Caractérisation du métabolisme des lipides de la diatomée marine Phaeodactylum tricornutum

L’océan domine la surface de notre planète et joue un rôle majeur dans la régulation de notre biosphère. Par exemple, les microorganismes photosynthétiques vivant dans l’océan produisent 50% de l’oxygène que nous respirons tous les ans, et une grande partie de notre alimentation et des ressources minérales en proviennent. En cette époque de crise écologique liée à l’accumulation anthropogénique de gaz à effet de serre dans l’atmosphère, il est impératif de développer des énergies plus durables que les carburants fossiles. Le biodiesel pourrait être une source de carburant viable et durable pour remplacer le pétrodiesel mais jusque-là, nos efforts visant à produire des lipides à base de microalgues se sont essentiellement concentrés sur des algues vertes. Dans cette thèse je propose des méthodes pour mieux caractériser une autre catégorie de microalgue : les diatomées. Les diatomées sont une composante importante du phytoplancton et contribuent 40% de la production marine de biomasse primaire. Les diatomées accumulent des lipides en réponse à la carence en nutriments, et même si elles semblent accumuler tout autant de lipides que les microalgues vertes, les voies métaboliques menant à l’accumulation de lipides sont encore peu connues.Dans cette thèse je décris notre caractérisation du glycerolipidome de la diatomée modèle Phaeodactylum tricornutum ainsi que notre étude du remodelage de lipides suite à la carence d’azote ou de phosphate. Des accessions de P. tricornutum isolées dans différentes régions de l’océan ont aussi été étudiées afin de comparer leurs réponses au stress nutritif. Nous avons trouvé que la réponse métabolique menant à l’accumulation de lipides en carence d’azote ou de phosphate est différente. En effet, la carence en azote semble déclencher le recyclage des galactoglycerolipides chloroplastiques ainsi qu’une augmentation de la biosynthèse de novo d’acides gras, alors que la carence en phosphate est plus sévère car nous avons observé une accumulation plus significative de triacylglycerols ainsi que la déplétion totale des phospholipides. De plus, nous avons observé des réponses au stress différentes entre les accessions de P. tricornutum, et en particulier concernant leur capacité à accumuler des lipides. Nous proposons l’hypothèse que ces différences sont liées à leur aptitude à recycler du carbone issu de molécules de stockage non lipidiques.Des approches génomiques ont permis de nombreuses avancées pour mieux comprendre le métabolisme des lipides de microalgues mais notre compréhension des voies de biosynthèse de lipides chez les diatomées est encore limitée. Il y a eu diverses tentatives de caractérisation de la réponse au stress de carence par approche transcriptomique mais l’étude de ces données est incomplète du fait de l’annotation insuffisante des gènes encodant les voies métaboliques pertinentes. Ainsi, dans cette thèse je décris nos tentatives d’annotation de gènes impliqués dans le métabolisme des lipides de P. tricornutum ainsi que les approches d’ingénierie génétique visant à mieux caractériser certains de ces gènes. J’ai également utilisé notre nouvelle annotation de gènes impliqués dans le métabolisme des lipides pour effectuer une étude comparative de plusieurs transcriptomes de P. tricornutum en conditions de carence trouvés dans la littérature. J’ai ainsi pu produire une liste de gènes potentiellement impliqués dans l’accumulation de lipides. Enfin, nous avons pu utiliser ces données pour aider l’interprétation de données génomiques et transcriptomiques issues de la diatomée oléagineuse Fistulifera solaris afin de mieux comprendre comment elle accumule des quantités importantes de lipides pour des applications dans le secteur des biotechnologies et des bioénergies.The ocean dominates the surface of our planet and plays a major role in regulating the biosphere. For example, the microscopic photosynthetic organisms living in the ocean provide 50% of the oxygen we breathe every year, and much of our food and mineral resources are extracted from the ocean. In a time of ecological crisis linked to the accumulation of anthropogenic greenhouse gases in the atmosphere, we must investigate more sustainable energies than fossil fuels. Much attention has been given to biodiesel but so far most efforts to efficiently produce triacylglycerols in microalgae have focused on green algae. In this thesis I propose approaches to better understand another type of microalgae that is significantly divergent from green lineages: diatoms. Diatoms are a major phylum of phytoplankton in the ocean and account for 40% of marine primary productivity. While diatoms appear to be at least as effective as green algae for producing lipids, the fatty acid and glycerolipid biosynthetic pathways leading to their production have not yet been well characterized. Therefore, I propose to better characterize these pathways in the model diatom Phaeodactylum tricornutum in order to help unlock the potential of diatoms for lipid-based biotechnological applications.In this thesis, I discuss our attempts to establish a reference for the glycerolipidome of P. tricornutum and of our assessment of the lipid remodeling and accumulation that occurs in response to nitrogen- and phosphorus-starvation. A range of accessions of P. tricornutum isolated from different parts of the ocean were also examined to compare their responses to nutrient deprivation. We found that the metabolic response leading to lipid accumulation in different nutrient-deprived conditions are distinct. Nitrogen-deprivation appears to trigger the recycling of chloroplastic galactoglycerolipids as well as a strong increase in de novo fatty acid synthesis while the response to phosphorus-deprivation was more severe as we observed a higher triacylglycerol pool and the complete depletion of phospholipids. Furthermore, we observed several differences among accessions of P. tricornutum regarding their ability to accumulate triacylglycerol in response to nutrient starvation and propose the hypothesis that these differences are linked to their ability to recycle intracellular carbon from non-lipid storage molecules.Genome-enabled approaches have also allowed significant steps towards elucidating the lipid metabolism of microalgae in the past decade, but our understanding of diatom metabolic pathways is still limited compared to that of other microalgae and higher plants. There have been several attempts to characterize the stress response in P. tricornutum by using transcriptomic approaches but this data is difficult to exploit to its full potential without a better annotation of genes encoding the relevant pathways. Therefore, in this thesis I discuss our attempts to annotate P. tricornutum lipid metabolism genes. Based on this annotation I have attempted to better characterize a selection of genes by genetic engineering and have pursued a comparative study of several published transcriptomes of P. tricornutum in nutrient deprived conditions to produce a list of candidate genes likely to be involved in triacylglycerol accumulation. Finally, we used this data to help interpret genome and transcriptome data of the newly sequenced oleaginous diatom Fistulifera solaris to help understand how it accumulates unusually high amounts of triacylglycerol for applications in the biotechnology and bioenergy industry

