Exploring Antibiotic Resistance in Diverse Homologs of the Dihydrofolate Reductase Protein Family through Broad Mutational Scanning

Conventional antibiotic resistance studies often focus on individual protein fitness and neglect broader protein family dynamics due to methodological limitations. However, understanding how resistance develops within protein families can provide crucial insights into its spread. Leveraging DropSynth, a scalable, low-cost multiplexed gene synthesis method, we constructed a phylogenetically diverse library of 1,536 synthetic gene homologs encoding dihydrofolate reductase (DHFR), a key antibiotic target. We then employed a multiplexed in-vivo assay to assess their ability to complement metabolic function in an E. coli knockout strain and their resistance to the antibiotic trimethoprim. Using Broad Mutational Scanning (BMS), we evaluated the functional effects of over 100,000 mutant variants across nine conditions, representing nearly 780 unique species within the DHFR protein family. Our DropSynth-assembled gene library provides unprecedented insights into the fitness landscape of DHFR evolution in a single experiment, with the potential to guide future strategies to combat antibiotic resistance.