Directed evolution is a highly effective strategy to generate proteins with tailored applications such as catalytic selectivity, thermostability, kcat etc. SpeedyGenes is a method of generating bespoke protein libraries through overlaping oligonucleotides. Oligonucleotides are designed computationally and in combination with targeted degenerate codons (CodonGenie) this method can yield very large and yet highly targeted variant libraries. As an initial demonstration of the application of SpeedyGenes at DTU Biosustain we generated a variant library of the aromatic amino acid transporter yddG. We exploited a pangenomic dataset for site selection, and designed a library with alterations at 45 positions each containing 2-20 variant amino acids for a total library size of 10²³. A growth based selection assay was used to isolate transporters with enhanced function and sequencing revealed specific sites of variation.

References:

Sadler et al. SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Variant Libraries for Directed Evolution. (2022)

Currin et al. SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution. (2014)