Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis

Cellular & Molecular Immunology volume 18, pages 2344–2357 (2021)Cite this article

Subjects

Abstract

Colitis is a common disease of the colon that is very difficult to treat. Probiotic bacteria could be an effective treatment. The probiotic Escherichia coli Nissle 1917 (EcN) was engineered to synthesize the ketone body (R)-3-hydroxybutyrate (3HB) for sustainable production in the gut lumen of mice suffering from colitis. Components of heterologous 3HB synthesis routes were constructed, expressed, optimized, and inserted into the EcN genome, combined with deletions in competitive branch pathways. The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions. The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days, elevating gut 3HB and short-chain-length fatty acid (SCFA) levels 8.7- and 3.1-fold compared to those of wild-type EcN, respectively. The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria, especially Akkermansia spp., to over 31% from the initial 2% of all the microbiome. As a result, the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium (DSS) in mice. Compared to wild-type EcN or oral administration of 3HB, oral EcNL4 uptake demonstrated better effects on mouse weights, colon lengths, occult blood levels, gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations. Thus, a promising live bacterium was developed to improve colonic microenvironments and further treat colitis. This proof-of-concept design can be employed to treat other diseases of the colon.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data availability

Data supporting the study are available from the corresponding authors by request. Source data are provided with this paper.

References

  1. Kurtz CB, Millet YA, Puurunen MK, Perreault M, Charbonneau MR, Isabella VM, et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci Transl Med. 2019;11:475.

    Article  CAS  Google Scholar 

  2. Aggarwal N, Breedon AME, Davis CM, Hwang IY, Chang MW. Engineering probiotics for therapeutic applications: recent examples and translational outlook. Curr Opin Biotech. 2020;65:171–9.

    Article  CAS  PubMed  Google Scholar 

  3. Pedrolli DB, Ribeiro NV, Squizato PN, de Jesus VN, Cozetto DA, Team AQA Unesp at iGEM. Engineering microbial living therapeutics: the synthetic biology toolbox. Trends Biotechnol. 2019;37:100–15.

    Article  CAS  PubMed  Google Scholar 

  4. Cao Z, Cheng S, Wang X, Pang Y, Liu J. Camouflaging bacteria by wrapping with cell membranes. Nat Commun. 2019;10:1–10.

    Article  CAS  Google Scholar 

  5. Bai L, Gao M, Cheng X, Kang G, Cao X, Huang H. Engineered butyrate-producing bacteria prevents high fat diet-induced obesity in mice. Microb Cell Fact. 2020;19:1–13.

    Article  Google Scholar 

  6. Piñero-Lambea C, Ruano-Gallego D, Fernández LÁ. Engineered bacteria as therapeutic agents. Curr Opin Biotech. 2015;35:94–102.

    Article  PubMed  CAS  Google Scholar 

  7. Isabella VM, Ha BN, Castillo MJ, Lubkowicz DJ, Rowe SE, Millet YA, et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat Biotechnol. 2018;36:857–64.

    Article  CAS  PubMed  Google Scholar 

  8. Riglar DT, Silver PA. Engineering bacteria for diagnostic and therapeutic applications. Nat Rev Microbiol. 2018;16:214–25.

    Article  CAS  PubMed  Google Scholar 

  9. Praveschotinunt P, Duraj-Thatte AM, Gelfat I, Bahl F, Chou DB, Joshi NS. Engineered E. coli Nissle 1917 for the delivery of matrix-tethered therapeutic domains to the gut. Nat Commun. 2019;10:1–14.

    Article  CAS  Google Scholar 

  10. Ho CL, Tan HQ, Chua KJ, Kang A, Lim KH, Ling KL, et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nat Biomed Eng. 2018;2:27–37.

