Streamlining tRNA-Synthetase Evolution for Genetic Code Expansion and Deep Sequencing Analyses of Its Evolved Variants.

Proteins are typically composed of 20 amino acids encoded by 61 codons. However, some bacteria and archaea have evolved to incorporate additional amino acids by repurposing stop codons, a phenomenon that led to the development of genetic code expansion (GCE) in the early 21st century. This approach introduces orthogonal tRNA and aminoacyl-tRNA-synthetase (aaRS) pairs into target organisms, enabling the incorporation of noncanonical amino acids (ncAAs) with distinct side chains into proteins. GCE has broad applications, including site-specific cross-linking, fluorescence labeling, and electron-transfer functionalities. Despite its versatility, improving the efficiency of ncAA incorporation remains a challenge. Directed evolution provides a powerful solution by introducing mutations into the aaRS sequence and applying selection to identify variants with enhanced activity. Here, we present a simplified directed evolution system designed to improve the activity of pyrrolysyl-tRNA synthetase (PylRS) from . Our approach is accessible, requiring only basic laboratory equipment, making it suitable and facile to implement by graduate students. We evolved PylRS variants toward three distinct substrates, each pathway yielding unique, substrate-specific mutations. We characterized the impact of these mutations on both PylRS activity and expression levels, demonstrating that tandem codon randomization can be an effective strategy for improving PylRS function through additive effects of the mutations. Additionally, deep sequencing validated our approach, confirming its efficiency, revealing conserved and mutationally flexible sites and reinforcing the advantage of tandem mutations in PylRS evolution. Collectively, these findings streamline the process of evolving PylRS and provide insights into strategies for enhancing ncAA incorporation in synthetic biology and protein engineering.