MaPylRS was cloned into M13 phage, replacing gene III. Phages were initially propagated without selection using the permissive host E. coli S1059. Cultures were grown in 2xYT media supplemented with the appropriate antibiotics at 37°C until reaching an OD₆₀₀ of 0.4–0.6. Cells were then infected with a viral load of phage ranging from 10⁴–10⁶ pfu/ml, and the culture was grown overnight at 37°C. The following day, a 1 ml aliquot of the overnight culture was centrifuged at 14,000 × g for 1 min, and the phage-containing supernatant was decanted and stored at 4°C. Three independent phage lineages were maintained throughout the PANCE process.

Positive selection was performed using E. coli S1030 cells transformed with the accessory plasmid pJT017 (gIII_1xTAG MatRNA^Pyl ₍₆₎), as well as mutagenesis plasmid MP4 during rounds of mutagenesis. 30 ml cultures were grown in 2xYT media supplemented with 20 mM glucose and the appropriate antibiotics to an OD₆₀₀ of 0.4–0.6. BocK (5 mM) was then added where indicated, and during rounds of mutagenesis, 5 mM arabinose was also added. Cultures were then infected with 10⁴–10⁶ pfu/ml M13ΔgIII:MaPylRS. The infected culture grew overnight at 37°C, and phages were harvested the next morning. In later rounds of higher stringency positive selection, plasmids pJT018 (gIII_2xTAG MatRNA^Pyl ₍₆₎) and pJT019 (gIII_3xTAG MatRNA^Pyl ₍₆₎) were used in place of pJT017.

Negative selection was carried out using the negative selection plasmids pJF011 (T7RNAP_2xTAG MatRNA^Pyl ₍₆₎) and pDB016 (carrying gIII under a T7 promoter). 30 ml cultures were grown in 2xYT media, and at an OD₆₀₀ of 0.4–0.6, the cells were infected with 10⁴–10⁶ pfu/ml of phage. The culture grew overnight at 37°C, and phages were harvested the following morning.

Phage titers were performed at the conclusion of every generation as a checkpoint to ensure that phage propagation remained robust and selective for BocK. Collected phage samples were serially diluted and plated on LB-top agar plates containing E. coli S1059 cells. 5 µL of each serial dilution were spotted on the plates, and the pfu/mL for each phage lineage +/− BocK was calculated.

Electrocompetent E. coli DH10B cells were transformed with a pMW plasmid encoding the PylRS variant, and a pBAD plasmid encoding sfGFP 2TAG and MatRNA^Pyl. Variations of the two plasmids were cloned and used where indicated. Colonies of freshly transformed DH10B cells harboring the indicated plasmids were picked and grown at 37°C overnight in 5 ml of LB media supplemented with appropriate antibiotics. 2 µL of the overnight cultures were diluted into 150 µL LB supplemented with 500 µM IPTG, 0.2% arabinose, appropriate antibiotics, and 1 mM of the indicated ncAA. Inoculated cultures were grown shaking at 37°C in a BioTek Synergy plate reader. Fluorescence intensity (λ _ex = 485nm, λ _em = 528 nm) and OD₆₀₀ were measured every 15 min, and fluorescence/OD₆₀₀ was calculated using the 12-h time point. Fluorescence/OD₆₀₀ measurements and standard deviations were calculated based on data collected from three biological and two technical replicates.

For the dual ncAA incorporation assay, a three-plasmid system was used: 1) pMW AzFRS.2. t1, 2) pULTRA encoding the PylRS variant and the ochre-suppressing mutant tRNA MatRNA^Pyl _(6)/UUA, and 3) pBAD sfGFP 2TAG 149TAA M. jannaschii tRNA^Tyr _CUA. The plasmids were co-transformed into electrocompetent E. coli DH10B cells. The rest of the procedure follows the above protocol.

