Genetic Code Expansion in the Engineered Organism Vmax X2: High Yield and Exceptional Fidelity
Sebasthian Santiago González, Omer Ad, Bhavana Shah, Zhongqi Zhang, Xizi Zhang, Abhishek Chatterjee, Alanna Schepartz
Abstract
strains 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.