Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity

[Image: see text] We report that the recently introduced commercial strain of Vibrio natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 noncanonical α-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recodedEscherichia colistrains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of misincorporated natural α-amino acids at the UAG-programmed position, especially in cases where the ncAA is a moderate substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that the use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve the selectivity of an aaRS toward highly desired but suboptimal ncAA substrates.