3 Results and Discussion
3.2.3 M. jannaschii TyrRS Library
In the MjTyrRS library constructed by Fabian Schildhauer, a total of 8 residues were mutated, resulting in a theoretical library size of 6.9 x 108 variants: Tyr32VNK (all aa except Cys, Phe, Tyr, Trp), Ala67GBC, His70CTC, Asp158CTC (small aa: Ala, Gly, Leu, Val), Leu65NNS, Phe108NNS, Gln109NDT, Leu162VHG (all aa except Trp). In Figure 42 the mutated residues are shown as sticks and highlighted in red. In addition to screening for variants capable of charging [3,2]Tp to the cognate tRNA, it was simultaneously screened for variants capable of charging 4-F-indole. In the meantime, in the Budisa lab, two more strains had been adapted to growth on the indole analogs 4-F-indole and 5-F-indole491, respectively, with the former strain being the more robust of the two.
Figure 42 I Active site of the M. jannaschii TyrRS with bound tyrosine (green). Residues chosen for mutation are shown as sticks and highlighted in red. PDB: 1J1U
Prior to selection, the maximal concentrations of indole analogs tolerated by NEB10-beta cells under selection conditions were tested by comparing colony growths of culture dilutions after the first round of positive selection on LB-agar plates containing increasing concentrations of [3,2]Tp and 4-F-indole (Figure 43).
Y32
L162
D158
L65
A67
H70 F108
Q109
Conc.
[mM]
Indole
analog Dilution series Indole
analog Dilution series 0.03
4-F-indole [3,2]Tp
0.1
4-F-indole [3,2]Tp
0.5
4-F-indole [3,2]Tp
1
4-F-indole [3,2]Tp
Figure 43 I Indole analog tolerance of NEB10-beta cells under selection conditions. After transformation with the MjTyrRS library, a dilution series was prepared from recovered NEB10-beta cells and 2 µL of each dilution were plated on LB agar plates containing 37 µg/mL Cm and increasing concentrations of 4-F-indole or [3,2]Tp.
While 4-F-indole only supports the growth of NEB10-beta cells in concentrations of up to 0.1 mM, the cells tolerated [3,2]Tp in all tested concentrations. Based on these results, concentrations of 0.1 mM 4-F-indole and 0.5 mM [3,2]Tp were chosen for the selection experiments. The concentration of [3,2]Tp was increased in comparison to prior selection experiments to increase the selection pressure towards [3,2]Tpa-incorporating aaRS variants. During the selection experiments with fluoro-indoles, a concentration of 0.03 µM 4-F-indole was sufficient to support the growth of the adapted strain to OD600
values of around 1.5. Thus 0.1 mM 4-F-indole should provide enough excess of the substrate to drive the selection experiments towards 4-F-Trp-incorporating aaRS variants.
Transformation efficiencies of 1.6 x 109 for [3,2]Tp and 6 x 108 for 4-F-indole were achieved for the first positive selection with the TyrRS library. The chloramphenicol concentrations were increased from 30 µg/mL in the first round of positive selection (P1) to 50 µg/mL in later rounds. After each positive selection, the number of colonies on the selection plates was counted and compared to the number of colonies on the control plate lacking the indole analog. The number of colonies on the selection plates was set as 100 % and the percentage of the colony numbers on the corresponding control plate was calculated. Figure 44 depicts the (mean) colony numbers for one agar plate each. A volume of 450 µL of recovered transformation was spread on each plate, except for the first positive selection with [3,2]Tp, where 1 mL of culture volume was spread on each plate. Therefore, the numbers of colonies for P1 were adjusted to a culture volume of 450 µL for comparison with the following positive selections.
Figure 44 I Number of colonies on positive selection plates supplemented with the indicated indole analog compared to control plates lacking the analog. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. P1-3: positive selection 1-3, ctrl: control plates. P1 represents the mean of 3 ([3,2]Tp) and 5 (4-F-indole) agar plates with the SD represented as the error bar. a) Double-sieve selection with [3,2]Tp. The colony numbers for P1 were adjusted to the volume of recovered transformations spread on P2 and P3 plates (450 µL / plate). b) Selection experiments with 4-F-indole.
