Development of orthogonal aminoacyl-tRNA synthetase mutant for incorporating a non-canonical amino acid

Abstract

Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-L-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to developing novel aaRS from various organisms.

Keywords: Methanosaeta concilii; Amber suppression; Aminoacyl-tRNA synthetase; Non-canonical amino acid; Para-azido-L-phenylalanine.

Fig. 1

Alignment for selecting target residues. Mj TyrRS and Mc TyrRS protein sequences were aligned with local alignment, preserving conserved sequences. Target residues for rational design (Y33, D162, and L166 of Mc TyrRS corresponding to Y32, D158, and L162 of Mj TyrRS) were presented as a triangle

Fig. 2

Comparison of amber suppression efficiency. Normalized fluorescence intensity was calculated by dividing fluorescence by OD 600 nm to determine the suppression efficiency of each aaRS. Mutant Mc TyrRS screened from a random mutagenesis library presented selectivity in AzF recognition, while Mc TyrRS wt and Mc TyrRS mutant 6 did not. n = 3; error bars, mean ± s.d

Fig. 3

Predicted structures of (a) Mc TyrRS wt, (b) Mc TyrRS mutant 6, and (c) mutant Mc TyrRS. Three residues in the active sites (Y33G, Y112F, and D162T) and substrate (tyrosine or AzF) were represented with modeling. Compared to Mc TyrRS wt charged the tyrosine as substrate, Mc TyrRS mutant 6 (Y33G, D162T) disfavored not only tyrosine, but also AzF. With the additional mutation in mutant Mc TyrRS (Y33G, Y112F, and D162T), it could recognize the AzF. The yellow dotted line represents a hydrogen bond