A Genetically\nEncoded Fluorescent Biosensor for Intracellular\nMeasurement of Malonyl-CoA

Malonyl-CoA is the essential building block of fatty\nacids and\nregulates cell function through protein malonylation and allosteric\nregulation of signaling networks. Accordingly, the production and\nuse of malonyl-CoA is finely tuned by the cellular energy status.\nMost studies of malonyl-CoA dynamics rely on bulk approaches that\ntake only a snapshot of the average metabolic state of a population\nof cells, missing out on heterogeneous differences in malonyl-CoA\nand fatty acid biosynthesis that could be occurring among a cell population.\nTo overcome this limitation, we have developed a genetically encoded\nfluorescent protein-based biosensor for malonyl-CoA that can be used\nto capture malonyl-CoA dynamics in single cells. This biosensor, termed\nMalibu (<b><u>mal</u></b>onyl-CoA <b><u>i</u></b>ntracellular <b><u>b</u></b>iosensor to <b><u>u</u></b>nderstand dynamics),\nexhibits an excitation-ratiometric change in response to malonyl-CoA\nbinding. We first used Malibu to monitor malonyl-CoA dynamics during\ninhibition of fatty acid biosynthesis using cerulenin in Escherichia coli, observing an increase in Malibu\nresponse in a time- and dose-dependent manner. In HeLa cells, we used\nMalibu to monitor the impact of fatty acid biosynthesis inhibition\non malonyl-CoA dynamics in single cells, finding that two inhibitors\nof fatty acid biosynthesis, cerulenin and orlistat, which inhibit\ndifferent steps of fatty acid biosynthesis, increase malonyl-CoA levels.\nAltogether, we have developed a new genetically encoded biosensor\nfor malonyl-CoA, which can be used to study malonyl-CoA dynamics in\nsingle cells, providing an unparalleled view into fatty acid biosynthesis.