Curiously, in both experiments, the number of colonies per plate drastically decreases during the second positive selection (P2) and increases again during the third round of positive selections (P3).
Usually, a steady increase in colony numbers would be expected with the progression of the selection, as functional library members should be enriched. The drastic decrease in colony numbers observed here, suggests a reduction of functional library members. Possible reasons for this observation could be the very stringent negative selection, which might have killed promising candidates in addition to those that incorporate only cAAs. For example, a variant that incorporates the desired ncAA might also incorporate one or more cAAs. This variant would still be a promising candidate, as long as the ncAA outcompetes its canonical contender(s), but would be killed during the negative selection in the absence of the non-canonical substrate. Another reason for the decrease in colony numbers might be a possible loss of library members during harvesting of the colonies from the selection plates, followed by plasmid preparations (midiprep), digestion of the selection plasmid, the subsequent purification of the library plasmids, and transformation of the next selection round. Finally, the high number of colonies per plate during the first positive selections might be attributed to a high number of variants that solely incorporate canonical amino acids, which are then removed during the negative selection.
This hypothesis might be supported by the similarly high colony numbers on the corresponding control plates. Furthermore, in the case of the experiment with [3,2]Tp the difference between colony numbers on the selection and control plate increases during the second round of positive selections and even more so during the third round, where the number of colonies drastically increases again.
While the number of colonies on the selection plate is approximately 1.3 fold higher for P3 compared to P1, the colony number on the corresponding control plate decreases from 86 % for P1 to 55 % for P3, indicating enrichment of MjTyrRS variants capable of charging [3,2]Tp to their cognate tRNA. In the case of the experiment with 4-F-indole, the colony number on the selection plate increases by around 7-fold in P3 compared to P1, but the colony number on the corresponding control plates stays at 90 % or higher compared to the selection plates.
After the second and third positive selections, 40 colonies were screened and plated on increasing Cm concentrations with and without the respective ncAA. Even after three rounds of positive selection and two rounds of negative selection, in the case of experiments with 4-F-indole still no promising candidates could be obtained.
0
Cm
[µg/mL] Variant - ncAA + 0.5 mM [3,2]Tp
37
33 39
70
33 39
100
33 39
150
33 39
Figure 45 I Serial dilutions of the two promising MjTyrRS variants 33 and 39 on increasing Cm concentrations with and without [3,2]Tp.
However, screening of the colonies from the third positive selection with [3,2]Tp yielded two promising candidates, colony 33 and colony 39. Serial dilutions of these cultures were spotted on plates with and without [3,2]Tp and Cm concentrations ranging from 37 µg/mL to 70 µg/mL, 100 µg/mL and finally 150 µg/mL. As can be seen in Figure 45, both isolates, 33 and 39, exhibit enhanced growth on plates supplemented with the indole analog [3,2]Tp, suggesting that these may be able to charge [3,2]Tp to their cognate tRNA and thus suppress the stop codons in the cat gene. Sequencing analysis of the two isolates revealed the mutations indicated in Table 4.
Table 4 I Summary of mutations in isolates 33 and 39 as revealed through sequencing analysis.
Residue 32 65 67 70 108 109 158 163 wt Tyr Leu Ala His Phe Gln Asp Leu 33 Gly Glu Gly Leu His Glu Leu Pro 39 Gly Gly Ala Gly Leu His Ala Asp
In order to further characterize the variants’ ability to suppress amber codons a fluorescence assay analogous to the one in chapter 3.2.1 (p. 63) was conducted. The two variants, 33 and 39, were transferred from the high copy library backbone (pBU18) to a low copy backbone (pBU16) harboring the cognate tRNA and co-transformed with sfGFP(R2TAG) into BL21(DE3) cells. Again, both absorption at 600 nm as well as fluorescence at 511 nm of six biological replicates were measured over 24 h. The cells were cultured in ZYP 5052 auto-induction media supplied with either 0.5 mM [3,2]Tp, 0.1 mM 4F-indole (to check for promiscuity of the variants), or no ncAA. As a control, wt sfGFP was expressed. This
time, all cultures exhibited similar growth curves with the exception of the wt sfGFP cultures reaching higher absorptions (almost twofold), which might be explained by these cells harboring only the sfGFP plasmid, while the other cells additionally harbor the MjTyrRS/tRNA plasmid. The cultures reached stationary phase after about 6 h of incubation at 37°C and in the case of the wildtype control approximately 12 h. The mean, as well as the standard deviation, were calculated and the fluorescence was normalized to the absorption. Figure 46 shows the normalized fluorescence after 15 h of incubation.
