Creation of Phaeodactylum tricornutum mutant strains with truncated light harvesting antennas through CRISPR/Cas9 mediated knockout of members of the CpSRP pathway
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- Institutt for biologi 
Production of marine fatty acids by diatom cultivation has previously been proposed as a way to create a new source of omega-3 to be used in aquafeed. However, low photosynthetic productivity of diatoms in mass cultivation hampers the economic viability of this type of industrial production. The creation of Truncated Light-harvesting Antenna (TLA) mutants can be used as a tool to maximize photosynthetic productivity by increasing the efficiency of the conversion of light energy to biomass in high-density cultures. The purpose of this thesis was to create TLA mutant strains of the diatom Phaeodactylum tricornutum (P. tricornutum ) that might be more suitable for industrial cultivation. Two target genes encoding members of the Chloroplast Signal Recognition Particle (CpSRP) pathway, Alb3b and CpSRP54, were knocked out in the diatom P. tricornutum by using CRISPR/ Cas9 technology to create putative TLA mutant strains. The CpSRP pathway has both a post-translational and a co-translational function that facilitate the integration of light harvesting antenna proteins into the thylakoid membrane and the integration of subunits of the photosystems into the thylakoid membrane, respectively. The mutants were characterized in light experiments where WT, three lines of Alb3b mutants and three lines of CpSRP54 mutants were acclimated to LL and then subjected to HL for 0.5 h, 6 h, 24 h, 48 h and 1 week. Pigment concentration, photosynthetic parameters, fluorescence excitation spectra, gene expression and growth rate were investigated in the harvested cell material. The Alb3b knockout (KO) mutants had a green coloration different from the golden brown color of the wild type (WT), indicating that the pigment concentrations of these mutants were altered. The results showed that the Alb3b KO mutants had a severely reduced pigment content compared to WT, and had a ~50 % and ~65 % reduction of Chl a and Fuco levels, respectively. In addition, the photosynthetic capacity and the light saturation index were ~20 % higher in HL acclimated Alb3b KO mutants compared to WT. The CpSRP54 KO mutants had a similar content of the light harvesting pigments Chl a and Fuco as WT, but had a ~70-80 % higher concentration of the photoprotective pigment Diato after transferal to HL. In addition, the CpSRP54 KO mutants showed up to ~50 % lower photosynthetic capacity and ~40 % lower photosynthetic efficiency compared to the WT. The combined results suggest that the Alb3b insertase plays a role in the insertion of antenna proteins into the thylakoid membrane, and that the Alb3b KO mutants can be classified as TLA mutants. The analysis of the CpSRP54 mutants does not indicate that these mutants has a truncated antenna, instead, they seem to be HL sensitive, possibly because of an inefficient replacement of damaged D1 protein. Based on the findings in the present thesis it can be concluded that the Alb3b gene probably function in the post-translational CpSRP pathway, while the CpSRP54 protein possibly is a member of the co-translational pathway.