Mechanistic understanding of the "gatekeeper" phosphofructokinase of the glycolytic pathway on the central carbon metabolism

Abstract Background: Glycolysis is one of the oldest and most fundamental metabolic pathways responsible for glucose breakdown and energy production in cells. The key regulatory enzyme in glycolysis is phosphofructokinase (Pfks), which catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate. The regulation of Pfks plays a crucial role in fine-tuning glycolytic flux to meet the cellular energy demands, making it a "gatekeeper" enzyme of glycolysis. In Saccharomyces cerevisiae, Pfks has evolved into a hetero-octameric phosphofructokinase, consisting of the Alpha and Beta subunits encoded by PFK1 and PFK2, respectively. Deletion of either PFK1 or PFK2 could significantly impair the organism's fitness, whereas the underlying metabolic mechanisms remain unclear. In this study, we investigated the specific effects of PFK1 and PFK2 on the cellular metabolism of S. cerevisiae. Results: Deletion of either PFK1 or PFK2 significantly reduced the cell growth, decreased ethanol and glycerol production and increased acetate production. Furthermore, through Flux Balance Analysis (FBA) and transcriptome analysis, we observed a significant upregulation of the Tricarboxylic Acid (TCA) cycle and the electron transfer chain in the PFK2 deletion strain. This upregulation indicated a shift towards enhanced oxidative respiration and energy production under the absence of the Beta subunit of Pfks. Furthermore, we demonstrated that the deletion of PFK2 led to a 33% improvement in the free fatty acid production, indicating its effectiveness as a key factor in regulation of lipid metabolism as well as bioproduction. Conclusions: Our physiological and transcriptomic data suggested that, as an isoenzyme, Pfk1 has a relatively limited influence on the metabolism of S. cerevisiae, whereas Pfk2 plays significant and vital roles in cellular metabolism. Deletion of PFK2 has the potential to greatly enhance respiration in yeast under aerobic conditions. These observations underscore the pivotal role of PFK2 in governing cellular energy metabolism and suggest that manipulation of PFK2 expression could hold promising implications for bioproduction processes.