Supplementary information to: Simpler is not always better: transplanting the Yarrowia lipolytica glycolytic pathway into Saccharomyces cerevisiae reveals essential synergetic regulatory mechanisms (dataset)

Supplementary information to: Simpler is not always better: transplanting the Yarrowia lipolytica glycolytic pathway into Saccharomyces cerevisiae reveals essential synergetic regulatory mechanisms

doi:10.4121/19228347.v1

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doi: 10.4121/19228347

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Ewout Knibbe; Francine J. Boonekamp; Rachel Stuij; Koen A.J. Pelsma; Liset Jansen et. al. (2022): Supplementary information to: Simpler is not always better: transplanting the Yarrowia lipolytica glycolytic pathway into Saccharomyces cerevisiae reveals essential synergetic regulatory mechanisms. Version 1. 4TU.ResearchData. dataset. https://doi.org/10.4121/19228347.v1

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Abstract

The Embden-Meyerof-Parnas pathway of glycolysis is a widely distributed and intensively investigated metabolic route. While allosteric regulation is thought to be essential for glycolytic flux dynamics in many organisms including yeast, to date single enzyme complementation studies with non-allosteric glycolytic enzymes have failed to experimentally demonstrate this essentiality and quantify the overall contribution of allosteric regulation in tuning the glycolytic flux. This study brings new insight in the synergetic metabolic role of allosteric regulation by implementing pathway swapping, a strategy enabling to remodel, in two simple genetic interventions, the entire glycolytic pathway of Saccharomyces cerevisiae. S. cerevisiae equipped with the full set of non-allosteric glycolytic enzymes from the oleaginous yeast Y. lipolytica lost the ability to grow on media containing 2% glucose and displayed dynamic responses suggesting metabolic imbalance between upper and lower glycolysis. Single and combined gene complementation demonstrated that this phenotype was caused by the simultaneous deregulation of the three key kinases: hexokinase, phosphofructokinase and pyruvate kinase. ‘Deregulated glycolysis’ S. cerevisiae strains could naturally restore glycolytic stability and growth on glucose by evolving mutations in the Y. lipolytica glucokinase, causing a strong decrease in glucokinase activity and glycolytic flux. This solution could be recapitulated in non-evolved deregulated glycolysis S. cerevisiae strains by experimentally tuning glucose import. Supported by kinetic modelling, the present work demonstrates the major synergetic role played by allosteric regulations in preventing metabolic imbalance in glycolysis and highlights the power of synthetic biology in addressing long-standing questions in systems biology.

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