Green algae are one of the biggest biomass producers in the world and are ubiquitous organisms in all types of environments. Due to their photosynthetic capabilities, high lipid accumulation and ability to grow in nonarable areas they are considered as potential third generation source of biofuels. The low costs of cultivation and fast growth make them attractive biotechnological organisms with applications in high-value compounds and pharmaceutical proteins production. In the published review, which is the first manuscript in my PhD thesis, we explored potentials of converting sunlight energy to industrially relevant products by genetically engineering cyanobacteria and chloroplasts. We showed the correlation between complexity of synthesized compounds and theoretical photosynthetic limitations, which suggests frequent limitations in carbon assimilation or in the biosynthetic pathways. These limitations can be overcome by applying metabolic engineering or synthetic biology strategies and I focused my research on the development of new synthetic biology tools for the model, eukaryotic, green algae Chlamydomonas reinhardtii. In this new field of biotechnology, researchers exploit the modular nature of biological systems and apply the iterative engineering cycle: design-build-test, to systematically assemble new-to-nature genetic elements and whole synthetic systems. In the second manuscript, I present the results of a collaborative project, where we developed a modular cloning strategy (MoClo) for C. reinhardtii, which allows to prototype and iterate new construct in a high-throughput manner. Despite the long history of research on C. reinhardtii, many molecular tools are inefficient in heterologous expression of genes.
Therefore, by gradual improvements and design iterations, we developed new robust expression systems based on internal ribosome entry site, which provide reliable transformations efficiencies and high protein accumulation. During biochemical characterization of the new constructs, we discovered a possible new way of controlling gene expression, which functionally resembles a resistor in electric circuit board. The details are presented in the third manuscript. The potential to use algae for biofuel production is still unexploited due to high production costs and unsatisfactory productivity. In the last manuscript, I present a possible strategy to accelerate adoption of this new technology, by developing added-value product formation, and showed the first functional designs for the assembly of biosynthetic pathways on the lipid droplets. High-content screening microscopy methods, applied in this project, are also opening new possibilities for quantitative, single-cell characterization of this alga.