Genome Engineering of Genomically Recoded Escherichia coli Enhances Site-Specific Incorporation of Non-Standard Amino Acids in Cell-Free Protein Synthesis

Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins and biopolymers by amber suppression makes possible new chemical properties, new structures, and new functions. However, competition between release-factor 1 (RF1) and orthogonal tRNAs have limited the technology. Here, we describe the development of a high yielding cell-free protein synthesis (CFPS) platform from crude extracts of genomically recoded Escherichia coli lacking release factor 1. Because this recoded strain has not been previously optimized for CFPS, we exploited multiplex automated genome engineering (MAGE) to design and construct synthetic genomes that, upon cell lysis, lead to improved extract performance. To stabilize template DNA, messenger RNA, amino acids, and protein products in CFPS, we targeted the deletion of 15 potential negative effectors. More than 50 strains were generated and tested in CFPS, allowing us to catalogue the systems impact by making numerous gene deletions both individually and in combinations. The protein synthesis activities of our most productive cell extracts were more than four-fold greater as compared to the extract from the parent strain, achieving more than 1.6 mg/ml of superfolder green fluorescent protein (sfGFP). We also show high efficiencies for producing modified sfGFP containing p-acetylphenylalanine at single and multiple positions. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.