A Highly Productive One-Pot System for the Incorporation of Non-Standard Amino Acids into Cell-Free Synthesized Proteins

The site-specific installation of non-standard amino acids (NSAAs) into proteins via amber suppression enables the synthesis of peptides and biopolymers with new chemical properties and functions. However, in most systems competition between release factor 1 (RF1) and aminoacylated orthogonal tRNAs results in the formation of a significant quantity of undesired truncated products. Recent efforts have addressed this limitation through the generation of a high-yielding cell-free protein synthesis (CFPS) platform using extracts derived from a recoded organism lacking RF1 that has been optimized for CFPS. These extracts, while highly productive, are dependent upon supplementation with purified T7 RNA polymerase (T7RNAP) to catalyze orthogonal transcription in vitro. Here, we describe the development of a truly one-pot CFPS system for synthesis of NSAA-containing proteins at high yields. As the aforementioned CFPS-optimized strain was incapable of synthesizing T7RNAP, we applied lambda Red recombination to site-specifically integrate the T7RNAP gene into the genome of the strain. We investigated different combinations of insertion loci and promoter strength to identify the combination that best balances strain health and T7RNAP overexpression levels. Further analysis of these strains revealed that T7RNAP produced was being cleaved by the membrane-bound periplasmic protease OmpT, potentially reducing the activity of the enzyme. To address this, we leveraged multiplex advanced genome engineering (MAGE) in the highest-performing T7RNAP-expressing strain, mutating the coding region of the T7RNAP gene to eliminate OmpT recognition and cleavage sites. This mutant T7RNAP remains highly productive, and is highly resistant to OmpT-mediated degradation. Our most highly productive cell extracts featuring this mutant T7RNAP achieve more than 2.0 g/L of active superfolder green fluorescent protein (sfGFP) in the presence of supplemental T7RNAP, and ~1.6 g/L of sfGFP in the absence of supplemental T7RNAP. We show that these extracts enable significantly higher levels of NSAA incorporation than current state-of-the-art one-pot CFPS systems produced from BL21 (DE3) and its derivatives. To our knowledge, this is the highest-yielding one-pot CFPS system developed to date. Our work has implications for using recombineering approaches for CFPS strain development, expanding the chemistry of biological systems, and cell-free synthetic biology.