Aix Marseille Université L'Ecole Doctorale 62 « Sciences de la Vie et de la Santé » Etude des sources de carbone et d’énergie pour la synthèse des lipides de stockage chez la microalgue verte modèle Chlamydomonas reinhardtii Yuanxue LIANG Soutenue publiquement le 17 janvier 2019 pour obtenir le grade de « Docteur en biologie » Jury Professor Claire REMACLE, Université de Liège (Rapporteuse) Dr. David DAUVILLEE, CNRS Lille (Rapporteur) Professor Stefano CAFFARRI, Aix Marseille Université (Examinateur) Dr. Gilles PELTIER, CEA Cadarache (Invité) Dr. Yonghua LI-BEISSON, CEA Cadarache (Directeur de thèse) 1 ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my advisor Dr. Yonghua Li-Beisson for the continuous support during my PhD study and also gave me much help in daily life, for her patience, motivation and immense knowledge. I could not have imagined having a better mentor. I’m also thankful for the opportunity she gave me to conduct my PhD research in an excellent laboratory and in the HelioBiotec platform. I would also like to thank another three important scientists: Dr. Gilles Peltier (co- supervisor), Dr. Fred Beisson and Dr. Pierre Richaud who helped me in various aspects of the project. I’m not only thankful for their insightful comments, suggestion, help and encouragement, but also for the hard question which incented me to widen my research from various perspectives. I would also like to thank collaboration from Fantao, Emmannuelle, Yariv, Saleh, and Alisdair. Fantao taught me how to cultivate and work with Chlamydomonas. Emmannuelle performed bioinformatic analyses. Yariv, Saleh and Alisdair from Potsdam for amino acid analysis. My sincere thanks also go to Ismael, Stéphan, Adrien, Bertrand, Stephanie, Pascaline, Audrey, Solène, Hélène and Marie-Christine. Ismael helped me in many aspects, including solving daily problems, improving my oral English, and also giving me guidance in my experiments. Stéphan and Stephanie solved many technical or machine problems in the lab, in addition, they also taught me how to do Western blot and lipid extraction/TLC analyses. Adrien and Solène taught me how to measure mitochondrial respiration and photosynthetic performances. Bertrand helped me with the LC-MS analyses and also taught me how to run GC-MS. Pascaline taught me how to do qRT-PCR experiment. Audrey, Hélène and Marie-Christine taught me how to use Confocal microscopy. I would especially like to thank Véronique who did a lot of behind-the-scene’s work, such as keeping the algal strains healthy, cleaning and preparing the equipment for experiment. Thanks also go to Marie and Cyril for being friendly in the lab. 2 Thanks again to everyone in the “Laboratoire de bioénergétique et biotechnologie des bactéries et microalgues” for creating an excellent working environment, thank you all for being so friendly and kind to me. Last but not the least, I would like to thank my family: my parents and my wife gave me moral support and care for my life. 3 OUTLINE “Avant-props” ABSTRACT ABBREVIATONS INTRODUCTION 1. Microalgae and microalgal biotechnology 1.1 The importance of biodiesel 1.2 Microalgal biotechnology 1.3 Conversion of algal lipid or biomass to biodiesel 2. Lipid definition and classes 2.1 Lipid definition 2.2 Lipid classes 2.3 Fatty acid (FA) and glycerolipids 3. Lipid metabolism 3.1 Carbon and energy supply for FA synthesis 3.2 De novo FA synthesis and export 3.3 Polar membrane lipid synthesis 3.4 Triacylglycerol (TAG) biosynthesis 3.5 Lipid droplets (LDs) 3.6 Lipid catabolism and the β-oxidation of FAs 4. Branched-chain amino acid (BCAA) metabolism 4.1 Occurrence and cellular functions 4.2 BCAA biosynthesis 4.3 BCAA degradation 5. A crosstalk between metabolism of lipids and BCAAs 6. Chlamydomonas reinhardtii OBJECTIVES OF THIS PHD THESIS MATERIALS AND METHODS 1. Strains and culture conditions 4 2. The use of Flow cytometry to evaluate cellular oil content 3. DNA and RNA isolation and cDNA preparation 4. Identification of the Aph8 insertion site in the Pb8C12 mutant by RESDA-PCR 5. Reverse transcription PCR (RT-PCR) 6. Cloning the full length gene BKDE1α and genetic complementation of Pb8C12 (i.e. bkdE1α) mutant 7. LD imaging 8. Protein extraction, quantification and SDS-PAGE 9. Antibody generation and immunoblot analysis 10. Lipid analyses 11. Chlorophyll and starch quantification 12. Isolation and validation of mutants from the Chlamydomonas library (CLIP) 13. Amino acid analyses by gas chromatography-mass spectrometry (GC-MS) 14. Respiration analysis using a Clark electrode 15. Total RNA extraction, RNA-seq library preparation and sequencing 16. Enzymatic activity assays RESULTS AND DISCUSSION Chapter 1: Branched-chain amino acid catabolism impacts triacylglycerol homeostasis in Chlamydomonas reinhardtii. Chapter 2: Chlamydomonas