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.

Biological conversion of methane to methanol through genetic reassembly of native catalytic domains

Abstract

Methane monooxygenase (MMO), which exists in particulate (pMMO) or soluble forms (sMMO) in methanotrophic bacteria, is an industrially promising enzyme that catalyses oxidation of low-reactive methane and other carbon feedstocks into methanol and their corresponding oxidation products. However, the simple, fast and high-yield production of functionally active MMO, which has so far been unsuccessful despite diverse approaches based on either native methanotroph culture or recombinant expression systems, remains a major challenge for its industrial applications. Here we developed pMMO-mimetic catalytic protein constructs by genetically encoding the beneficial reassembly of catalytic domains of pMMO on apoferritin as a biosynthetic scaffold. This approach resulted in high-yield synthesis of stable and soluble protein constructs in Escherichia coli, which successfully retain enzymatic activity for methanol production with a turnover number comparable to that of native pMMO.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Native pMMO protomer and pMMO-mimics.
Fig. 2: MD simulations of designed pMMO-mimics.
Fig. 3: Heterologous expression of designed pMMO-mimics.
Fig. 4: Catalytic activity of designed pMMO-mimics and mutants.
Fig. 5: Spectroscopic analyses of function and catalytic sites of pMMO-mimics.
Fig. 6: Verification of metal contents in pMMO-mimics and its effect on catalytic activity.

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

References

  1. Elliott, S. J. et al. Regio- and stereoselectivity of particulate methane monooxygenase from Methylococcus capsulatus (Bath). J. Am. Chem. Soc. 119, 9949–9955 (1997).

    Article  CAS  Google Scholar 

  2. Merkx, M. et al. Dioxygen activation and methane hydroxylation by soluble methane monooxygenase: a tale of two irons and three proteins. Angew. Chem. Int. Ed. 40, 2782–2807 (2001).

    Article  CAS  Google Scholar 

  3. Park, D. & Lee, J. Biological conversion of methane to methanol. Korean J. Chem. Eng. 30, 977–987 (2013).

    Article  CAS  Google Scholar 

  4. Wang, V. C.-C. et al. Alkane oxidation: methane monooxygenase, related enzymes, and their biomimetics. Chem. Rev. 117, 8574–8621 (2017).

    Article  CAS  Google Scholar 

  5. Chan, S. I. & Yu, S. S.-F. Controlled oxidation of hydrocarbons by the membrane-bound methane monooxygenase: the case for a tricopper cluster. Acc. Chem. Res. 41, 969–979 (2008).

    Article  CAS  Google Scholar 

  6. Danciu, T. et al. Direct synthesis of propylene oxide with CO2 as the solvent. Angew. Chem. Int. Ed. 42, 1140–1142 (2003).

    Article  CAS  Google Scholar 

  7. Hua, Q. et al. Crystal-plane-controlled selectivity of Cu2O catalysts in propylene oxidation with molecular oxygen. Angew. Chem. Int. Ed. 53, 4856–4861 (2014).

    Article  CAS  Google Scholar 

  8. Clomburg, J. M., Crumbley, A. M. & Gonzalez, R. Industrial biomanufacturing: the future of chemical production. Science 355, aao0804 (2017).

    Article  Google Scholar 

  9. Hakemian, S. & Rosenzweig, A. C. The biochemistry of methane oxidation. Annu. Rev. Biochem. 76, 223–241 (2007).

    Article  CAS  Google Scholar 

  10. Lieberman, R. L. et al. Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster. Proc. Natl Acad. Sci. USA 100, 3820–3825 (2003).

    Article  CAS  Google Scholar 

  11. Krause, S. M. et al. Lanthanide-dependent cross-feeding of methane-derived carbon is linked by microbial community interaction. Proc. Natl Acad. Sci. USA 114, 358–363 (2017).

    Article  CAS  Google Scholar 

  12. Gilman, A. et al. Bioreactor performance parameters for an industrially-promising methanotroph Methylomicrobium buryatense 5GB1. Microb. Cell Fact. 14, 182 (2015).

    Article  Google Scholar 

  13. Islam, R. S., Tisi, D., Levy, M. S. & Lye, G. J. Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k L a. Biotechnol. Bioeng. 99, 1128–1139 (2008).

