1,721,071 research outputs found
Dynamique des réserves lipidiques chez la microalgue modèle Chlamydomonas reinhardtii
No full textD'importants efforts de recherche ont été déployés pour domestiquer les microalgues afin de produire des biocarburants durables et d'autres composés à haute valeur ajoutée. Les conditions utilisées pour enrichir la biomasse de microalgues avec des réserves de carbone, triacylglycérols (TAG ou huiles) et l'amidon, cependant, nuisent gravement à la croissance cellulaire et compromettent donc la productivité. Le but de cette thèse est d’analyser le lien entre la division cellulaire et le stockage du carbone, ainsi que de comprendre la biogénèse des gouttelettes de lipides (LDs), le principal site subcellulaire de stockage des TAGs.Ainsi, nous avons d'abord étudié l'incompatibilité entre le stockage du carbone et la croissance cellulaire en caractérisant génétiquement et biochimiquement des mutants de Chlamydomonas reinhardtii dépourvus de protéine CDC5. Nous démontrons son implication dans le cycle cellulaire et montrons qu'un ralentissement de la division cellulaire entraîne un flux d'énergie et de carbone vers la synthèse des TAGs et de l'amidon sans arrêter la croissance cellulaire. Deuxièmement, nous avons identifié et caractérisé une α/β hydrolase putative (CrABHD1), l'une des principales protéines associées aux LDs chez Chlamydomonas. La protéine recombinante CrABHD1 purifiée chez Escherichia coli hydrolyse du lyso-DGTS pour produire un acide gras libre et une glycérol-N,N,N-triméthylhomosérine (GTH). Nous avons découvert une nouvelle protéine associée à la LD et démontré sa capacité à augmenter la teneur en lipides des microalgues, ce qui devrait avoir des implications importantes pour une bioéconomie plus verte.Large research efforts have been put to domesticate microalgae for production of sustainable biofuels and other valuable compounds. Triacylglycerols (TAGs, or oils) and starch are the major forms of carbon storage in green algal cells. However, the conditions used to enrich microalgal biomass with these carbon reserves severely undermine cell growth therefore compromising productivity. An economically viable production of lipids from microalgae requires a deeper and integrated understanding of lipid synthesis, storage and cell division. The goal of this thesis is to dissect the connection between cell division and carbon storage, and to understand the biogenesis of the lipid droplet (LD), the major subcellular site where TAGs are stored. Toward this goal, we first investigated the incompatibility between carbon storage and cell growth. By characterizing genetically and biochemically mutants of Chlamydomonas reinhardtii deficient in CDC5 protein, we demonstrate its implication in the cell cycle and show that a slowdown in cell division entails a diverted flow of energy and carbon towards the synthesis of TAGs and starch without arresting cell growth. Secondly, we identified and characterized a putative α/β-fold hydrolase (CrABHD1), one of the major proteins associated to LDs in Chlamydomonas. The CrABHD1 recombinant protein purified from Escherichia coli hydrolyzes lyso-DGTS to produce a free fatty acid and a glycerol-N,N,N-trimethylhomoserine (GTH). We have discovered a novel LD-associated protein and demonstrated its capacity in increasing lipid content in microalgae, which should have important implications for a greener bioeconomy
Study of carbon and energy sources for storage lipid synthesis in model green microalga Chlamydomonas reinhardtii
No full textLes triacylglycérols d'algues (TAG) représentent une source prometteuse de biocarburants. Les principales étapes de la synthèse des acides gras et du métabolisme du TAG des algues ont été déduites de celles des plantes terrestres, mais on en sait peu sur les sources de carbones et d’énergie intervenant dans la synthèse de lipides de réserve. Nous avons donc étudié la synthèse des acides gras chez l’algue modèle Chlamydomonas reinhardtii en utilisant une combinaison d'approches génétiques, biochimiques et microscopiques. Plus précisément, j'ai d'abord examiné la localisation subcellulaire de gouttelettes de lipides dans des cellules d'algues exposées à une forte lumière, conditions où une plus grande quantité de pouvoir réducteur est produite. J'ai ensuite contribué à mettre en évidence que la bêta-oxydation des acides gras est un processus peroxysomal, et que pendant une carence en azote réalisée en conditions photoautotrophe, des mutants dépourvus de la malate déshydrogénase 2 peroxysomale (mdh2) accumulent 50% plus TAG que les souches parentales. Ces résultats nous ont permis de mettre en évidence l'importance du contexte redox cellulaire sur la synthèse