Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (~50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7, and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here, we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase (aaRS) pair into the chromosome of a GRO that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering (MAGE)12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harboring 3 TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for seven days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6-10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternate genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

Peptide drugs are an exciting class of pharmaceuticals increasingly used for the treatment of a variety of diseases; however, their main drawback is a short half-life, which dictates multiple and frequent injections and an undesirable "peak-and-valley" pharmacokinetic profile, which can cause undesirable side-effects. Synthetic prolonged release formulations can provide extended release of biologically active native peptide, but their synthetic nature can be an obstacle to production and utilization. Motivated by these limitations, we have developed a new and entirely genetically encoded peptide delivery system-Protease Operated Depots (PODs)-to provide sustained and tunable release of a peptide drug from an injectable s.c. depot. We demonstrate proof-of-concept of PODs, by fusion of protease cleavable oligomers of glucagon-like peptide-1, a type-2 diabetes drug, and a thermally responsive, depot-forming elastin-like-polypeptide that undergoes a thermally triggered inverse phase transition below body temperature, thereby forming an injectable depot. We constructed synthetic genes for glucagonlike peptide-1 PODs and demonstrated their high-yield expression in Escherichia coli and facile purification by a nonchromatographic scheme we had previously developed. Remarkably, a single injection of glucagon-like peptide-1 PODs was able to reduce blood glucose levels in mice for up to 5 d, 120 times longer than an injection of the native peptide drug. These findings demonstrate that PODs provide the first genetically encoded alternative to synthetic peptide encapsulation schemes for sustained delivery of peptide therapeutics.drug delivery | sustained release | peptide polymers W ith more than 40 approved peptide drugs worldwide and over 650 peptides in clinical and preclinical development,

Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format.

Peptide drugs are an exciting class of pharmaceuticals for the treatment of a variety of diseases; however, their short half-life dictates multiple and frequent injections causing undesirable side-effects. Herein, we describe a novel peptide delivery system that seeks to combine the attractive features of prolonged circulation time with a prolonged release formulation. This system consists of glucagon-like peptide-1, a type-2 diabetes drug fused to a thermally responsive, elastin-like-polypeptide (ELP) that undergoes a soluble-insoluble phase transition between room temperature and body temperature, thereby forming an injectable depot. We synthesized a set of GLP-1-ELP fusions and verified their proteolytic stability and potency in vitro. Significantly, a single injection of depot forming GLP-1-ELP fusions reduced blood glucose levels in mice for up to 5 days, 120 times longer than an injection of the native peptide. These findings demonstrate the unique advantages of using ELPs to release peptide-ELP fusions from a depot combined with enhanced systemic circulation to create a tunable peptide delivery system.

Conventional methods for synthesizing protein/peptide–polymer conjugates, as a means to improve the pharmacological properties of therapeutic biomolecules, typically have drawbacks including low yield, non-trivial separation of conjugates from reactants, and lack of site-specificity, which results in heterogeneous products with significantly compromised bio activity. To address these limitations, the use of sortase A from Staphylococcus aureus is demonstrated to site-specifically attach an initiator solely at the C-terminus of green fluorescent protein (GFP), followed by in situ growth of a stealth polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) by atom transfer radical polymerization (ATRP). Sortase-catalyzed initiator attachment proceeds with high specificity and near-complete (≈95%) product conversion. Subsequent in situ ATRP in aqueous buffer produces 1:1 stoichiometric conjugates with > 90% yield, low dispersity, and no denaturation of the protein. This approach introduces a simple and useful method for high yield synthesis of protein/peptide–polymer conjugates.

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELPprotein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.