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.

Involvement of Rad51 paralogs in Rad51 filament formation in DNA repair

Homologous recombination (HR) is a major repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA generated from these breaks. We have shown that strict control of these filaments is essential, so that HR does not itself induce chromosomal rearrangements (eLife 2018, Cells 2021). In humans, functional homologs of control proteins are tumor suppressors. Thus, the control of HR appears to be as important as the HR mechanism itself. Our project involves the use of new molecular tools enabling a real breakthrough in the study of these controls. We will be using a functional fluorescent version of the Rad51 protein developed for the first time by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will enable us to observe the influence of control proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein megacomplexes in association with Rad51 filaments. This study also led to the identification of specific domains for each paralog protein, outside the structurally conserved Rad51-like core, that might define the specificity of each paralog proteins. We will use a multidisciplinary approach based on genetic, molecular biology, biochemistry, protein structure and live microscopy methods and yeast as model organism to study the consequences of the ablation of these specific domains. We will also search for proteins specifically binding these domains. Their identification would be crucial to understand the function of Rad51 paralog complexes and help to develop new therapeutic approaches.

Biosynthesis and functional evaluation of novel antimicrobial peptides from mammalian gut microbiomes

The WHO has identified antibiotic resistance as one of the major threats to human health. According to predictions, the number of deaths related to antibiotic resistance is estimated at 10 million a year by 2050. This situation is prompting scientists to find new molecules, ideally natural ones, whose structures and mode of action differ from those of conventional antibiotics, to overcome the phenomena of resistance. One promising alternative concerns antimicrobial peptides of the RiPPs family (ribosomally synthesized and post-translationally modified peptides) produced by bacteria. Numerous studies show that the intestinal microbiome plays a very important role in the health of the host. Among the mechanisms involved, the production of antimicrobial peptides appears to be of particular importance. Part of our collaborative work aims to identify new antimicrobial peptides from complex biological ecosystems using metagenomic methods. To date, we have identified ten potentially interesting sequences. In this project, we will focus on the biosynthesis as well as the biochemical and structural characterizations of the antimicrobial peptides. An important part of the project will be devoted to the biological activity of these compounds on resistant and multi-resistant pathogens to conventional antibiotics. Moreover, the mode of action and the toxicity of the most effective peptides will be addressed.