lipidique. Cette étude a également permis de révéler l’existence d'un échange d’énergie entre le peroxysome et le chloroplaste. Enfin, en caractérisant des mutants déficients dans la dégradation des acides aminés à chaîne ramifiée (BCAA), j'ai montré que le catabolisme des BCAAs joue un double rôle dans la synthèse de TAG en fournissant des précurseurs carbonés et de l'ATP. L'ensemble de ces travaux ouvert de nouvelles pistes pour l'amélioration génétique future de souches d'algues pour la production de biocarburants.Algal triacylglycerols (TAG) represent a promising source for biofuel. The major steps for fatty acid synthesis and TAG metabolism have been deduced based on that of land plants, but little is known about carbon and energy sources. To address this question, we investigated fatty acid synthesis in algal cells using a combination of genetic, biochemical and microscopic approaches in the model microalga Chlamydomonas reinhardtii. Specifically, I first examined subcellular localization of lipid droplets in algal cells exposed to high light, a condition favoring production of reducing power. Secondly, I contributed to put on evidence that the beta-oxidation of fatty acids is a peroxisomal process, and that during photoautotrophic nitrogen starvation, knock-out mutants of the peroxisomal malate dehydrogenase 2 (mdh2) made 50% more TAG than parental strains, highlighting the importance of cellular redox context on lipid synthesis. This study also revealed for the first time the occurrence of an energy trafficking pathway from peroxisome to chloroplast. And finally, by characterizing mutants defected in degradation of branched-chain amino acids (BCAAs), I showed that BCAA catabolism plays a dual role in TAG synthesis via providing carbon precursors and ATP. Taken together, this work highlighted the complex interplay between carbon and energy metabolism in green photosynthetic cells, and pointed future directions for genetic improvement of algal strains for biofuel productions
Biosynthesis of fatty acid-derived hydrocarbons in microalgae
No full textLes alcanes et les alcènes sont des hydrocarbures non cycliques important dans l’industrie. Ils sont synthétisés à partir d'acides gras par une grande variété d’organismes mais les connaissances à ce sujet sont très limitées chez les microalgues. Le but de ces travaux était donc de rechercher la présence d’alcanes ou d’alcènes dans diverses microalgues modèles, et d’essayer d’identifier la ou les enzymes responsables de la synthèse de ces composés. Nous avons mis en évidence la présence d’hydrocarbures linéaires en C15-C17 chez les microalgues Chlorella et Chlamydomonas. Ces composés étaient synthétisés uniquement en présence de lumière. L’absence dans le génome de ces microalgues d’homologues de gènes codant pour des enzymes connues de synthèse d’alcanes/alcènes a permis de conclure à la présence d’un nouveau système de synthèse d’hydrocarbures. Des purifications enzymatique et des analyses protéomique ont permis d’identifier une enzyme candidate qui exprimée chezE. coli est suffisante à la synthèse d’hydrocarbures. L'étude de cette enzyme révella qu'il s'agissait d'une photoenzyme utilisant l'énergie des photons bleue pour décarboxyler les acides grass en alca(e)ne. La structure de cette photoenzyme montre la présence un tunnel hydrophobe contenant l’acide gras et le cofacteur FAD. Cette nouvelle enzyme nommée « alcane photosynthase » amène de nombreuses question: qu'elle est la fonction des hydrocarbures chez ces microorganismes? Quel est le mécanisme catalytique de l’alcane photosynthase? Enfin, elle offre de nouvelles possibilités pour la production de biocarburants utilisant directement l’énergie solaire.Alkanes and alkenes are important in industry. Alkanes and alkenes are synthesized from fatty acids by a variety of organisms, such as plants and insects. However, the presence in microalgae of enzymes converting fatty acids into hydrocarbons has been poorly studied. The aim of this work was to investigate the presence of alkanes and alkenes in various microalgae models, and try to identify the enzymes responsible for the synthesis of these compounds.We have first demonstrated the presence of linear hydrocarbons C15-C17 in microalgae Chlorella and Chlamydomonas. Then we have shown that the main hydrocarbon formed in Chlorella and Chlamydomonas was derived from cis-vaccenic acid and was synthesized only in the presence of light. Absence of homologues of genes coding for known alkane/alkene biosynthetic enzymes in the genome of Chlorella and Chlamydomonas indicate the presence of an unknown pathway. Enzymatic purification and proteomic analysis allowed to identify a candidate enzyme which, expressed in E. coli lead to the formation of hydrocarbons with variable chain lengths, thus demonstrating that it was really an synthase alkane. Characterization showed that the enzyme was a photoenzyme, which used blue light to catalyse the decarboxylation of fatty acid to an alka(e)ne. The three-dimensional structure of this enzyme revealed a hydrophobic tunnel containing the fatty acid and the FAD cofactor
