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Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases

Nature Chemistry (2024)Cite this article

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Abstract

Despite their intriguing photophysical and photochemical activities, naturally occurring photoenzymes have not yet been repurposed for new-to-nature activities. Here we engineered fatty acid photodecarboxylases to catalyse unnatural photoredox radical C–C bond formation by leveraging the strongly oxidizing excited-state flavoquinone cofactor. Through genome mining, rational engineering and directed evolution, we developed a panel of radical photocyclases to facilitate decarboxylative radical cyclization with excellent chemo-, enantio- and diastereoselectivities. Our high-throughput experimental workflow allowed for the directed evolution of fatty acid photodecarboxylases. An orthogonal set of radical photocyclases was engineered to access all four possible stereoisomers of the stereochemical dyad, affording fully diastereo- and enantiodivergent biotransformations in asymmetric radical biocatalysis. Molecular dynamics simulations show that our evolved radical photocyclases allow near-attack conformations to be easily accessed, enabling chemoselective radical cyclization. The development of stereoselective radical photocyclases provides unnatural C–C-bond-forming activities in natural photoenzyme families, which can be used to tame the stereochemistry of free-radical-mediated reactions.

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Fig. 1: Repurposing and directed evolution of FAPs as new-to-nature stereoselective RAPs.
Fig. 2: Development of chemo- and stereoselective RAPs: mining and engineering of FAPs.
Fig. 3: Substrate scope of RAP-catalysed asymmetric decarboxylative radical cyclization.
Fig. 4
Fig. 5: Utility of evolved CvRAP1–CvRAP6.
Fig. 6: MD simulations on radical cyclization within the enzyme active site.

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All data are available in the main text and the Supplementary Information.

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Acknowledgements

This research is supported by the National Institutes of Health (NIH; R35GM128779 to P.L., R35GM147387 to Y.Y.), American Chemical Society Petroleum Research Fund (ACS PRF; 65807-DNI1) and the National Science Foundation (NSF; OCE-1756947 to D.L.V.). We acknowledge the NSF BioPACIFIC Materials Innovation Platform (MIP; DMR-1933487) and NSF Materials Research Science and Engineering Centers (MRSEC) at University of California, Santa Barbara (UCSB; DMR-2308708) for access to instrumentation. Computational studies were carried out at the University of Pittsburgh Center for Research Computing and the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) programme, supported by NSF award numbers OAC-2117681 and OAC-2138259. We thank Y. Wang for critical reading of this manuscript.

Author information

Author notes

  1. These authors contributed equally: Shuyun Ju, Dian Li.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA, USA

    Shuyun Ju, Dian Li, Xin Liu, Jianping Wu & Yang Yang

  2. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Binh Khanh Mai & Peng Liu

  3. Interdepartmental Graduate Program for Marine Science, University of California Santa Barbara, Santa Barbara, CA, USA

    Alec Vallota-Eastman

  4. Marine Science Institute, University of California Santa Barbara, Santa Barbara, CA, USA

    David L. Valentine

  5. Department of Earth Science, University of California Santa Barbara, Santa Barbara, CA, USA

    David L. Valentine

  6. Biomolecular Science and Engineering (BMSE) Program, University of California Santa Barbara, Santa Barbara, CA, USA

    Yang Yang

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Contributions

Y.Y. conceived and directed the project. S.J. performed all the enzyme engineering and substrate scope studies. S.J. and A.V.-E. performed enzyme mining and molecular cloning with Y.Y. and D.L.V., and J.W. provided guidance. D.L., S.J. and X.L. synthesized all the substrates and racemic products for analysis. B.K.M. carried out the computational studies with P.L. providing guidance. Y.Y., P.L., S.J. and B.K.M. wrote the manuscript with the input of all other authors.

Corresponding authors

Correspondence to Peng Liu or Yang Yang.

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Competing interests

Y.Y. and S.J. are inventors on a patent application submitted by the University of California Santa Barbara (UC case no. 2024-843) that covers compositions, methods and applications of evolved RAPs derived from natural FAPs. The remaining authors declare no competing interests.

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Nature Chemistry thanks Dunming Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Origin of stereoconvergent photobiocatalytic radical cyclisation with (E)- and (Z)-1a.

a, Stereoconvergent photobiotransformation of (E)-1a and (Z)-1a. b, Photoisomerisation of (Z)-1a with FAD (free cofactor). c, Initial rate and photoisomerisation with CvRAP1.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Discussion and Tables 1–26.

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Ju, S., Li, D., Mai, B.K. et al. Stereodivergent photobiocatalytic radical cyclization through the repurposing and directed evolution of fatty acid photodecarboxylases. Nat. Chem. (2024). https://doi.org/10.1038/s41557-024-01494-0

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