    Article  CAS  PubMed  Google Scholar 

  11. Chen Z, Guo L, Zhang Y, Walzem RL, Pendergast JS, Printz RL, et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J Clin Investig. 2014;124:3391–406.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hendrikx T, Duan Y, Wang Y, Oh JH, Alexander LM, Huang W, et al. Bacteria engineered to produce IL-22 in intestine induce expression of REG3G to reduce ethanol-induced liver disease in mice. Gut. 2019;68:1504–15.

    Article  CAS  PubMed  Google Scholar 

  13. Ozdemir T, Fedorec AJ, Danino T, Barnes CP. Synthetic biology and engineered live biotherapeutics: toward increasing system complexity. Cell Syst. 2018;7:5–16.

    Article  CAS  PubMed  Google Scholar 

  14. Charbonneau MR, Isabella VM, Li N, Kurtz CB. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nat Commun. 2020;11:1–11.

    Article  CAS  Google Scholar 

  15. Rottinghaus AG, Amrofell MB, Moon TS. Biosensing in smart engineered probiotics. Biotechnol J. 2020;15:10.

    Article  CAS  Google Scholar 

  16. Westendorf AM, Gunzer F, Deppenmeier S, Tapadar D, Hunger JK, Schmidt MA, et al. Intestinal immunity of Escherichia coli Nissle 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Mic. 2005;43:373–84.

    Article  CAS  Google Scholar 

  17. Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, et al. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut. 2004;53:1617–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tan Y, Shen J, Si T, Ho CL, Li Y, Dai L. Engineered Live biotherapeutics: progress and challenges. Biotechnol J. 2020;15:2000155.

    Article  CAS  Google Scholar 

  19. Sonnenborn U. Escherichia coli strain Nissle 1917-from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol Lett. 2016;363:1–6.

  20. Sonnenborn U, Schulze J. The non-pathogenic Escherichia coli strain Nissle 1917-features of a versatile probiotic. Microb Ecol Health Dis. 2009;21:122–58.

    CAS  Google Scholar 

  21. Wehkamp J, Harder J, Wehkamp K, Wehkamp-von Meissner B, Schlee M, Enders C, et al. NF-κB- and AP-1-mediated induction of human beta defensin-2 in intestinal epithelial cells by Escherichia coli Nissle 1917: a novel effect of a probiotic bacterium. Infect Immun. 2004;72:5750–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Grabig A, Paclik D, Guzy C, Dankof A, Baumgart DC, Erckenbrecht J, et al. Escherichia coli strain Nissle 1917 ameliorates experimental colitis via toll-like receptor 2-and toll-like receptor 4-dependent pathways. Infect Immun. 2006;74:4075–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kai-Larsen Y, Lüthje P, Chromek M, Peters V, Wang X, Holm A, et al. Uropathogenic Escherichia coli modulates immune responses and its curli fimbriae interact with the antimicrobial peptide LL-37. PLoS Pathog. 2010;6:e1001010.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Choudhary R, Mahadevan R. Toward a systematic design of smart probiotics. Curr Opin Biotech. 2020;64:199–209.

    Article  CAS  PubMed  Google Scholar 

  25. Fan J-X, Li ZH, Liu XH, Zheng DW, Chen Y, Zhang XZ. Bacteria-mediated tumor therapy utilizing photothermally-controlled TNF-α expression via oral administration. Nano Lett. 2018;18:2373–80.

    Article  CAS  PubMed  Google Scholar 

  26. Huyghebaert N, Vermeire A, Neirynck S, Steidler L, Remaut E, Remon JP. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000;289:1352–5.

    Article  Google Scholar 

  27. Oh J-H, Schueler KL, Stapleton DS, Alexander LM, Yen CLE, Keller MP, et al. Secretion of recombinant interleukin-22 by engineered Lactobacillus reuteri reduces fatty liver disease in a mouse model of diet-induced obesity. Msphere. 2020;5:3.

    Article  Google Scholar 

  28. Amiri-Jami M, Abdelhamid AG, Hazaa M, Kakuda Y, Griffths MW. Recombinant production of omega-3 fatty acids by probiotic Escherichia coli Nissle 1917. FEMS Microbiol Lett. 2015;362:20.

