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

Vögeli, Bastian; Schulz, Luca; Garg, Shivani; Tarasava, Katia; Clomburg, James M.; Lee, Seung Hwan; Gonnot, Aislinn; Moully, Elamar Hakim; Kimmel, Blaise R.; Tran, Loan; Zeleznik, Hunter; Brown, Steven D.; Simpson, Sean D.; Mrksich, Milan; Karim, Ashty S.; Gonzalez, Ramon; Köpke, Michael; Jewett, Michael C.

doi:10.1038/s41467-022-30571-6

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

Journal Article · 31 May 2022 · Nature Communications

DOI: https://doi.org/10.1038/s41467-022-30571-6 · OSTI ID:1870722

;

; Garg, Shivani; Tarasava, Katia; Clomburg, James M.;

; Gonnot, Aislinn; Moully, Elamar Hakim;

; Tran, Loan; Zeleznik, Hunter; Brown, Steven D.; Simpson, Sean D.;

;

Abstract Carbon-negative synthesis of biochemical products has the potential to mitigate global CO ₂ 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.

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Research Organization:: Northwestern Univ., Evanston, IL (United States); Northwestern University, Evanston, IL (United States); University of California, Oakland, CA (United States)

Sponsoring Organization:: David and Lucile Packard Foundation; Joint Genome Institute Community Science Program; Swiss National Science Foundation; USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; EE0008354; SC0018249

OSTI ID:: 1870722

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 13; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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