Electrocompetent DH10B cells were co-transformed with pBAD sfGFP 2TAG MatRNA^Pyl and pMW MaPylRS or PylRS_opt. Fresh colonies were used to inoculate 20 ml LB media supplemented with 0.2% arabinose, 500 µM IPTG, 1 mM BocK, 25 μg/ml spectinomycin, and 100 μg/ml ampicillin. Cultures were grown shaking at 37°C overnight. The following morning, the cultures were centrifuged at 4,000 × g for 10 min, and the pellets were resuspended in 1X BugBuster protein extraction reagent (Millipore-Sigma). The lysate was clarified by centrifugation (15,000 × g for 20 min) and the cleared lysate was incubated with nickel-NTA agarose beads for 20 min. The beads were washed five times with wash buffer (50 mM Tris pH 8.0, 50 mM NaCl, 10 mM imidazole), and eluted with elution buffer (50 mM Tris pH 8.0, 50 mM NaCl, 250 mM imidazole. The purified proteins were concentrated and buffer-exchanged into 25 mM Tris pH 8.0, 25 mM NaCl using an Amicon Ultra Centrifugal Filters (10 kDa MWCO).

Mass spectroscopy was performed by Bioinformatics Solutions Inc. in Waterloo, ON, Canada. LC-MS analysis of DTT reduced samples were performed on a Thermo Scientific Orbitrap Exploris 240 mass spectrometer, equipped with a heated electrospray ionization source (H-ESI) in positive ion mode with a Thermo Fisher Ultimate 3000 RSLCnano HPLC System. On the H-ESI source, sheath gas was set to 2 arbitrary units (arb), and auxiliary gas was set to 6 arb. The ion transfer tube was set at 275°C. The vaporizer temp was at 200°C. The sample was analyzed on a MAbPac RP, 4 µM, 3.0 × 50 mm analytical column (ThermoFisher, San Jose, CA, United States), held at 60°C. The protein was eluted at a rate of 500 μL/min for a 10-min gradient, where 0–7 min: 10–70% acetonitrile +0.1% formic acid; 7–8.2 min: 95% acetonitrile +0.1% formic acid, 8.2–10 min: 20% acetonitrile +0.1% formic acid. MS spectra were acquired using full scans at 15,000 resolution in the orbitrap within a range of 700–2,200 m/z. The maximum injection time was set at auto with a standard AGC target. Ten micro scans were employed, and the RF lens was set to 60%. 15 V of insource CID was applied. Thermo BioPharma Finder 4.1 was used for intact mass deconvolution and peak identification.

Electrocompetent DH10B cells were transformed with a pMW plasmid encoding the PylRS variant, and a pCAM plasmid encoding chloramphenicol acetyltransferase 112TAG and MatRNA^Pyl. Colonies of freshly transformed DH10B cells harboring the indicated plasmids were picked and grown at 37°C overnight in 5 ml of LB media supplemented with appropriate antibiotics. The overnight cultures were serially diluted, and 2 µL of each dilution were spotted onto LB-agar plates supplemented with the appropriate antibiotics for plasmid maintenance, 100 µM IPTG, 1 mM of the ncAA where indicated, and either 100 (PrK plates) or 200 (BocK and ALocK) µg/mL chloramphenicol. The spotted plates were grown at 37°C overnight. Images were taken after 16 h of incubation.