    Article  CAS  Google Scholar 

  29. Newman JC, Verdin E. β-hydroxybutyrate: much more than a metabolite. Diabetes Res Clin Pr. 2014;106:173–81.

    Article  CAS  Google Scholar 

  30. Kashiwaya Y, Takeshima T, Mori N, Nakashima K, Clarke K, Veech RL. d-β-Hydroxybutyrate protects neurons in models of Alzheimer’s and Parkinson’s disease. P Natl Acad Sci USA. 2000;97:5440–4.

    Article  CAS  Google Scholar 

  31. Zhang J, Cao Q, Li S, Lu X, Zhao Y, Guan JS, et al. 3-Hydroxybutyrate methyl ester as a potential drug against Alzheimer’s disease via mitochondria protection mechanism. Biomaterials. 2013;34:7552–62.

    Article  CAS  PubMed  Google Scholar 

  32. Olson CA, Vuong HE, Yano JM, Liang QY, Nusbaum DJ, Hsiao EY. The gut microbiota mediates the anti-seizure effects of the ketogenic diet. Cell. 2018;173:1728–41. e1713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kossoff EH. More fat and fewer seizures: dietary therapies for epilepsy. Lancet Neuro. 2004;3:415–20.

    Article  Google Scholar 

  34. Chakraborty S, Galla S, Cheng X, Yeo JY, Mell B, Singh V, et al. Salt-responsive metabolite, β-hydroxybutyrate, attenuates hypertension. Cell Rep. 2018;25:677–89. e674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Youm Y-H, Nguyen KY, Grant RW, Goldberg EL, Bodogai M, Kim D, et al. The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease. Nat Med. 2015;21:263–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rahman M, Muhammad S, Khan MA, Chen H, Ridder DA, Müller-Fielitz H, et al. The β-hydroxybutyrate receptor HCA 2 activates a neuroprotective subset of macrophages. Nat Commun. 2014;5:1–11.

    Article  CAS  Google Scholar 

  37. Cao Q, Zhang J, Liu H, Wu Q, Chen J, Chen GQ. The mechanism of anti-osteoporosis effects of 3-hydroxybutyrate and derivatives under simulated microgravity. Biomaterials. 2014;35:8273–83.

    Article  CAS  PubMed  Google Scholar 

  38. Wang Y, Jiang XL, Peng SW, Guo XY, Shang GG, Chen JC, et al. Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates. Biomaterials. 2013;34:3737–46.

    Article  CAS  PubMed  Google Scholar 

  39. Xiao XQ, Zhao Y, Chen GQ. The effect of 3-hydroxybutyrate and its derivatives on the growth of glial cells. Biomaterials. 2007;28:3608–16.

    Article  CAS  PubMed  Google Scholar 

  40. Obruca S, Sedlacek P, Mravec F, Samek O, Marova I. Evaluation of 3-hydroxybutyrate as an enzyme-protective agent against heating and oxidative damage and its potential role in stress response of poly (3-hydroxybutyrate) accumulating cells. Appl Microbiol Biot. 2016;100:1365–76.

    Article  CAS  Google Scholar 

  41. Liu Q, Ouyang S-P, Chung A, Wu Q, Chen G-Q. Microbial production of R-3-hydroxybutyric acid by recombinant E. coli harboring genes of phbA, phbB, and tesB. Appl Microbiol Biot. 2007;76:811–8.

    Article  CAS  Google Scholar 

  42. Tseng H-C, Martin CH, Nielsen DR, Prather KLJ. Metabolic engineering of Escherichia coli for enhanced production of (R)-and (S)-3-hydroxybutyrate. Appl Environ Microb. 2009;75:3137–45.

    Article  CAS  Google Scholar 

  43. Singh V, Yeoh BS, Walker RE, Xiao X, Saha P, Golonka RM, et al. Microbiota fermentation-NLRP3 axis shapes the impact of dietary fibres on intestinal inflammation. Gut. 2019;68:1801–12.

