A genetically encoded fluorescent biosensor for visualization of acetyl-CoA in live cells

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Acetyl-coenzyme A (acetyl-CoA) is a crucial metabolite in numerous cellular pathways.
• Acetyl-CoA metabolism is known to be compartmentalized within mammalian cells.
• Current methods for measuring acetyl-CoA in living cells are limited.

Purpose of the Study:

• To engineer a novel biosensor for detecting acetyl-CoA in living cells.
• To enable subcellular measurements of acetyl-CoA dynamics.
• To overcome limitations of existing acetyl-CoA detection methods.

Main Methods:

• Engineered an acetyl-CoA biosensor, "PancACe," using the bacterial protein PanZ and circularly permuted green fluorescent protein (cpGFP).
• Characterized sensor performance, including dynamic range (∼10 μM-2 mM) and selectivity (>7-fold over related molecules).
• Expressed the biosensor in E. coli and human cells (cytoplasm, nucleus, mitochondria) for dynamic and subcellular measurements.

Main Results:

• The PancACe biosensor exhibits a dynamic range of approximately 10 μM to 2 mM acetyl-CoA with a ~2-fold change.
• Demonstrated high selectivity for acetyl-CoA over other CoA esters, including coenzyme A, butyryl-CoA, malonyl-CoA, succinyl-CoA, and propionyl-CoA.
• Successfully detected rapid changes in acetyl-CoA levels in E. coli and enabled subcellular measurements in human cells, consistent with orthogonal assays.

Conclusions:

• The PancACe biosensor provides a valuable tool for measuring acetyl-CoA in living cells.
• Enables real-time, subcellular analysis of acetyl-CoA metabolism.
• Facilitates deeper understanding of compartmentalized metabolic pathways involving acetyl-CoA.