To evolve a hyperactive variant of MaPylRS that also discriminates against canonical amino acids, we adapted a variation of the previously described PANCE system (Figure 1A) (Suzuki et al., 2017). For each generation of phage evolution, we performed a three-step selection process: positive selection with mutagenesis, followed by negative selection, and finally positive selection without mutagenesis. In the first step, E. coli S1030 cells are transformed with the MP4 mutagenesis plasmid and an accessory plasmid (AP), pJT017 (Badran and Liu, 2015; Bryson et al., 2017). The AP encodes the essential phage protein pIII with one to three TAG codons, as well as MatRNA^Pyl ₍₆₎, an engineered tRNA^Pyl variant that is orthogonal to the M. mazei PylRS/tRNA^Pyl system (Supplementary Figure S1) (Willis and Chin, 2018). We utilized MatRNA^Pyl ₍₆₎ in our PANCE system to determine if the tRNA binding domain of MaPylRS would evolve a greater affinity for MatRNA^Pyl ₍₆₎. The transformed E. coli S1030 cells are grown to mid-log phase, supplemented with BocK (Figure 1B) and arabinose, and infected by the phage M13 ΔgIII::MaPylRS. Phage propagation is dependent on successful aminoacylation of tRNA^Pyl by MaPylRS and subsequent suppression of the TAG codon(s) in gIII (the gene encoding pIII), thus enabling selection for phages carrying the most active PylRS variants.

To avoid the unwanted evolution of a promiscuous synthetase that is active towards canonical amino acids, after each round of positive selection with mutagenesis, we performed negative selection in the absence of BocK. Originally developed as an additional selection mechanism in the continuous directed evolution technique PACE, negative selection is highly effective in maintaining the amino acid selectivity of evolving aminoacyl-tRNA synthetases (Carlson et al., 2014; Bryson et al., 2017). The negative selection system relies on the production of pIII-neg, a dominant-negative version of pIII. Production of pIII-neg inhibits phage propagation, thus depleting the population of phages carrying PylRS mutants that are active towards canonical amino acids and can produce pIII-neg in the absence of BocK. In our pilot experiments to optimize the PANCE system for evolving MaPylRS, we found that without negative selection, significant phage propagation occurs in the absence of BocK after consecutive generations of positive selection with mutagenesis (Supplementary Figure S2). This indicates that MaPylRS is sensitive to mutations that enable activity towards a canonical amino acid, most likely Phe. This is predictable, as M. mazei PylRS has been shown to readily incorporate Phe when the critical active site residues N346 (N166 in M. alvus) and C348 (V168) are mutated (Wang et al., 2011). We therefore adopted the negative selection step to maintain the selectivity of evolving MaPylRS variants.

As a final selection step, the surviving phage population isolated from the prior negative selection was passed through a round of positive selection without mutagenesis. This final step amplifies phages that survived negative selection and are still active towards BocK without the added stringency of mutagenesis. We performed this selection in both the presence and absence of BocK, and used the phages isolated from these two conditions as a checkpoint to verify that evolution was proceeding successfully. We measured the phage titers of both conditions to ensure that phage propagation remained robust in the presence of BocK, and low when BocK was not added to the media (Figure 2A). The significant difference between the phage titers indicates that promiscuous variants are not persisting through the negative selection step, and variants that are active towards BocK are indeed being selected. The phage population recovered from this final round of positive selection in the presence of BocK was carried over to the next generation of selection, and the three-step cycle was repeated.

We performed a total of 20 generations of directed evolution and maintained three independent lineages of evolved phages. The stringency of selection was increased as evolution proceeded. Initially, gIII carried a single in-frame TAG codon to be suppressed by MaPylRS/tRNA^Pyl ₍₆₎. After 7 generations of selection, we increased the stringency to two TAG codons in gIII. The evolved phage population efficiently propagated in this higher stringency selection when cultures were supplemented with BocK, while maintaining low levels of propagation in the absence of BocK. After 6 rounds of selection with two TAG codons, we again increased the stringency to three TAG codons. Even at this highest level of stringency, phage propagation remained robust and highly selective for BocK. At this stage, we compared the propagation of our evolved phage lineages to that of the ancestral variant (generation zero) that encodes wild-type MaPylRS. We measured propagation under the high-stringency three TAG codon system in the absence of mutagenesis. The phage titer indicated that the evolved phage lineages propagated far more efficiently than generation zero in the presence of BocK, suggesting that more active variants of PylRS had emerged (Figure 2B).

