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

## TL;DR

This study shows that PFK2 in yeast plays a key role in redirecting carbon metabolism toward respiration and fatty acid production, more so than PFK1.

## Contribution

The study reveals the distinct regulatory roles of PFK1 and PFK2 in yeast central carbon metabolism and identifies PFK2 as a target for metabolic engineering.

## Key findings

- Deleting PFK2 causes greater growth and ethanol production defects than deleting PFK1.
- PFK2 deletion increases carbon flux to the TCA cycle and respiration-related gene expression.
- PFK2 deletion boosts free fatty acid production by 33.3% compared to wild-type yeast.

## 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.

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.