Evidence that Fermentable Sugar Intolerance in Yeast Lacking Trehalose Biosynthesis is Caused by Shifts in the Free Energy of Glycolytic Enzymes

Chen, Jonathan C. [Browse]
Senior thesis
91 pages


Rabinowitz, Joshua D. [Browse]
Princeton University. Department of Chemistry [Browse]
Class year
Restrictions note
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Summary note
The deletion of TPS1, which synthesizes trehalose-6-phosphate in the trehalose biosynthesis pathway, drastically alters cellular metabolism and glycolytic flux in the model system Saccharomyces cerevisiae. tps1Δ results in the loss of robustness in glycolysis at the respiro-fermentative transition. I present evidence that this is caused in part by a thermodynamic bottleneck in lower glycolysis that prevents entering the energy payoff phase. This rapidly depletes adenylate energy charge, through a positive feedback loop and futile cycle. As ATP is invested in upper glycolysis, lack of ATP production in lower glycolysis only serves to further activate phosphofructokinase, Pfk1. The subsequent buildup of upper glycolytic intermediates shifts the free energy of glycolytic enzymes like 6-phosphofructo-2-kinase, Pfk26. This moves the equilibrium away from the product fructose-2,6-bisphosphate (F26BP) towards the reactant fructose-6-phosphate(F6P). As F26BP is an inhibitor of fructose-1,6-bisphosphatase (FBPase), this lifts FBPase inhibition, resulting in a futile cycle between Pfk1 and FBPase. Introduction of Li\(^{+}\), a potent FBPase inhibitor, to tps1Δ during growth on galactose confers the ability to survive the respiro-fermentative transition. Metabolomics indicates that this is a function of decreased FBPase consumption of ATP. However, the preservation of ATP concentration comes at the cost of ADP levels, resulting in significantly decreased glycolytic flux. Deletion of AMP deaminase (AMD1) attempts to rectify this by elevating intracellular ADP concentrations. Although ATP and ADP concentrations in amd1Δtps1Δ following glucose upshift are more stable, the resulting increase in AMP concentrations may directly interfere with proper glycolytic regulation, resulting in less efficient glycolytic flux and energy production.

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