Throughout the course of the directed evolution process, phage titers at the end of each generation consistently showed greater phage propagation when BocK was present in the media (Figure 2C). After the 20th round of evolution, several plaques from each evolved lineage were sequenced to determine the identity of PylRS mutations. Five mutations were identified, with very little crossover between each of the three independent lineages (Figure 2D). The most frequently occurring mutations that arose from our system were V42I, V75I, V168A, K181E, and A190V. V42I and K181E were found as concomitant mutations, while the rest of the variants were point mutations. V168A is the only active site mutation that was observed. This mutation has been identified previously and exploited for its role in substrate specificity (Wang et al., 2011; Wang et al., 2012; Wan et al., 2014; Seki et al., 2020). As such, we hypothesized that this mutation to the smaller alanine residue may confer altered substrate specificity favoring larger ncAAs such as BocK. V75I, K181E, and A190V are located in the catalytic domain just outside of the active site, while V42I is found in the tRNA binding domain (Figure 2E). Interestingly, multiple sequence alignment shows that the K181E mutation matches the sequence of several PylRS homologs that also encode Glu at this position (E360 in M. mazei PylRS) (Supplementary Figure S3) (Yanagisawa et al., 2008). To the best of our knowledge, aside from V168, none of these residues have previously been identified or targeted for engineering in MaPylRS or any other PylRS homologs.

To test the activity of our evolved MaPylRS mutants, we cloned the mutant genes into a pMW expression vector and co-transformed them along with a pBAD reporter plasmid carrying sfGFP (2TAG) and tRNA^Pyl into E. coli DH10B. Read-through of the TAG codon at position 2 is dependent on PylRS aminoacylating its cognate tRNA^Pyl with a ncAA. Aminoacyl-tRNA^Pyl is then utilized in translation to suppress TAG and incorporate the ncAA into the protein (Figure 3A). Thus, fluorescence from sfGFP production can be used to measure the activity of PylRS. We measured the fluorescence/OD₆₀₀ of cells expressing our PylRS variants and compared this to those expressing wild-type MaPylRS, both in the presence and absence of BocK (Figure 3B). The results show that each of the four mutant constructs as well as wild-type MaPylRS are highly active and selective for BocK. The lack of background activity in the absence of a ncAA for all four mutants substantiates the importance of the negative selection step in our PANCE system. We tested the activity of the variants using both wild-type MatRNA^Pyl and MatRNA^Pyl ₍₆₎. The data indicate that both MatRNA^Pyl and MatRNA^Pyl ₍₆₎ are efficiently charged with BocK by all four mutant variants and wild-type MaPylRS. The activity of the evolved variants towards MatRNA^Pyl ₍₆₎ is consistent with that of wild-type MaPylRS, suggesting that the variants did not evolve enhanced recognition of MatRNA^Pyl ₍₆₎. However, compared to wild-type MaPylRS, the activity towards BocK is improved for three of the four mutants, with the exception being V75I which is approximately equal to wild-type.

We hypothesized that in addition to having robust activity towards the directed evolution substrate BocK, our mutants may also have improved activity towards other Lys- and Phe-ncAAs. We screened four additional ncAAs: N ^ε-propargyloxycarbonyl-l-lysine (PrK), N ^ε-allyloxycarbonyl-l-lysine (ALocK), 4-azido-l-phenylalanine (pAzF), and 3-iodo-l-phenylalanine (mIF) (Figure 3C). The most active variants, V42I/K181E and A190V, have dramatically improved activity towards PrK, ALocK, and mIF. Interestingly, the activity of the active-site mutant V168A is significantly decreased to all the ncAAs we tested except BocK, highlighting the previously established role of this mutation in governing the size and selectivity of the PylRS active site (Wang et al., 2011; Wang et al., 2012; Wan et al., 2014; Seki et al., 2020). The activity of V75I is moderately higher towards PrK compared to the most active variants, and we omitted V75I from further screening. None of the mutants have significant activity towards the para-substituted phenylalanine derivative pAzF. As such, we hypothesized that like MaPylRS, the evolved PylRS variants should also be orthogonal to the M. jannaschii-derived AzFRS.2. t1, enabling them to be used jointly to incorporate multiple, distinct ncAAs into a single protein (Amiram et al., 2015; Tharp et al., 2021).