Caractérisation de la synthèse d'hydrocarbures chez les microalgues
No full textLes hydrocarbures (HCs) sont prédominants dans notre économie actuelle (carburants, cosmétiques, chimie, etc.) mais sont quasi-exclusivement issus des ressources fossiles. Les problématiques de changement climatique et d’épuisement des ressources poussent les recherches vers l’étude et la domestication des voies de synthèse naturelles d’HCs. Lorsque j’ai commencé ma thèse, une enzyme de biosynthèse d’HC, l’acide gras photodécarboxylase (FAP) venait d’être découverte chez la microalgue Chlorella. J’ai d’abord caractérisé son homologue chez la microalgue modèle Chlamydomonas. Une étude phylogénétique de la famille des GMC oxidoréductases à laquelle appartient la FAP a permis d’identifier un large réservoir de de 200 FAPs putatives. La caractérisation biochimique de plusieurs d’entre elles a permis de montrer qu’une FAP fonctionnelle a été conservée lors des endosymbioses secondaires. Cela suggère que la FAP joue un rôle important chez les algues. Ce rôle a été étudié par une approche de génétique inverse chez Chlamydomonas. La caractérisation physiologique de mutants knockout a permis de démontrer le rôle de la FAP dans la synthèse d’HCs dans le chloroplaste et de mettre en évidence des modifications physiologiques transitoires. Des mécanismes de compensation à l’absence d’HCs restent donc à découvrir. Dans une dernière partie, j’ai développé une souche d’E. coli exprimant la FAP et une thioestérase. Cette souche produit en continu des HCs dans la phase gaz des cultures, ce qui permet une récolte facilitée du produit d’intérêt sous forme pure. Cette étude constitue une preuve de concept que la FAP pourrait être utilisée pour la production biosourcée d’HCs.Hydrocarbons (HCs) are predominant in our current economy (fuels, cosmetics, chemicals, etc.) but are almost exclusively derived from fossil resources. Climate change and resource depletion concerns are pushing research towards the study and domestication of natural HC synthesis pathways. When I started my thesis, a HC forming enzyme, the fatty acid photodecarboxylase (FAP) had just been discovered in the microalgae Chlorella. I first characterised its homolog in the model microalgae Chlamydomonas. A phylogenetic study of the GMC oxidoreductase family to which the FAP belongs has allowed identification of a large reservoir of 200 putative FAPs. Biochemical characterisation of several of them showed that a functional FAP was maintained during secondary endosymbiosis. This suggests that FAP plays an important role in algae. This role has been studied by a reverse genetic approach in Chlamydomonas. The physiological characterisation of knockout mutants demonstrated the role of FAP in the synthesis of HCs in chloroplasts as well as transient physiological changes. Mechanisms to compensate for the absence of HCs therefore remain to be discovered. In a last part, I developed a strain of E. coli expressing the FAP and a thioesterase. This strain continuously produces HCs in the gas phase of the cultures, which allows an easier harvesting of the product of interest in a pure form. This study is a proof of concept that FAP could be used for the biobased production of HCs
Functional study of oil assembly pathway in oil palm (Elaeis guineensis Jacq.) fruits
No full textLe palmier à huile est la première culture oléagineuse, avec environ 40% de la production mondiale, et son fruit accumule deux huiles de composition très différente dans le mésocarpe et l’amande. Chez les plantes, les acides gras sont assemblés en huile dans le réticulum endoplasmique, ceci par la voie dite de Kennedy à laquelle s’ajoutent des mécanismes d’édition impliquant le métabolisme de la phosphatidylcholine. Nous avons utilisé les outils de la lipidomique pour analyser la variabilité au sein de différentes populations de palmier ainsi que pour caractériser l’accumulation d’huile durant le développement du mésocarpe et de l’amande. Puis, nous avons entrepris de tester, dans le système du double hybride de levure, les interactions entre toutes les enzymes de la voie de Kennedy et celles responsables des mécanismes d’édition, et mis en évidence 241 interactions, dont 132 sont fortes, 73 moyennes et 36 faibles. Ces résultats suggèrent que ces enzymes pourraient