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

Phosphofructokinase (Pfk), a key regulatory enzyme in glycolysis, is composed of Pfk1 and Pfk2 subunits in <i>Saccharomyces cerevisiae</i>. However, the distinct roles of these subunits in central carbon metabolism remain unclear. Here, we examined the metabolic consequences of deleting <i>PFK1</i> or <i>PFK2</i>. The <i>pfk2Δ</i> strain exhibited more severe defects than <i>pfk1Δ</i>. Its maximum specific growth rate was reduced by approximately 54 % in <i>pfk2Δ</i> and by about 15 % in <i>pfk1Δ</i>, both relative to the reference strain. Ethanol production decreased by 36 % and 82 % in <i>pfk1Δ</i> strain and <i>pfk2Δ</i> 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 <i>pfk1Δ</i> strain and 82 % in the <i>pfk2Δ</i> strain. Flux balance analysis (FBA) revealed a markedly increased carbon flux to the tricarboxylic acid cycle (TCA) in the <i>pfk2Δ</i> strain, with respiration-associated carbon flux elevated 1.5-fold compared to the <i>pfk1Δ</i> strain. Consistently, transcriptomic profiling showed significant upregulation of respiration-related genes in the <i>pfk2Δ</i> strain compared to the reference strain. Notably, deletion of <i>PFK2</i> 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 <i>PFK2</i> 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.