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A Fluorescent L-2-Hydroxyglutarate Biosensor

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A Fluorescent L-2-Hydroxyglutarate Biosensor bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.187567; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 fluorescent L-2-hydroxyglutarate biosensor 2 3 Zhaoqi Kang1, Manman Zhang3, Kaiyu Gao1, Wen Zhang4, Yidong Liu1, Dan Xiao1, Shiting 4 Guo1, Cuiqing Ma1, Chao Gao1, *, Ping Xu2, * 5 6 1State Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s 7 Republic of China 8 2State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of 9 Metabolic & Developmental Sciences, and School of Life Sciences & Biotechnology, 10 Shanghai Jiao Tong University, Shanghai, People’s Republic of China 11 3Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, 12 Department of Radiobiology, Institute of Radiation Medicine of Chinese Academy of Medical 13 Science & Peking Union Medical College, Tianjin, People’s Republic of China 14 4Center for Gene and Immunotherapy, The Second Hospital of Shandong University, Jinan, 15 People’s Republic of China 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, E- 20 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/10.1101/2020.07.07.187567; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 L-2-Hydroxyglutarate (L-2-HG) plays important roles in diverse physiological processes, 26 such as carbon starvation response, tumorigenesis, and hypoxic adaptation. Despite its 27 importance and intensively studied metabolism, regulation of L-2-HG metabolism remains 28 poorly understood and a regulator specifically responded to L-2-HG has never been 29 identified. Based on the genomic neighborhood analysis of the gene encoding L-2-HG 30 oxidase (LhgO), LhgR, which represses the transcription of lhgO, was identified in 31 Pseudomonas putida W619 in this study. LhgR was demonstrated to recognize L-2-HG as its 32 specific effector molecule, and this allosteric transcription factor was then used as a 33 biorecognition element for construction of L-2-HG-sensing FRET sensor. The newly 34 developed L-2-HG sensor can conveniently monitor the concentrations of L-2-HG in various 35 biological samples. In addition to bacterial L-2-HG generation during carbon starvation, 36 biological functions of the L-2-HG dehydrogenase and hypoxia induced L-2-HG 37 accumulation were also revealed by using the L-2-HG sensor in human cells. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.187567; this version posted July 7, 2020. 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. 38 Introduction 39 L-2-Hydroxyglutarate (L-2-HG) is an important metabolite in various domains of life. In 40 mammals and plants, it is produced by lactate dehydrogenase (LDH) and malate 41 dehydrogenase (MDH)-mediated 2-ketoglutarate (2-KG) reduction under hypoxic 42 conditions1-5. In microorganisms, it is a metabolic intermediate of glutarate catabolism 43 produced by a glutarate hydroxylase, CsiD6-8. L-2-HG dehydrogenase (L2HGDH) or L-2-HG 44 oxidase (LhgO), an FAD-containing oxidoreductase that converts L-2-HG to 2-KG, plays an 45 indispensable role in the catabolism of L-2-HG9. While extensive efforts have been devoted 46 to investigate L-2-HG anabolism and catabolism, the molecular machinery that specifically 47 senses L-2-HG and regulates its metabolism has not been clarified until now. 48 L-2-HG is an inhibitor of 2-KG dependent dioxygenases with specific pro-oncogenic 49 capabilities10,11. Thus, this oncometabolite is viewed as a biomarker for a variety of cancers 50 and its rapid and sensitive measurement in body fluids is of clinical significance12-15. 51 Importantly, L-2-HG also has endogenous functions in healthy animal cells. For example, this 52 compound was recently identified to aid in the proliferation and antitumorigenic abilities of 53 CD8+ T-lymphocytes16, to contribute to relieving the cellular reductive stress3, and to 54 coordinate glycolytic flux with epigenetic modifications17. Considering the diversity of the 55 roles of L-2-HG in cell metabolism, development and optimization of assays for real-time 56 tracking of this metabolite in living cells is required. 