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.

中文翻译：

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使用正交合成生物传感器测量单个酵母细胞中的糖酵解通量。

群体个体细胞之间的代谢异质性给基础和应用研究带来了重大挑战。识别代谢异质性并研究其出现需要工具来放大单个细胞的代谢。虽然存在测量单细胞代谢物水平的方法，但我们缺乏在单细胞水平上测量代谢通量（即代谢活动的最终功能输出）的能力。在这里，我们结合启动子工程、计算蛋白质设计、生化方法、蛋白质组学和代谢组学，开发了一种生物传感器来测量单个酵母细胞中的糖酵解通量。因此，利用糖酵解通量与 1,6-二磷酸果糖 (FBP) 水平之间强大的细胞内在相关性，我们将枯草芽孢杆菌 FBP 结合转录因子 CggR 移植到酵母中。利用开发的生物传感器，当进行流式细胞术和显微镜检查时，我们可以可靠地识别混合培养物中具有不同 FBP 水平的细胞亚群。利用微流体，我们还能够评估细胞周期期间的时间 FBP/糖酵解通量动态。我们预计我们的生物传感器将成为识别和研究细胞群代谢异质性的宝贵工具。