## 2 Results
### A Novel Pathway of Glycerol Formation in E. coli  
As described in the introduction, cells of _E. coli_ are not known to form and excrete glycerol when growing on glucose [38; 39]. As we showed that GL3/pJF119_fsaA_A1295 (Figure 5), and to a lesser extent also GL4 (Table 4), excrete DHA during growth on glucose, we intended to stop the observed catabolism of DHA in order to favor DHA and glycerol formation from sugars. We therefore deleted the chromosomal _dhaKLM_ genes in strain GL4 to yield strain GL5. The deletion in this case was performed by the novel CRISPR-Cas9 technology (see Table 1) [48].  
GL5 grew slower on glucose (\(\mu\) of 0.02 h\({}^{-1}\)) than strain GL4 (see Table 4) and only after a prolonged growth lag. GL5 formed DHA (maximal 3.1 mM) transiently but eventually consumed it again, presumably via the inherent _gldA_ (glycerol dehydrogenase) and _glpkK_ (glycerol kinase) gene activities. The deletion of _dhaKLM_ genes apparently had not yet abolished DHA consumption. Next, the _glpk_ gene was knocked out in strain GL5 to prevent any glycerol consumption (strain GL6, see Table 1). Subsequently, _gldA_ was cloned and integrated in the chromosomal _rbSK_ locus (under the control of P\({}_{\text{tac}}\)) to come up with strain GL7 (see Table 1 for construction details).  
Then we studied the growth behavior of strains GL6 and GL7 and their product spectra. When incubated on MM containing glucose and IPTG, GL6 required approx. 288 h to reach maximal optical density, while GL7 took about 240 h (see Table 4 and Figure S2). Heat-treated cfe of GL6 and GL7 contained FSA activities each of about 0.1 U/mg. Glycerol dehydrogenase (GLDA) activity in cfe of GL7 was approx. 0.4 (\(\pm\) 0.1 U/mg), whereas GLDA activity in GL6 was below detection level. In the supernatant of GL6, approx. 8.8 mM DHA were found, in GL7 approx. 3.5 mM (see Table 4). As a novel product, glycerol, was detected in GL6 with a concentration of 1.9 \(\pm\) 0.2 mM, in GL7 this value increased about 11-fold to 21.8 (see Table 4). Both products (DHA and glycerol) remained in the supernatant until the end of the incubation. In the case of GL7, the sum of both C3 products is about 25 mM and thus about 84% of the theoretical maximal yield (based on C molarity of glucose, a C6 compound, which was consumed).  
We were then interested to see whether strain GL7 could also utilize other sugars for glycerol production, especially those which are constituents of hemicellulose. Therefore, D-xylose, L-arabinose, and D-galactose were used as single C-sources (see Table 4). Initial pentose (C5 compounds) concentrations were set at approx. 33 mM to be comparable to the initial 28 mM of hexoses (165 mM C atom compared to 168 mM C atom). With D-galactose, GL7 needed only half the time to reach the maximal OD\({}_{600\,\text{nm}}\) than on glucose (120 h against 240 h). The pentoses D-xylose and L-arabinose (constituents of hemicellulose in plants) were also used (see Figure 7 and Table 4) for growth and glycerol was yielded as product.  
After growth on D-galactose, approximately 18 mM glycerol was found which is about 4 mM less glycerol in comparison to growth on D-glucose. Glycerol concentrations were 13.3 mM for D-xylose and 13.9 mM for L-arabinose. In terms of atom molarity (expressed in the amounts of glycerol produced) this gives the order:  
D-glucose \(>\) D-galactose \(>\) L-arabinose\(\sim\)D-xylose.  
[Calculation 21.8 \(\times\) 3 = 65.4 mM C from 90.6 mM C for D-glucose (\(\approx\)72% theoretical maximal value), 17.9 \(\times\) 3 = 53.7 mM C for 84.6 mM C of D-galactose (\(\approx\)63%); for D-xylose: 39.9 mM C from 98.7 mM C (\(\approx\)40%), and 41.7 from 105 mM C (\(\approx\)40%) for L-arabinose].  
Please note that the molar yield on glycerol/DHA is less than theoretically possible. For possible reasons see Section 3.