Team:Bielefeld-CeBiTec/Project/unnatural base pair/uptake and biosynthesis

Uptake and Biosynthesis of iso-CmTP and iso-GTP

Strategies to Supply the Cell with iso-CmTP and iso-GTP

The first step to ensure that unnatural base pairs are retained over a long period of time is to provide the cell with the unnatural bases needed for the replication of the unnatural base pair. There are two different strategies to provide the cell with the unnatural bases. The first one is to supplement the media with the nucleoside triphosphates of the desired unnatural bases. To allow uptake of the nucleoside triphosphates, the cell needs to possess a transporter that can facilitate a sufficient flux into the cell. While this strategy seems to be the easiest one, it also has some disadvantages. First, E. coli does not possess nucleoside triphosphate transporters suitable for the transport of unnatural nucleoside triphosphates. Given that nucleoside triphosphates such as ATP are essentially absent from the extracellular environment, such transporters have not evolved. Another reason why nucleoside triphosphate transporters are basically absent from the cell membrane is that the existence of such could potentially put the intracellular ATP pool at risk. Therefore, a heterologous transporter has to be introduced. Secondly, chemically synthesized nucleotides are very expensive, so feeding them to the cells would make bioprocesses uneconomically for scale up. On the other hand, this strategy allows the testing of highly unique unnatural bases if uptake can be ensured. The second strategy is based on the in vivo biosynthesis of the desired unnatural bases. This strategy would allow scaling up of processes without making them uneconomical and would eliminate the need for a heterologous transport system. But especially the biosynthesis of fully synthetic bases is, if possible at all, extremely challenging. To allow the incorporation of those bases into the DNA, phosphorylation of the nucleosides needs to be ensured which is facilitated by in part highly specific kinases. Therefore, this strategy represents an ideal system which as of now is extremely challenging to implement.

Figure 1: Two strategies for making iso-CmTP and iso-GTP available to the cell.
The first strategy is based on a heterologous transporter which can facilitate the transport of iso-CmTP and iso-GTP. The media is supplemented with the unnatural nucleosidtriphosphates which are then transported into the cell and incorporated into the DNA. The second strategy is more complex and is based on the de novo synthesis of the unnatural nucleosidtriphosphates. Therefore, existing pathways from natural sources or newly designed pathways have to be introduced into the cell. The nucleosidtriphosphates are then incorporated into the DNA.

The Nucleotide Transporter from Phaeodactylum tricornutum PtNTT2

Given their structural similarity to the natural bases, iso-dCm and iso-dG might be taken up through the existing nucleoside transporters in sufficient amounts. But phosphorylation of the nucleosides remains a challenge. Therefore, the introduction of a heterologous nucleoside triphosphate transporter into the desired strain represents a promising alternative.

Phaeodactylum tricornutum, a diatom of the genus Phaedactylum, features six putative nucleotide transporters (NTTs). Two isoforms of these NTTs have been characterized by Ast et al. 2009 and it was shown that both isoforms facilitate transport across the plastid membrane. While isoform 1 (NTT1) acts as a proton-dependent adenine nucleotide importer, NTT2 facilitates the counter exchange of (deoxy-)nucleoside triphosphates (Ast et al., 2009).

Figure 2: Uptake of α32-labeled nucleotides by the two isoforms PtNTT1 and PtNTT2 when expressed in E. coli (Ast et al., 2009)
The uptake of α32-labeled nucleotides was measured in E. coli. Isoform 1 (A) was shown to transport adenosine mono-, di- and triphosphates, while isoform 2 (B) shows a specificity for nucleoside triphosphates.

Ast and colleauges demonstrated the subcellular localization by tagging the transporters with GFP. Figure 2 shows the subcellular localization of the two isoforms PtNTT1 and PtNTT2. The fluorescence signal can only be detected within the cell around the plastid and not within the cell membranes. The isoform 2 of the nucleotide transporter was shown to be an unspecific (deoxy-)nucleoside transporter, facilitating the uptake of CTP, GTP, dCTP, ATP, UTP, dGTP, dATP and TTP when expressed in E. coli, as shown in figure 3 (Ast et al., 2009). Uptake was measured using α32-labeled nucleotides. While isoform 1 (A) can transport adenosine mono-, di- and triphosphates, isoform 2 (B) shows a specificity for nucleoside triphosphates. The fact that PtNTT2 can accept a broad range of different nucleotides makes the transporter interesting for the transport of unnatural nucleotides, as long as they are provided as nucleoside triphosphates.

Figure 3: Subcellular localization of PtNTT1 and PtNTT2 in Phaeodactylum tricornutum
PtNTT1 and PtNTT2 were fused to GFP to study the subcellular localization (Ast et al., 2009).

The fact that PtNTT2 can accept a broad range of different nucleotides makes the transporter interesting for the transport of unnatural nucleotides.

Zhang et al. 2017 integrated PtNTT2 chromosomally in E. coli BL21 (DE3) under control of the PlacUV5 promoter. To demonstrate its feasibility for the uptake of nucleotides, uptake of [α-32P]-dATP was measured. The native sequence of PtNTT2 features an N-terminal signal sequence directing the subcellular localization to the plastid membrane. In E. coli, this signal sequence is likely to be retained, leading to a growth defect in cells expressing the native PtNTT2 transporter. Therefore, a truncated version of PtNTT2, PtNTT2(65-575), was used. The chromosomally integrated, truncated, and codon optimized PtNTT2 (65-575) under control of PlacUV5 was shown to be an optimal compromise between efficient uptake and the growth limitation resulting from expression of the heterologous protein (Zhang et al., 2017).

References

Ast, M., Gruber, A., Schmitz-Esser, S., Neuhaus, H.E., Kroth, P.G., Horn, M., and Haferkamp, I. (2009). Diatom plastids depend on nucleotide import from the cytosol. Proc. Natl. Acad. Sci. U. S. A. 106: 3621–3626.
Zhang, Y., Lamb, B.M., Feldman, A.W., Zhou, A.X., Lavergne, T., Li, L., and Romesberg, F.E. (2017). A semisynthetic organism engineered for the stable expansion of the genetic alphabet. Proc. Natl. Acad. Sci. 114: 1317–1322.