High-Throughput Evolution of in Vitro Orthogonal Translation Systems Via an Integrated Platform Merging Cell-Free Protein Synthesis and Microfluidics

The extraordinary power of protein biosynthesis in conjunction with site-specific non-standard amino acid (nsAA) incorporation into a growing peptide chain has opened a new frontier in the chemistry of life. Site-specific nsAA incorporation provides not only new protein function and structure, but also endows unique chemical properties. However, nsAA incorporation efficiency is about 1000x lower than that of natural amino acids in translation due to the challenge of reengineering the protein translation apparatus, a complex biochemical orchestra composed of nearly 80 macromolecules including proteins, RNAs, and cofactors. To address this challenge, we have developed a high-throughput platform for the evolution of an in vitro orthogonal translation system (OTS) that improves nsAA incorporation efficiency. For the synthesis of proteins containing nsAAs, we utilized the crude-lysate cell-free protein synthesis (CFPS) system. Coupling this protein biosynthesis platform to efficient microfluidic devices capable of droplet generation, merging, and sorting, we established a high-throughput evolution platform capable of screening a large library of translation machinery variants. We evolved one residue of the tRNA binding interface and two residues of the amino acid binding pocket of an aminoacyl-tRNA synthetase (aaRS) from M. jannaschii for para-azido-L-phenylalanine (pAzF) incorporation. Each droplet containing a variant of the aaRS was merged with a CFPS droplet. The resulting droplet synthesized super folder green fluorescence protein with 5 amber codons (sfGFP 5xUAG). Droplets were then sorted based on fluorescence. We achieved high incorporation efficiency (>95%) by screening 5 randomized consecutive amino acids for each residue hypothesized to be critical active sites for the aaRS. We expect this work to meet the growing demands for OTS evolution as well as accelerate broader efforts in protein engineering.