Acetate, a major by‐product of glycolytic metabolism in Escherichia coli and many other microorganisms, has long been considered a toxic waste compound that inhibits microbial growth. This counterproductive auto‐inhibition represents a major problem in biotechnology and has puzzled the scientific community for decades. Recent studies have however revealed that acetate is also a co‐substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism in E. coli. Computational and experimental analyses demonstrate that decreasing the glycolytic flux enhances co‐utilization of acetate with glucose. Acetate metabolism thus compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, rather than being toxic, actually enhances E. coli growth under these conditions. We validated this mechanism using three orthogonal strategies: chemical inhibition of glucose uptake, glycolytic mutant strains, and alternative substrates with a natively low glycolytic flux. In summary, acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth.

As the use of antioxidant compounds in the domains of health, nutrition and well-being is exponentially rising, there is an urgent need to quantify antioxidant power quickly and easily, ideally within living cells. We developed an Anti Oxidant Power in Yeast (AOPY) assay which allows for the quantitative measurement of the Reactive Oxygen Species (ROS) and free-radical scavenging effects of various molecules in a high-throughput compatible format. Key parameters for Saccharomyces cerevisiae were investigated, and the optimal values were determined for each of them. The cell density in the reaction mixture was fixed at 0.6; the concentration of the fluorescent biosensor (TO) was found to be optimal at 64 µM, and the strongest response was observed for exponentially growing cells. Our optimized procedure allows accurate quantification of the antioxidant effect in yeast of well-known antioxidant molecules: resveratrol, epigallocatechin gallate, quercetin and astaxanthin added in the culture medium. Moreover, using a genetically engineered carotenoid-producing yeast strain, we realized the proof of concept of the usefulness of this new assay to measure the amount of β-carotene directly inside living cells, without the need for cell lysis and purification.

Acetate is a major by-product of glycolytic metabolism in Escherichia coli and many other microorganisms. It has long been considered a toxic waste compound that inhibits microbial growth, but this counterproductive auto-inhibition, which represents a major problem in biotechnology, has puzzled the scientific community for decades. Recent studies have revealed that acetate is also a co-substrate of glycolytic nutrients and a global regulator of E. coli metabolism and physiology. However, most of these insights were obtained at high glycolytic flux and little is known about the role of acetate at lower glycolytic fluxes, conditions that are nevertheless frequently experienced by E. coli in natural, industrial and laboratory environments. Here, we used a systems biology strategy to investigate the mutual regulation of glycolytic and acetate metabolism. Computational and experimental results demonstrate that reducing the glycolytic flux enhances co-utilization of acetate and glucose through the Pta-AckA pathway. Enhanced acetate metabolism compensates for the reduction in glycolytic flux and eventually buffers carbon uptake so that acetate, far from being toxic, actually enhances E. coli growth under these conditions. The same mechanism of increased growth was also observed on glycerol and galactose, two nutrients with a natively low glycolytic flux. Therefore, acetate makes E. coli more robust to glycolytic perturbations and is a valuable nutrient, with a beneficial effect on microbial growth. Finally, we show that some evolutionarily conserved design principles of eukaryotic fermentative metabolism are also present in bacteria.