Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System.

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Site-directed and global incorporation of orthogonal and isostructural noncanonical amino acids into the ribosomal lasso peptide capistruin.

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc-L-lysine) instead of Ala. A second-generation Hoveyda-Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.