Figure 46 I Normalized fluorescence of MjTyrRS variant 33 and 39 after 15 h of incubation. Six biological replicates were cultivated in ZYP 5052 media supplied with the appropriate antibiotics, as well as either 0.5 mM [3,2]Tp, 0.1 mM 4-F-indole, or no ncAA at all. Fluorescence was normalized to absorption at 600 nm.
The controls cultivated in the absence of any ncAA exhibit a rather high fluorescence compared to the wt control, indicating background suppression. No (distinct) increase in fluorescence can be observed upon 4-F-indole supplementation, however, an approximately twofold increase in fluorescence can be seen upon [3,2]Tp supplementation. To identify the amino acid incorporated in response to the amber codon the mass was determined. BL21(DE3) cells harboring pET28a_sfGFP(R2TAG)-His and the MjTyrRS variants 33 and 39 were cultivated in DYT supplemented with the appropriate antibiotics and 0.1 % glucose to suppress leaky sfGFP expression before induction. At a cell density of OD600 = 1 the indole analog [3,2]Tp was added at a concentration of 0.5 mM and it was incubated for another 30 min at 37°C to allow uptake of [3,2]Tp and conversion to the amino acid [3,2]Tpa. sfGFP expression was induced with 1 mM IPTG and expressed for 4 h at 37°C. After purification via immobilized metal affinity chromatography (IMAC) the mass was detected via electrospray ionization mass spectrometry (ESI-MS). The MS spectra were extracted from the highest peak of the total ion count (TIC) chromatogram and deconvoluted using the Agilent Mass Hunter BioConfirm software (Figure 47).
0 5 10 15 20 25 30
Figure 47 I Identification of the amino acid incorporated in response to the amber codon in sfGFP(R2TAG). The reporter protein sfGFP(R2TAG) was co-expressed with MjTyrRS variants 33 or 39 in the presence of [3,2]Tp. The ESI-MS spectra were deconvoluted using the Agilent Mass Hunter BioConfirm software for masses between 27 kDa and 30 kDa. The highest peak was normalized to 100. a) Deconvoluted spectrum of sfGFP(R2TAG) co-expressed with variant 33. The highest peak corresponds to phenylalanine incorporation. The smaller peaks are each approximately 22 Da apart and likely correspond to sodium adducts (Na = 22.99 Da). b) Deconvoluted spectrum of sfGFP(R2TAG) co-expressed with variant 39. The highest peak corresponds to glutamine incorporation. The smaller peaks are each approximately 22 Da apart and likely correspond to sodium adducts (Na = 22.99 Da).
In the case of stop-codon suppression with variant 33, the main peak corresponds to phenylalanine incorporation at position 2 of the reporter protein (predicted mass: 27744.2 Da, observed mass:
27744.8 Da). In the experiment with variant 39, the main peak corresponds to Gln (predicted mass:
27744.85 Da, observed mass: 27744.84 Da). Incorporation of glutamine at amber positions is common in the absence of an amber suppressor503 as its CAG codon is similar to the amber codon (UAG). The other peaks are each approximately 22 Da apart and likely represent sodium adducts (Na = 22.99 Da).
The third peak in Figure 47 a (27788.4 da) and the fourth peak in Figure 47 b (27790.9 Da) exhibit masses similar to that expected for [3,2]Tpa incorporation (27789.3). These results might indicate a TyrRS variant promiscuous for aromatic side chains in the former case and a variant that charges [3,2]Tpa to its cognate tRNA so inefficiently, that the main expression product stems from background suppression of the UAG codon with glutaminyl-tRNA in the latter case, resulting in protein mixtures with different amino acids at position 2. However, due to the regular pattern of the peaks, it is more likely that these represent sodium adducts. Nevertheless, as both, the Cm assay and the fluorescence assay, show a clear difference between the control and [3,2]Tp-supplemented samples, these variants were deemed a promising starting point for further improvement.
