PORTFOLIO

Green algae are photosynthetic organisms that can easily be cultivated on a large scale. Some members have evolved as valuable food sources due to their high content of essential fatty acids. The development of biotechnological tools to modify these organisms is currently of great interest. In this project, we will engineer the surface of the green alga Chlamydomonas reinhardtii (C. reinhardtii) to transform the organism into a carrier for the specific delivery of active compounds. Already existing cell-surface engineering has found a broad range of applications. For human and animal health, cell-surface engineering is used for vaccine development, and more recently, for drug delivery. As enzymes can be immobilized on the surface, it has also found numerous applications within the food industry, energy production and others. Additionally, cell-surface engineering has aided basic research by unveiling the mechanism of cell wall formation. The overall aim of the ‘Green Surfing’ project is to establish a platform for drug delivery application by transforming the alga C. reinhardtii with cell-surface engineering. We propose to modify the photosynthetic organism into a carrier of therapeutically active compounds by expressing specific anchors at its surface. We developed in the first year a new surface engineering strategy for C. reinhardtii. This method represents the first example of the expression of a designed anchor at the cell wall of this complex organism. More specifically the objectives are to identify and characterize functional anchoring domains; to evaluate displayed proteins' accessibility to external molecules and particles; to evaluate modified C. reinhardtii strains as detoxification organisms. Finally, we are targeting detoxification of metal as a final application of our methodology. Our system showed promising results using contaminated soil samples.

The alga, C. reinhardtii, is a model organism possessing a photosynthetic system, is generally recognized as safe (GRAS) by the FDA and thereof is a potential food source. However, up to date, it is not possible to display protein at its surface using a recombinant surface. The ‘Green Surfing’ project tackle this unmet need by applying a systematic set of experiments to generate functional anchoring domains in the algal system. This key technology unlocks several biotechnological applications, by either algae biological or economical traits both required for the translation of academic knowledge to society. For instance, the GRAS status of C. reinhardtii renders it applicable for human and animal health, as well as for food-processing technologies, drug delivery and whole-cell biocatalyst. The photosynthetic capacity of C. reinhardtii combined with its aquatic nature is an attractive characteristic for tackling environmental problems. This modified organism could be used for cleaning polluted bodies of water bodies contaminated by compounds with poor membrane permeability such as glyphosate. The low production cost and the possibility of surface engineering are attractive for biocatalytic processes, such as lipase-produced biofuels. Finally, the autotrophic-engineered organism combined with a controlled drug-release strategy could be used to treat chronic and traumatic wounds in over 400 million people worldwide.

During the project, we identified the protein GP1 as an ideal anchor for building a surface display system on the alga Chlamydomonas reinhardtii. This specific protein is highly attractive since its attachment to the algae cell wall is governed by non-covalent interaction, which makes its N- and C-terminal free for further transformation. The system was first evaluated with a fluorophore attached at one side of GP1 and a clear labeling of the alga surface was observed under the microscope. Furthermore, we demonstrated that the N- and C-terminals of GP1 could be functionalized at the same time resulting in a doubled fluorescently labeled GP1.
In the second part of the project, we investigated the conditions for releasing the labeled GP1 from the cell wall. Two specific stimuli were discovered to promote the removal of the anchor protein. In the first one, a chemical strategy using chaotropic salts was used to disrupt non-covalent interaction within the extracellular part of the cell wall, which resulted in the removal of the fluorescently labeled GP1. In the second application, the sexual reproduction of Chlamydomonas reinhardtii was artificially promoted resulting in the release of the labeled anchor.
Finally, the last part of the project focused on applying the developed protocol from the first and second stages to create a system to catch and release metal from contaminated samples. Several metal chelators were fused to a fluorescently labeled GP1 and a screening procedure was performed to identify the best hit. Our preliminary data led to the identification of a suitable system for bioremediation and we are currently evaluating a release procedure as well as a potential patent application for the further financial development of the system.

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Molino JVD, Carpine R., Gademann K., Mayfield S., Sieber S. Development of a Cell Surface Display System in Chlamydomonas reinhardtii, biorxiv.org. 2021;
Carpine R., Sieber S. Antibacterial and antiviral metabolites from cyanobacteria: Their application and their impact on human health, CRBIOT 2021. 3: 65–81.
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