An enzymic L-2-hydroxyglutarate biosensor based on L-2-hydroxyglutarate dehydrogenase from Azoarcus olearius

L-2-Hydroxyglutarate (L-2-HG) is a critical signaling and immune metabolite but its excessive accumulation can lead to L-2-hydroxyglutaric aciduria, renal cancer, and other diseases. Development of efficient and highthroughput methods for selective L-2-HG detection is urgently required. In this study, L-2-HG dehydrogenase in Azoarcus olearius BH72 (AoL2HGDH) was screened from ten homologs and identified as an enzyme with high specificity and activity toward L-2-HG dehydrogenation. Then, an enzymatic assay-based L-2-HG-sensing fluorescent reporter, EaLHGFR which consists of AoL2HGDH and resazurin, was developed for the detection of L-2HG. The response magnitude and limit of detection of EaLHGFR were systematically optimized using a singlefactor screening strategy. The optimal biosensor EaLHGFR-2 exhibited a response magnitude of 2189.25 +/- 26.89% and a limit of detection of 0.042 mu M. It can accurately detect the concentration of L-2-HG in bacterial and cellular samples as well as human body fluids. Considering its desirable properties, EaLHGFR-2 may be a promising alternative for quantitation of L-2-HG in biological samples.