3.2.3.1 Error-prone PCR library
The semi-rational design of mutant aaRS libraries usually focuses on first shell active site residues, as transformation efficiencies limit the library size and thereby the number of mutable residues.
However, these libraries often result in enzymes with low affinities for their ncAA substrates, as well as low overall aminoacylation237,504–506. Furthermore, the active site is sensitive to perturbations; first shell residues interact with other residues in a way that is not always fully understood and those interactions may be inadvertently perturbed by mutated sidechains310. Hence, only mutating first shell
275000 28000 28500
residues may not be enough to yield efficient enzymes507. The creation of randomly mutagenized libraries via error-prone PCR has therefore proven beneficial508.
Thus, variant 33 was used as a template for error-prone PCR (epPCR), where the mutation rate of the Taq polymerase is further enhanced by the addition of MnCl2, resulting in a MjTyrRS library with random point mutations distributed throughout the entire enzyme. Variant 33 was chosen, as it produced higher fluorescence and increased colony growth on Cm in comparison to variant 39. Eight single colonies were sequenced and their mutations are summarized in Table 5.
Table 5 I Summary of the number and type of mutations found in eight colonies of the epPCR-based MjTyrRS library.
Type(s) of mutations Frequency Proportion of total
Transitions 48 39.36 %
A --> G, T --> C 34 27.88 %
G --> A, C --> T 14 11.48 %
Transversions 30 24.6 %
A --> T, T --> A 22 18.04 %
A --> C, T --> G 6 4.92 %
G --> C, C --> G 1 0.82 %
G --> T, C --> A 1 0.82 %
Insertions and deletions 4 3.28 %
Insertions 3 2.46 %
Deletions 1 0.82 %
Summary of bias
Transitions / transversions 1.6 NA AT --> GC / GC --> AT 2.43 NA
A --> N, T --> N 62 50.84 %
G --> N, C --> N 16 13.12 %
Mutation rate
Mutations per kb 11.13 NA
Mutations per MjTyrRS gene 10.25 NA
This data was used to estimate the library characteristics using PEDEL-AA509, the results are summarized in Table 6.
Table 6 I Summary of library characteristics of the error-prone PCR-based MjTyrRS library estimated with PEDEL-AA509.
Property Estimate
Total library size 1 x 107
Number of variants with no indels or stop codons 4.48 x 106 Mean number of amino acid substitutions per variant 7.28 Unmutated (wildtype) sequences (% of library, Poisson est.) 0.03091 % Number of distinct full-length proteins in the library (Poisson est.) 4.46 x 106
With a transformation efficiency of 4.2 x 107 the estimated library size of 1 x 107 was covered. Double-sieve selection was performed as described above and a total of three consecutive rounds of positive and negative selections were conducted with Cm concentrations increasing from 30 µg/mL to 45 µg/mL and finally 70 µg/mL. From the beginning, a clear difference in colony numbers between the control plate and the selection plates could be observed with the number of colonies on the control plate only 9 % that of those on the corresponding selection plates (Figure 48). With the second positive selection, the overall number of colonies increased, indicating the accumulation of functional library members. However, with 26 % the ratio of colonies on the control plate compared to the selection plates also noticeably increased, indicating a possible enrichment of variants charging canonical amino acids to their cognate tRNAs. During the third positive selection, the number of colonies drastically decreased to levels lower than during the first round of selection with the number of colonies on the control plate also slightly decreasing to 17 % of those on the selection plates. This decrease in colony numbers could suggest that 70 µg/mL Cm might have posed too high of a selection pressure. However, during this third positive selection, a new selection plasmid was used, which additionally harbors the gene for sfGFP(R2TAG) to facilitate screening with the fluorescence assay without the need for cloning and transformations between Cm and fluorescence assays. It is possible that leaky sfGFP expression from the lac promoter in addition to chloramphenicol acetyl-transferase and MjTyrRS expression might have posed too much strain on the cells.
Figure 48 I Number of colonies on positive selection plates supplemented [3,2]Tp compared to control plates lacking the analog during selections with the epPCR MjTyrRS 33 library. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. P1-3: positive selection 1-3, ctrl: control plates. The numbers for the selection plates represent the mean of 5 (P1), 3 (P2), and 2 (P3) agar plates with the SD represented as error bars. The colony numbers were normalized to the culture volume.
