Using enantioselective biosensors to evolve asymmetric biocatalysts

Published

January 30, 2025

Author(s)

d’Oelsnitz Simon, Wantae Kim, Haley Hardtke, Svetlana Ikonomova, Nina Alperovich, Olga Vasilyeva, Michael James, Eric Zigon, Charlie Johnson, Andrew Ellington, Quincey Justman, Michael Springer, Jessie Zhang, Pamela Silver, David Ross

Abstract

Biocatalysts are championed for their exquisite stereochemistry, but slow chromatographic separations necessary to measure enantiomeric excess can bottleneck their development. Prokaryotic transcription factors can address this limitation by transducing enantiomer-specific chemical concentrations into programmable gene expression outputs. We describe a method for high-throughput chiral screening of asymmetric biocatalysts using enantioselective transcription factors. Using growth-coupled massively parallel sequencing, we quantitatively profile the sensitivity, selectivity, and dynamic range of >300,000 transcription factor variants generated by random, site-saturation, and gene shuffling mutagenesis. Ultimately, we identify biosensors that are both highly sensitive (EC50 as low as 2 µmol/L) and selective for enantiomer products and an achiral precursor of an intermediate used to manufacture the licensed pharmaceutical Solifenacin. High-resolution structures reveal how sterics enforce enantioselectivity and charge interactions differentiate the precursor imine from product amines. Finally, we apply our biosensors to invert the enantioselectivity of an imine reductase by screening a large enzyme library using fluorescence activated cell sorting. We demonstrate that enantioselective transcription factors enable rapid screening of asymmetric reactions, which may dramatically accelerate the pace of biocatalyst development for pharmaceutical manufacture.

Citation

Nature Biotechnology

Pub Type

Journals

Keywords

Synthetic biology, living measurement systems, protein engineering

Citation

Simon, D. , Kim, W. , Hardtke, H. , Ikonomova, S. , Alperovich, N. , Vasilyeva, O. , James, M. , Zigon, E. , Johnson, C. , Ellington, A. , Justman, Q. , Springer, M. , Zhang, J. , Silver, P. and Ross, D. (2025), Using enantioselective biosensors to evolve asymmetric biocatalysts, Nature Biotechnology (Accessed October 15, 2025)