Physcomitrella patens (P. patens) was chosen as host organism, because of various advantages and challenges in synthetic biology. P. patens is a member of the bryophyte family used as a model plant system for the analysis of several aspects of modern plant biology. This species is a promising candidate for synthetic biology, since it combines diverse characteristic traits that provide advantages over other model plant systems. The bryophyte is photoautotrophic and does not require a carbon source in the medium and can be grown in either solid medium or liquid culture. A unique feature of P. patens is its high degree of homologous recombination, which allows the precise editing of genomic DNA (Frank, Decker, and Reski 2005).
The main goal of Mossphate is to provide a sustainable synthetic biology approach to close the phosphate cycle by genetically engineering the moss P. patens for an enhanced uptake and accumulation of phosphate. To achieve this aim, we established 3 specific milestones.
used fertilizer in wastewater and new fertilizer on the field
Improve phosphate storage in the form of polyphosphate granules.
P. patens has the ability to accumulate the excessive Pi in the form of osmotically inert polyphosphate granules (Seufferheld & Curzi, 2010). Those are synthesized by the polyphosphate kinase (PPK, Gene id: 112291913), which catalyses the transfer of the terminal phosphate of ATP to a growing polyphosphate chain (McMahon, Dojka, Pace, Jenkins, & Keasling, 2002).
Despite the presence of PPK activity, wild-type strains of P. patens do not accumulate high amounts of polyphosphate (Seufferheld & Curzi, 2010). With a synthetic biology-based approach, it is possible to increase the accumulation of polyphosphate by overexpression of PPK enzymes. It has been reported that random mutagenesis experiments performed on the PPK from
E. coli identified the E173K variant, which has been shown to have greater polyphosphate processivity than the native PPK (Rudat et al. 2018). We tested both the native PPK and the PPK variant E173K from E. coli to achieve maximal activity.
Test the accumulation of polyphosphate in the cytosol and vacuole.
It has been reported that in some cases the high accumulation of polyphosphate leads to toxicity problems due to sequestration of Magnesium in biologically unavailable polyphosphate complexes (Rudat, Pokhrel, Green, Gray 2018). Therefore, the localization of polyphosphate granules in the cytosol and vacuole will be tested by expressing the PPKs with and without the presence of a vacuolar import signal to test the effect on toxicity.
Enhance phosphate uptake capacity of P. patens.Higher plants take up phosphate from the environment in the form of inorganic organophosphate (Pi) (Nussaume et al. 2011). In Arabidopsis thaliana, phosphate transport and homeostasis is mainly performed by the PHO1 gene family members, which have a predominant expression in both vascular and non-vascular tissues. Seven PHO1 homologs have been characterised in P. patens. Those have distinct expression patterns in different tissues. Thus, indicates they play different, yet overlapping, functions in Pi transport and homeostasis in the tissues of the moss (Wang, Secco, Poirier 2008). PpPHO1;1 and PpPHO1;7 are specifically up-regulated under Pi deficiency (Wang, Secco, Poirier 2008). Our goal is to enhance phosphate uptake in P. patens by the overexpression of the PpPHO1;1 and PpPHO1;7 genes.
The expression of the genes will be done in different variations as seen in in more detailed in the “ Parts ” section and integrated into the protoplasts of P. patens by transient expression. The plasmid used is pSHDY (Behle, Saake, & Axmann, 2019), which has a broad host range and therefore is a suited candidate as shuttle vector in the P. patens. It is compatible with the biobrick system and contains the Spectinomycin and Kanamycin resistance cassettes.
|I2-10 Promoter||Synthetic promoter for gene expression in P. patens||(Peramuna et al., 2018)|
|RBS P. patens||Sequence confirmed to lead to Ribosome Binding Site||BBa_M45214|
|NOS Terminator||Common terminator from Agrobacterium used in plant science||(Sarrion-Perdigones et al., 2013)|
|PpAP1 Signal Peptide||Vacuolar signal peptide for the aspartic proteinase used for targeting PPKs to the vacuole of P. patens||(Shaaf et al., 2005)|
|PPK P. patens||Polyphosphate kinase. Catalyzes the formation of polyphosphate chains from ATP.||The gene sequence was acquired from NCBI (Gene ID: 112291913)|
|PPK Escherichia coli||Polyphosphate kinase. Catalyzes the formation of polyphosphate chains from ATP.||BBa_K2789020|
|PPK E173K Escherichia coli||Polyphosphate kinase variant with greater polyphosphate processivity||(Rudat, Pokhrel, Green, & Gray, 2018)|
The ultimate goal of Mossphate is to create a new last-stage in wastewater treatment facilities (WWF). A bioreactor array with the genetically engineered P. patens to reduce the phosphate content to a hypotrophic level, which current WWF fail to accomplish (McGrath and Quinn 2003). A reduction in the phosphate content of the outgoing clean water would have a positive impact and probably lead to a reduction of eutrophication and its negative impacts in the environment. At the same time, the phosphate-rich P. patens could be dried and used as a fertilizer.
This concept would provide a considerable enhancement to the phosphate cycle, since the phosphate would constantly be removed from wastewater and put back to its source. Mossphate would provide a more sustainable and environmentally-friendly methodology for phosphate recycling by using moss as a fertilizer and at the same time, contribute to the establishment of P. patens as a biotechnological chassis.
McGrath, John W., and John P. Quinn. 2003. “Microbial Phosphate Removal and Polyphosphate Production from Wastewaters.” Advances in Applied Microbiology52: 75–100.
McMahon, K. D., Dojka, M. A., Pace, N. R., Jenkins, D., & Keasling, J. D. (2002). Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal. Applied and Environmental Microbiology, 68(10), 4971–4978. https://doi.org/10.1128/AEM.68.10.4971-4978.2002
Nussaume, Laurent et al. 2011. “Phosphate Import in Plants: Focus on the PHT1 Transporters.” Frontiers in Plant Science2(NOV): 1–12.
Rudat, Amanda K., Arya Pokhrel, Todd J. Green, and Michael J. Gray. 2018. “Mutations in Escherichia Coli Polyphosphate Kinase That Lead to Dramatically Increased in Vivo Polyphosphate Levels.” Journal of Bacteriology200(6): 1–20.
Seufferheld, M. J., & Curzi, M. J. (2010). Recent discoveries on the roles of polyphosphates in plants. Plant Molecular Biology Reporter, 28(4), 549–559. https://doi.org/10.1007/s11105-010-0187-z
Wang, Yong, David Secco, and Yves Poirier. 2008. “Characterization of the PHO1 Gene Family and the Responses to Phosphate Deficiency of Physcomitrella Patens.” Plant Physiology146(2): 646–56.