Mechanistic and applied study of phosphofructokinases, the “gatekeeper” of the glycolytic pathway on the central carbon metabolism

Journal article, 2026

Phosphofructokinase (Pfk), a key regulatory enzyme in glycolysis, is composed of Pfk1 and Pfk2 subunits in Saccharomyces cerevisiae . However, the distinct roles of these subunits in central carbon metabolism remain unclear. Here, we examined the metabolic consequences of deleting PFK1 or PFK2 . The pfk2Δ strain exhibited more severe defects than pfk1Δ . Its maximum specific growth rate was reduced by approximately 54 % in pfk2Δ and by about 15 % in pfk1Δ , both relative to the reference strain. Ethanol production decreased by 36 % and 82 % in pfk1Δ strain and pfk2Δ strain, respectively, relative to the reference strain. Both deletion strains accumulated higher acetate levels compared to the reference strain, increasing by 25.4 % in the pfk1Δ strain and 82 % in the pfk2Δ strain. Flux balance analysis (FBA) revealed a markedly increased carbon flux to the tricarboxylic acid cycle (TCA) in the pfk2Δ strain, with respiration-associated carbon flux elevated 1.5-fold compared to the pfk1Δ strain. Consistently, transcriptomic profiling showed significant upregulation of respiration-related genes in the pfk2Δ strain compared to the reference strain. Notably, deletion of PFK2 enhanced acetyl-CoA-derived product formation, with free fatty acid (FFA) titers increasing from 412 mg L⁻¹ to 517 mg L⁻¹ (a 33.3 % increase). These findings establish PFK2 as a key regulatory node redirecting carbon flux from fermentation toward respiration and biosynthesis, offering new opportunities for metabolic engineering of acetyl-CoA-derived products.