s’assembler en complexes supra-moléculaires susceptibles de former des métabolons. Certaines isoformes d’une même enzyme ont des profils d’interaction distincts, ce qui ouvre des perspectives pour de futures recherches. De plus, nous avons caractérisé, par expression fonctionnelle dans un mutant de levure dépourvu de TAG, une acyltransférase présumée (EgWSD1-like) ainsi que les trois formes majeures de diacylglycérol acyltransférases du mésocarpe. EgWSD1-like ne restaure que l’activité de synthèse d’esters de cire dans le mutant, tandis que les trois DGAT complémentent toutes la déficience en TAG du mutant, avec d’apparentes spécificités distinctes vis-à-vis des acides gras.Oil palm is the highest oil-yielding crop-plant, accounting for approximately 40% of the total world vegetable oil production. The fruit accumulates oil, made of triacylglycerol (TAG) molecules, in both mesocarp and kernel with totally different fatty acid profiles. Fatty acids are assembled into oil through Kennedy pathway in the endoplasmic reticulum, which is complicated by editing processes involving phosphatidylcholine metabolism. To investigate oil assembly in oil palm, we use lipidomics as a tool to analyze different populations of palm to search for TAG structural diversity, and to further characterize changes in lipid content and composition in mesocarp and kernel during fruit ripening. We used yeast two-hybrid system (split ubiquitin) to test protein-protein interactions for almost all the enzymes (32) involved in oil assembly pathway, and we demonstrated 241 interactions, including 132 strong interactions, 73 medium interactions and 36 weak interactions. Our results suggest that all enzymes might assemble into one or several complexes that may form metabolons. In addition, different isoforms of enzymes showed distinct interaction profiles, providing hints for future studies. Moreover, we also characterized the in vivo function of a putative acyltransferase (designated EgWSD1-like) possibly involved in oil assembly and the three major diacylglycerol acyltransferase (DGAT) isoforms of palm mesocarp in the mutant yeast H1246, which is devoid of neutral lipid synthesis. EgWSD1-like only shows wax ester synthase activity in yeast, while three EgDGATs all can restore TAG biosynthesis in yeast with different substrate specificities
Editorial feature: Meet the PCP editor—Yonghua Li-Beisson
No full textInternational audienceYonghua Li-Beisson received a B.Sc. from Henan University of Technology, China, and a Ph.D. from the University of Hull (England), where her work highlighted the importance of reducing power for lipid synthesis in oleaginous fungi. She then conducted postdoctoral research at Michigan State University focusing on understanding seed oil biosynthesis as well as dissecting the molecular pathways of lipid polyester synthesis and assembly. Since 2009, Yonghua is a staff scientist at the French Atomic and Alternative Energies Commission in France. Her current work focuses on dissecting lipid metabolism in microalgae, in particular using the model green microalga Chlamydomonas reinhardtii (Li-Beisson et al. 2019). In addition to leading her research team, she is also a director of the lipidomics platform HelioBiotec (https://www.cite-des-energies.fr/biam/plateformes-technologiques/heliobiotec/). Since 2016, Yonghua is a serving editor for Plant and Cell Physiology and handles papers in the area of plant and algal lipid metabolism and physiolog
Third-generation biofuels: current and future research on microalgal lipid biotechnology
No full textOne pressing issue faced by modern societies is to develop renewable energy for
transportation. Microalgal biomass offers an attractive solution due to its high (annual)
surface biomass productivity, efficient conversion of solar energy into chemical energy
and the ability to grow on non-agricultural land. Despite these considerable advantages,
microalgal biofuels are not yet commercially sustainable. Major challenges lie in
improving both cultivation technologies and microalgal strains. A microalgal crop species
is yet to emerge. In this review, we focus on researches aiming at understanding and
harnessing lipid metabolism in microalgae in view of producing lipid-based biofuels such
as biodiesel. Current biotechnological challenges and key progresses made in the
development of algal models, genetic tools and lipid metabolic engineering strategies are
reviewed. Possible future research directions to increase oil yields in microalgae are
also highlighted
Biogenesis and fate of lipid droplets
