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Metabolic engineering of Escherichia coli with electron channelling for the production of natural products

Nature Catalysis volume 5, pages 726–737 (2022)Cite this article

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Abstract

The biosynthesis of natural products often requires eukaryotic cytochrome P450s (P450s) in combination with P450 reductase, in physical proximity, to perform electron-transfer reactions. Unfortunately, functional expression of eukaryotic P450s in bacteria remains generally difficult. Here we report an electron channelling strategy based on the application of Photorhabdus luminescens CipB scaffold protein, which allows efficient electron transfer between P450s and reductases by bringing these enzymes in close proximity. The general applicability of this electron channelling strategy is proved by developing recombinant Escherichia coli strains producing lutein, (+)-nootkatone, apigenin and l-3,4-dihydroxyphenylalanine (l-DOPA), each of which requires P450s in its biosynthetic pathway. The production titres are then further enhanced by increasing the haem pathway flux or by optimization of the culture conditions. Remarkably, the final lutein strain produced 218.0 mg l−1 of lutein with a productivity of 5.01 mg l−1 h−1 in fed-batch fermentation under optimized culture conditions.

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Fig. 1: The primary lutein biosynthetic pathway.
Fig. 2: Results of flask culture for lutein production.
Fig. 3: Biosynthetic pathways of (+)-nootkatone, apigenin and L-DOPA.
Fig. 4: Applying the electron channelling strategy to produce natural products in E. coli.
Fig. 5: Enhancement of lutein production by increasing haem supply.
Fig. 6: Enhancement of lutein production by optimization of culture conditions.
Fig. 7: Fed-batch fermentation of LUT5MH1 for lutein production.

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Data availability

The data to reproduce the findings in this study are presented in the Paper and the Supplementary Information or are available from the authors upon reasonable request. Source data are provided with this Paper.

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Acknowledgements

We thank J. Bang, K. R. Choi, S. Cho, D. Huccetogullari, G. B. Kim, G. Ryu and T. Yu for helpful discussions and valuable advice. This work was supported by the Cooperative Research Program for Agriculture Science & Technology Development (project no. PJ01550602), Rural Development Administration, Republic of Korea. This work was further supported by the Development of Next-Generation Biorefinery Platform Technologies for Leading Bio-based Chemicals Industry Project (2022M3J5A1056072) and by the Development of Platform Technologies of Microbial Cell Factories for the Next-Generation Biorefineries Project (2022M3J5A1056117) from the National Research Foundation supported by the Korean Ministry of Science and ICT.

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Authors and Affiliations

  1. Metabolic and Biomolecular Engineering National Research Laboratory and Systems Metabolic Engineering and Systems Healthcare Cross-Generation Collaborative Laboratory, Department of Chemical and Biomolecular Engineering (BK21 four), Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Seon Young Park, Hyunmin Eun, Mun Hee Lee & Sang Yup Lee

  2. KAIST Institute for the BioCentury and KAIST Institute for Artificial Intelligence, KAIST, Daejeon, Republic of Korea

    Seon Young Park, Hyunmin Eun, Mun Hee Lee & Sang Yup Lee

  3. BioProcess Engineering Research Center and BioInformatics Research Center, KAIST, Daejeon, Republic of Korea

    Sang Yup Lee

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Contributions

S.Y.L. conceived the project. S.Y.L. and S.Y.P. designed the experiments. S.Y.P. and H.E. conducted experiments and analysed the data. M.H.L. contributed to HPLC and HPLC-MS analysis. S.Y.L., S.Y.P. and H.E. wrote the manuscript, and all authors read and approved the final manuscript.

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Correspondence to Sang Yup Lee.

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S.Y.L. and S.Y.P. declare competing financial interests as the work described in this Paper is covered by a patent filed including, but not limited to, KR1020210005806, and is of commercial interest. The remaining authors declare no competing interests.

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Nature Catalysis thanks Kristina Haslinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Park, S.Y., Eun, H., Lee, M.H. et al. Metabolic engineering of Escherichia coli with electron channelling for the production of natural products. Nat Catal 5, 726–737 (2022). https://doi.org/10.1038/s41929-022-00820-4

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