Proof of concept
As proof of concept, we tried to prove that an enhanced phosphate accumulation in the moss Physcomitrella patens can be achieved by introduction of specific importers and polyphosphate kinases. This is necessary to reach the required phosphate concentration for the later application as a fertilizer.
During the research and project development, we experienced an inconsistency regarding possible limits of phosphate accumulation in the cytosol of plants due to a possible toxicity of polyphosphate (Rudat et al, 2018). To solve this potential problem, we decided to test an accumulation of polyphosphate in the cytosol and in the vacuole of the moss and to compare both concepts with each other. Therefore, we planned to design 3 different constructs which we wanted to test by integration into the genome of P. patens.
The first modification of P. patens is based on the introduction of 2 different proteins to the moss. We used the highly effective I2-10 promoter, which was supplied by Prof. Henrik Toft Simonsen from the Technical University of Denmark (Peramuna et al, 2018) to drive the expression of our genes of interest. To store phosphate in a more inert and durable form, polyphosphate kinases, which synthesize polyphosphate were expressed. Polyphosphate in higher concentrations forms granules, which are a durable, but easily degradable form of phosphate. These are very beneficial for the later application as fertilizer. The PPKs from P. patens itself and the PPK from E. coli strain K12, were interesting for the planned application, since the PPK from E. coli is well researched and there is already a lot of data to compare the native PPK of P. patens to. Additionally, there is a known mutation of the PPK from E. coli which leads to a higher yield of polyphosphate granules that can be used (Rudat et al, 2008)..
These two designs have the purpose of comparing a heterologous expression strategy with a highly characterized PPK to a homologous strategy, where the native gene is overexpressed.
In addition, overexpression of phosphate importers from the PHO1;1-PHO1;7 family can enhance phosphate accumulation. The phosphate importers are stimulated by a lack of phosphate (Wang et al, 2008), so an introduction of PPKs can keep the importers activated by sequestering phosphate into the polyphosphate granules, leading to a higher cellular phosphate concentration. The first modification can be seen in Figure 1.
The second modification is very similar to the first one, with the exception that the PPK enzymes are targeted to the vacuole through a fused peptide signal. The reason for this is a potential toxicity of polyphosphate due to magnesium starvation, since both reactants chelate with each other (Rudat et al, 2018). To prevent this, we use a specific vacuolar import peptide which is characterized in more detail here. The second modification can be seen in Figure 2.
The third modification consists of the expression of the importer PHO1;1-PHO1;7 family, but instead of the PPK enzymes the Vacuolar Transporter Chaperone 4 (VTC4) from Saccharomyces cerevisiae is expressed and tested in P. patens (Uniprot 25.10.2020). This protein translocates phosphate through the tonoplast and synthesizes polyphosphate simultaneously (Gerasimaite et al, 2014). Since the VTC4 complex is stimulated by the inositol diphosphate 5-Insp-P7, an overexpression of the inositol pyrophosphate kinase Ins-P6 KCS1 leads to an enhanced phosphate translocation and polyphosphate synthesis (Wild et al, 2014). This modification can be seen in Figure 3.
We also thought about implementing mechanisms to cope with the effects of phosphate starvation our host organism would experience when focusing on the production of polyphosphate, as this drains phosphate from the rest of the metabolism (Wei et al. 2020). These mechanisms are:
1) the lipid remodeling which consists of the replacement of phosphate containing membrane lipids, predominantly galactolipids and betaine lipids (Abida et al. 2015), (Li & Yu 2018), (Wang et al. 2008).
2) is the expression of RNase NE, which acts extracellularly, which could help in harvesting organically bound phosphate in the final implementation in wastewater treatment plants (Plaxton & Tran 2011).
Due to the recent Covid-19 situation and restrictions from the university and institute, we were not able to perform our experiments as planned with the expression strategies above. Instead, we decided to express only 4 different gene constructs in protoplasts of P. patens, to prove our concept. Further, we decided that transient expression is the most feasible method as we have limited time in the laboratory. In order to reduce the lab work as much as possible, we focussed on the step we consider as the most important: The introduction of PPKs into the cytosol and the vacuole of the plant.
We started our lab work with the cultivation of P. patens protoplasts. We thankfully received the moss from another work group at the University Duesseldorf and prepared all stock solutions we needed. Subsequently, we began to assemble our constructs via overlap extension PCR (OE-PCR). The constructs we actually used were the following:
| Construct | Promoter & RBS | Vacuolar Signal Peptide | PPK | Plant-Specific Insert | Terminator |
|---|---|---|---|---|---|
| 1 | I2-10 Promoter + RBS | No | E. coli PPK | No | NOS Terminator |
| 2 | I2-10 Promoter + RBS | No | E. coli E173K PPK | No | NOS Terminator |
| 3 | I2-10 Promoter + RBS | No | P. patens PPK | No | NOS Terminator |
| 4 | I2-10 Promoter + RBS | Yes | E. coli PPK | Yes | NOS Terminator |
| 5 | I2-10 Promoter + RBS | Yes | E. coli E173K PPK | Yes | NOS Terminator |
| 6 | I2-10 Promoter + RBS | Yes | P. patens PPK | Yes | NOS Terminator |
Due to a repeat in the I2-10 promoter and size restrictions affecting the P. patens PPK, we were forced to split those parts into two pieces each and perform OE-PCR’s in several cycles until we got our full BioBrick-constructs. In Figure 4 the results of a successful PCR of the individual promoter parts can be seen.
The OE-PCR yielded useful results at first, as can bee seen in Figure 5.
Unfortunately, we experienced some unexpected issues during the OE-PCR of both E. coli PPKs with vacuolar import sequence and were unable to find the expected fragment length after gel electrophoresis. The failed PCR can be seen in figure 6.
