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Shifting redox reaction equilibria on demand using an orthogonal redox cofactor

Nature Chemical Biology (2024)Cite this article

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Abstract

Nature’s two redox cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN+) as a noncanonical cofactor orthogonal to NAD(P)+. Here we present the development of Nox Ortho, a reduced NMN+ (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN+ ratio together with an NMN+-specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~103–106-fold cofactor specificity switch from NAD(P)+ to NMN+. We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)’s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).

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Fig. 1: Overview of the four proposed BDO stereo-upgrading systems for testing the crosstalk of two orthogonal cofactors.
Fig. 2: Directed evolution of Ll Nox to exclude NADH.
Fig. 3: Engineering Bdhs to use NMN(H).
Fig. 4: Orthogonal redox driving forces enable BDO stereo-upgrading in four cell-free systems.
Fig. 5: Resting cell stereo-upgrading of m-BDO to (SS)-BDO in E. coli.
Fig. 6: Visualizing emerging design principles for noncanonical redox cofactor-specific enzyme design.

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Data availability

Information on the strains and plasmids used in this study is available in Supplementary Data. Accession codes for genes investigated in this study can be found in Supplementary Tables 4 and 5. All DNA sequence and structural information used is publicly available (Ser Bdh2, UniProt A0A7U3Z3T2; Kp Dar, UniProt D7RP28; Bs BdhA, UniProt O34788; Cs Bdh, UniProt M1MWX5; Bs Gdh, UniProt P12310; Ll Nox, UniProt A2RIB7; Lb Nox, UniProt Q03Q85, PDB 5VN0; Tp Nox (Lb Nox G159A;D177A;A178R;M179S;P184R); Ecl Dar, GenBank JN035909; Kp BudC, PDB 1GEG; Ka BudC, National Center for Biotechnology Information (NCBI) Ref WP_015366942; Ka Dar, GenBank VEC79507; Ko BudC, GenBank AEX06195; As Bdh, GenBank NLH91458; Cr Bdh, GenBank AEI90716; Ca Bdh, NCBI Ref WP_073006451; Pb Bdh, NCBI Ref WP_148550966; Cbo Bdh, NCBI Ref WP_075141790; Km Bdh, NCBI Ref WP_102401827; Cbu Bdh, NCBI Ref WP_104675707; Zp Bdh, NCBI Ref WP_027704711). All data generated in this study are provided in the Supplementary Information. Source data are provided with this paper.

Code availability

The input files and source code for the Rosetta molecular simulation and MD simulations are publicly accessible on Zenodo (https://doi.org/10.5281/zenodo.11478967)80.

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Acknowledgements

H.L. acknowledges support from the University of California, Irvine, the National Science Foundation (NSF) (award nos. 1847705 and MCB-2328145), the National Institutes of Health (NIH) (award no. DP2 GM137427), an Alfred Sloan research fellowship and the Advanced Research Projects Agency—Energy (award no. DE-AR0001508). Y.C., E.L. and J.B.S. acknowledge the funding of the National Institute of Environmental Health Sciences (grant no. P42ES004699), the NIH (grant no. R01 GM 076324-11) and the NSF (grant nos. 1627539, 1805510 and 1827246). R.L. acknowledges support from the NIH (award no. R35 GM130367). D.A acknowledges support from the NSF Graduate Research Fellowship Program (grant no. DGE1839285). We acknowledge valuable technical support provided by AAT Bioquest for the redox ratio kits.

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Author notes

  1. These authors contributed equally: Derek Aspacio, Yulai Zhang, Youtian Cui.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA

    Derek Aspacio, Yulai Zhang, William B. Black, Sean Perea, Qiang Zhu, Yongxian Wu, Ray Luo & Han Li

  2. Genome Center, University of California, Davis, Davis, CA, USA

    Youtian Cui, Emma Luu & Justin B. Siegel

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA

    Edward King, Qiang Zhu, Yongxian Wu & Ray Luo

  4. Department of Material Science and Engineering, University of California, Irvine, Irvine, CA, USA

    Qiang Zhu, Yongxian Wu & Ray Luo

  5. Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA

    Qiang Zhu, Yongxian Wu, Ray Luo & Han Li

  6. Department of Chemistry, University of California, Davis, Davis, CA, USA

    Justin B. Siegel

  7. Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, CA, USA

    Justin B. Siegel

  8. Department of Biological Chemistry, University of California, Irvine, Irvine, CA, USA

    Han Li

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Contributions

D.A., Y.Z., E.K. and H.L. designed the experiments. Y.C. and E.L. performed Rosetta modeling. E.L. and D.A. performed bioinformatic analysis of the NAD(P)-binding Rossmann-like domain superfamily. E.K. and Y.Z. performed rational protein engineering experiments. Y.Z. performed growth-based selection of Nox. Q.Z., Y.W. and R.L. performed MD simulations and analyzed the results. D.A. and E.K. bioprospected Bdh homologs. D.A. performed the Michaelis–Menten kinetic experiments for Bdhs. Y.Z. performed the Michaelis–Menten kinetic experiments for Nox. D.A. and S.P. performed cell-free biotransformation experiments. D.A. performed the resting cell biotransformation experiments. W.B.B. and D.A. reformulated the colorimetric redox ratio assay. D.A. performed the colorimetric redox ratio assays. Y.C., E.L. and J.B.S. analyzed the modeling results. All authors analyzed the data and wrote the paper.

Corresponding author

Correspondence to Han Li.

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The authors declare no competing interests.

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Nature Chemical Biology thanks Zhen Chen, Steffen Lindner and Carine Vergne-Vaxelaire for their contribution to the peer review of this work.

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Aspacio, D., Zhang, Y., Cui, Y. et al. Shifting redox reaction equilibria on demand using an orthogonal redox cofactor. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01702-5

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