Fig. 4: The ncAAs biosynthetic pathway was scalable and efficient for coupling with various orthogonal aaRS/tRNA.

a Plasmid design for coupling ncAA synthesis and incorporation into sfGFP. The plasmids used for ncAA synthesis and incorporation into sfGFP were the same as those described in Fig. 2b. These included plasmids encoding CsLTA and RpTD for ncAA synthesis, and plasmids encoding sfGFP_Y151TAG as the reporter protein. Mutants of two types of PylRS/tRNA pairs were constructed in the pCDF vector, while mutants of the MjTyrRS/tRNA pair were constructed in the pUltra vector. tRNA expression was driven by a constitutive promoter, whereas other genes were expressed under IPTG induction. b Detection of sfGFP_Y151pIF fluorescence intensity in strains expressing different LTAs. Fluorescence intensity of sfGFP_Y151pIF in strains expressing different LTAs was measured after 24 h of incubation. The fluorescence intensity of cells cultured with 1 mM pIF was set as 100%. For the experimental group, 1 mM pIBzH, 50 mM glycine, and 20 μM PLP were added to the culture at the same time as IPTG induction. Error bars represent the mean ± s.d. of n = 3 independent samples. c Fluorescence intensity of sfGFP containing different ncAAs. The fluorescence intensity of sfGFP_Y151TAG was measured in E. coli RARE (CsLTA-RpTD) strains containing various OTSs, after 24 h of fermentation. The culture was supplemented with either 1 mM ncAAs or 1 mM aromatic aldehyde substrates. Error bars represent the mean ± s.d. of n = 3 independent samples. d Yields of sfGFP containing ncAAs after purification. The yield of sfGFP containing ncAAs was determined after purification. To the culture, 1 mM aromatic aldehydes were added, and after 24 h of induction, the proteins were purified using a Ni-NTA column. The yield of wild-type sfGFP was set as 100% for comparison. e Mass spectra of purified sfGFP with p-ethynylphenylalanine (pENF) or p-azidephenylalanine (pAzF) and corresponding detection of fluorescence intensity. For fluorescence intensity, error bars represent the mean ± s.d. of n = 3 independent samples. Mass spectra of purified sfGFP incorporated with other ncAAs were shown in Supplementary Fig. 19. PG, positive group with 1 mM ncAAs; EG, experimental group with 1 mM aromatic aldehydes; NG, negative group without ncAAs or aromatic aldehydes. Source data of (b–e) are provided in the Source Data file.