Opening future biotechnological possibilities by revealing the acclimation and biogenesis of the photosynthetic apparatus in marine algae

Diatoms are the ecologically most significant group of microalgae (phytoplankton) contributing 20-25% of the global primary productivity. These organisms also have the potential to be exploited commercially for biofuels, feed for the aquaculture industry, as a source of nanomaterials and in bioremediation. Despite the importance of diatoms in nature and biotechnology, functional studies of diatom biology are scarce. In the DiaPhotosynth project we focused on understanding the molecular mechanisms controlling how the photosynthetic apparatus is assembled and regulated. In Part I of the project, we investigated the function of a chloroplast localized kinase that we predicted to be a key sensor and signaling molecule of the photosynthetic status of the cell. We successfully knocked out the selected chloroplast kinase using the gene editing technology CRISPR/Cas9 and subjected the mutant lines to a range of different light conditions to reveal if the protein had an important role in photoacclimation. Surprisingly, the mutants behaved as wild type cells in almost all light conditions. Only very modest differences in growth, photosynthetic productivity and content of photoprotective pigments could be observed in cells treated with high intensity light. Whole genome transcriptional profiling of cells shortly after a shift from low to high intensity light detected only a very small number of regulated genes, and the few differentially expressed genes had mostly unknown functions and did not provide clues to the role of the chloroplast kinase. We interpreted these findings as that other chloroplast kinases can compensate for the loss of the one that had been investigated so far in this project. Gene mining of the diatom genome revealed another family of chloroplast kinases that also has the potential to be important for photoacclimation. Because of the very weak phenotype of the chloroplast kinase initially selected for functional characterization, we shifted our attention to other undescribed chloroplast kinases with possible important roles in photoacclimation. We have now successfully identified a chloroplast kinase necessary for handling prolonged high light exposure. Lack of this kinase causes severe changes to photosynthetic membranes (thylakoids), slow growth, reduced level of carotenoids and a decline of photosynthetic fitness. To identify the specific function of the kinase and the reason for the observed phenotypic changes we have looked at changes in gene and protein expression, changes in protein phosphorylation status and effects on the lipid composition of the thylakoids. The results are currently being analyzed and prepared for publication. In Part II of the project, we attempted to determine which protein complexes are involved in transporting pigment-binding proteins from the chloroplast membrane to the inner membrane structures, the thylakoid membranes, which is where the light reactions of photosynthesis take place. We have previously characterized a protein involved in inserting these pigment-binding proteins into the thylakoid membrane, the ALB3b protein. We have successfully expressed and purified the protein interaction domain of this insertase in Escherichia coli to use as bait to capture its interaction partners from protein extracts of the diatom. However, no candidate proteins could be reliably identified. As these protein interactions are only transient and not very strong, they most likely need to be stabilized to achieve successful identification of target proteins. Therefore, we have adapted a novel approach, which has only recently been developed for our model diatom species. Using this methodology, interactions can be fixed upon addition of an inducing molecule. Thus, much more harsh conditions during the purification steps can be employed without risk of washing away the protein of interest while keeping the level of background low. Furthermore, interactions are captured in native conditions in the living organism instead of relying on protein extracts generated by lysis of the cells, which can potentially lead to bias. Using this approach, we could identify around 50 proteins, which were strongly overrepresented in the dataset. As expected, this candidate list consists to a large extent of previously uncharacterized proteins with a known conserved domain or proteins containing only domains of unknown functions. The number of candidate genes was narrowed down using literature searches, evaluating gene expression patterns and published proteomics datasets and phylogenetic analyses. The best candidates for being ALB3b interaction partners were targeted for creation of knockout strains. Phenotypic characterization of mutants where proteins believed to be possible interaction partners of ALB3b were lacking, did not show the expected results. We concluded that none of the selected proteins are ALB3b interaction partners.

Project outcomes and impacts in addition to the research results produced during the project period include 1) learning of new skills and methods that will important for future research projects, 2) securing of a permanent position for the project leader as a senior researcher at SINTEF Ocean as a microalgae expert, 3) expansion of the general knowledge base of marine algae that is currently lagging far behind that of terrestrial plants, 4) proof of differences in the biogenesis of the photosynthetic apparatus between plants and diatoms that can be used to argue that more basic research should be performed in this group of organisms. A greater knowledge of diatom biology is important to develop microalgae strains suitable for industrial cultivation increasing the chances of products and biomass from diatoms to contribute to the world's energy, food, and material needs, 5) discovery of the importance of chloroplast kinases in photoacclimation that can form the basis for future research and research applications in the field.

Diatoms are the ecologically most significant group of microalgae contributing 20-25% of the global primary productivity. These organisms also promise a multitude of potential biotechnological applications. Despite the importance of diatoms in nature and biotechnology, functional studies of diatom biology are scarce. In the proposed project we aim to determine the molecular mechanisms that facilitate regulation and biogenesis of the photosynthetic apparatus of diatoms using the model species P. tricornutum. Such information will be valuable for understanding the evolutionary success of diatoms, and also for performing future strain optimization aiming to create an algae strain more suitable for growth in photobioreactors. In Part I of the project we aim to identify the role of reversible phosphorylation of chloroplast proteins in short- and long-term responses to changes in light intensity. We have identified a single copy gene encoding a chloroplast protein containing both a kinase and a phosphatase domain, that we believe can act as a key sensor and signaling molecule of the photosynthetic status of the cell. The kinase domain show homology to STN7 and STN8 that are important for light acclimation in plants/green algae. In Part II we will investigate how diatom pigment-binding proteins (FCPs) are guided to thylakoid membranes. In green algae and higher plants, the molecular players behind this process are known and function in the chloroplast signal recognition particle (CpSRP) pathway. Preliminary analysis of diatom homologs of the CpSRP pathway strongly indicate that transport of FCPs to thylakoid membranes are independent of this pathway. We have previously identified an insertase necessary for efficient insertion of FCP proteins in thylakoid membranes. By determining the interaction partners of this insertase we will identify novel players guiding FCP proteins to thylakoid membranes. Such proteins might be future targets for strain optimization of diatoms.