Measuring glycolytic flux in single yeast cells with an orthogonal synthetic biosensor

Abstract

Metabolic heterogeneity between individual cells of a population harbors significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence require tools to zoom into metabolism of individual cells. While methods exist to measure metabolite levels in single cells, we lack capability to measure metabolic flux, i.e., the ultimate functional output of metabolic activity, on the single‐cell level. Here, combining promoter engineering, computational protein design, biochemical methods, proteomics, and metabolomics, we developed a biosensor to measure glycolytic flux in single yeast cells. Therefore, drawing on the robust cell‐intrinsic correlation between glycolytic flux and levels of fructose‐1,6‐bisphosphate (FBP), we transplanted the B. subtilis FBP‐binding transcription factor CggR into yeast. With the developed biosensor, we robustly identified cell subpopulations with different FBP levels in mixed cultures, when subjected to flow cytometry and microscopy. Employing microfluidics, we were also able to assess the temporal FBP/glycolytic flux dynamics during the cell cycle. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.

Another Consequence of the Warburg Effect? Metabolic Regulation of Na+/H+ Exchangers May Link Aerobic Glycolysis to Cell Growth

“…It has been hypothesized that sensing of metabolic flux may depend on the accumulation of metabolites, which tightly correlate with pathway activity and trigger the appropriate cellular response ( 106 ). For example, the abundance of fructose-1,6-bisphosphate (FBP) tightly correlates with glycolytic flux in yeast and bacteria, and binding of FBP to a transcription factor allows for coupling of glycolytic flux to gene expression ( 107 , 108 ). Similarly, in pancreatic beta-cells, ATP correlates with glucose concentration and glycolytic activity and triggers insulin secretion by binding to ATP-sensitive K + -channels ( 109 ).…”

Metabolic engineering for plant natural products biosynthesis: new procedures, concrete achievements and remaining limits

“…3). 40,41 Having said this, for microbial production of PNPs to become a relevant supply chain of human therapeutics, efforts (i) to maximise activity of hard-to-express plant enzymes, (ii) relieve effects of toxic PNP intermediates, (iii) limit shunt product formation, (iv) stabilize titers/rates/yields of microbial PNP cell factories over prolonged cultivations, and (v) efficient down-stream processing, are all aspects needing further attention in the near future.…”

Single-cell image analysis to explore cell-to-cell heterogeneity in isogenic populations

“…A step beyond quantifying metabolite concentration is measuring flux, the relevant functional output of metabolic activity. Since fructose-1,6-bisphosphate (FBP) levels strictly correlate with glycolytic flux, a modified Bacillus subtilis CggR transcription factor that binds FBP, together with a synthetic promoter, were used to report on flux in budding yeast (Monteiro et al, 2019). Single-cell image analysis revealed that FBP concentration, and thus glycolytic flux, changes during the cell cycle, peaking around cytokinesis and in G1 phase.…”