Balancing An Expanded Genetic Code System

One could suppose that the low efficiency of UAA incorporation could be easily adjusted by simply overexpressing the required components, i.e., the tRNA/aaRS pairs, and/or by increasing the UAA concentration in the media. However, the overexpression of endogenous components could also provoke toxic ancillary effects and exhaust biosynthetic energy. Moreover, UAAs, especially those that are not commercially obtainable, have to be used as economically as possible.

In order to determine optimal expression and aminoacylation levels of the tRNA/aaRS pairs, which guarantee sufficient suppression of the blank codons without distressing the cells, we developed assays for their detection. The absence of specific antibodies against the aaRS PylS and MjYRS led us introduce the well-characterized small His6-tag into these proteins but neither the addition to the N- nor the C-terminus allowed for clear detection of the synthetases by western blot using antibodies against the His6-tag. Since the cell lysates

were treated under denaturing conditions for the blot, using SDS and heat, the accessibility of the terminal epitopes by antibodies should not be restricted. This was proven by the Schultz group which obtained a relatively weak but distinct signal of a C-terminal His6 -tagged BPARS, a mutant MjYRS that decodes for BPA, in cell lysates by western blot analysis^[69]. The detection of His6-tagged PylS also failed for them, until they used a variant that was codon-optimized for expression in E. coli^[72]. Considering the crystal structures of both aaRS (Figure 4.2 and 4.10), their C-termini are exposed to the protein surfaces and in case of MjYRS not directed towards the tRNA. This holds also true for the N-terminus of MjYRS, but concerning PylS no statement can be made because structural data in combination with PylT and for the N-terminal domain is missing. However, unless there are unknown interactions with other components and as long as the proteins are properly folded, the His6-epitope should be accessible even under native conditions. Srinivasan et al.

were therefore able to purify PylS using an N-terminal His6-tag^[33]. An observation made by Chatterjee et al.^[72] and, independently by ourselves, is that PylS is mostly found in the insoluble fraction, probably due to misfolding in E. coli. Improper folded proteins are prime targets for proteolysis^[209] and could thereby represent one explanation for the poor detectability of (tagged) PylS. We tried to solve this problem by using chaperones, reducing the temperature during expression and deleting parts of the proline-rich linker (Figure 4.1, 4.5 and 4.6) without success (data not shown).

In addition to these findings, we discovered that MjYRS accepted a His6-tag at the C-terminus without losing catalytic activity (Figure 4.11), whereas PylS revealed a grave loss of activity upon addition of this tag (Figure 4.3). By contrast, shifting the His6-tag into the flexible linker region of PylS, between the N-terminus tRNA binding domain and the C-terminus catalytic domain, proved beneficial because we were then able to detect the non-codon-optimized protein by western blot without severely impairing its enzymatic functionality (Figure 4.3 and 4.4). The introduction of an unconventional internal His6-tag also helped us to determine MjYRS expression levels by western blot, which were many times lower than PylS levels, although under the control of the same glnS promoter (Figure 4.12). When expressed from the same tacI promoter Chatterjee et al. observed MjYRS to be stronger expressed than PylS^[72]. This demonstrates how important different promoters can be for the regulation of certain protein levels and, in the case of these two aaRSs, very likely for the suppression efficiency of the blank codons.

When we quantified PylS and MjYRS expression levels at different stages of growth in rich medium under the control of a glnS promoter, we found both proteins exhibited

diminished expression levels when the cultures reached high cell densities (Figure 4.4 and 4.12). This was consistent with previous reports stating that, in E. coli, the enzyme level of the naturally regulated glutaminyl-tRNA synthetase increases with increasing growth rate^[210], because cultures that approach saturation exhibit decreased growth rates. Thus, the aaRSs may become limiting in their ability to charge their cognate tRNAs with UAAs, and thereby for suppression of blank codons in recombinant proteins, during prolonged expressions. This, in turn, renders the wild type glnS promoter amendable for this purpose.

