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Peer-reviewedCC BY-NC-ND 4.0Nature Chemical Biology

Using enantioselective biosensors to evolve asymmetric biocatalysts

  • Simon d’Oelsnitz1,2,3
  • Wantae Kim4
  • Nicole N. Zhao3
  • Haley Hardtke5
  • Svetlana P. Ikonomova6
  • Nina Alperovich6
  • Olga Vasilyeva6
  • Michael J. James7
  • Eric S. Zigon2
  • Michael B. Cory1,3
  • Charlie D. Johnson5
  • Andrew D. Ellington5,8
5 more authors
  • Quincey A. Justman1,3
  • Michael Springer1,2,3
  • Y. Jessie Zhang5
  • Pamela A. Silver1,2,3
  • David Ross6
8 affiliations
  1. 1Synthetic Biology HIVE, Department of Systems Biology, Harvard Medical School, Boston, MA USA
  2. 2Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA USA
  3. 3Department of Systems Biology, Harvard Medical School, Boston, MA USA
  4. 4McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX USA
  5. 5Department of Molecular Biosciences, University of Texas at Austin, Austin, TX USA
  6. 6National Institute of Standards and Technology, Gaithersburg, MD USA
  7. 7Department of Microbiology, Harvard Medical School, Boston, MA USA
  8. 8Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX USA
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Abstract

Biocatalysts are prized for their enantioselectivity, but slow chromatographic separations required to measure enantiomeric excess bottleneck their development. To overcome this limitation, we evolve enantioselective transcription factors (eTFs) that convert enzyme-catalyzed enantiomer concentrations into programmable gene expression outputs, focusing on imine reductases. Here, using a massively parallel reporter assay, we measure dose–response curves for over 300,000 transcription factor variants in response to an imine precursor and chiral amine products. We quantify the sensitivity, selectivity and dynamic range across variants generated by random, site-saturation and shuffling mutagenesis, isolating variants with exceptional specificity. High-resolution structures of evolved eTFs elucidate how steric effects enforce enantioselectivity, while charge interactions distinguish the imine from the amines. Using two eTFs, we create an ultrahigh-throughput chiral screen to evolve an imine reductase with inverted enantioselectivity. To support generalizability and speed, we design a genetic circuit that enables TF generation within weeks. Our methods enable rapid measurement of asymmetric reactions, supporting innovation in chemical manufacturing.

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Cite

BibTeXRIS
BibTeX
@article{doelsnitz2026using,
  title = {{Using enantioselective biosensors to evolve asymmetric biocatalysts}},
  author = {Simon d’Oelsnitz and Wantae Kim and Nicole N. Zhao and Haley Hardtke and Svetlana P. Ikonomova and Nina Alperovich and Olga Vasilyeva and Michael J. James and Eric S. Zigon and Michael B. Cory and Charlie D. Johnson and Andrew D. Ellington and Quincey A. Justman and Michael Springer and Y. Jessie Zhang and Pamela A. Silver and David Ross},
  journal = {Nature Chemical Biology},
  year = {2026},
  month = jul,
  doi = {10.1038/s41589-026-02275-1},
  url = {https://doi.org/10.1038/s41589-026-02275-1},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - Using enantioselective biosensors to evolve asymmetric biocatalysts
AU  - Simon d’Oelsnitz
AU  - Wantae Kim
AU  - Nicole N. Zhao
AU  - Haley Hardtke
AU  - Svetlana P. Ikonomova
AU  - Nina Alperovich
AU  - Olga Vasilyeva
AU  - Michael J. James
AU  - Eric S. Zigon
AU  - Michael B. Cory
AU  - Charlie D. Johnson
AU  - Andrew D. Ellington
AU  - Quincey A. Justman
AU  - Michael Springer
AU  - Y. Jessie Zhang
AU  - Pamela A. Silver
AU  - David Ross
PY  - 2026
DA  - 2026/07/23
JO  - Nature Chemical Biology
DO  - 10.1038/s41589-026-02275-1
UR  - https://doi.org/10.1038/s41589-026-02275-1
AB  - Biocatalysts are prized for their enantioselectivity, but slow chromatographic separations required to measure enantiomeric excess bottleneck their development. To overcome this limitation, we evolve enantioselective transcription factors (eTFs) that convert enzyme-catalyzed enantiomer concentrations into programmable gene expression outputs, focusing on imine reductases. Here, using a massively parallel reporter assay, we measure dose–response curves for over 300,000 transcription factor variants in response to an imine precursor and chiral amine products. We quantify the sensitivity, selectivity and dynamic range across variants generated by random, site-saturation and shuffling mutagenesis, isolating variants with exceptional specificity. High-resolution structures of evolved eTFs elucidate how steric effects enforce enantioselectivity, while charge interactions distinguish the imine from the amines. Using two eTFs, we create an ultrahigh-throughput chiral screen to evolve an imine reductase with inverted enantioselectivity. To support generalizability and speed, we design a genetic circuit that enables TF generation within weeks. Our methods enable rapid measurement of asymmetric reactions, supporting innovation in chemical manufacturing.
SN  - 1552-4450
SN  - 1552-4469
ER  -