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

Abstract

Metabolic heterogeneity between individual cells of a population harbors offers significant challenges for fundamental and applied research. Identifying metabolic heterogeneity and investigating its emergence requires 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, by drawing on the robust cell-intrinsic correlation between glycolytic flux and levels of fructose-1,6-bisphosphate (FBP), and by transplanting the B. subtilis FBP-binding transcription factor CggR into yeast. As proof of principle, using fluorescence microscopy, we applied the sensor to identify metabolic subpopulations in yeast cultures. We anticipate that our biosensor will become a valuable tool to identify and study metabolic heterogeneity in cell populations.