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

Nature Catalysis volume 2, pages 342–353 (2019)Cite this article

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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.

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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.

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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.

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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).

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  1. These authors contributed equally: Hyun Jin Kim, June Huh.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Korea University, Seoul, Republic of Korea

    Hyun Jin Kim, June Huh, Donghyun Park, Yeonhwa Yu, Young Eun Jang, Bo-Ram Lee, Eunji Jo & Jeewon Lee

  2. KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul, Republic of Korea

    Young Wan Kwon

  3. Department of Chemical Engineering, School of Applied Chemical Engineering, Kyungpook National University, Daegu, Republic of Korea

    Eun Jung Lee

  4. Department of Biochemistry, Yonsei University, Seoul, Republic of Korea

    Yunseok Heo & Weontae Lee

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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.

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Correspondence to Jeewon Lee.

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Supplementary information

Supplementary Information

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

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Supplementary Data 1

Cartesian coordinates of Figure 1b

Supplementary Movie 1

MD simulations of pmoB with duroquinol and hydroquinone

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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

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