, formation of glycerol by _E. coli_ cells from glucose via DHA is a novel feature (\"new-to-nature pathway\"). This pathway was reached by expression or alteration of genes which are already present in _E. coli_. To be efficient, a single amino acid substitution (A129S in FSAA) and a change of promoters (P\({}_{\text{tc}}\) instead of the native promoter of _faA_) were necessary. A comparison of the novel glycerol pathway with the \"yeast glycerol pathway\" made possible by introduction of DARI/GPD1 and GPP genes from yeast in _E. coli_ shows that the novel pathway (from F6P to glycerol) consumes 1 mol ATP less than the yeast pathway from F6P via F1,6 BP to DHAP, glycerol 3-P and finally glycerol [41; 42; 43]. The novel glycerol pathway, however, requires NADH as reducing agent. In its present state, it has an upper theoretical limit of 1 mole glycerol/mole of glucose; the remaining C atoms of F6P flow into G3P and the lower glycolytic pathway or into the PPP. As already stated above, PEP is needed for glucose uptake via the PTS. In our best glycerol producer (strain GL7), glycerol and DHA are end products and together almost make up half (\(\approx\)42%) of the carbon which is generated from the fed glucose. The residual carbon can be used for generation of only approximately 1.2 moles of G3P (from 1 mole of glucose) and subsequently only 1.2 moles of PEP should be gained from glucose. On the other hand, 1 mole PEP is necessary as phosphoryl group donor for glucose uptake via the PTS. As discussed above, several enzymes involved in catabolic, anaplerotic, or anabolic reactions would compete for the PEP provided by the EMP. Therefore, it does not come as a surprise that generation times on glucose are much higher in GL7 (\(\approx\)25 h) and the growth yield from this C source is only about 30% of that of wild type (Table 4). The many challenges and imbalances (discussed above) which lead to the observed slow growth in strains GL5 and GL7, however, can on the other hand be seen as starting point of adaptive evolution. Through selection and analysis of faster growing descendants we want to find out which metabolic solutions will be found by these evolved strains ([PERSON], [PERSON], [PERSON], manuscript in preparation).