Using enantioselective biosensors to evolve asymmetric biocatalysts

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Chemical Biology

Background:

• Biocatalysts offer high enantioselectivity but are hindered by slow chiral separation methods.
• Measuring enantiomeric excess is crucial for developing enantioselective biocatalysts, particularly imine reductases.

Purpose of the Study:

• To develop a rapid, high-throughput method for measuring enantiomer concentrations.
• To evolve enantioselective transcription factors (eTFs) for quantifying chiral amine products from imine reduction.

Main Methods:

• Massively parallel reporter assays were used to screen over 300,000 transcription factor variants.
• Mutagenesis strategies including random, site-saturation, and shuffling were employed.
• High-resolution structural analysis elucidated the mechanisms of enantioselectivity.

Main Results:

• Identified transcription factor variants with high sensitivity and specificity for chiral amines.
• Evolved eTFs demonstrated the ability to distinguish imine precursors from chiral amine products.
• A genetic circuit enabled rapid generation of transcription factors within weeks.

Conclusions:

• The developed eTFs provide a rapid readout for asymmetric reactions, accelerating biocatalyst development.
• This approach facilitates ultrahigh-throughput screening for enzyme evolution, including achieving inverted enantioselectivity.
• The method supports innovation in chemical manufacturing by enabling faster optimization of biocatalytic processes.