Liposome-Based in Vitro Evolution of Aminoacyl-tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation

Graphical Abstract

Enzyme evolution: Using a liposome-based in vitro compartmentalization approach, we screened a randomly mutagenized library of N-benzyloxycarbonyl-L-lysine (LysZ) tRNA synthetases and obtained one that was able to increase LysZ incorporation both in vitro and in vivo. The ease and flexibility of this approach should enable evolution of not only the aminoacyl-tRNA synthetases but also of other enzymes involved in protein translation.

Abstract

Methanosarcina species pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ-RS, that was able to attach N-benzyloxycarbonyl-L-lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ-RS engineering has not been performed; consequently, we aimed to generate LysZ-RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome-based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ-RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome-based IVC should enable the evolution of not only LysZ-RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.