Expanding the Chemistry of Lifevia Genome Engineering and Cell-Free Protein Synthesis

Site-specific incorporation of non-standard amino acids (NSAAs) into proteins and biopolymers makes possible new chemical properties, new structures, and new functions. However, competition between release factor 1 (RF1) and NSAA incorporation during protein translation have limited the technology. Hong will describe the development of a high yielding cell-free protein synthesis (CFPS) platform from crude extracts of genomically recoded Escherichia coli lacking RF1. Because this recoded strain has not been previously optimized for CFPS, he exploited multiplex automated genome engineering to design and construct synthetic genomes that, upon cell lysis, lead to improved extract performance. He sought to stabilize DNA, mRNA, amino acids, and protein products in CFPS by targeting the inactivation of fifteen putative negative protein effectors. Twenty-seven strains were generated and tested in CFPS, allowing him to catalogue the systemic impact of making numerous gene disruptions both individually and in combinations. The protein synthesis activities of his most productive cell extracts were more than five-fold greater as compared to the extract from the parent strain, achieving synthesis of more than 1.6 mg/ml of active superfolder green fluorescent protein (sfGFP). He achieved high incorporation efficiency of the NSAA p-acetylphenylalanine (pAcF) in sfGFP at single and multiple positions. Upon optimizing the levels of the orthogonal transalation system components, he synthesized elastin-like polypeptides containing up to 40 pAcFs with high fidelity (>95%) and yield (~100 mg/L). To his knowledge, this is the highest level of site-specific NSAA incorporation in pure proteins to date. His work has implications for using whole-genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.