Indeed, the Schultz laboratory achieved a fivefold improved suppression efficiency using a mutant glnS promoter (glnS’) that led to a twofold increase in BPARS expression^[69]. Additional improvements were made by further raising the expression levels of different MjYRS derivatives using a dual glnS′/araBAD promoter system or a single tacI-driven aaRS expression cassette^[51,70,72].

We decided to use promoter libraries for optimization, which have been shown to provide a broad range of promoter activities[158–160,211]

. We determined, that for the PylS, there was a direct positive dependency upon promoter strength for enzyme level, tRNA aminoacylation and suppression efficiency (Figure 4.19). Whereas Jensen and Hammer directly measured the effect of their synthetic promoter library in Lactococcus lactis and E. coli by β-galactosidase activity, obtaining up to 400-fold variation in promoter strength^[160], we performed indirect measurements and observed a fivefold variation, only. This was determined by the ability of the PylS to suppress an amber codon in the genes of CAT or histone H3, with the most efficient promoter being two to threefold more efficient than the wild type glnS variant. In the case of the MjYRS the promoter libraries did not reveal a similar tendency (Figure 4.22), leading to the assumption that the PylS constrains the performance of the current system for the incorporation of two distinct UAAs. The replacement of the wild type glnS promoter in front of the PylS gene with some well-defined standard promoters of different strength, including the most promising candidate used by the Schultz group, i.e., tacI^[72], permitted us to enhance the performance of the system once more (Figure 4.25). Furthermore, we could characterize the glnS promoter and also its strongest library variant, 3E, as relatively weak. However, we discovered modest increases in PylS levels already being advantageous, yielding higher amounts of UAA containing proteins. Promoters with stronger activities, like tacI, did not prove more useful and likely consumed biosynthetic resources, particularly if more than one component is controlled by these regulatory elements. To this end, we performed subsequent experiments using the constitutive promoter Pcon or the IPTG inducible Plac to carry out PylS expression.

In order to optimize the expression levels of the appropriate tRNAs, PylT and MjYT_UCCU, the original lpp promoters were also exchanged by promoter libraries. DIG labeled hybridization probes were created for the simultaneous detection of their abundance and loading status by northern blot analysis. We obtained specific signals for both tRNAs and were able to distinguish their aminoacylated form from their free form by means of a mobility shift in acid urea gels (Figure 4.9 and 4.16), as demonstrated by Polycarpo et al.^[39,212] or Köhrer and Rajbhandary^[142]. However, the effect of the tRNA promoter libraries on the suppression efficiency has not yet been investigated by western and northern blot.

They were merely used in combination with the two aaRS promoter libraries to construct a four component suppressor plasmid (see Ch. 6.2). According to the results of the Schultz laboratory, where they replaced the weak lpp promoter of MjYT_CUA by the stronger proK promoter^[69], one could expect improved suppression efficiencies by increasing the tRNA expression levels. While they initially observed a positive correlation between suppression efficiency and increased copies of tRNA, up to six tRNA copies per plasmid organized in two clusters under the control of proK, they later stated that these increases caused toxicity effects to rise to significant levels, leading to considerably slowed growth rates of E. coli.

They could show that a single copy of an optimized M. jannaschii amber suppressor tyrosyl-tRNA^[71] exceeded the performance of multiple non-optimized tRNAs, while the cell growth was less affected^[51].

In summary, it was shown by us, and others, that raising expression levels of both aaRS and tRNA to a certain limit provides benefits to the genetic code expansion system. Expression levels beyond these limits are either producing toxic side effects or do not further enhance suppression efficiencies and are probably exhausting biosynthetic energy. Instead of increasing the component’s abundance, additional opportunities to optimize the system could focus on the functionalities of the components. This is exemplified by the work of Kobayashi et al. who created a MjYRS mutant that aminoacylates its cognate tRNA 65 times more efficiently than its wild type variant, mainly due to improved recognition of the amber anticodon^[73].