Genetic code expansion.

Another in vivo approach for incorporation of ncAAs is genetic code expansion (GCE), which is in a site-specific manner. This strategy is similar to the natural translation process. An aaRS charges an ncAA onto the tRNA with a specific anticodon. Then, the charged tRNA is delivered to the ribosome, leading to the incorporation of the corresponding amino acid (Fig. 1D). GCE relies on the use of an orthogonal aaRS-tRNA pair. Four aaRS-tRNA pairs are most common used for ncAA incorporation: the tyrosyl-tRNA synthetase (MjTyrRS)-tRNA_CUA pair from Methanococcus jannaschii, tyrosyl-tRNA synthetase (EcTyrRS)-tRNA_CUA pair from E. coli, leucyl-tRNA synthetase (EcLeuRS)-tRNA_CUA pair from E. coli, and pyrrolysyl-tRNA synthetase (PylRS)-tRNA_CUA pair from Methanosarcina spp. (76). Each of these orthogonal pairs can be used in certain organisms. To date, the suppression of nonsense codons, usually the amber codon TAG, has been successfully applied to multiple organisms (77,–82). Although stop-codon suppression (SCS) has become increasingly sophisticated, its competition with RF1-mediated termination of peptide chain and low heterogeneous enzyme activity result in low integration efficiency. Several studies have been performed on improving its efficiency, such as elimination of RF1 (83, 84), directed evolution of aaRSs (85, 86) and ribosome (87), optimization of the orthogonal system (88), and modification of elongation factor Tu (89). A recent study reported a genetically recoded E. coli C321.ΔA in which all 321 amber codons were recoded to ochre codons and RF1 was deleted (90). Two synonymous serine codons were further removed to create a new chassis using 59 codons to encode the 20 cAAs (91). The reassignment of sense codons allows incorporation of multiple ncAAs. In addition, quadruplet codons have been utilized to encode ncAAs, which, in principle, might provide 256 bland codons (92, 93). Rodriguez et al. (94) successfully introduced multiple ncAAs in a neuroreceptor expressed in vivo through stop codon and quadruplet codon suppression. Quadruplet codons have also been applied to Caenorhabditis elegans, paving the way toward in vivo multi-incorporation of ncAAs in a multicellular organism (95). Over the past 2 decades, GCE has been extensively used to incorporate ncAAs into AMPs. For example, four ncAAs have been used to replace four positions in the lasso peptide microcin J25, a 21-residue RiPP produced by E. coli strains (96), via SCS (97). Using this approach, ncAAs have also been incorporated into lanthipeptides (78, 98), thiopeptides (a group of sulfur-containing macrocyclic peptides [81, 99]), and cyanobactins (a group of ribosomal cyclic peptides produced by cyanobacteria [100, 101]). These efforts led to a batch of AMP derivatives with novel chemical and/or functional activities.

As described above, the incorporation efficiency of GCE is dependent on several parameters and requires specific genetic engineering of the host strain. At present, E. coli is the most investigated platform for GCE, and is available for heterologous expression of ncAA-incorporating AMPs. However, it is still challenging because the corresponding PTM enzymes and immune proteins must be coexpressed. Moreover, some proteases in the E. coli cytoplasm can degrade heterologous proteins, which may impede proper folding and biological activity of the proteins (102, 103). A recent study reported the incomplete dehydration of the heterologously expressed NisA in E. coli BL21(DE3). The endonuclease rne and the proteases ompT and lon were deleted to ensure the complete modification of NisA (104). Because AMPs have been successfully heterologously expressed in different host strains, such as Lactococcus, Bacillus, and yeast, some of which showed better AMP expression than E. coli (105,–107), it is necessary to develop new chassis, based on the principles of GCE, for suitable bioproduction of ncAA-incorporating AMPs. In addition, the in vivo GCE approaches are only available for non-toxic ribosomal synthesized peptides. To address this limitation, in vitro platforms combining CFPS and GCE have been developed (108).