Genistein, a bioactive isoflavone with therapeutic potential in treating oxidative stress, cardiovascular diseases,
and cancer, faces production limitations during microbial biosynthesis due to product feedback inhibition of
isoflavone synthase (IFS). To overcome this bottleneck, a biosensor-assisted continuous directed evolution
platform was developed. Specifically, a genistein-specific biosensor was engineered using the Bradyrhizobium
japonicum transcription factor FrrA, capable of distinguishing genistein from its precursor, (2S)-naringenin. The
biosensor was systematically optimized to eliminate background fluorescence at inhibitory genistein concen
trations while maintaining strong responses to elevated product levels, enabling precise detection during high- throughput screening. By coupling this biosensor with droplet microfluidic sorting, a high-quality mutant library
of Trifolium pratense IFS (TpIFS) generated through deaminase-T7 RNA polymerase fusion-mediated continuous
evolution, was screened. This approach successfully identified TpIFS
M6
, a feedback-resistant mutant exhibiting a
6.6-fold increase in the product inhibition constant and a 3.8-fold reduced binding affinity for genistein.
Consequently, the genistein yield obtained with TpIFS
M6
was 3.1 times that of the wild-type. Molecular dynamics
simulations revealed that mutations I187R and F303A prevented inhibitor-induced conformational displacement
of the catalytic I-helix. This work establishes a generalizable high-throughput screening strategy to mitigate
enzyme feedback inhibition, facilitating the robust biosynthesis of plant-derived natural products.