Development Of D-2-hydroxyglutarate Biosensors Based On Transcriptional Regulator DhdR

D-2-Hydroxyglutarate（D-2-HG）is an iconic metabolite in patients with D-2hydroxyglutaric aciduria（D-2-HGA）or isocitrate dehydrogenases（IDH1 and IDH2）mutation-related cancers.The detection of D-2-HG is of great significance for the diagnosis and treatment of D-2-HGA and various cancers.Nowadays,liquid chromatography-mass spectrometry（LC-MS）and gas chromatography-mass spectrometry（GC-MS）are often used to quantitatively assess D-2-HG levels.However,chiral derivatization with proper reagents is necessary to distinguish D-2-HG from its mirror-image enantiomer L-2-HG,and these methods are time-consuming and laborious,restricting the development of diagnosis and treatment technologies for D-2-HG-related diseases.Allosteric transcription factors in bacteria are able to sense a variety of chemicals.They have been widely used as biorecognition elements in the development of biosensors for the detection of small molecules.In this study,the transcriptional regulator DhdR from Achromobacter denitrificans NBRC 15125 has been used as a biorecognition element and combined with different transducer systems for the development of two novel D-2-HG biosensors.Both of them are able to quantify the concentrations of D-2-HG in different.types of biological samples and investigate the D-2-HG-related metabolic pathways.Amplified luminescent proximity homogeneous assay（AlphaScreen）technology is a highly sensitive homogeneous assay technology based on microbeads,In the second chapter of this thesis,we perform the fluorescence-based thermal shift assay and isothermal titration calorimetry to identify the ligand that binds to DhdR.It is found that D-2-HG can specifically bind to DhdR.Inspired by the allosteric effect of DhdR,we develop a D-2-HG biosensor by combining DhdR with AlphaScreen technology.It can convert the concentrations of D-2-HG to measurable luminescence signals.Thirteen mutations are generated to improve the sensitivity and linear detection range of the biosensor by changing the affinity between DhdR and biotinylated DNA fragment Bio-dhdO that contains the binding site of DhdR.The optimized biosensor BD2HG-1 exhibits a limit of detection of 0.10 μM and a linear range of 0.3-20 μM.It can effectively determine the concentrations of D-2-HG in serum,urine,and cell culture medium with high specificity,sensitivity,and accuracy.The results are highly concordant with those of LC-MS/MS.In addition,we identify a novel D-2-HG anabolic enzyme WbpB in Pseudomonas aeruginosa PAO1 by using BD2HG-1 and confirm that the accumulation of D-2-HG can inhibit the lipopolysaccharide synthesis by inhibiting the activity of WbpB,revealing the role the D-2-HG metabolism in the synthesis of lipopolysaccharide in P.aeruginosa PAO1.Forster resonance energy transfer（FRET）is an energy transfer process between a pair of light-sensitive molecules.In the third chapter of this thesis,we develop a ratiometric D-2HG biosensor based on FRET technology by inserting DhdR into green fluorescent protein（Clover）and red fluorescent protein（mRuby2）.The emission ratio of the biosensor at 600 nm and 515 nm can be reduced by D-2-HG in a dose-dependent manner at 500 nm excitation.To improve the response magnitude(ΔR_max),the biosensor is optimized by truncation of Nterminal and C-terminal amino acids of DhdR and the addition of artificial linkers.The AR_max has been improved from 6.05%to 25.28%.The optimized variant DHGFR1.0 exhibits excellent sensitivity and specificity with a half-maximal effective concentration（ECso）of 2.40μM and a dynamic detection range of 0.18-30.09 μM.Additionally,we predict the ligand-binding site of DhdR based on bioinformatic analysis.The affinity of the biosensor to D-2-HG has been reduced by site-directed mutagenesis of DhdR.This study lays the foundation for the real-time detection of D-2-HG in vivo.In summary,we develop two different D-2-HG biosensors based on specific transcriptional regulator DhdR.The biosensors are able to quantify the concentrations of D2-HG in various types of biological samples including human serum,urine,cell medium,and cell lysate.Based on the developed biosensor,a novel pathway of D-2-HG anabolism in P.aeruginosa PAO1 has been identified and the role of D-2-HG metabolism in the biosynthesis of lipopolysaccharide has been revealed.The development of D-2-HG biosensors provides new experimental tools and methods for the screening inhibitors of mutated IDH,investigation of D-2-HG-related metabolic pathways,and diagnosis of D-2-HG-associated diseases.