57 Liquid chromatography-tandem mass spectrometry (LC-MS/MS)18,19 and gas 58 chromatography-tandem mass spectrometry (GC-MS/MS)20,21 are often used to assess the 59 extracellular concentrations of L-2-HG. These common methods are, at present, time 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.187567; this version posted July 7, 2020. 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. 60 consuming, expensive to perform, and require highly skilled personnel. In addition, these 61 destructive methods are also incompatible with real-time monitoring of the fluctuations of L- 62 2-HG concentrations in intact living cells. In this study, we identified and characterized 63 LhgR, an L-2-HG catabolism regulator in Pseudomonas putida W619. Mechanistically, LhgR 64 represses the transcription of LhgO encoding gene lhgO. L-2-HG is a specific effector 65 molecule of LhgR and prevents LhgR binding to the promoter region of lhgO. Then, we 66 report the development and application of the LhgR based L-2-HG biosensor via Förster 67 resonance energy transfer (FRET), a technology widely applied in the investigation of 68 temporal dynamics of various small molecules, such as potassium22,23, glycine24, and 69 cAMP25,26. The newly developed sensor was used to test L-2-HG concentrations in various 70 biological samples and achieved competitive accuracy and precision. We also took advantage 71 of this biosensor to test predictions about the carbon starvation-induced L-2-HG production in 72 bacteria and to demonstrate hypoxia-induced L-2-HG production by LDH and MDH in 73 human cells. Therefore, the biosensor can also act as a useful tool for real-time measurement 74 of the L-2-HG concentrations in living cells. 75 76 Results 77 LhgR regulates L-2-HG catabolism 78 In this study, bacteria containing LhgO encoding gene lhgO were selected to study the 79 regulation of L-2-HG metabolism. Homologs of LhgO can be found in 612 diverse bacterial 80 strains. Similarly organized chromosomal clusters can be found in many bacterial genomes, 81 which contain various combinations of genes related to glutarate metabolism (csiD, lhgO, 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.187567; this version posted July 7, 2020. 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. 82 gabT, gabD, and gabP) (Fig. 1a). In Pseudomonas putida KT2440, the glutarate regulon is 83 regulated by allosteric transcription factor CsiR, which is encoded upstream of csiD27. The 84 glutarate sensing allosteric transcription factor CsiR and its cognate promoter have been 85 cloned into broad host range vectors to create a glutarate biosensor28. Interestingly, a different 86 pattern of lhgO gene neighborhood is observed in a few species that do not contain csiD 87 homologs (Fig. 1a). For example, a gene encoding a GntR family protein, lhgR, is found 88 directly upstream of lhgO in P. putida W619. The absence of csiD gene related to glutarate 89 catabolism made us to anticipate that lhgO of P. putida W619 might be solely involved in L- 90 2-HG metabolism and be L-2-HG inducible. 91 The lhgO gene in P. putida W619 was cloned into pME6032 vector, and the resulting 92 plasmid was transferred into P. putida KT2440 (∆lhgO). As shown in Fig. 1b-c, complement 93 of lhgO in P. putida W619 could restore glutarate and L-2-HG utilization abilities of P. 94 putida KT2440 (∆lhgO), confirming that lhgO encodes a functional L-2-HG catabolic 95 enzyme. To identify the function of LhgR in P. putida W619, the gene segment F2-lhgR-F1- 96 lhgO, which contains the promoter of lhgR (F2), lhgR, the promoter of lhgO (F1), and lhgO, 97 was cloned into pME6032 vector, and the resulting plasmid was transferred into different 98 derivatives of P. putida KT2440 (Fig. 1d). As shown in Fig. 1e, exogenous L-2-HG, but not 99 its mirror-image enantiomer D-2-HG, can induce the expression of lhgO in the gene segment 100 F2-lhgR-F1-lhgO and restore LhgO activity in P. putida KT2440 (∆csiR∆lhgO). In addition, 101 the activity of LhgO was also detected in P. putida KT2440 (∆csiR∆lhgO) harboring 102 pME6032-F2-lhgR-F1-lhgO when cultured with glutarate as the sole carbon source. 103 However, no activity of LhgO was detected in P. putida KT2440 (∆csiR∆csiD∆lhgO), in 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.07.187567; this version posted July 7, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
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