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

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. Brown, Sean D. Simpson, Milan Mrksich, Ashty S. Karim, Ramon Gonzalez, Michael Köpke, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article number3058
JournalNature communications
Volume13
Issue number1
DOIs
StatePublished - Dec 2022

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • General
  • Physics and Astronomy(all)

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