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

42 Scopus citations

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

Funding

This work was supported by the Office of Energy Efficiency and Renewable Energy (EERE) under Contract No. DE-EE0008343 and the U.S. Department of Energy (DOE) Biological and Environmental Research Division (BER), Genomic Science Program (GSP) for funding of this project under Contract No. DE-SC0018249. DNA synthesis for the gene libraries was supported by the Joint Genome Institute Community Science Program under award no. CSP-503280; https://doi.org/10.46936/10.25585/60001121 (M.C.J. and M.K.). The work conducted by the U.S. Department of Energy Joint Genome Institute ( https://ror.org/04xm1d337 ), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under contract no. DE-AC02-05CH11231. M.C.J. acknowledges the David and Lucile Packard Foundation. We also thank the following investors in LanzaTech’s technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Novo Holdings A/S, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China, and Suncor. B.V. acknowledges support from the Swiss National Science Foundation via a SNSF Early Postdoc.Mobility fellowship (P2SKP3_184036). B.R.K. acknowledges support from the Ryan Fellowship, the International Institute for Nanotechnology at Northwestern University. L.S. was supported by the Zeno Karl Schindler Foundation. This work was supported by the Office of Energy Efficiency and Renewable Energy (EERE) under Contract No. DE-EE0008343 and the U.S. Department of Energy (DOE) Biological and Environmental Research Division (BER), Genomic Science Program (GSP) for funding of this project under Contract No. DE-SC0018249. DNA synthesis for the gene libraries was supported by the Joint Genome Institute Community Science Program under award no. CSP-503280; https://doi.org/10.46936/10.25585/60001121 (M.C.J. and M.K.). The work conducted by the U.S. Department of Energy Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy operated under contract no. DE-AC02-05CH11231. M.C.J. acknowledges the David and Lucile Packard Foundation. We also thank the following investors in LanzaTech’s technology: BASF, CICC Growth Capital Fund I, CITIC Capital, Indian Oil Company, K1W1, Khosla Ventures, the Malaysian Life Sciences, Capital Fund, L. P., Mitsui, the New Zealand Superannuation Fund, Novo Holdings A/S, Petronas Technology Ventures, Primetals, Qiming Venture Partners, Softbank China, and Suncor. B.V. acknowledges support from the Swiss National Science Foundation via a SNSF Early Postdoc.Mobility fellowship (P2SKP3_184036). B.R.K. acknowledges support from the Ryan Fellowship, the International Institute for Nanotechnology at Northwestern University. L.S. was supported by the Zeno Karl Schindler Foundation.

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy

Fingerprint

Dive into the research topics of 'Cell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria'. Together they form a unique fingerprint.

Cite this