We performed the experiment two more times with temperature gradients, but didn’t get our desired result. Due to the little time we had left in the lab, we decided to rather focus on the things that worked and to discard both samples. The OE-PCR of the E. coli PPKs targeted into the cytosol worked such as the OE-PCR of both P. patens PPKs.
Each of the 4 constructs was inserted by restriction cloning into a pSHDY plasmid via restriction and ligation using the restriction enzymes Ecori and Pst1. The plasmid has previously been described by Behle et al. in 2019.
The ligation mix was used for a transformation of E. coli DH5ɑ, but no colonies were found on the selection plates, as it can be seen in figure 7. Since full access to a lab was only available to us from 21.09.2020 to 16.10.2020, we did not have time to repeat the cloning, so we could not move on to the transient expression.
It was planned to measure the phosphate concentration in the protoplasts via DAPI-staining and to compare the effects of the different PPKs with each other but due to the failed transformation we did not get to that point. Nevertheless we gained some data and were able to confirm that vancomycin is better for elimination of bacterial contamination in in vitro mosses than chloramphenicol, which can be seen in Figure 8, like it was described by Carey et al in 2005. The Cultivation of our moss is described below.
For the Cultivation of P. patens we resorted to the protocol made by Thévenin et al. 2016. The eventual creation of protoplasts as well as the subsequent transformation would have been made based on this protocol as well. The liquid medium was made with the demanded concentrations of the nutrients and mixed with agar. Before plating, the mix solution was divided into two sample series to compare the effect of antiobiotics to supress bacteria growth on the petri dishes: 5 plates contain vancomycin and 5 others chloramphenicol. The agar plates were covered with cellophane disks cutted to the appropriate size beforehand and then the moss itself was plated onto the plates after being gently homogenized. In Table 2 the composition of the medium is depicted, including the antibiotics concentration of the two sample series.
| Class | Compound | Concentration |
|---|---|---|
| Macronutrients | CaNO3×4 H2O | 0,8 g/L |
| MgSO4×7 H2O | 0,25 g/L | |
| FeSO4×7 H2O | 0,0125 g/L | |
| Micronutrients | CuSO4×5 H2O | 0,055 mg/L |
| ZnSO4×7 H2O | 0,055 mg/L | |
| H3BO3 | 0,614 mg/L | |
| MnCl2×4 H2O | 0,389 mg/L | |
| CoCl2×6 H2O | 0,055 mg/L | |
| KI | 0,028 mg/L | |
| Na2MoO4×2 H2O | 0,025 mg/L | |
| KH2PO4 | 250 mg/L | |
| Nitrogen Source | Ammonium tartrate (NH4)2C4H4O6 | 500 mg/L |
| Series 1 | Vancomycin | 250 µg/mL |
| Series 2 | Chloramphenicol | 1 mg/mL |
Behle, A., Saake, P., Germann, A. T., Dienst, D., & Axmann, I. M. (2020). Comparative Dose-Response Analysis of Inducible Promoters in Cyanobacteria. ACS synthetic biology, 9(4), 843–855. https://doi.org/10.1021/acssynbio.9b00505
Carey, S.B., Payton, A.C., & McDaniel, S.F. (2015). A method for eliminating bacterial contamination from in vitro moss cultures1. Applications in Plant Sciences, 3.
Gerasimaitė, R., Sharma, S., Desfougères, Y., Schmidt, A., & Mayer, A. (2014). Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity. Journal of cell science, 127(Pt 23), 5093–5104. https://doi.org/10.1242/jcs.159772
Li, H. M., & Yu, C. W. (2018). Chloroplast galactolipids: the link between photosynthesis, chloroplast shape, jasmonates, phosphate starvation and freezing tolerance. Plant and Cell Physiology, 59(6), 1128-1134.
Peramuna, A., Bae, H., Rasmussen, E. K., Dueholm, B., Waibel, T., Critchley, J. H., Brzezek, K., Roberts, M., & Simonsen, H. T. (2018). Evaluation of synthetic promoters in Physcomitrella patens. Biochemical and biophysical research communications, 500(2), 418–422. https://doi.org/10.1016/j.bbrc.2018.04.092
Plaxton, W. C., & Tran, H. T. (2011). Metabolic adaptations of phosphate-starved plants. Plant physiology, 156(3), 1006-1015.
Rudat, A. K., Pokhrel, A., Green, T. J., & Gray, M. J. (2018). Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels. Journal of Bacteriology, 200(6). https://doi.org/10.1128/JB.00697-17
Thévenin, J., Xu, W., Vaisman, L., Lepiniec, L., Dubreucq, B., & Dubos, C. (2016). The Physcomitrella patens system for transient gene expression assays. In Plant Synthetic Promoters (pp. 151-161). Humana Press, New York, NY.
Uniprot (25.10.2020) https://www.uniprot.org/uniprot/P47075
Wang, Y., Secco, D., & Poirier, Y. (2008). Characterization of the PH01 Gene Family and the Responses to Phosphate Deficiency of Physcomitrella patens. Plant Physiology, 146(2), 646-56. https://doi.org/10.1104/pp.107.108548
Wei, R., Wang, X., Zhang, W., Shen, J., Zhang, H., Gao, Y., & Yang, L. (2020). The improved phosphorus utilization and reduced phosphorus consumption of ppk-expressing transgenic rice. Field Crops Research, 248, 107715.
Wild, R., Gerasimaite, R., Jung, J. Y., Truffault, V., Pavlovic, I., Schmidt, A., Saiardi, A., Jessen, H. J., Poirier, Y., Hothorn, M., & Mayer, A. (2016). Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science (New York, N.Y.), 352(6288), 986–990. https://doi.org/10.1126/science.aad9858