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Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria

Author

Listed:
  • Bastian Vögeli

    (Northwestern University)

  • Luca Schulz

    (Northwestern University)

  • Shivani Garg

    (LanzaTech Inc.)

  • Katia Tarasava

    (University of South Florida)

  • James M. Clomburg

    (LanzaTech Inc.
    University of South Florida)

  • Seung Hwan Lee

    (University of South Florida)

  • Aislinn Gonnot

    (LanzaTech Inc.)

  • Elamar Hakim Moully

    (Northwestern University
    Northwestern University)

  • Blaise R. Kimmel

    (Northwestern University)

  • Loan Tran

    (LanzaTech Inc.)

  • Hunter Zeleznik

    (LanzaTech Inc.)

  • Steven D. Brown

    (LanzaTech Inc.)

  • Sean D. Simpson

    (LanzaTech Inc.)

  • Milan Mrksich

    (Northwestern University
    Northwestern University
    Northwestern University)

  • Ashty S. Karim

    (Northwestern University)

  • Ramon Gonzalez

    (University of South Florida)

  • Michael Köpke

    (LanzaTech Inc.)

  • Michael C. Jewett

    (Northwestern University)

Abstract

Carbon-negative synthesis of biochemical products has the potential to mitigate global CO2 emissions. An attractive route to do this is the reverse β-oxidation (r-BOX) pathway coupled to the Wood-Ljungdahl pathway. Here, we optimize and implement r-BOX for the synthesis of C4-C6 acids and alcohols. With a high-throughput in vitro prototyping workflow, we screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity. Implementation of these pathways into Escherichia coli generates designer strains for the selective production of butanoic acid (4.9 ± 0.1 gL−1), as well as hexanoic acid (3.06 ± 0.03 gL−1) and 1-hexanol (1.0 ± 0.1 gL−1) at the best performance reported to date in this bacterium. We also generate Clostridium autoethanogenum strains able to produce 1-hexanol from syngas, achieving a titer of 0.26 gL−1 in a 1.5 L continuous fermentation. Our strategy enables optimization of r-BOX derived products for biomanufacturing and industrial biotechnology.

Suggested Citation

  • Bastian Vögeli & Luca Schulz & Shivani Garg & Katia Tarasava & James M. Clomburg & Seung Hwan Lee & Aislinn Gonnot & Elamar Hakim Moully & Blaise R. Kimmel & Loan Tran & Hunter Zeleznik & Steven D. Br, 2022. "Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-30571-6
    DOI: 10.1038/s41467-022-30571-6
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    References listed on IDEAS

    as
    1. Clementina Dellomonaco & James M. Clomburg & Elliot N. Miller & Ramon Gonzalez, 2011. "Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals," Nature, Nature, vol. 476(7360), pages 355-359, August.
    2. Blake J. Rasor & Xiunan Yi & Hunter Brown & Hal S. Alper & Michael C. Jewett, 2021. "An integrated in vivo/in vitro framework to enhance cell-free biosynthesis with metabolically rewired yeast extracts," Nature Communications, Nature, vol. 12(1), pages 1-9, December.
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