Characterization of Fluorescent Reporters for Flow Cytometry-Based Single-Cell Studies in <i>Saccharomyces cerevisiae</i>

Fluorescent proteins (FPs) are commonly used as reporters to examine intracellular genetic, molecular, and biochemical status. Flow cytometry is a powerful technique for accurate quantification of single-cell fluorescent levels. Here, we characterize green, red, and blue FPs for use in yeast <i>Saccharomyces cerevisiae</i>. Fluorophore-containing FPs and fluorogen-activating FPs (YFAST and FrFAST) were characterized dynamically in batch cultivation. FPs with a low p<i>K</i>_a, StayGold, E2-Crimson, mTagBFP2, and mScarlet-I3, showed relatively stable fluorescence in diauxic growth when they were expressed under the control of the <i>TEF1</i> promoter. A pH sensor, based on the fusion of mNeonGreen and mTagBFP2, was used to investigate the dynamic change of intracellular pH in batch cultivation. High-concentration acetate (200 mM) interfered with intracellular pH dramatically, whereas low-concentration acetate (20 mM) could not. Using StayGold and mScarlet-I3, an Epac-based cAMP sensor was constructed, showing varied Förster resonance energy transfer (FRET) patterns in different growth phases in <i>S. cerevisiae</i> CEN.PK113-7D and EBY100 backgrounds. In summary, the change in intracellular pH can significantly affect the brightness of pH-sensitive FPs. It is important to use FPs with a low p<i>K</i>_a, neutralize intracellular pH, or compensate pH impacts when FPs are used as reporters.