A new synthetic pathway transforms C5 & C6 sugars into glycolic acid without carbon loss.
Résumé
Microbial glycolic acid (GA) production from lignocellulosic sugars can be achieved by engineering the natural glyoxylate shunt, by synthetic pathways and combinations of both pathways. Recently, we developed a cyclic non-natural pathway (Glycoptimus) to transform C5 & C6 sugars into GA with a challenge of no loss of carbon in Escherichia coli. This pathway proceeds via isomerization of D-ribulose 5-P to D-arabinose 5-P (KdsD), aldolytic cleavage of the latter to glyceraldehyde-3-P and glycolaldehyde (FsaA) and oxidation of glycolaldehyde into GA (AldA). Various operon construct bearing kdsD, fsaA and aldA gene were inserted into E. coli cells that were otherwise deleted for key genes encoding competing pathways (ie lower part of glycolysis, MGA pathways) in order to recycle GAP into the non-oxidative pathway to refill into Ara5P. Performance for GA production of the various constructed strains were evaluated. The strategy was to decouple growth and production phase, which was obtained by firstly growth on succinate/ gluconate, collect cells and resuspend at high cell concentration in fresh M9 medium containing 20 g.L-1 D-xylose, without nitrogen to promote the GA accumulation. We successfully obtained a mutant strain able to produce YGA close to the theoretical yield 1.26 g GA per g Xylose. However, this mutant produced very low amount of GA due to very weak consumption of xylose, raising further optimization of the pathway by both combination of genetic modifications and process fermentation