Improving Cell-Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

Site-specific incorporation of non-standard amino acids into proteins by amber suppression grants access to novel therapeutics and new types of biomaterials. 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 (rEc.E13.DprfA). We exploited genome engineering to design and construct synthetic genomes that upon cell lysis lead to improved extract performance. We targeted the deletion of five potential negative effectors, the nuclease genes rna, rnb, csdA, mazF, and endA. Our most productive extract, resulting from a csdA and endA double mutant, synthesized 550 ± 40 µg/mL of modified superfolder green fluorescent protein containing p-acetyl-L-phenylalanine and achieved ~84% suppression efficiency. The modified protein yield was further improved to 1300 ± 100 µg/mL using a semi-continuous cell-free reaction format, which is the highest reported for extracts derived from RF1-deficient strains. Our work has implications for using whole genome editing for CFPS strain development and cell-free synthetic biology.