Activating silent glycolysis bypasses inEscherichia coli

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, 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 immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. 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. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

A Genetically Encoded System to Quantify and Evolve RuBisCO-Catalyzed Carbon Fixation

“…1g), which regulates allostery and cooperativity during catalysis 29 . Since both strains can robustly grow in medium containing glucose but did not acquire mutations predicted to limit glucose uptake (Extended Data Table 4), we speculate that the methylglyoxal pathway now supports an effective glycolytic bypass, a hypothesis corroborated by a recent finding that MgsA overexpression permitted growth on glycerol in an EMP-deficient E. coli 30 . Finally, we completed our strain development efforts by engineering an F' plasmid, pOX38 int (based on pOX38::Tc) 31 , that expresses key proteins that would streamline our reporter assays: the transcriptional repressors LacI and TetR for tunable promoter control, LuxCDE to generate the LuxAB substrate decanal in situ, and the membrane integrity-responsive LacZ cassette 32 (Fig.…”

Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools

“…Works performed in the model organism E. coli demonstrated its ability to harness the underground metabolism to discover new metabolic functions ( Guzmán et al, 2015 ). Using this organism, specific examples are in recent works where (i) new metabolite links (“serine shunt”) were identified, allowing to rewire central metabolism, particularly the Embden–Meyerhof–Parnas glycolysis ( Iacometti et al, 2021 ) and (ii) elucidated the appearance of new pathways for isoleucine biosynthesis ( Cotton et al, 2020 ). E. coli was also demonstrated that GlcN-6P intracellular concentration increases two orders of magnitude (up to 9 mM) when the bacterium grows in GlcN as a sole carbon source ( Álvarez-Añorve et al, 2009 ), where specifically a GlcN-6P utilization enzyme [NagB (EC 3.5.99.6)] is activated to channel the hexosamine-P to the central glycolytic pathway ( Álvarez-Añorve et al, 2016 ).…”