Engineering Microalgae: Novel Tools for Genetic Manipulation of Chlamydomonas and Chlorella sp., from Light Harvesting to Cell Wall Remodelling

Microalgae are microscopic photosynthetic organisms living in aquatic environments widely recognized as promising “green bio-factories” due to their metabolic flexibility and capacity to convert solar energy into biomass. Their extensive biodiversity, which comprises approximately 72,500 species, and adaptability provide a robust foundation for biotechnological exploitation. Among green microalgae, species such as Chlamydomonas reinhardtii and Chlorella vulgaris have emerged as key models for both fundamental research and industrial-scale cultivation, owing to their genetic knowledge and metabolic potential. Nonetheless, industrial cultivation of microalgae remains largely limited to the production of value-added products, primarily due to high cultivation and processing costs. To address these limitations, this thesis explores advanced genetic engineering strategies, with a focus on CRISPR-Cas9 genome editing, to enhance the domestication and productivity of microalgae. The work is structured around the development and application of genome editing tools in C. reinhardtii and C. vulgaris, aiming to dissect photosynthetic pathways, improve light-harvesting efficiency, and facilitate downstream bioprocessing. In particular, photosynthesis in microalgae occurs through oxygenic mechanisms in the thylakoid membranes and is mediated by pigment–protein complexes. The absorption and utilization of light energy are influenced by the structure and composition of light-harvesting complexes (LHCs), which have evolved in aquatic species to optimize energy capture and photoprotection. Indeed, modifications in LHC composition and pigment-binding properties can significantly impact photosynthetic performance, particularly under variable light conditions. To investigate those physiological processes, we developed a DNA-free CRISPR-Cas9 system in C. reinhardtii to enhance genome-editing efficiency while avoiding the drawbacks of DNA integration and antibiotic marker dependence. This enabled functional analyses of LHC-related genes, including targeted knock-outs of LHCBM1, a key antenna protein implicated in non-photochemical quenching (NPQ), a photoprotective mechanism activated under excess light conditions. Complementation with mutated variants of LHCBM1 further allowed investigation into its interaction with stress-responsive protein LHCSR3. Additionally, we used CRISPR-Cas9 in C. reinhardtii to produce gene knock-outs of specific monomeric antenna proteins (LHCB4 and LHCB5) and applied site-directed mutagenesis aimed at enhancing far-red light absorption. To do this, we targeted chlorophyll-binding residues in light-harvesting proteins to shift light absorption towards the far-red spectrum—a range not naturally absorbed by algal PSII– by mutating a key histidine residue to asparagine to induce red-shifted chlorophyll forms, similar to those observed in higher plants. This red shift could improve growth under low light and increase productivity in photobioreactors affected by self-shading. Parallel efforts focused on C. vulgaris, a robust non-model species limited by a highly recalcitrant cell wall that hinders genetic transformation and metabolite extraction. We developed a domestication pipeline combining random mutagenesis, flow cytometry-based phenotypic screening, and ultrastructural analysis to isolate cell wall mutants with enhanced permeability. These advances provide a viable route to facilitate genetic manipulation and downstream processing in industrial settings. In conclusion, this thesis contributes to the advancement of microalgal biotechnology by developing precise, efficient gene-editing tools and applying them to key physiological pathways and industrial constraints. Through an integrative approach encompassing both model and non-model species, this work lays the groundwork for optimizing microalgae-based production systems.