    Article  CAS  PubMed  Google Scholar 

  44. Riglar DT, Giessen TW, Baym M, Kerns SJ, Niederhuber MJ, Bronson RT, et al. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation. Nat Biotechnol. 2017;35:653–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Rembacken B, Snelling A, Hawkey P, Chalmers D, Axon A. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet. 1999;354:635–9.

    Article  CAS  PubMed  Google Scholar 

  46. Leventhal DS, Sokolovska A, Li N, Plescia C, Kolodziej SA, Gallant CW, et al. Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity. Nat Commun. 2020;11:1–15.

    Article  CAS  Google Scholar 

  47. O’Toole PW, Marchesi JR, Hill C. Next-generation probiotics: the spectrum from probiotics to live biotherapeutics. Nat Microbiol. 2017;2:1–6.

    Article  CAS  Google Scholar 

  48. Van der Vossen J, van der Lelie D, Venema G. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters. Appl Environ Microb. 1987;53:2452–7.

    Article  Google Scholar 

  49. De Graef MR, Alexeeva S, Snoep JL, de Mattos MJT. The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J Bacteriol. 1999;181:2351–7.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Simon R, Priefer U, Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat Biotechnol. 1983;1:784–91.

    Article  CAS  Google Scholar 

  51. Kruger NJ. The Bradford Method For Protein Quantitation. In: Walker J.M. editor. The Protein Protocols Handbook. Totowa, NJ: Springer Protocols Handbooks. Humana Press; 2009.

  52. Nishimura T, Saito T, Tomita K. Purification and properties of β-ketothiolase from Zoogloea ramigera. Arch Microbiol. 1978;116:21–27.

    Article  CAS  PubMed  Google Scholar 

  53. Senior PJ, Dawes EA. The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J. 1973;134:225–38.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zheng Z, Gong Q, Liu T, Deng Y, Chen JC, Chen GQ. Thioesterase II of Escherichia coli plays an important role in 3-hydroxydecanoic acid production. Appl Environ Microb. 2004;70:3807–13.

    Article  CAS  Google Scholar 

  55. Sassone-Corsi M, Nuccio SP, Liu H, Hernandez D, Vu CT, Takahashi AA, et al. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature. 2016;540:280–3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101–8.

    Article  CAS  PubMed  Google Scholar 

  57. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987;160:47–56.

    Article  CAS  PubMed  Google Scholar 

  58. Alexander DB, Zuberer DA. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol Fertil Soils. 1991;12:39–45.

    Article  CAS  Google Scholar 

  59. Smith P, Mangan NE, Walsh CM, Fallon RE, McKenzie AN, van Rooijen N, et al. Infection with a helminth parasite prevents experimental colitis via a macrophage-mediated mechanism. J Immunol. 2007;178:4557–66.

    Article  CAS  PubMed  Google Scholar 

  60. Amrofell MB, Rottinghaus AG, Moon TS. Engineering microbial diagnostics and therapeutics with smart control. Curr Opin Biotech. 2020;66:11–17.

    Article  CAS  PubMed  Google Scholar 

  61. Volbeda A, Darnault C, Renoux O, Nicolet Y, Fontecilla-Camps JC. The crystal structure of the global anaerobic transcriptional regulator FNR explains its extremely fine-tuned monomer-dimer equilibrium. Sci Adv. 2015;1:e1501086.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Puchalska P, Crawford PA. Multi-dimensional roles of ketone bodies in fuel metabolism, signaling, and therapeutics. Cell Metab. 2017;25:262–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Smith PM, Howitt MR, Panikov N, Michaud M, Gallini CA, Bohlooly-Y M, et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science. 2013;341:569–73.

