Paleobiochemistry of the Calvin Benson Cycle to assess functionality of ancestral enzymes in Chlamydomonas reinhardtii

Climate change and increasing population are two main challenges of the present and of the future. Increasing the efficiency of carbon fixation is a promising solution as it can sequester CO2 from the atmosphere and convert it into organic compounds, which can be then used in a green-economy context. Moreover, improving carbon fixation efficiency can increase crop yield answering to the increased food demand associated with population growth. Carbon fixation is performed by photosynthetic organisms and mostly (>90%)1,2 through the Calvin Benson Cycle, which is a complex metabolic process involving eleven enzymes. These enzymes have evolved in very different conditions compared to modern ones. Therefore, some of them are not fully efficient and act as bottlenecks for carbon fixation in the organism. The strategy to improve the enzymes’ efficiency is to go back in their evolutionary history to remove possible evolutionary constraints, and then apply in vivo directed evolution to explore different evolutionary paths and possibly achieve better performances. To go back in the enzyme’s evolutionary history, viable photosynthetic mutants are necessary. Hence, the well-studied microalgae Chlamydomonas reinhardtii was chosen as model organism in the present research project. This thesis project regards the expression of inferred ancestral genes in mutant strains of Chlamydomonas reinhardtii and suggests possible solutions for optimisation of Chlamydomonas gene expression.