Fig. 5: Generalized ncAA uptake using Z-AisoK tripeptides.

a, Structure of tested Z residues within the Z-AisoK scaffold. b, SDS–PAGE analysis of sfGFP-N150TAG expression in the presence of 2 mM of BocK or Z-AisoK tripeptides with wt-MbPylRS/PylT. Expression levels of full-length sfGFP indicate successful transport of the Z-AisoK tripeptides, subsequent cleavage to Z and AisoK and AisoK incorporation. Top, expression in wild-type K12. Bottom, expression in the evolved strain K12-Z2. Consistent results were obtained over three independent replicate experiments. Arrow indicates full-length sfGFP, asterisk indicates truncated sfGFP. c, Scheme for OppA evolution to accommodate novel Z-AisoK substrates. Successful transport by OppA library variants was evaluated by incorporation of AisoK into sfGFP-N150TAG. Variants with high sfGFP fluorescence were enriched via three rounds of FACS. d, X-ray structure of the OppA–G-SisoK complex, highlighting four residues surrounding the N-terminal glycine of G-SisoK that were targeted for site-saturation mutagenesis to enable recognition of novel Z-AisoK substrates. e, Left, SDS–PAGE analysis of sfGFP-N150TAG expression with 0.25 mM LipK or the corresponding Z-AisoK tripeptide 13 with a LipK-specific Methanosarcina mazei PylRS/PylT (MmPylRS/PylT) variant (Y306A/Y384F). Expression levels are highest with peptide 13 in the K12-Z1 strain, which expresses OppA-Z1, evolved specifically for the transport of 13. Right, LC–MS analysis of sfGFP purified from K12-Z1 cultures grown with 13. Observed mass confirms incorporation of LipK. Consistent results were obtained over three independent replicate experiments. f, Dual stop codon suppression using a single isopeptide-linked tripeptide. Left, chemical structure of the tripeptide AcK-pLisoK (16). Middle, SDS–PAGE analysis of sfGFP-N40TAA-N150TAG expression in the presence of either AcK and G-pLisoK (or pLisoK) separately added to medium or in the presence of tripeptide 16 in IsoK12. Right, LC–MS analysis of purified sfGFP confirms dual ncAA incorporation (AcK and pLisoK) after the addition of tripeptide 16. Consistent results were obtained over three independent replicate experiments.