Work

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

Year: 2025

Type: article

Abstract: 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. • PFK1 and PFK2 play distinct regulatory roles in yeast central carbon metabolism, differentially affecting growth and fermentation. • PFK2 deletion redirects carbon flux toward mitochondria, enhancing oxidative respiration and energy production. • PFK2 deletion increases free fatty acid production, highlighting its potential as a metabolic engineering target for cell factories.

Source: Metabolic Engineering Communications

DOI: 10.1016/j.mec.2025.e00268