Team:Sorbonne U Paris/Plant


Plant Synthetic Biology

Chamydomonas reinhardtii is a promising algae species for many appliances. It is easy to culture and is considered a generally recognised as safe (GRAS) species. On top of that, the sequence of the entire C. reinhardtii genome is available. Relatively recent developments make it more receptive to manipulation of both the nuclear and the chloroplast genome. As an autotroph, Clamydomonas can survive on a minimum diet and in challenging conditions. This together opens up avenues to pursue in virtually any application.

We can now attempt to adapt C. reinhardtii to even harsher conditions. This is what we set out to do in our project: can we tune this alga so it survives in space? Paradoxically, space is limited in space and moving materiel from the interior to the exterior can mean a world of a difference. We therefore set out to cultivate C. reinhardtii in an extravehicular module. Here, the alga will have to combat γ and UV radiation, both leading to the formation of reactive oxygen species (ROS). Sequestering ROS is at the core of our project

Three branches guarantee a space tight approach

To assess the capacities of C. reinhardtii to thrive in the challenging environment of a bioreactor on the exterior of the ISS, we built our approach on three branches. These branches were designed to test the different premises in this project: the in vitro effectiveness of the antioxidant, the in vivo effectiveness of the antioxidant, and the capacity of this antioxidant to function when expressed from the C. reinhardtii genome.

Figure 1: Three branches of our workflow. The in vitro experiment assesses the capacity of the undecapeptide to recover the function of LDH in an oxidative environment. The proof of principle experiment tests the effect of the external undecapeptide in vivo. The in vivo experiment assesses whether undecapeptide transcribed from the genome has the desired effect. Membrane in white, nucleus in purple. [Figure description: The peptide/manganese/phosphate complex is thought to act as ROS scavengers within cells. To verify this postulate, two approaches are conducted: an in vitro experiment on LDH and an in vivo experiment. In the in vitro experiment, the presence of reactive oxygen species inhibits LDH activity and thus NAD+ production, the concentration of which can be monitored by UV-vis spectrometry. In the in-vivo experiment, the amount of ROS is directly measured within the cell.]

The antioxidant we used as a basis is a decapeptide with antioxidant capacities when in complex with manganese and orthophosphate. In order to express this decapeptide in Chlamydomonas reinhardtii, we had to add a start codon in fact creating an undecapeptide. To ensure that this novel undecapeptide has the same properties, we tested its in vitro capacities as an antioxidant. We did this in an assay that tested the efficiency of lactate dehydrogenase to oxidise NADH to NAD+ in oxidative conditions. Sadly, we were only able to optimise our experimental set up in the final stages of the project.

To test the properties of the undecapeptide in vivo, we made use of the allowance of diffusion of small peptides through membranes in cell wall-free Chlamydomonas reinhardtii. Examining the properties of the antioxidant in this way would have allowed us to understand the ROS scavenging properties of our antioxidant in vivo. This could have been assessed without the possible expression problems and would serve as a de facto proof of concept. Unfortunately, the experimental conditions we used lead to the formation of aggregates. This made assessment impossible.

We designed a module to constitutively express the undecapeptide. The module was built using the Golden Gate cloning technique and with C. reinhardtii codon optimization. The peptide is expressed under the control of the PSAD promoter. The module also confers hygromycin resistance. We found conflicting results since the algae expressing our peptide were not the ones with higher resistance to oxidative stress. We expect that a digestion event in the transformation conferred resistance without delivering the gene of interest. Future avenues could exploit indirect cloning methods to prevent exposing naked DNA in the alga.

Our project finds itself at the interface of manipulating an alga to thrive in an extravehicular bioreactor in space and designing a protocol to assess novel functionality. Following the conference of resistance, functions required on the ISS could be implemented. We encourage future teams to continue working on exobiology and we hope that our ideas will advance your project. The importance of C. reinhardtii as a versatile resident in bioreactors is not limited to space. It may as well serve in reactors supplying bio fuels, biopolymers and even medicine. We laid down an approach that helps to show the effectiveness of your ideas.