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A synthetic cell-free 36-enzyme reaction system for vitamin B12 production

Author

Listed:
  • Qian Kang

    (University of Chinese Academy of Sciences
    Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Huan Fang

    (University of Chinese Academy of Sciences
    Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Mengjie Xiang

    (University of Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Kaixing Xiao

    (Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Pingtao Jiang

    (Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Chun You

    (University of Chinese Academy of Sciences
    Chinese Academy of Sciences)

  • Sang Yup Lee

    (Korea Advanced Institute of Science and Technology (KAIST))

  • Dawei Zhang

    (University of Chinese Academy of Sciences
    Chinese Academy of Sciences
    Chinese Academy of Sciences)

Abstract

Adenosylcobalamin (AdoCbl), a biologically active form of vitamin B12 (coenzyme B12), is one of the most complex metal-containing natural compounds and an essential vitamin for animals. However, AdoCbl can only be de novo synthesized by prokaryotes, and its industrial manufacturing to date was limited to bacterial fermentation. Here, we report a method for the synthesis of AdoCbl based on a cell-free reaction system performing a cascade of catalytic reactions from 5-aminolevulinic acid (5-ALA), an inexpensive compound. More than 30 biocatalytic reactions are integrated and optimized to achieve the complete cell-free synthesis of AdoCbl, after overcoming feedback inhibition, the complicated detection, instability of intermediate products, as well as imbalance and competition of cofactors. In the end, this cell-free system produces 417.41 μg/L and 5.78 mg/L of AdoCbl using 5-ALA and the purified intermediate product hydrogenobyrate as substrates, respectively. The strategies of coordinating synthetic modules of complex cell-free system describe here will be generally useful for developing cell-free platforms to produce complex natural compounds with long and complicated biosynthetic pathways.

Suggested Citation

  • Qian Kang & Huan Fang & Mengjie Xiang & Kaixing Xiao & Pingtao Jiang & Chun You & Sang Yup Lee & Dawei Zhang, 2023. "A synthetic cell-free 36-enzyme reaction system for vitamin B12 production," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-40932-4
    DOI: 10.1038/s41467-023-40932-4
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    References listed on IDEAS

    as
    1. Meaghan A. Valliere & Tyler P. Korman & Nicholas B. Woodall & Gregory A. Khitrov & Robert E. Taylor & David Baker & James U. Bowie, 2019. "A cell-free platform for the prenylation of natural products and application to cannabinoid production," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    2. Tyler P. Korman & Paul H. Opgenorth & James U. Bowie, 2017. "A synthetic biochemistry platform for cell free production of monoterpenes from glucose," Nature Communications, Nature, vol. 8(1), pages 1-8, August.
    3. Huan Fang & Dong Li & Jie Kang & Pingtao Jiang & Jibin Sun & Dawei Zhang, 2018. "Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12," Nature Communications, Nature, vol. 9(1), pages 1-12, December.
    4. Saken Sherkhanov & Tyler P. Korman & Sum Chan & Salem Faham & Hongjiang Liu & Michael R. Sawaya & Wan-Ting Hsu & Ellee Vikram & Tiffany Cheng & James U. Bowie, 2020. "Isobutanol production freed from biological limits using synthetic biochemistry," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    5. Dario Mizrachi & Yujie Chen & Jiayan Liu & Hwei-Ming Peng & Ailong Ke & Lois Pollack & Raymond J. Turner & Richard J. Auchus & Matthew P. DeLisa, 2015. "Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy," Nature Communications, Nature, vol. 6(1), pages 1-10, November.
    6. Meaghan A. Valliere & Tyler P. Korman & Nicholas B. Woodall & Gregory A. Khitrov & Robert E. Taylor & David Baker & James U. Bowie, 2019. "Author Correction: A cell-free platform for the prenylation of natural products and application to cannabinoid production," Nature Communications, Nature, vol. 10(1), pages 1-1, December.
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