Bioprospecting Marine Plankton

The ocean dominates the surface of our planet and plays a major role in regulating the biosphere. For example, the microscopic photosynthetic organisms living within provide 50% of the oxygen we breathe, and much of our food and mineral resources are extracted from the ocean. In a time of ecological crisis and major changes in our society, it is essential to turn our attention towards the sea to find additional solutions for a sustainable future. Remarkably, while we are overexploiting many marine resources, particularly the fisheries, the planktonic compartment composed of zooplankton, phytoplankton, bacteria and viruses, represents 95% of marine biomass and yet the extent of its diversity remains largely unknown and underexploited. Consequently, the potential of plankton as a bioresource for humanity is largely untapped. Due to their diverse evolutionary backgrounds, planktonic organisms offer immense opportunities: new resources for medicine, cosmetics and food, renewable energy, and long-term solutions to mitigate climate change. Research programs aiming to exploit culture collections of marine micro-organisms as well as to prospect the huge resources of marine planktonic biodiversity in the oceans are now underway, and several bioactive extracts and purified compounds have already been identified. This review will survey and assess the current state-of-the-art and will propose methodologies to better exploit the potential of marine plankton for drug discovery and for dermocosmetics

Kinase Inhibitor Specialized Screening facility (KISSf), Station Biologique de Roscoff, Centre

marine drug

Evolution of galactoglycerolipid biosynthetic pathways - From cyanobacteria to primary plastids and from primary to secondary plastids.

International audience: Photosynthetic membranes have a unique lipid composition that has been remarkably well conserved from cyanobacteria to chloroplasts. These membranes are characterized by a very high content in galactoglycerolipids, i.e., mono- and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Galactoglycerolipids make up the bulk of the lipid matrix in which photosynthetic complexes are embedded. They are also known to fulfill specific functions, such as stabilizing photosystems, being a source of polyunsaturated fatty acids for various purposes and, in some eukaryotes, being exported to other subcellular compartments. The conservation of MGDG and DGDG suggests that selection pressures might have conserved the enzymes involved in their biosynthesis, but this does not appear to be the case. Important evolutionary transitions comprise primary endosymbiosis (from a symbiotic cyanobacterium to a primary chloroplast) and secondary endosymbiosis (from a symbiotic unicellular algal eukaryote to a secondary plastid). In this review, we compare biosynthetic pathways based on available molecular and biochemical data, highlighting enzymatic reactions that have been conserved and others that have diverged or been lost, as well as the emergence of parallel and alternative biosynthetic systems originating from other metabolic pathways. Questions for future research are highlighted

Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum.

International audienceDiatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol

Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum.

International audienceDiatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

International audienceOleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga