Strategies to Supply the Cell with iso-CmTP and iso-GTP
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 the biosynthesis of fully synthetic bases is, if possible at all, extremely challenging. Furthermore, to allow the incorporation of those bases into the DNA, phosphorylation of the nucleosides has to be ensured which is facilitated by in part highly specific kinases. Therefore, this strategy represents an optimal 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 (left) is based on a heterologous transporter which can facilitate the transport of iso-CmTP and iso-GTP. The media is supplemented with the unnatural (deoxy)nucleoside triphosphates, which are then transported into the cell and incorporated into the DNA. The second strategy (right) is more complex and is based on the de novo synthesis of the unnatural (deoxy)nucleoside triphosphates. Therefore, existing pathways from natural sources or newly designed pathways have to be introduced to the cell. The nucleoside triphosphates are then incorporated into the DNA.
The Nucleotide Transporter from Phaeodactylum tricornutum PtNTT2
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.
Figure 3: Subcellular localization of PtNTT1 and PtNTT2 in Phaeodactylum tricornutum (Ast et al., 2009). PtNTT1 and PtNTT2 were fused to GFP to study the subcellular localization.
Zhang et al. 2017 integrated PtNTT2 into the chromosome of E. coli BL21(DE3) under control of the lacUV5 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(66-575), 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).
Figure 4: Uptake of α32labeled ATP by the different versions of PtNTT2 (Zhang et al., 2017).
The expression of PtNTT2 was investigated in different strains, under control of different promotors, and plasmid-bound as well as integrated into the chromosome. In their final design, Zhang and colleagues integrated PtNTT2 chromosomally in E.coli BL21(DE3) under control of the lacUV5 promoter
Sec- and Tat Secretion PathwaysPtNTT2 comes from an eukaryote and is integrated into the plastid membrane in its native algael host. Therefore, expression of PtNTT2 in E. coli might not lead to efficient integration into the inner membrane. Therefore, we wanted to test different signal peptides to find the one best suited for efficient integration of PtNTT2 into the inner membrane. In E. coli , proteins can be secreted via two main pathways, namely the general secretion (Sec) and the twin-arginine translocation (Tat) pathway . Proteins which are intended to be secreted are targeted by signal peptides which are located at the N-terminus of a pre-protein and share one common tripartite structure: a positively charged N-terminal domain (N-domain) followed by a nonpolar hydrophobic domain (H-domain) and the more polar C-domain (De Keyzer et al., 2003; von Heijne, 1998). Signal peptides vary in length, but are usually between 18 and 30 amino acids long. During the translocation process, the signal peptide is cleaved from the pre-protein by a signal peptide peptidase, which recognizes a cleavage site within the C-domain of the signal peptide (De Keyzer et al., 2003). Signal peptides targeting the Sec pathway do not feature a common consensus sequence or motif (Caspers et al., 2010). However, the binding of the secretion specific chaperone SecB or the signal recognition particle SRP (which represents the first step of the Sec pathway) to the signal peptide is influenced by the hydrophobicity of the H-domain (De Keyzer et al., 2003). Signal peptides targeting the Tat pathway are usually around 30 amino acids long (Berks et al., 2014). On the contrary to Sec pathway targeting signal peptides, Tat pathway targeting signal peptides do feature a consensus sequence. The crucial element for Tat recognition is a twin-arginine motif (RR) located at the N/H-domain junction. In bacteria, the RR is usually followed by phenylalanine, leucine and lysine at the RR+2 position with especially the phenylalanine being of high importance (Cline, 2015).