    Article  CAS  PubMed  Google Scholar 

  64. Bian X, Yang L, Wu W, Lv L, Jiang X, Wang Q, et al. Pediococcus pentosaceus LI05 alleviates DSS‐induced colitis by modulating immunological profiles, the gut microbiota and short‐chain fatty acid levels in a mouse model. Microb Biotechnol. 2020;13:1228–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Parker BJ, Wearsch PA, Veloo AC, Rodriguez-Palacios A. The genus Alistipes: Gut bacteria with emerging implications to inflammation, cancer, and mental health. Front Immunol. 2020;11:906.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Kovatcheva-Datchary P, Nilsson A, Akrami R, Lee YS, De Vadder F, Arora T, et al. Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of Prevotella. Cell Metab. 2015;22:971–82.

    Article  CAS  PubMed  Google Scholar 

  67. Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013;500:232–6.

    Article  CAS  PubMed  Google Scholar 

  68. Chassaing B, Aitken JD, Malleshappa M, Vijay‐Kumar M. Dextran sulfate sodium (DSS)‐induced colitis in mice. Curr Protoc Immunol. 2014;104:11–15.

    Article  Google Scholar 

  69. Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat Rev Gastro Hepat. 2009;6:306–14.

    Article  CAS  Google Scholar 

  70. Tripathi A, Debelius J, Brenner DA, Karin M, Loomba R, Schnabl B, et al. The gut-liver axis and the intersection with the microbiome. Nat Rev Gastro Hepat. 2018;15:397–411.

    Article  CAS  Google Scholar 

  71. Yu J, Plackett D, Chen LX. Kinetics and mechanism of the monomeric products from abiotic hydrolysis of poly [(R)-3-hydroxybutyrate] under acidic and alkaline conditions. Polym Degrad Stab. 2005;89:289–99.

    Article  CAS  Google Scholar 

  72. Tan D, Xue YS, Aibaidula G, Chen GQ. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour Technol. 2011;102:8130–6.

    Article  CAS  PubMed  Google Scholar 

  73. Pradhan S, Weiss AA. Probiotic properties of Escherichia coli Nissle in human intestinal organoids. Mbio. 2020;11:e01470–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Hancock V, Vejborg RM, Klemm P. Functional genomics of probiotic Escherichia coli Nissle 1917 and 83972, and UPEC strain CFT073: comparison of transcriptomes, growth and biofilm formation. Mol Genet Genomics. 2010;284:437–54.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was financially supported by a grant from the Chunfeng Foundation (2020Z99CFG002) of Tsinghua University. Other support includes the National Natural Science Foundation of China (Grant Nos. 31870859, 21761132013, 31771886, and 31971170).

Author information

Authors and Affiliations

  1. Center for Synthetic and Systems Biology, Tsinghua University, Beijing, China

    Xu Yan, Xin-Yi Liu, Dian Zhang, Yu-Dian Zhang, Zi-Hua Li, Xu Liu, Fuqing Wu & Guo-Qiang Chen

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Xu Yan, Xin-Yi Liu, Xu Liu, Fuqing Wu & Guo-Qiang Chen

  3. MOE Key Lab for Industrial Biocatalysis, Dept. Chemical Engineering, Tsinghua University, Beijing, China

    Guo-Qiang Chen

Authors
  1. Xu Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xin-Yi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yu-Dian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zi-Hua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xu Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fuqing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guo-Qiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

XY designed and conducted the study. XYL and DZ performed the experiments. YDZ and ZHL assisted with the animal experiments. XL helped draw the illustrations. XY wrote the manuscript. FQW and GQC supervised the project and helped write the manuscript. Source data are provided with this paper

Corresponding author

Correspondence to Guo-Qiang Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, X., Liu, XY., Zhang, D. et al. Construction of a sustainable 3-hydroxybutyrate-producing probiotic Escherichia coli for treatment of colitis. Cell Mol Immunol 18, 2344–2357 (2021). https://doi.org/10.1038/s41423-021-00760-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41423-021-00760-2

Keywords

This article is cited by

Search

Advanced search

Quick links