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1. Non‐canonical Amino Acid Substrates of E. coli Aminoacyl‐tRNA Synthetases

   https://doi.org/10.1002/cbic.202100299  

   Hartman, Matthew C. T. ( September 2021 , ChemBioChem)

   In this comprehensive review, I focus on the twentyE. coliaminoacyl‐tRNA synthetases and their ability to charge non‐canonical amino acids (ncAAs) onto tRNAs. The promiscuity of these enzymes has been harnessed for diverse applications including understanding and engineering of protein function, creation of organisms with an expanded genetic code, and the synthesis of diverse peptide libraries for drug discovery. The review catalogues the structures of all known ncAA substrates for each of the 20E. coliaminoacyl‐tRNA synthetases, including ncAA substrates for engineered versions of these enzymes. Drawing from the structures in the list, I highlight trends and novel opportunities for further exploitation of these ncAAs in the engineering of protein function, synthetic biology, and in drug discovery.
2. Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

   https://doi.org/10.1002/anie.201914671  

   Tharp, Jeffery M. ; Ad, Omer ; Amikura, Kazuaki ; Ward, Fred R. ; Garcia, Emma M. ; Cate, Jamie H. D. ; Schepartz, Alanna ; Söll, Dieter ( January 2020 , Angewandte Chemie International Edition)

   By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMetfrom the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
3. Initiation of Protein Synthesis with Non‐Canonical Amino Acids In Vivo

   https://doi.org/10.1002/ange.201914671  

   Tharp, Jeffery M. ; Ad, Omer ; Amikura, Kazuaki ; Ward, Fred R. ; Garcia, Emma M. ; Cate, Jamie H. D. ; Schepartz, Alanna ; Söll, Dieter ( January 2020 , Angewandte Chemie)

   By transplanting identity elements into E. coli tRNA^fMet, we have engineered an orthogonal initiator tRNA (itRNA^Ty2) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA^Ty2can initiate translation in vivo with aromatic non‐canonical amino acids (ncAAs) bearing diverse sidechains. Although the initial system suffered from low yields, deleting redundant copies of tRNA^fMetfrom the genome afforded an E. coli strain in which the efficiency of non‐canonical initiation equals elongation. With this improved system we produced a protein containing two distinct ncAAs at the first and second positions, an initial step towards producing completely unnatural polypeptides in vivo. This work provides a valuable tool to synthetic biology and demonstrates remarkable versatility of the E. coli translational machinery for initiation with ncAAs in vivo.
4. The Role of Orthogonality in Genetic Code Expansion

   https://doi.org/10.3390/life9030058  

   Arranz-Gibert, Pol ; Patel, Jaymin R. ; Isaacs, Farren J. ( September 2019 , Life)

   The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.
5. Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro

   https://doi.org/10.1038/s41467-020-18001-x  

   Lee, Joongoo ; Schwarz, Kevin J. ; Kim, Do Soon ; Moore, Jeffrey S. ; Jewett, Michael C. ( August 2020 , Nature Communications)

   Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.