Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, UT 84112, USA.
Int J Mol Sci. 2024 Oct 16;25(20):11098. doi: 10.3390/ijms252011098.
Malate is a key intermediate in the citric acid cycle, an enzymatic cascade that is central to cellular energy metabolism and that has been applied to make biofuel cells. To enable real-time sensing of malate levels, we have engineered a genetically encoded, protein-based fluorescent biosensor called Malon specifically responsive to malate by performing structure-based mutagenesis of the Cache-binding domain of the Citron GFP-based biosensor. Malon demonstrates high specificity and fluorescence activation in response to malate, and has been applied to monitor enzymatic reactions in vitro. Furthermore, we successfully incorporated Malon into redox polymer hydrogels and bacterial cells, enabling analysis of malate levels in these materials and living systems. These results show the potential for fluorescent biosensors in enzymatic cascade monitoring within biomaterials and present Malon as a novel tool for bioelectronic devices.
苹果酸是柠檬酸循环（柠檬酸循环是细胞能量代谢的核心酶级联反应，已被应用于生物燃料细胞的制造）中的关键中间产物。为了实现对苹果酸水平的实时感测，我们通过对基于Citron GFP 的生物传感器的 Cache 结合结构域进行基于结构的突变，设计了一种名为 Malon 的基因编码、基于蛋白质的荧光生物传感器，使其对苹果酸具有特异性响应。Malon 对苹果酸表现出高特异性和荧光激活，并且已被应用于监测体外酶反应。此外，我们成功地将 Malon 整合到氧化还原聚合物水凝胶和细菌细胞中，从而能够分析这些材料和活系统中的苹果酸水平。这些结果表明荧光生物传感器在生物材料中的酶级联监测中的应用潜力，并将 Malon 作为生物电子设备的新型工具。