carries out fatty acid β-oxidation in ancestral peroxisomes using a bona fide acyl-CoA oxidase. Chapter 3: Interorganelle communication: peroxisomal MALATE DEHYDROGENASE 2 connects lipid catabolism to photosynthesis through redox coupling in Chlamydomonas. Chapter 4: Saturating light induces sustained accumulation of oil primarily stored in lipid droplets of plastidial origin in Chlamydomonas reinhardtii. CONCLUSION AND PERSPECTIVES REFERENCE Annex: CURRICULUM VITAE 5 “Avant-propos” I was awarded a PhD studentship by the China Scholarship Council (CSC). I joined the “Laboratoire de bioénergétique et biotechnologie des bactéries et microalgues (LB3M)” at CEA Cadarache the 1st of March 2015. I finished my contract at the end of Febrary 2018. My research interests are to understand lipid biosynthesis and turnover processes in microalgae and investigate the possible occurrence of metabolic links between lipids and other subcellular metabolisms. To this end, I took advantage of the recent isolation of a few mutants defected in oil turnover in the model microalga Chlamydomonas reinhardtii in our laboratory (Cagnon et al., 2013). In the past three years, I have been involved in molecular genetic, biochemical and physiological characterization of three mutants, and contributed to one other work in the laboratory (detailed below). 1. A mutant defected in a gene encoding a putative E1α subunit of the branched chain -keto acid dehydrogenase (BCKDH). [Liang et al submitted to Plant Physiology] This is my major work. The mutant bkdE1α, deficient in the E1α subunit of the BCKDH complex, is found to accumulate less oil during nitrogen (N) starvation, and also is compromised in oil breakdown upon N resupply. We showed via quantification of the amount of branched chain amino acids (BCAAs) that BCKDH is indeed involved in BCAA degradation in Chlamydomonas reinhardtii. Through biochemical, genetic and physiological characterization of this mutant, we put on evidence that BCAA degradation contributes to TAG metabolism via the provision of carbon precursors and ATP, thus highlighting the complex interplay between distinct subcellular metabolisms for oil storage in green microalgae. 6 2. A mutant defected in an acyl-CoA oxidase (ACX) catalyzing the first step in the -oxidation of fatty acid in peroxisomes. [Kong, Liang et al Plant J 2017 90:358] This work helped to bring peroxisome to a center stage on lipid homeostasis in a primitive alga, i.e. the Chlamydomonas reinhardtii. We showed for the first time that H2O2 generating reaction occurs in algal peroxisomes and that Chlamydomonas carries out β-oxidation of fatty acids mainly in this subcellular organelle. Specifically, I: - Cloned the 11 kb genomic DNA encoding the ACX2 protein for genetic complementation of the acx2 mutant. - Did lipid extractions and fatty acid composition analysis. - Carried out quantitative RT-PCR analysis of the expression level of the gene under various cultivation conditions. - Tested the effect of perturbation in fatty acid turnover on senescence and in prolonged darkness. 3. Contributed to characterize the peroxisomal malate dehydrogenase 2 (MDH2) mutant. [Kong, Burlacot, Liang et al Plant Cell 2018 30:1824] This paper showed for the first time, the occurrence of a redox communication between peroxisome and chloroplast, and we showed further that this communication is not only important for carbon reserve accumulation but also is critical for cells to avoid photo- oxidative damage during changing environmental conditions such as nutrient deficiency or high light (HL). Specifically, I: - did RT-PCR. - performed MDH activity assays. - tested growth parameters under various cultivation conditions. - carried out lipid and starch analysis during a day/night cycle. 7 In addition: Microscopy characterization of subcellular localization of lipid droplets in cells starved for N in comparison to cells exposed to HL. [Goold, Cuine, Legeret, Liang et al Plant Physiology 2016 171:2406] This work reported the synthesis of TAGs upon HL exposure, and HL can induce an overall higher lipid productivity than the most commonly used N starvation. Furthermore, by studying and comparing the lipidome and proteome of lipid droplets (LDs) isolated from HL-exposed cells versus N-starved cells, we infer a possibility that distinct sub-populations of LDs or different biogenesis origins of LDs could accumulate in HL-exposed cells compared to N-starved cells. This is further supported by microscopy observations. 8 RESUME Etude des sources de carbone et d’énergie pour la synthèse des lipides de stockage chez la microalgue verte modèle Chlamydomonas reinhardtii Les 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. Pour répondre à cette question, nous avons 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.