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A synthetic cell-free pathway for biocatalytic upgrading of formate from electrochemically reduced CO2

Nature Chemical Engineering volume 3, pages 57–69 (2026)Cite this article

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Abstract

Electrochemical reduction of carbon dioxide (CO2) can produce important one-carbon (C1) feedstocks for sustainable biomanufacturing, such as formate. Unfortunately, natural formate assimilation pathways are inefficient and constrained to organisms that are difficult to engineer. Here we establish a synthetic reductive formate pathway (ReForm) in vitro. ReForm is a six-step pathway consisting of five engineered enzymes catalyzing nonnatural reactions to convert formate into the universal biological building block acetyl-CoA. We establish ReForm by selecting enzymes among 66 candidates from prokaryotic and eukaryotic origins. Through iterative cycles of engineering, we create and evaluate 3,173 sequence-defined enzyme mutants, tune cofactor concentrations and adjust enzyme loadings to increase pathway activity toward the model end product malate. We demonstrate that ReForm can accept diverse C1 substrates, including formaldehyde, methanol and formate produced from the electrochemical reduction of CO2. Our work expands the repertoire of synthetic C1 utilization pathways, with implications for synthetic biology and the development of a formate-based bioeconomy.

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Fig. 1: The synthetic reductive formate pathway.
Fig. 2: Engineering of a phosphoketolase into an acetyl-Pi synthase.
Fig. 3: Engineering distinct substrate-specific acyl-CoA reductases.
Fig. 4: Enzyme engineering campaigns for specific acyl-CoA reductases and an active acyl-CoA synthetase.
Fig. 5: Chemoenzymatic conversion of C1 substrates into acetyl-CoA.

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Data availability

All data are available in the Article or its Supplementary Information. Source data are provided with this paper. Atomic structures reported in this Article are deposited to the Protein Data Bank under accession codes 9CD3 and 9CD4. The cryo-EM data were deposited to the Electron Microscopy Data Bank under EMD-45461 and EMD-45462.

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Acknowledgements

Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. This work made use of the IMSERC MS facility at Northwestern University, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-2025633), the State of Illinois and the International Institute for Nanotechnology (IIN). We thank Stanford University Cryo-EM center (cEMc) and particularly B. Singal for providing support for cryo-EM grid preparation, data collection, processing and structure determination pipeline. We thank F. ‘Ralph’ Tobias for his help in developing analytical methods for malate detection and K. Seki for his help in gathering intact protein MS data on the deacetylated acyl-CoA synthetases. We also thank J. W. Bogart for conversations regarding this work. Funding was provided by the Department of Energy (DE-SC0023278) (G.M.L., B.V., K.Z., I.M., A.G., R.L., C.T., E.H.S., A.S.K. and M.C.J.), NSF GRFP (G.M.L.) and Stanford University Cryo-electron Microscopy Center (cEMc) (B.S.).

Author information

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Grant M. Landwehr, Bastian Vogeli, Anika Gupta, Rebeca Lion, Ashty S. Karim & Michael C. Jewett

  2. Center for Synthetic Biology, Northwestern University, Evanston, IL, USA

    Grant M. Landwehr, Bastian Vogeli, Anika Gupta, Rebeca Lion, Ashty S. Karim & Michael C. Jewett

  3. Department of Chemistry, Northwestern University, Evanston, IL, USA

    Cong Tian & Edward H. Sargent

  4. Stanford SLAC CryoEM Initiative, Stanford University, Stanford, CA, USA

    Bharti Singal

  5. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Kyle Zolkin, Irene Martinez & Michael C. Jewett

Authors
  1. Grant M. Landwehr
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Contributions

Conceptualization: A.S.K., B.V., G.M.L. and M.C.J. Methodology: B.V. and G.M.L. Investigation: A.G., C.T., G.M.L., I.M., K.Z. and R.L. Cryo-EM: B.S. Supervision: A.S.K., M.C.J. and E.H.S. Funding acquisition: A.S.K., M.C.J. and E.H.S. Writing: A.S.K., G.M.L. and M.C.J.

Corresponding authors

Correspondence to Ashty S. Karim or Michael C. Jewett.

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Competing interests

G.M.L., B.V., A.S.K. and M.C.J. have filed an invention disclosure based on the work presented. M.C.J. has a financial interest in National Resilience, Gauntlet Bio, Pearl Bio, Inc., and Stemloop Inc. M.C.J.’s interests are reviewed and managed by Northwestern University and Stanford University in accordance with their competing interest policies. All other authors declare no competing interests.

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Nature Chemical Engineering thanks Mattheos Koffas, Zaigao Tan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Landwehr, G.M., Vogeli, B., Tian, C. et al. A synthetic cell-free pathway for biocatalytic upgrading of formate from electrochemically reduced CO2. Nat Chem Eng 3, 57–69 (2026). https://doi.org/10.1038/s44286-025-00315-6

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