Research Article | Open Access

Volume 2022 |Article ID 9859643 | https://doi.org/10.34133/2022/9859643

Activating Silent Glycolysis Bypasses in Escherichia coli

Camillo Iacometti,¹ Katharina Marx,¹ Maria Hönick,¹ Viktoria Biletskaia,¹ Helena Schulz-MirbachiD ,¹ Beau DronsellaiD ,¹ Ari SatanowskiiD ,¹ Valérie A. Delmas,² Anne Berger,² Ivan Dubois,² Madeleine BouzoniD ,² Volker DöringiD ,² Elad NooriD ,^3,4 Arren Bar-EveniD ,¹ Steffen N. Lindner iD ^1,5

¹Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
²Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry-Courcouronne, France
³Institute of Molecular Systems Biology, ETH Zürich, Otto-Stern-Weg 3, 8093 Zürich, Switzerland
⁴Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israe
⁵Department of Biochemistry, Charité Universitätsmedizin, Virchowweg 6, 10117 Berlin, Germany

Abstract

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal and the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the “serine shunt” which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution study using an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.