Once again, single colonies were screened with and without [3,2]Tp on increasing Cm concentrations after the second and the third positive selection. Promising candidates were isolated and their serial dilutions were plated under the same conditions. Figure 49 provides an overview of promising variants isolated after the second positive selection.
0 1000 2000 3000 4000 5000 6000 7000 8000 9000
number of colonies
P1 P2ctrl P3
ctrl ctrl
Cm
[µg/mL] Variant - ncAA + 0.5 mM [3,2]Tp
37
3 4 8 24 25 30
70
3 4 8 24 25 30
120
3 4 8 24 25 30
Figure 49 I Serial dilutions of promising variants on increasing Cm concentrations with and without [3,2]Tp after the second round of positive selection with the epPCR MjTyrRS 33 library.
These variants grow in the absence of the analog even at very high Cm concentrations, but the number of CFU somewhat increases upon [3,2]Tp addition. The pronounced growth in the absence of the indole analog is in line with the high fluorescence of variant 33 in the absence of [3,2]Tp, which was used as a template for the error-prone PCR based library used here, and indicates incorporation of cAAs in response to the amber codons in the CAT. Nevertheless, the increase of cell growth upon [3,2]Tp supplementation also suggests incorporation of the target ncAA.
However, in the case of variants isolated after the third round of positive selection, almost no difference between the serial dilutions plated in the absence or presence of [3,2]Tp could be seen (Figure 50).
Cm
[µg/mL] Variant - ncAA + 0.5 mM [3,2]Tp
37
16 17 19 20 38 39 40 41 42
100
16 17 19 20 38 39 40 41 42
Figure 50 I Representative serial dilutions of promising variants on increasing Cm concentrations with and without [3,2]Tp after the third round of positive selection with the epPCR MjTyrRS 33 library.
Nevertheless, some of the more promising candidates picked after the second and third round were screened in the fluorescence assay with sfGFP(R2TAG) and are shown in Figure 51 a. BL21(DE3) cells transformed with sfGFP(R2TAG) but not with any of the MjTyrRS variants served as a control for background suppression. Unfortunately, no significant increase in fluorescence intensity upon analog addition could be observed for any of the tested variants.
In parallel, another epPCR library was constructed and screened for variants capable of charging 5-F-Trp to their cognate tRNA. This library was based on a promiscuous MjTyrRS variant (termed 14.2) found during selections for another Trp analog (β-(1-Azulenyl)-L-Alanine) in the Budisa laboratory. In addition to charging the target ncAA to the suppressor tRNACUA
Tyr , variant 14.2 was found to exhibit some activity for F-Trp as well. Therefore, this variant was employed in a test to compare the efficacy of 5-F-indole and 5-F-Trp in the sfGFP(R2TAG) assay employed for the identification of promising aaRS variants throughout this study. During the numerous selection experiments with [3,2]Tp (and 4-F-indole), only the precursor was used rather than the amino acid [3,2]Tpa itself. The indole analog diffuses through the cell membrane and is intracellularly converted to the corresponding amino acid231. While this approach worked well for the adaptation experiments (chapter 1.3.1, p. 22), it might not work as well during double-sieve selection and the screening experiments described here. Expression of the orthogonal aaRS/tRNA pair, as well as the selection marker (CmR/sfGFP), already poses significant stress to the host cells. Having to additionally convert the precursor to the amino acid prior to selection marker expression might put too much strain on the cells. Hence, having access to an aaRS
variant capable of incorporating Trp analogs was a good opportunity to test this hypothesis in a fluorescence assay. Figure 51 b presents the fluorescence of sfGFP(R2TAG) co-expressed with variant 14.2 in the absence and presence of increasing 5-F-indole and 5-F-Trp concentrations. Indeed, fluorescence is considerably higher when 5-F-Trp is supplied than with supplementation of the indole precursor.
Figure 51 I Fluorescence assay of sfGFP(R2TAG) with different MjTyrRS variants in the absence and presence of ncAAs. a)
Figure 51 I Fluorescence assay of sfGFP(R2TAG) with different MjTyrRS variants in the absence and presence of ncAAs. a)