    Article  CAS  Google Scholar 

  14. Jahng, D. et al. Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria. Biotechnol. Bioeng. 51, 349–359 (1996).

    Article  CAS  Google Scholar 

  15. Balasubramanian, R. et al. Oxidation of methane by a biological dicopper centre. Nature 465, 115–119 (2010).

    Article  CAS  Google Scholar 

  16. Gau, Z. et al. Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis. FEMS Microbiol. Lett. 263, 136–141 (2006).

    Article  Google Scholar 

  17. Afiah, T. S. N. & Rusmana, I. Cuperedoxin domain of particulate methane monooxygenase (pMMO) gene expression in recombinant Escherichia coli. Malays. J. Microbiol. 12, 56–61 (2016).

    CAS  Google Scholar 

  18. Tie-nam, Z. et al. Heterologous expression of pmoB in Escherichia coli and catalytic oxygenation of methane to methanol. J. Mol. Catal. 30, 177–181 (2016).

    Google Scholar 

  19. Chan, S. I. et al. Redox potentiometry studies of particulate methane monooxygenase: support for a trinuclear copper cluster active site. Angew. Chem. Int. Ed. 46, 1992–1994 (2007).

    Article  CAS  Google Scholar 

  20. Piku, B., Katterle, B. & Andersson, K. K. The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein. Biochem. J. 369, 417–427 (2003).

    Article  Google Scholar 

  21. Yoshizwa, K. & Shiota, Y. Conversion of methane to methanol at the mononuclear and dinuclear copper sites of particulate methane monooxygenase (pMMO): a DFT and QM.MM study. J. Am. Chem. Soc. 128, 9873–9881 (2006).

    Article  Google Scholar 

  22. Lieberman, R. L. & Rosenzweig, A. C. Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane. Nature 434, 177–182 (2005).

    Article  CAS  Google Scholar 

  23. Smith, S. M. et al. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry 50, 10231–10240 (2011).

    Article  CAS  Google Scholar 

  24. Cao, L., Caldararu, O., Rosenzweig, A. C. & Ryde, U. Quantum refinement does not support dinuclear copper sites in crystal structure of particulate methane monooxygenase. Angew. Chem. Int. Ed. 57, 162–166 (2017).

    Article  Google Scholar 

  25. Sirajuddin, S. et al. Effects of zinc on particulate methane monooxygenase activity and structure. J. Biol. Chem. 289, 21782–21794 (2014).

    Article  Google Scholar 

  26. Culpepper, M. A. & Rosenzweig, A. C. Architecture and active site of particulate methane monooxygenase. Crit. Rev. Biochem. Mol. Biol. 47, 483–492 (2012).

    Article  CAS  Google Scholar 

  27. Luzzago, A. & Cesareni, G. Isolation of point mutations that affect the folding of the H chain of human ferritin in E. coli. EMBO J. 8, 569–576 (1989).

    Article  CAS  Google Scholar 

  28. Wang, L. et al. Structure-based design of ferritin nanoparticle immunogens displaying antigenic loops of Neisseria gonorrhoeae. FEBS Open Bio 7, 1196–1207 (2017).

    Article  CAS  Google Scholar 

  29. Duwe, S. et al. Expression-enhanced fluorescent proteins based on enhanced green fluorescent protein for super-resolution microscopy. ACS Nano 9, 9528–9541 (2015).

    Article  CAS  Google Scholar 

  30. Lee, S. H. et al. A novel approach to ultrasensitive diagnosis using supramolecular protein nanoparticles. FASEB J. 21, 1324–1334 (2007).

    Article  CAS  Google Scholar 

  31. Lee, B. R. et al. Engineered human ferritin nanoparticles for direct delivery of tumor antigens to lymph node and cancerimmunotherapy. Sci. Rep. 6, 35182 (2016).

    Article  CAS  Google Scholar 

  32. Kwon, K. C. et al. Enhanced in vivo tumor detection by active tumor cell targeting using multiple tumor receptor-binding peptides presented on genetically engineered human ferritin nanoparticles. Small 12, 4241–4253 (2016).

    Article  CAS  Google Scholar 

  33. Lee, J. H. et al. Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles. Biosens. Bioelectron. 69, 213–225 (2015).

    Article  CAS  Google Scholar 

  34. Lee, J. H. et al. Proteinticle engineering for accurate 3D diagnosis. ACS Nano 7, 10879–10886 (2013).

    Article  CAS  Google Scholar 

  35. Cornell, T. A. et al. The crystal structure of a maxi/mini-ferritin chimera reveals guiding principles for the assembly of protein cages. Biochemistry 56, 3894–3899 (2017).

