Genome Engineering of Genomically Recoded Escherichia coli enables Multi-Site Non-Standard Amino Acid Incorporation in Cell-Free Synthesized Proteins at High Yield and Purity

Site-specific incorporation of non-standard 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 during protein translation 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 RF1. 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. We 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 us to catalogue the systemic impact of making numerous gene disruptions 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 synthesis of more than 1.6 g/L of active superfolder green fluorescent protein (sfGFP). We also show high incorporation efficiency (>95%) of the NSAA p-acetylphenylalanine (pAcF) in sfGFP at single and multiple positions. Upon optimizing the levels of the orthogonal translation system components, we further show the ability to synthesize elastin-like polypeptides containing up to 40 pAcFs with high fidelity (>95%) and yield (~100 mg/L). To our knowledge, this is the highest level of site-specific incorporation in pure proteins to date. Our work has implications for using whole-genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.