Study of the natural alkane/alkene synthesis pathway in the model microalga Chlamydomonas reinhardtii and genetic engineering of this microalga to produce and excrete these compounds

Microalgae are fast-growing eukaryotic photosynthetic microorganisms that have developed efficient mechanisms to harvest and transform solar energy into energy-rich molecules such as lipids. They are thus potentially great cell factories for production of fuels and biomaterials for the chemical industries. These processes are however still not profitable, mostly because of low oil productivity and high costs of biomass harvest and oil extraction. A major breakthrough would be to create microalgal strains producing and excreting fatty acid-derived hydrocarbons (alkanes and alkenes). Toward this goal, the host team has recently made significant advances: i) Discovery of a novel alka(e)ne-forming enzyme, the photoenzyme fatty acid photodecarboxylase (FAP), which converts free fatty acids to an alka(e)nes, and characterization of the way FAP interacts with its fatty acid substrates at lipid-water interfaces, (ii) Identification of novel players governing the accumulation and turnover of fatty acids in oil reserves (lipid droplets). In this thesis, our goal will be twofold: i) from a fundamental point of view, gain some insights into the way fatty acids are delivered to FAP in the chloroplast as well as into the fate of the hydrocarbon products in the model green microalga Chlamydomonas reinhardtii, ii) from a biotechnological point of view, boost hydrocarbon synthesis and excretion in this microalga. To achieve this goal, specifically, we aim to: i), identify the lipase(s) that is delivering free fatty acids to FAP and determine if any specific proteins bind the hydrocarbon products in membranes, ii) try to boost alka(e)ne synthesis by overexpressing FAP in the cytosol and addressing it to lipid droplets where most of the fatty acids are stored (together with a lipase or as a lipase-FAP chimera), (iii) try to increase hydrocarbon volatility, and thus excretion, by shortening the chain length of the fatty acids without increasing their degradation, (iv) express known hydrocarbon soluble transporters and known ABC transporters at the plasma membrane. This work should therefore contribute to increase our understanding of the detailed mechanism of the generation of alka(e)nes by FAP and of their cellular fate. At a biotechnological level, innovative strategy based on lipid droplets, FAP and hydrocarbons are proposed to build a first version of a eukaryotic photosynthetic cell factory excreting lipids.

The role of the signaling nucleotide ppGpp in plant resilience to climate change.

Amidst the growing challenges of climate change, crops face threats from rising temperatures and prolonged droughts, leading to reduced photosynthetic efficiency and the need for rapid stress acclimation. In this PhD project we will investigate the role of the nucleotide guanosine tetraphosphate (ppGpp) signalling pathway, a known regulator of plastid function and photosynthesis. Recent preliminary work from our and other labs indicate that ppGpp plays a pivotal role in plant stress acclimation, and we have indications that perturbation of ppGpp signalling affects plant responses to heat stress. This research aims to explore how ppGpp is involved in plant acclimation to heat and drought stress. Using a combination of physiological evaluations, biochemical techniques, transcriptomics, and biosensors this study will investigate the modulation of ppGpp levels under stress conditions, its impact on plastid genome expression, and its intersection with other signalling pathways. The ultimate goal is to enhance our understanding of ppGpp's role in plant acclimation, offering insights for improving crop resilience in a climate-challenged world.

characterization of acyltransferases in two oleaginous marine algae

The project aims to study the plastidial lipid metabolism of two microalgae using the CRISPR-Cas9 system. This project is in the context of increasing atmospheric CO2 and climate change. Photosynthetic microalgae can capture CO2 and convert it into lipids, which is useful for various industrial applications. However, stramenopile microalgae have a unique cellular structure, making it challenging to apply knowledge from simpler models. Therefore, we need a better understanding of lipid synthesis in these microalgae to enhance their ability to sequester CO2 and produce lipids for biotechnological applications. Our research focuses on the biosynthesis pathway of plastidial galactoglycerolipids, the dominant lipids in photosynthetic organisms, in two model microalgae, Phaeodactylum tricornutum and Microchloropsis gaditana. These lipids consist of a glycerol backbone, fatty acids (FAs), and galactose residues. FAs are produced in the plastids, then modified in other parts of the cell to form long-chain polyunsaturated FAs, such as eicosapentaenoic acid (EPA). The transport pathway of EPA to the plastid is still poorly understood. Then, EPA is esterified onto glycerol-3-phosphate (G3P) through enzymes called acyltransferases (AT). This study is one of the key research areas of our team. The synthesis of galactoglycerolipids in stramenopiles involves several steps, including esterifying an FA onto G3P, forming phosphatidic acid, converting it to diacylglycerol, and adding galactose residues to form the final lipids. In summary, our research aims to understand how these microalgae produce important lipids for CO2 capture and industrial applications. This research is relevant for combating climate change and reducing our dependence on fossil fuels.

RootExu-C : Plant genetic control of root exudation and microbiome assembly

Ongoing breeding programs selected for crop varieties with high yield under favorable conditions: sufficient water supply (irrigation) and high levels of chemical fertilizers (N, P). However, increased abiotic stresses (drought, salinity, high temperatures) as well as ecological concerns demand for new traits to transition to more sustainable production systems. One approach may consist in better control and exploitation of root microbiota, which have the potential to protect their host plants from abiotic and biotic stresses, and to improve nutrition and productivity. It is assumed that plant innate immunity and root exudates scale and structure root microbiota, but exact mechanisms remain unknown. In this project, I propose to analyze “root-adhering soil” (RAS), the soil aggregated around roots, as a global proxy for shoot-to-root carbon allocation, root exudation and recruitment of exopolysaccharide-producing microbiota in Solanum lycopersicum (tomato). A respective PhD student shall analyze the RAS trait in a tomato natural variation panel towards the identification of underlying genes. Further, he/she shall directly inactivate candidate genes assumedly involved in root exudation (multiplex CRISPR/Cas). Lines with contrasting RAS phenotypes, from natural and/or induced variation, will be analyzed for microbiota recruitment and exudate composition. This will provide fundamental knowledge on the genetic control of root exudation and microbiome assembly and scaling cues. RAS may represent a valuable trait for the adaptation and performance of plants under lower input conditions, and may also facilitate enhanced storage of carbon in agricultural soils.

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