We suspected that combining the mutations that conferred the greatest increases in activity may result in an additive effect, yielding a hyperactive, ncAA-specific PylRS variant. Based on our results from the initial fluorescence experiments, we cloned a MaPylRS variant containing the mutations V42I, K181E, and A190V, and named the construct PylRS_opt. We tested the activity of MaPylRS, PylRS_V42I/K181E, PylRS_A190V, and PylRS_opt towards several ncAAs, and found that the activity of PylRS_opt was higher than both wild-type and the individual mutants for every ncAA we tested (Figure 4A). Notably, background fluorescence in the absence of ncAA remains close to basal levels for PylRS_opt. Like MaPylRS, PylRS_opt is highly selective for ncAAs over canonical amino acids. To support this, we co-transformed DH10B cells with sfGFP 2TAG and either MaPylRS or PylRS_opt. The cells were supplemented with 1 mM BocK, and sfGFP was purified. The yield of sfGFP was robust at approximately 20 mg/L in cells expressing either MaPylRS or PylRS_opt (Figure 4B). We analyzed these samples by intact mass spectroscopy, which confirmed that BocK is solely present at position 2 of the protein (Figure 4C).

To gain further insight into the robustness of the activity of PylRS_opt, we titrated BocK, ALocK, and PrK and measured the fluorescence in cells expressing either MaPylRS or PylRS_opt (Supplementary Figure S4). We tested concentrations as low as 62.5 µM, which is slightly above the K_m that was reported for M. mazei PylRS and its natural substrate, pyrrolysine (Guo et al., 2014). We found that the activity of PylRS_opt is significantly greater than MaPylRS at all ncAA concentrations tested.

To validate our in vivo fluorescence data, we implemented a chloramphenicol acetyltransferase assay as a second reporter system. DH10B cells expressing either MaPylRS or PylRS_opt, MatRNA^Pyl, and the chloramphenicol acetyltransferase gene with a single in-frame TAG codon were challenged to grow on plates containing bacteriostatic levels of chloramphenicol in the presence and absence of BocK, PrK, and ALocK (Figure 4D). The results corroborated our observations from the fluorescence data. Upon diluting saturated overnight cultures 1/100, cells expressing MaPylRS are unable to grow on chloramphenicol plates regardless of the presence or absence of a ncAA. However, under these same conditions, PylRS_opt supports strong colony growth in the presence of BocK, PrK, or ALocK. Neither enzyme supports growth on 200 μg/ml chloramphenicol plates in the absence of a ncAA. We note, however, that on the less stringent 100 μg/ml chloramphenicol plates, spotting undiluted cells expressing PylRS_opt enables slow growth in the absence of a ncAA, whereas the same cells expressing MaPylRS cells are unable to grow under these conditions. This phenomenon is not observed at 200 μg/ml chloramphenicol, where growth is only apparent for PylRS_opt in the presence of 1 mM BocK or ALocK. These results confirm the previous observation that PylRS_opt is more active than MaPylRS towards BocK, ALocK, and PrK, with the caveat that under low stringency conditions, PylRS_opt can apparently incorporate a canonical amino acid in the absence of a ncAA and facilitate modest colony growth.