    Article  CAS  Google Scholar 

  36. Jetz, G. et al. Ferritin: a versatile building block for bionanotechnology. Chem. Rev. 115, 1653–1701 (2015).

    Article  Google Scholar 

  37. Lee, E. J. et al. A novel bioassay platform using ferritin based nanoprobe hydrogel. Adv. Mater. 24, 4739–4744 (2012).

    Article  CAS  Google Scholar 

  38. Masuda, T., Gota, F., Yoshifara, T. & Mikami, B. The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site. Biochem. Biophys. Res. Commun. 400, 94–99 (2010).

    Article  CAS  Google Scholar 

  39. Lontoh, S. et al. Differential inhibition in vivo of ammonia monooxygenase, soluble methane monooxygenase and membrane-associated methane monooxygenase by phenylacetylene. Environ. Microbiol. 2, 485–494 (2000).

    Article  CAS  Google Scholar 

  40. Lee, S. W. et al. Effect of nutrient and selective inhibitor amendments on methane oxidation, nitrous oxide production, and key gene presence and expression in landfill cover soils: characterization of the role of methanotrophs, nitrifiers, and denitrifiers. Environ. Microbiol. 85, 389–403 (2009).

    CAS  Google Scholar 

  41. Paszczynski, A. J. et al. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase. Biodegradation 22, 1045–1059 (2011).

    Article  CAS  Google Scholar 

  42. Sirajuddin, S. & Rosenzweig, A. C. Enzymatic oxidation of methane. Biochemistry 54, 2283–2294 (2015).

    Article  CAS  Google Scholar 

  43. Choi, D. W. et al. The membrane-associated methane monooxygenase (pMMO) and pMMO-NADH: quinone oxidoreductase complex form Methylococcus capsulatus Bath. J. Bacteriol. 185, 5755–5764 (2003).

    Article  CAS  Google Scholar 

  44. Yu, S. S. F. et al. Production of high-quality particulate methane monooxygenase in high yields from Methylococcus capsulatus (Bath) with a hollow-fiber membrane bioreactor. J. Bacteriol. 185, 5915–5924 (2003).

    Article  CAS  Google Scholar 

  45. Martinho, M. et al. Mössbauer studies of the membrane-associated methane monooxygenase from Methylococcus capsulatus Bath: evidence for a diiron center. J. Am. Chem. Soc. 129, 15783–15785 (2007).

    Google Scholar 

  46. Zahn, J. & DiSpiriop, A. A. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath). J. Bacteriol. 178, 1018–1029 (1996).

    Article  CAS  Google Scholar 

  47. Lieberman, R. L. & Rosenzweig, A. C. Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase. Crit. Rev. Biochem. Mol. Biol. 39, 147–164 (2004).

    Article  CAS  Google Scholar 

  48. Tonge, G. M., Harrison, D. E. F. & Higgins, I. J. Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b. Biochem. J. 161, 333–344 (1977).

    Article  CAS  Google Scholar 

  49. Lee, D. S. et al. A protein nanofiber hydrogel for sensitive immunoassays. Analyst 138, 4786–4794 (2013).

    Article  CAS  Google Scholar 

  50. Pollak, A. et al. Enzyme immobilization by condensation copolymerization into crosslinked polyacrylamide gels. J. Am. Chem. Soc. 102, 6324–6336 (1980).

    Article  CAS  Google Scholar 

  51. Chen, Y. Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review. J. Ind. Microbiol. Biotechnol. 38, 581–597 (2011).

    Article  CAS  Google Scholar 

  52. Gonzalez-Saiz, J. M. & Pizarro, C. Polyacrylamide gels as support for enzyme immobilization by entrapment. Effect of polyelectrolyte carrier, pH and temperature on enzyme action and kinetics parameters. Eur. Polym. J. 37, 435–444 (2001).

    Article  CAS  Google Scholar 

  53. Kumar, S., Yadav, R. V. & Negi, S. A Comparative study of immobilized lipase produced from Penicillium chrysogenum SNP5 on two different anionic carriers for its pH and thermostability. Indian J. Biotechnol. 13, 301–305 (2014).

