Improving protein expression in E. coli B‑strains using rapid genome engineering

E. coli B‑strains, such as BL21, and their derivatives are often used for protein expression due to their optimized amino acid production, ATP utilization, lon and ompT protease deficiency, and reduced acetate accumulation. However, optimizing B‑strains for increased protein production using modern approaches to genome engineering has not been reported. Here we apply CRISPR-based, high-throughput genome-wide and pathway-specific editing to improve the expression of an enzyme. With shallow sampling of the genome-edited libraries, we identify variants with greater than 2‑fold improvement in active protein expression. This work demonstrates how rapid genome engineering can be applied to significantly enhance protein expression in E. coli.