Sec PathwayIn bacteria, most proteins are secreted via the conserved and essential Sec pathway in an unfolded state (Schneewind and Missiakas, 2014; Caspers and Freudl, 2008). The key component of the Sec pathway is the translocase which is located in the plasma membrane and composed of the SecYEG heterotrimeric protein as well as the SecA ATPase. SecYEG consists of three components, SecY, SecE and SecG. SecY and SecE form the core of the transmembrane channel, while SecG is an additional integral subunit that seems to be not essential for the translocation process (Schneewind and Missiakas, 2014; de Keyzer et al., 2003; Caspers and Freudl, 2008). SecA serves as the molecular motor of the translocation process by consecutively binding and hydrolyzing ATP and hence initializing the translocation. The proton motive force (PMF) helps translocating the protein once the process is initialized by SecA (Caspers and Freudl, 2008). SecYEG can associate with another heterotrimeric complex involved in the translocation process consisting of SecD, SecF, and YajC. The exact role of this additional heterotrimeric complex is yet unknown but seems to optimize secretion (de Keyzer et al., 2003). It was proposed that SecDFYajC might be involved in the proton motive force dependent steps of the translocation process (Denks et al., 2014). Proteins are targeted in two different ways, being mediated by the chaperone SecB, or the signal recognition particle (SRP) . SecB and SRP both bind the nascent pre-protein but seem to bind pre-proteins of different lengths. Proteins that are to be secreted are usually targeted via the SecB mechanism (de Keyzer et al., 2003). No matter which mechanism is involved in the targeting process, the proteins eventually interact with the translocase. Gradual translocation of the pre-protein is catalyzed by the translocase, during which the pre-protein is further processed by the signal peptide peptidase (SPase). The SPase is embedded in the periplasmic membrane and removes the signal peptide of the pre-protein (Denks et al., 2014; de Keyzer et al., 2003).
Figure 5: Schematic overview over the Sec-pathway and the involved components.(de Keyzer et al., 2003). Pre-proteins are either targeted by SRP or SecB and translocated by the translocase. The translocase consists of SecYEG, forming the transmembrane channel, and SecA, the ATP-driven molecular motor of the translocation. The signal peptide is eventually removed by the SPase (here Lep) .
Tat PathwayOpposite to the Sec pathway, which secretes proteins while they are not fully folded, proteins secreted via the twin-arginine translocase (Tat) pathway are fully folded (Cline, 2015). The Tat pathway is an active transport system with the transmembrane proton motive force being the driving force of the translocation (Palmer and Berks, 2012). Three proteins from only two structural families form the machinery required for Tat mediated secretion (Berks et al., 2014). The first family called TatA is described as a single transmembrane helix with a short N-terminal transmembrane domain and an unstructured C-tail (Berks et al., 2014; Cline, 2015). TatB belongs to the TatA family but was shown to be abdicable for the basic functionality. The second structural family involved in the Tat pathway is TatC, consisting of six transmembrane helices with the N- and C-termini being exposed to the cytoplasm. The structure of TatC was determined for Aquifex aeolicus and is shown together with the structure of TatA from E. coli in Figure 6 (Berks et al., 2014; Cline, 2015). A fourth component, TatE provides an overlapping function to TatA, hence making TatE dispensable (Kikuchi et al., 2009).
Figure 6: Structures of TatA from E. coli and TatC from Aquifex aeolicus
While TatA forms a single transmembrane helix, TatC consists of six transmembrane helices (Berks et al., 2014).
Figure 7: Steps of the Tat translocation system
After the signal peptide is bound by TatBC TatA assembles and forms the translocation pore. Using the transmembrane proton motive force, the substrate is translocated and the signal peptide cleaved by a signal peptidase (Palmer and Berks 2012)
Origin of Croton Tiglium
Usages of Croton Tiglium
Secondary Metabolism of C. Tiglium
De novo Synthesis of Purine Bases
Figure 1: De novo Synthesis of Pyrimidine Bases
Conversion of Ribonucleosid Diphosphates to Deoxyribonucleotides
Figure 1: De novo Synthesis of Purine Bases
Adaptions of the purine pathway used in C. Tiglium for the production of iso-Guanosine
The ordinary form of Guanosine is produced within the purine pathway. Thus, it is likely that the production of iso-guanosine is realised inbetween these reactions.
Kanehisa, M. and Goto, S. (2000). Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28: 27–30.
Isolation of Isoguanosine from Croton tiglium and Its Antitumor Activity. Jung Han Kim, Sang Jun Lee, Young Bok Han, Jung Jo Moon, Jong Bae Kim; Arch. Pharm. Res. Vol. 17, No.2 , pp. 115-118, 1994
Cherbuliez, E. and Bernhard, K., Croton seed(1)crotonoside. Helv. Chim. Act&, 15, 464, 978-982 (1932).
Huang, M., Shimizu, H. and Daly, J. W., Accumulation of cyclic adenosine monophosphate in incubated slices of brain tissue. J. Med. Chem., 15, 462-468 (1972)
Lowry, B. A. and Brown, G. B., The utilization of purine nucleosides for nucleic acid synthesis in the rat. J. BioL Chem., 197, 591 (1952).
Vasu, N. and David, A. Y., A New Synthesis of Isogua- nosine. J. Org. Chem., 50, 406-408 (1985).