Strains and media

E. coli DH10B (Invitrogen, Waltham, MA) was used for plasmid cloning and recombinant protein expression. LB media (BD-Difco, Franklin Lakes, NJ) was utilized as the culture media. The working antibiotic concentrations were as follows: 50 μg/mL chloramphenicol (Daejung Chemicals, Siheung, Republic of Korea) and 50 μg/mL gentamicin (KisanBio, Seoul, Republic of Korea). Primers used in this study were synthesized and purchased from Macrogen (Seoul, Republic of Korea). AzF (Chem-Impex, Wood Dale, IL), a non-canonical amino acid, was included as a substrate for aaRS. Arabinose (Alfa Aesar, Haverhill, MA) and isopropyl β-d-1-thiogalactopyranoside (IPTG, Bioneer, Daejeon, Republic of Korea) were applied as inducers.

Selection of aaRS from new resource

BLAST was employed on the NCBI database to align mutant tyrosyl-tRNA synthetase genes of M. jannaschii (Mj TyrRS), which was previously developed to introduce non-canonical amino acids (Chin et al. 2002b).

Site-directed mutagenesis of Mc TyRS

From previous reports on the Mj TyrRS mutant with amber suppression, specific sites in proteins are known to be essential for substrate recognition. Mj TyrRS and Mc TyrRS protein sequences were aligned with the pairwise sequence alignment of EMBL-EBI (Madeira et al. 2022) to identify the significant sites in Mc TyrRS, and visualization was performed with ESPript 3 (Robert and Gouet 2014). Tyrosine 33, aspartic acid 162, and lysine 166 in Mc TyrRS were selected for site-directed mutation. Rationally designed genes were synthesized by Genophile (Gwangju, Republic of Korea).

Random mutagenesis for library construction

Random mutagenesis was conducted using the Genemorph II random mutagenesis kit (Agilent, Santa Clara, CA) according to the manufacturer's instructions. DNA fragments for library creation were cloned into BglII/PstI sites of the plasmid backbone. The plasmid backbone was generated via PCR on the pEVOL-pAzF plasmid (Addgene: 31186), including suppressor tRNA under the proK promoter, the chloramphenicol resistance gene under the CAT and aaRS gene under ara promoter (Lee et al. 2023). As a result, the pEVOL-McTyrRSmut plasmid was constructed for mutant McTyrRS expression under the ara promoter. The amplified plasmid backbone was confirmed using Sanger sequencing.

sfGFP expression with the amber codon

Superfolder Green Fluorescence Proteins (sfGFPs) bearing the TAG codon at position 204 were expressed with the aaRS mutant library to quantify aaRS activity (Kim and Choi 2022). In previous study, several positions in sfGFPs were tested to bear TAG codon. Among them, sfGFPs mutated at position 204 showed the intended fluorescence. In this system, the presence of the amber codon (TAG) at the midpoint of the sfGFP sequence plays a critical role. If an amino acid is not incorporated at the amber codon site due to the lack of a compatible aminoacyl-tRNA synthetase, translation is terminated early, resulting in a truncated sfGFP that does not exhibit fluorescence. Conversely, successful incorporation of the AzF allows for full-length sfGFP synthesis, rendering the protein fluorescent. E. coli DH10B was co-transformed with the pEVOL-McTyrRSmut plasmid and pSEVA631pt-sfGFP204amb, including the sfGFP gene with the TAG codon under the Tac promoter and a gentamicin resistance gene under the Pc promoter.

As a control, pEVOL-pAzF or pEVOL-McTyrRSwt were co-transformed with pSEVA631pt-sfGFP204amb and cultured under the same conditions. A single colony was pre-cultured in 5 mL LB media supplemented with antibiotics at 37 °C overnight. Pre-cultured cells were inoculated into 50 mL LB media supplemented with antibiotics, 0.5 mM AzF, 0.2% (w/v) arabinose, and 0.5 mM IPTG at an initial OD600 nm = 0.1, and cultured at 37 °C for 9 h. Experiments were performed three times, and standard deviations were presented.

Microscope

sfGFP expressed culture was centrifuged to prepare the sample. Collected cell was deposited on a glass slide and covered by cover glass. Each specimen was observed at 1000× magnification by ZEISS Axio Scope.A1 Microscope (ZEISS, Oberkochen, Baden-Württemberg, Germany) and Microscope illuminating system-HBO 100 (ZEISS).

Microplate reader

Optical density (600 nm) and fluorescence intensity (excitation 485 nm, emission 528 nm) were measured in 96-well black clear-bottom plates (SPL Life Science Co., Gyeonggi-do, South Korea) with a Synergy H1 Hybrid Multi-Mode Reader (BioTek, Winooski, VT). Fluorescence intensity was normalized by dividing the fluorescence value by the optical density value.

Fluorescence-activated cell sorting (FACS)

The sfGFP-expressed cell culture with the random mutagenesis library was diluted to OD600 nm = 1, centrifuged, and washed twice with 1 mL of 1 × phosphate-buffered saline (PBS). MoFlo XDP Flow Cytometer & Sorter (Beckman Coulter, Brea, CA) sorted cells with the top 1 ~ 3% fluorescence for three rounds.

Prediction of the mutant Mc TyrRS structure

Mc TyrRS wt, Mc TyrRS mutant 6, and mutant Mc TyrRS protein sequences were submitted to the SWISS-MODEL server for homology modeling (Waterhouse et al. 2018). Tertiary structures were predicted based on the crystal structure of Mj TyrRS (PDB: 1J1U) as a template. Docking for AzF was conducted with AutoDock Vina (v.1.1.2) (Eberhardt et al. 2021). Side chains near the substrate around 5 Å were specified as flexible for recognizing substrate. The figure’s tertiary structure was prepared using PyMOL (v2.5.2) (Schrodinger 2021).