Activating Silent Glycolysis Bypasses in .

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 , we complementarily employed 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 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.