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Acetyl-CoA synthesis through a bicyclic carbon-fixing pathway in gas-fermenting bacteria

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Abstract

Gas-fermenting acetogens can upgrade one-carbon (C1) compounds (such as CO2 and CO) to the two-carbon (C2) metabolite acetyl coenzyme A (CoA) and convert sugar feedstocks to acetyl-CoA with minimal CO2 emissions. Fulfilling the biosynthetic potential of these microbes requires overcoming challenges in pathway engineering. Here we design a synthetic acetyl-CoA bi-cycle—in addition to the natural carbon-fixing pathways—for C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in sequence: carbon fixation, gluconeogenesis and non-oxidative glycolysis. The pathway was examined by in silico thermodynamic and kinetic analyses. The prototypic pathway was implemented in a syngas-fermenting organism, Clostridium ljungdahlii DSM 13528, by expressing a heterologous phosphoketolase that can work with other native enzymes in the host acetogen. The carbon conversion pathway is possible under various growth conditions and is independent of the Wood–Ljungdahl pathway for the valorization of H2 and CO2. This study reports the improvement of carbon conversion using a reductive acetyl-CoA bi-cycle and the potential impact of redox homoeostasis in the acetogenic host for industrial applications of gas fermentation.

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Fig. 1: Reductive acetyl-CoA bi-cycle for one-carbon fixation.
Fig. 2: Catalytic properties and robustness of the reductive acetyl-CoA bi-cycle pathway.
Fig. 3: Contour profiling of C2 metabolites productivity as a function of the WLP and the reductive acetyl-CoA bi-cycle activity.
Fig. 4: Functionality of carbon fixation and gluconeogenesis modules proved by labelling experiment using [U-13C2] acetate.
Fig. 5: Pathway enzyme allocation.
Fig. 6: Functionality of gluconeogenesis and NOG modules proved by labelling experiment using [1-13C] pyruvate.
Fig. 7: Growth and production of the host C. ljungdahlii strains carrying acetyl-CoA bi-cycle.
Fig. 8: Phylogenetic heat map.

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Data availability

The authors declare that all data supporting the findings of this study are available within this paper and the Supplementary Information.

Code availability

The scripts and models used in this paper are available at https://doi.org/10.5281/zenodo.4548907. The code for thermodynamics, enzyme protein cost and robustness analysis can be found at https://github.com/Chaowu88/PathParser.

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Acknowledgements

We acknowledge A.M. Grunden at North Carolina State University for providing an ACS-deficient C. ljungdahlii OTA1. This work was authored in part by the National Renewable Energy Laboratory, operated by the Alliance for Sustainable Energy for the US Department of Energy (DOE) under contract no. DE-AC36-08GO28308. This work is supported by a Laboratory Directed Research and Development (LDRD) project at the National Renewable Energy Laboratory to W.X., C.W., J.L., X.G. and K.J.C., partially by Shell International Exploration and Production to W.X., P.M. and J.L., a DOE Bioenergy Technologies Office (BETO) Co-Optimization of Fuels and Engines (Co-Optima) project to W.X., C.W., B.Y., J.H., C.U., D.P. and N.T., as well as the Start-up Fund of Miami University to X.W. and S.S. The views expressed in the article do not necessarily represent the views of the DOE or the US Government. The US Government retains and the publisher, by accepting the article for publication, acknowledges that the US Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for US Government purposes.

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Author notes

  1. These authors contributed equally: Chao Wu, Jonathan Lo.

Authors and Affiliations

  1. Biosciences Center, National Renewable Energy Laboratory, Golden, CO, USA

    Chao Wu, Jonathan Lo, Chris Urban, Xiang Gao, Bin Yang, Jonathan Humphreys, Katherine J. Chou, PinChing Maness & Wei Xiong

  2. Department of Microbiology, Miami University, Oxford, OH, USA

    Shrameeta Shinde & Xin Wang

  3. Shell International Exploration and Production Inc, Houston, TX, USA

    Nicolas Tsesmetzis & David Parker

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Contributions

W.X. projects conception. C.W., W.X. designed and performed all computational biology analysis. J.L. designed and performed genetic engineering on C. ljungdahlii. J.L., C.U., X.G., B.Y., J.H. and W.X. performed experiments including cell culture, enzyme assay, gas fermentation and product measurement. C.W., C.U., J.H., X.G. and W.X. designed and performed 13C-labelling experiments and data analysis. S.S., X.W., C.W., and W.X. generated and analysed proteomics data. C.W. and W.X. wrote the manuscript with input from all co-authors and revisions from K.J.C., P.M., N.T. and D.P.

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Correspondence to Wei Xiong.

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Nature Synthesis thanks Stephen Ragsdale, Michael Köpke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling editor: Thomas West, in collaboration with the Nature Synthesis team.

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Wu, C., Lo, J., Urban, C. et al. Acetyl-CoA synthesis through a bicyclic carbon-fixing pathway in gas-fermenting bacteria. Nat. Synth 1, 615–625 (2022). https://doi.org/10.1038/s44160-022-00095-4

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