BioProject

A synthetic pathway for (D)-xylose assimilation was stoichiometrically evaluated regarding its potential to produce selected value-added compounds and implemented in Escherichia coli strains. More...

A synthetic pathway for (D)-xylose assimilation was stoichiometrically evaluated regarding its potential to produce selected value-added compounds and implemented in Escherichia coli strains. The pathway proceeds via the isomerization of (D)-xylose to (D)-xylulose, the phosphorylation of (D)-xylulose to obtain (D)-xylulose-1-phosphate (Xyl1P), and the aldolase cleavage of the latter to yield glycolaldehyde and DHAP. Both compounds can be further processed via the annotated natural metabolic network. Stoichiometric analyses showed that the synthetic pathway provides an efficient access to the value-added two-carbon (C2) compounds ethylene glycol and glycolic acid, which are either inaccessible via the annotated metabolic network of E. coli or produced at 20 % lower theoretical yield, respectively. The simultaneous expression of xylulose-1 kinase and Xyl1P aldolase activities, which were provided by human ketohexokinase-C and human aldolase-B, respectively, was necessary and sufficient to restore growth of a (D)-xylulose-5-kinase (ΔxylB) mutant on xylose. In this strain, ethylene glycol was the major metabolic end-product and produced at a molar yield of 0.47. Further metabolic engineering provided strains that assimilated the entire C2 fraction back into the central metabolism, or that produced 4.3 g/l glycolic acid at a molar yield of 0.9 in shake flasks. Overall design: The wild-type (WT) culture and synthetic strain (PEN205) were used for the microarray analysis. PEN205 is a ΔxylB deleted strain that express the synthetic pathway. WT and PEN205 were grown to the logarithmic phase of growth in M9 minimal medium supplemented with xylose. PEN205 culture RNA was extracted when their OD was around 1. WT culture were split into two equal aliquots at the OD of ~1 and further cultivated in the presence or absence of 10 mM glycolaldehyde. After 30 min of incubation, cells were used for RNA extraction as well. Less...