Next, we sought to determine whether the mutations found in PylRS_opt can improve the activity of a previously engineered variant of MaPylRS that readily incorporates meta- and ortho-substituted Phe (Tharp et al., 2021). The highly conserved active site residue N166 (N346 in M. mazei PylRS) plays a critical role as the “gatekeeper residue” of the PylRS active site, as the amide nitrogen is involved in the recognition of pyrrolysine and its analogs (Yanagisawa et al., 2008; Wang et al., 2011; Wang et al., 2012). Mutating N166 alters the substrate specificity of MaPylRS, effectively eliminating recognition of lysine-derived ncAAs while greatly increasing its activity towards several phenylalanine derivatives. MaPylRS_N166S was recently shown to aminoacylate a variety of meta- and ortho-substituted phenylalanine derivatives (Tharp et al., 2021). Thus, we created PylRS_N166S/opt and compared its activity to MaPylRS_N166S to assess whether combining the PylRS_opt mutations with N166S can enhance its activity towards Phe derivatives without compromising its selectivity (Figure 5A). The results show that PylRS_N166S/opt has increased activity towards mCNF and oIF, which are both difficult substrates for MaPylRS_N166S. However, the PylRS_opt mutations do not improve activity towards the well-recognized substrate mIF. As MaPylRS_N166S is already highly active towards mIF, the lack of improvement in activity towards this substrate is predictable. It should be noted that the background activity of PylRS_N166S/opt is also elevated in the absence of a ncAA. It is plausible that the canonical amino acid may be sufficiently outcompeted when a recognized ncAA is present, as is the case with PylRS_opt in the presence of BocK. Overall, the data indicate that the mutations in PylRS_opt improve the activity of MaPylRS_N166S towards difficult Phe-ncAAs. However, elevated background activity is also apparent for this PylRS variant and may be a drawback for applications that require a homogeneous protein sample. Lower background levels may be attainable when combining the PylRS_opt mutations with other active site mutants variants, such as those reported previously for incorporating bulky Lys-ncAAs (Seki et al., 2020).

As a final assessment of the usefulness of PylRS_opt, we tested whether the enhanced activity and substrate specificity of PylRS_opt could be used to improve the incorporation of two distinct ncAAs into a single protein. Translation efficiency drops dramatically when two or more distinct ncAAs are to be inserted into a protein, a problem that is exacerbated when aminoacylation of either of the ncAAs is particularly challenging (Hoesl and Budisa, 2011). Thus, improved activity is crucial to attaining substantial yields of proteins containing multiple ncAAs. To investigate the applicability of PylRS_opt for improving dual incorporation of difficult ncAAs, we cloned a system to measure the production of sfGFP featuring two in-frame stop codons: 2TAG and 149TAA. We utilized the M. jannaschii TyrRS variant AzFRS.2. t1 and its cognate MjtRNA^Tyr _CUA to incorporate pAzF, and either MaPylRS or PylRS_opt along with MatRNA^Pyl _(6)/UUA to incorporate PrK. Importantly, PylRS does not discriminate against the anticodon of tRNA^Pyl (Ambrogelly et al., 2007), and tRNA^Pyl _UUA does not suppress UAG codons (Odoi et al., 2013), thus enabling PylRS/tRNA^Pyl _UUA to function as an orthogonal pair with the MjTyrRS/tRNA^Tyr _CUA system (Wan et al., 2010). When cultures were supplemented with both pAzF and PrK, we observed over 120% increased fluorescence in cells expressing PylRS_opt as opposed to MaPylRS (Figure 5B). Background activity in the absence of one or both ncAAs was comparable for PylRS and PylRS_opt. Interestingly, low levels of expression were seen when PrK was present but pAzF was omitted, consistent with earlier observations of the PylRS/MjTyrRS dual ncAA incorporation system (Wan et al., 2010; Chatterjee et al., 2013). As has been suggested previously, this artifact is likely due to low-level recognition of canonical amino acids by AzFRS.2. t1 in the absence of pAzF. Ultimately, utilizing PylRS_opt to incorporate a second ncAA into a protein leads to over a two-fold improvement in the apparent protein yield compared to wild-type MaPylRS. PylRS_opt should therefore be quite useful in applications requiring site-specific incorporation of multiple reactive moieties in a single protein, such as bioorthogonal click chemistry (Neumann et al., 2010; Wan et al., 2010).