Isolamento di mutanti fotorespiratori in Nannochloropsis gaditana

The growing climate emergency requires a rapid change of the current systems for the production of bio-commodities at the base of our economy (e.g. food), via the identification of suitable alternatives to the industrial processes currently based on fossil resources. It is necessary not only to adopt strategies to mitigate climate change, but also to innovate and develop production processes that fits in a circular economy perspective. Microalgae could represent a sustainable source of biomass for biofuels and bio-commodities production. Microalgae are photosynthetic microorganisms, capable of synthetizing and storing a wide number of molecules, such as pigments, starch and triacylglycerols that find many applications in the current economy. The cultivation of those microorganisms, whose biomass can ultimately be converted into valuable resources, offers many environmental benefits, compared to crops, since the latter requires much more freshwater and land per unit of biomass. Despite the advantages that microalgae cultivation offers, the commercial scale production is limited by unsatisfactory biomass yields and high operation costs. One of the main issues to overcome those limitations is maximize biomass production, by increasing carbon fixation efficiency. This approach includes the application of genetic engineering techniques, to obtain microalgal strains that ensure higher biomass productivity, guaranteeing an economically suitable production at an industrial cultivation scale. The microalgae organism studied in this thesis is Nannochloropsis gaditana, a unicellular eukaryotic alga that belongs to the phylum of Heterokonta and is part of the Eustigmatophyceae class. It has acquired a considerable interest in biotechnology, given its ability to produce large amounts of lipids for biofuels and valuable bioactive compounds. In order to achieve higher cultivation efficiency, the target of the study is the photorespiration pathway. In fact, studies showed that photorespiration potentially decreases the conversion of CO2 into biomass by about 25%, a percentage likely to increase under high-temperature or drought stress conditions. This pathway takes place when the enzyme ribulose-1,5-bisphosphate (RuBP) reacts with oxygen instated of CO2, producing 2-phosphoglycolate (2PG). This molecule appears to be toxic for the organism since it can inhibit some Calvin-Benson Cycle and glycolysis enzymes, so it needs to be detoxified. In order to do that, the organism puts in place many enzymatic reactions with high cost in terms of chemical energy and reducing equivalents. Moreover, the pathway generates CO2 and Nitrogen, which need to be re-integrated in the central metabolism, via additional energy-consuming reactions. According to these considerations, photorespiration appears to be an inefficient process, that limits the achievement of higher biomass volume. The aim of my thesis project is twofold. First, to collect phenotypical data about the behavior of N. gaditana knock-out strains that lack for genes that encode for some proteins involved in this pathway: PGLP, BASS6 and GOX. 2PG phosphatase (PGLP) is a chloroplast enzyme, the first one to take part in the process. It hydrolyses 2PG to Glycolate, which is than carried into the peroxisome by the transporter BASS6. In this subcellular compartment, Glycolate Oxidase (GOX) oxidizes Glycolate to Glyoxylate and forming the by-product H2O2. The second goal is to implement a metabolic bypass to increase the detoxification efficiency of 2PG, produced by RuBisCO oxygenase reaction.