(276a) Genome Engineering of Escherichia Coli Enhances Site-Specific Incorporation of Unnatural Amino Acids in Cell-Free Protein Synthesis

Site-specific incorporation of unnatural amino acids (uAAs) 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 genomically recoded Escherichia coli strains lacking RF1. Specifically, occurrences of the amber stop codon TAG are reassigned to the synonymous TAA codon. Because the parent 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 17 potential negative effectors. More than 50 strains were generated and tested in CFPS, allowing us to catalogue the systems impact of making numerous gene deletions both individually and in combinations. The protein synthesis activities of our most productive cell extracts were more than five-fold greater as compared to the extract from the parent strain, achieving more than 1.2 mg/ml of superfolder green fluorescent protein (sfGFP). The yields of modified sfGFP containing p-acetylphenylalanine at single and multiple positions in our in vitro system were more than ten-fold higher than the reported in vivo results. Our work demonstrates improved CFPS activity using whole genome editing for extract strain development and promises CFPS systems as suitable platforms for generating unnatural biopolymers.