No full textInternational audienceEditorial Special issue "Biogenesis and Fate of Lipid Droplets" Intracellular lipid droplets (LDs) are receiving increasing interest from the scientific community, notably because of their link with metabolic diseases (obesity, diabetes) and the production of lipid-derived biofuels in microorganisms. Understanding the biogenesis and fate of LDs upon energy mobilization is essential to monitor fat storage in adipose tissue, but also to improve lipid productivity in microalgae under various conditions of growth [1]. Intracellular LDs are today considered as individual organelles since they host specific metabolic functions such as the biosynthesis of triacylglycerols (TAGs) and phospholipids [2]. In addition to the enzymes involved in these pathways, several proteins have been identified as associated with LDs and their number has been increasing with the progress of proteomics. Some of these proteins presenting hydrophobic patches and a high amphiphilicity are involved in the structure of LDs and together with polar lipids they form the membrane surrounding the neutral lipid core. Some others are involved in the budding of LDs from lipid bilayers and the interactions of LDs with membranes and other organelles. These interactions are important for the transfer of fatty acids and lipid remodelling and homeostasis. The combination of lipidomics and proteomics has led to a better description of LD composition, while microscopy has allowed the visualisation of the specific location of proteins at the surface of LDs. Nevertheless, a better knowledge of the lipid-protein interactions that lead to the biogenesis and fate of LDs is still required. In this context, cellular biologists now adopt principles and methods from biophysics and physical chemistry of lipid emulsions and colloids [3]. Emulsions made of TAGs, polar lipids and proteins can serve as models for intracellular LDs. The biodiversity of LDs in animals, plants and microorganisms and their various functions in lipid storage and transport also allows the comparison of data and knowledge from various fields that will help build a global understanding of structure-function relationships. The original articles and reviews of this special issue are a collection of topics presented at the 14th GERLI Lipidomics meeting, which was be held at St Maximin-la-Sainte Baume, France, from September 30th to October 3rd, 2018, with the aim of gathering researchers from various areas with a common interest in LDs, their physical chemistry and metabolism, their association with diseases and various applications in biotechnology, pharmacology and nutrition. The two first mini-reviews are related to lipid metabolism in microalgae. Lupette et al. cover the biosynthesis of fatty acids, acyl-glycero-lipids and sterols in diatoms and show how a metabolic intermediate common to these pathways, like acetyl-CoA, can play an essential role in directing the carbon flux from acyl-lipid to sterol biosynthesis and be determinant for the balance between TAGs and sterols. Prioretti et al. investigate how the target of rapamycin (TOR) signalling pathway could be modulated to enhance TAG production in microalgae. The use of TOR inhibitors increases TAG productivity in the marine diatom Phaeodactylum tricornutum, without stopping growth, while TAG accumulation in microalgae is usually observed under stress conditions with a low growth rate. Yuan et al. report on the characterization of three diacylglycerol acyltransferases (DGATs) from oil palm (Elaeis guineensis), the main source of vegetable oil on Earth. DGATs are key enzymes for the biosynthesis of TAGs and the formation of LDs. Using the heterologous expression of these DGATs in the yeast Saccharomyces cerevisiae, they show that two of them are true DGATs, that could restore TAG synthesis in a yeast TAG-deficient mutant, while the third one is a wax ester synthase
Plant unusual fatty acids: learning from the less common
No full textInternational audienceThe plant kingdom contains an abundance of structurally diverse fatty acids referred to as unusual fatty acids. Unusual fatty acids on plant surfaces can form polyesters that contribute to the function of cutin as a barrier for water loss and pathogen protection. Unusual fatty acids are also found as abundant components of seed oils of selected species and often confer desirable properties for industrial and nutritional applications. Here, we review recent findings on the biosynthesis and metabolism of unusual fatty acids in cutin and seed oils and use of this information for enzyme structure-function studies and seed oil metabolic engineering. We also highlight the recent discovery of unusual fatty acids that are formed from a previously undescribed variation of fatty acid elongation
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