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2-Hydroxyacyl-CoA lyase catalyzes acyloin condensation for one-carbon bioconversion

Nature Chemical Biology volume 15, pages 900–906 (2019)Cite this article

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Abstract

Despite the potential of biotechnological processes for one-carbon (C1) bioconversion, efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts, here we report that 2-hydroxyacyl CoA lyase (HACL), an enzyme involved in mammalian α-oxidation, catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity, including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechanism. The use of formyl-CoA as a C1 donor provides kinetic advantages and enables C1 bioconversion to multi-carbon products, demonstrated here by engineering an Escherichia coli whole-cell biotransformation system for the synthesis of glycolate and 2-hydroxyisobutyrate from formaldehyde and formaldehyde plus acetone, respectively. Our work establishes a new approach for C1 bioconversion and the potential for HACL-based pathways to support synthetic methylotrophy.

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Fig. 1: Known and hypothesized reactions catalyzed by HACL.
Fig. 2: Implementation of HACL-based C1 biocatalysis.
Fig. 3: Homology-guided site-directed mutagenesis of RuHACL.

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All data supporting the findings of this study are included in the paper and its supplementary information files.

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Acknowledgements

We thank S. Garg for assistance with protein purification methods and S. Cheong for assistance with genetic methods. This work was partially supported by a grant from the US National Science Foundation (no. CBET-1605999).

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Author notes

  1. Shuai Qian

    Present address: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA

    Alexander Chou, James M. Clomburg, Shuai Qian & Ramon Gonzalez

  2. Department of Chemical and Biomedical Engineering, University of South Florida, Tampa, FL, USA

    Alexander Chou, James M. Clomburg & Ramon Gonzalez

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Contributions

R.G. conceptualized the research and supervised the project. A.C., J.M.C., S.Q. and R.G. designed the methodology. A.C. identified and characterized HACL variants and performed cell-free experiments. S.Q. modeled and analyzed the structure of RuHACL and designed mutations. J.M.C. performed whole-cell experiments. A.C. and S.Q. constructed E. coli strains. A.C., J.M.C. and S.Q. analyzed the data. A.C. and R.G. prepared the manuscript with feedback from all authors.

Corresponding author

Correspondence to Ramon Gonzalez.

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Competing interests

A.C., J.M.C. and R.G. are co-inventors and assignees on a patent application (PCT/US2015/058121), which relates to the reported research. Correspondence and requests for materials should be addressed to ramongonzale@usf.edu.

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Supplementary information

Supplementary Information

Supplementary Table 1–3, Supplementary Figs. 1–11

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Supplementary Dataset

RuHACLmodel

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Chou, A., Clomburg, J.M., Qian, S. et al. 2-Hydroxyacyl-CoA lyase catalyzes acyloin condensation for one-carbon bioconversion. Nat Chem Biol 15, 900–906 (2019). https://doi.org/10.1038/s41589-019-0328-0

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