    CAS  Google Scholar 

  54. Blanchette, C. D. et al. Printable enzyme-embedded materials for methane to methanol conversion. Nat. Commun. 7, 11900 (2016).

    Article  CAS  Google Scholar 

  55. Culpepper, M. A. et al. Identification of the valence and coordination environment of the particulate methane monooxygenase copper centers by advanced EPR characterization. J. Am. Chem. Soc. 136, 11767–11775 (2014).

    Article  CAS  Google Scholar 

  56. Page, C. C., Moser, C. C., Chen, X. & Dutton, P. L. Natural engineering principles of electron tunneling in biological oxidation reduction. Nature 402, 47–52 (1999).

    Article  CAS  Google Scholar 

  57. Hempstead, P. D. et al. Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 268, 424–448 (1997).

    Article  CAS  Google Scholar 

  58. Ro, S. Y. et al. From micelles to bicelles: effect of the membrane on particulate methane monooxygenase activity. J. Biol. Chem. 293, 10457–10465 (2018).

    Article  Google Scholar 

  59. Mayne, C. G., Saam, J., Schulten, K. & Tajkhorshid, E. Rapid parameterization of small molecules using the Force Field Toolkit. J. Comput. Chem. 34, 2757–2770 (2013).

    Article  CAS  Google Scholar 

  60. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).

    Article  CAS  Google Scholar 

  61. Kale, L. et al. NAMD2: greater scalability for parallel molecular dynamics. J. Comput. Phys. 151, 283–312 (1999).

    Article  CAS  Google Scholar 

  62. Feller, S. E., Zhang, Y., Pastor, R. W. & Brooks, B. R. Constant-pressure molecular-dynamics simulation – the Langevin piston method. J. Chem. Phys. 103, 4613–4621 (1995).

    Article  CAS  Google Scholar 

  63. Darden, T., York, D. & Pederson, L. Particle mesh Ewald: an N·log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089–10092 (1993).

    Article  CAS  Google Scholar 

  64. Andersen, H. C. Rattle: a “velocity” version of the shake algorithm for molecular dynamics calculations. J. Comput. Phys. 52, 24–34 (1983).

    Article  CAS  Google Scholar 

  65. Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005).

    Article  CAS  Google Scholar 

  66. Stoll, S. & Schweiger, A. EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J. Magn. Reson. 178, 42–55 (2006).

    Article  CAS  Google Scholar 

  67. Liu, X., Yu, M., Kim, H., Mameli, M. & Stellacci, F. Determination of monolayer-protected gold nanoparticle ligand–shell morphology using NMR. Nat. commun. 3, 1182 (2012).

    Article  Google Scholar 

Download references

Acknowledgements

This study was supported by the 2015 NLRL (National Leading Research Lab.) Project (grant no. NRF (National Research Foundation Korea)-2015R1A2A1A05001861), Bio & Medical Technology Development Program (grant no. NRF‐2017M3A9F5032628), and also partly by NRF-Korea (NRF-2016R1A6A3A11933393).

Author information

Authors and Affiliations

Authors

Contributions

J.L. conceived the design and synthesis of pMMO-mimics, conducted the data analysis and wrote the paper. H.J.K. performed the synthesis and activity analyses of the designed pMMO-mimics and collected the data. J.H. performed the MD simulation based on the design of pMMO-mimics, collected the data and co-wrote the paper. Y.W.K. performed the data collection and analyses of XANES, EXAFS and EPR spectroscopy. D.P., Y.Y., Y.E.J., B.-R.L. and E.J. performed the experiments of pMMO-mimics synthesis. Y.H. and W.L. performed SEC chromatographic and CD spectroscopic analyses. E.J.L. analysed the experimental data. We also appreciate the contribution of P. Shrestha at Yonsei University in data collection.

Corresponding author

Correspondence to Jeewon Lee.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Methods, Supplementary Figures 1–21, Supplementary Tables 1 and 2, Supplementary Note 1 and Supplementary References

Reporting Summary

Supplementary Data 1

Cartesian coordinates of Figure 1b

Supplementary Movie 1

MD simulations of pmoB with duroquinol and hydroquinone

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kim, H.J., Huh, J., Kwon, Y.W. et al. Biological conversion of methane to methanol through genetic reassembly of native catalytic domains. Nat Catal 2, 342–353 (2019). https://doi.org/10.1038/s41929-019-0255-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-019-0255-1

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing