bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A D-2-hydroxyglutarate biosensor based on specific 2 transcriptional regulator DhdR 3 4 Dan Xiao1,5, Wen Zhang2,5, Xiaoting Guo3, Yidong Liu1, Chunxia Hu1, Shiting Guo1, Zhaoqi 5 Kang1, Xianzhi Xu1, Cuiqing Ma1, Chao Gao1,*, Ping Xu4,* 6 7 1State Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s 8 Republic of China 9 2Institute of Medical Sciences, The Second Hospital, Cheeloo College of Medicine, Shandong 10 University, Jinan, People’s Republic of China 11 3Eye Hospital of Shandong First Medical University, Jinan, People’s Republic of China 12 4State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of 13 Metabolic & Developmental Sciences, and School of Life Sciences & Biotechnology, 14 Shanghai Jiao Tong University, Shanghai, People’s Republic of China 15 5These authors contributed equally: Dan Xiao, Wen Zhang. 16 17 *Corresponding authors: 18 Mailing address for C. Gao: State Key Laboratory of Microbial Technology, Shandong 19 University, Qingdao 266237, People’s Republic of China, Tel/Fax: +86-532-58631561, 20 E-mail: [email protected]. 21 Mailing address for P. Xu: State Key Laboratory of Microbial Metabolism, and School of 22 Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s 23 Republic of China, Tel/Fax: +86-21-34206723, E-mail: [email protected]. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 D-2-Hydroxyglutarate (D-2-HG) is a metabolite in many physiological metabolic processes. 26 When D-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenases or 27 D-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a 28 therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter 29 denitrificans NBRC 15125 was identified as an allosteric transcription factor that negatively 30 regulates D-2-HG dehydrogenase expression and responds to presence of D-2-HG. It is the 31 first known transcription regulator specifically responding to D-2-HG across all domains of 32 life. Based on the allosteric effect of DhdR, a D-2-HG biosensor was developed by combining 33 DhdR with amplified luminescent proximity homogeneous assay technology. The biosensor 34 was able to detect D-2-HG in serum, urine, and cell culture with high specificity and 35 sensitivity. Additionally, this biosensor was also successfully used to identify the role of 36 D-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, 37 demonstrating its broad usages. 38 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 39 Introduction 40 D-2-Hydroxyglutarate (D-2-HG) has traditionally been considered an abnormal metabolite 41 associated with the neurometabolic disorder D-2-hydroxyglutaric aciduria1. However, D-2-HG 42 accumulation, due to mutations of isocitrate dehydrogenase (IDH), has been observed in 43 many tumor cells, and thus D-2-HG is also considered an oncometabolite2-6. Recent work has 44 revealed that D-2-HG may also be an important metabolic intermediate involved in different 45 core metabolic processes, including L-serine synthesis7, lysine degradation8, and 46 4-hydroxybutyrate catabolism9. D-2-HG dehydrogenase (D2HGDH) catalyzes the conversion 47 of D-2-HG to 2-ketoglutarate (2-KG) and is the key enzyme involved in D-2-HG catabolism7. 48 D-2-HG is harmful to cells and is present at rather low levels under physiological conditions, 49 suggesting that organisms may have evolved specific mechanisms to recognize D-2-HG 50 accumulation and enhance D2HGDH expression to catabolize D-2-HG10-13. However, the 51 regulation of D2HGDH expression is not fully understood and how organisms respond to the 52 presence of D-2-HG has not yet been elucidated. 53 D-2-HG levels are increased in patients with D-2-hydroxyglutaric aciduria (D-2-HGA) 54 and IDH mutation-related cancers1,2. As such, the detection of D-2-HG is relevant for the 55 diagnosis and monitoring of these diseases14-16. Gas chromatography-tandem mass 56 spectrometry (GC-MS/MS)17,18 and liquid chromatography-tandem mass spectrometry 57 (LC-MS/MS)19,20 are often used to quantitatively assess D-2-HG levels. However, chiral 58 derivatization with proper reagents is necessary to distinguish D-2-HG from its mirror-image 59 enantiomer L-2-HG, and these methods are time-consuming and laborious. Allosteric 60 transcription factors (aTFs) in bacteria have evolved to sense a variety of chemicals21. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 Various aTFs, such as HucR, HosA, TetR, and SRTF1, have been well characterized, and then 62 coupled with different transduction systems to develop convenient biosensors to assay for 63 uric acid, 4-hydroxybenzoic acid, tetracycline, and progesterone22-25. Until now, no aTF that 64 specifically responds to D-2-HG has been identified, restricting the development of D-2-HG 65 biosensors. 66 In this study, the D-2-HG catabolism regulator DhdR was identified in Achromobacter 67 denitrificans NBRC 15125. This aTF can depress the expression of the D2HGDH-encoding 68 gene d2hgdh and specifically responds to D-2-HG. Then, DhdR was combined with amplified 69 luminescent proximity homogeneous assay (AlphaScreen) technology, a bead-based 70 immunoassay, to develop a D-2-HG biosensor with high specificity and sensitivity. Various 71 biological samples were selected to demonstrate the utility of the biosensor. In addition, the 72 biosensor was used to identify the UDP-2-acetamido-2-deoxy-D-glucuronic acid 73 (UDP-GlcNAcA) 3-dehydrogenase WbpB of Pseudomonas aeruginosa PAO1 as a D-2-HG 74 anabolic protein that also participates in the intracellular generation of D-2-HG. 75 76 Results 77 Genome analysis predicts a D-2-HG catabolism operon 78 Bacteria have evolved various aTFs to respond to different stimuli. To identify the possible 79 aTFs that respond to D-2-HG and regulate the expression of D2HGDH, two open reading 80 frames (ORFs) upstream and two ORFs downstream of D2HGDH in bacteria containing the 81 D2HGDH were subjected to gene occurrence profile analysis. GntR family transcriptional 82 regulator, electron transfer flavoprotein (ETF), 3-phosphoglycerate dehydrogenase (SerA), 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 carbohydrate diacid regulator (CdaR), lactate permease (LldP) and glycolate oxidase 84 iron-sulfur subunit (GlcF) appear to be the most frequently observed in the neighborhood of 85 D2HGDH (Supplementary Table 1). 86 Subsequent chromosomal gene clustering analysis identified five typical patterns of 87 organized chromosomal clusters (Fig. 1a). GntR and CdaR are located upstream of D2HGDH 88 in Parageobacillus thermoglucosidasius DSM 2542 and Bacillus cereus NJ-W, respectively. 89 However, LldP and GlcF, responding for the metabolism of two other hydroxycarboxylic 90 acids, lactate and glycolate, are also located adjacent to D2HGDH in P. thermoglucosidasius 91 DSM 2542 and B. cereus NJ-W. Despite that these two transcriptional regulators might 92 respond to D-2-HG and regulate D2HGDH expression, their possible effector 93 non-specificities should not be overlooked. Interestingly, in A. denitrificans NBRC 15125, 94 GntR is located directly upstream of D2HGDH and no adjacent hydroxycarboxylic acid 95 metabolism-related protein is encoded. Thus, in A. denitrificans NBRC 15125, GntR and 96 D2HGDH might comprise an operon that is specifically responsible for D-2-HG catabolism 97 and warrant further investigation. The uncharacterized transcriptional regulator GntR was 98 tentatively designated as D-2-hydroxyglutarate dehydrogenase regulator, DhdR. 99 100 D2HGDH is required for extracellular D-2-HG metabolism 101 D2HGDH of A. denitrificans NBRC 15125 was expressed as a His-tagged protein in 102 Escherichia coli BL21(DE3) and purified by affinity chromatography. Sodium dodecyl 103 sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography 104 revealed that D2HGDH was a dimer (Fig. 1b and Supplementary Fig. 1). Substrate screening 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.18.430539; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 revealed that D2HGDH had narrow substrate specificity and only exhibited distinct activity 106 towards D-2-HG (Fig. 1c). The product of D2HGDH-catalyzed dehydrogenation of D-2-HG 107 was identified to be 2-KG by high performance liquid chromatography (HPLC) analysis (Fig. 108 1d). The apparent Km and Vmax of purified D2HGDH for D-2-HG were 31.16 ± 1.40 µM and 109 3.95 ± 0.09 U mg−1, respectively (Supplementary Fig. 2). 110 SerA is the key enzyme for L-serine biosynthesis in various species, such as P.