Protein allostery, present in all three domains of life, is key to the regulation of metabolism by allowing fast and precise control of catalysis in response to cellular demands. While metabolic pathways are frequently equipped with multiple allosterically regulated catalytic steps, experimental studies often focus on a single step, failing to capture how regulations exerted at multiple steps interact with each other for tuning pathways. Using the nearly ubiquitous Embden-Meyerhof-Parnas pathway of glycolysis as a paradigm, the present study unveils a remarkable regulatory synergy between multiple allosteric proteins of a metabolic pathway and demonstrates its impact on cell survival in dynamic environments. By using complete pathway complementation, as well as single-gene complementation, the essential regulatory steps were identified to be glucokinase, phosphofructokinase, and pyruvate kinase. Expression of these enzymes together, even in the context of the Saccharomyces cerevisiae pathway, led to imbalances in glycolysis that could only be overcome by lowering the glucokinase activity. Integrating these results with kinetic modeling and microfluidics experiments, the present work reveals the key synergistic role played by allosteric regulations in preventing glycolytic imbalance in the model eukaryote Saccharomyces cerevisiae and highlights the power of synthetic biology in addressing long-standing questions in systems biology.IMPORTANCEAll forms of life are equipped with intricate molecular mechanisms that tune their cellular responses to external and internal cues. These mechanisms are key to cells' survival in natural environments and important for the performance of bioprocesses, which are characterized by variable environments (e.g., nutrient availability). One of these molecular mechanisms, protein allostery, enables rapid fine-tuning of the rate of cellular processes by modulating protein activity in response to metabolites in vivo. Using the industrial yeast and model eukaryote Saccharomyces cerevisiae as a paradigm, the present work reveals that, in the major route for sugar utilization known as glycolysis, three distinct allosteric regulations are critical to yeast cell survival when transitioning between carbon sources. These three regulations, while not required for pathway operation per se, allow efficient and balanced pathway operation under dynamic conditions. These findings, therefore, reveal a new aspect of yeast glycolysis, one of the best-studied metabolic pathways.