Relationship between acyl-lipid and sterol metabolisms in diatoms.

Diatoms are a phylum of unicellular photosynthetic eukaryotes living in oceans and fresh waters, characterized by the complexity of their plastid, resulting from a secondary endosymbiosis event. In the model diatom Phaeodactylum tricornutum, fatty acids (FAs) are synthesized from acetyl-CoA in the stroma of the plastid, producing palmitic acid. FAs are elongated and desaturated to form very-long chain polyunsaturated fatty acids (VLC-PUFAs) in domains of the endomembrane system that need to be identified. Synthesis of VLC-PUFAs is coupled with their import to the core of the plastid via the so-called "omega" pathway. The biosynthesis of sterols in diatoms is likely to be localized in the endoplasmic reticulum as well as using precursors deriving from the mevalonate pathway, using acetyl-CoA as initial substrate. These metabolic modules can be characterized functionally by genetic analyzes or chemical treatments with appropriate inhibitors. Some 'metabolic modules' are characterized by a very low level of metabolic intermediates. Since some chemical treatments or genetic perturbation of lipid metabolism induce the accumulation of these intermediates, channeling processes are possibly involved, suggesting that protein-protein interactions might occur between enzymes within large size complexes or metabolons. At the junction of these modules, metabolic intermediates might therefore play dramatic roles in directing carbon fluxes from one direction to another. Here, acetyl-CoA seems determinant in the balance between TAGs and sterols. Future lines of research and potential utilization for biotechnological applications are discussed.

Maréchal E ，Lupette J 《-》

Tracking the sterol biosynthesis pathway of the diatom Phaeodactylum tricornutum.

Diatoms are unicellular photosynthetic microalgae that play a major role in global primary production and aquatic biogeochemical cycling. Endosymbiotic events and recurrent gene transfers uniquely shaped the genome of diatoms, which contains features from several domains of life. The biosynthesis pathways of sterols, essential compounds in all eukaryotic cells, and many of the enzymes involved are evolutionarily conserved in eukaryotes. Although well characterized in most eukaryotes, the pathway leading to sterol biosynthesis in diatoms has remained hitherto unidentified. Through the DiatomCyc database we reconstructed the mevalonate and sterol biosynthetic pathways of the model diatom Phaeodactylum tricornutum in silico. We experimentally verified the predicted pathways using enzyme inhibitor, gene silencing and heterologous gene expression approaches. Our analysis revealed a peculiar, chimeric organization of the diatom sterol biosynthesis pathway, which possesses features of both plant and fungal pathways. Strikingly, it lacks a conventional squalene epoxidase and utilizes an extended oxidosqualene cyclase and a multifunctional isopentenyl diphosphate isomerase/squalene synthase enzyme. The reconstruction of the P. tricornutum sterol pathway underscores the metabolic plasticity of diatoms and offers important insights for the engineering of diatoms for sustainable production of biofuels and high-value chemicals.

Fabris M ，Matthijs M ，Carbonelle S ，Moses T ，Pollier J ，Dasseville R ，Baart GJE ，Vyverman W ，Goossens A ... -  《-》

Inventory of fatty acid desaturases in the pennate diatom Phaeodactylum tricornutum.

Dolch LJ ，Maréchal E 《Marine Drugs》

Plastidial acyl carrier protein Δ9-desaturase modulates eicosapentaenoic acid biosynthesis and triacylglycerol accumulation in Phaeodactylum tricornutum.

The unicellular marine diatom Phaeodactylum tricornutum accumulates up to 35% eicosapentaenoic acid (EPA, 20:5n3) and has been used as a model organism to study long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis due to an excellent annotated genome sequence and established transformation system. In P. tricornutum, the majority of EPA accumulates in polar lipids, particularly in galactolipids such as mono- and di-galactosyldiacylglycerol. LC-PUFA biosynthesis is considered to start from oleic acid (18:1n9). EPA can be synthesized via a series of desaturation and elongation steps occurring at the endoplasmic reticulum and newly synthesized EPA is then imported into the plastids for incorporation into galactolipids via an unknown route. The basis for the flux of EPA is fundamental to understanding LC-PUFA biosynthesis in diatoms. We used P. tricornutum to study acyl modifying activities, upstream of 18:1n9, on subsequent LC-PUFA biosynthesis. We identified the gene coding for the plastidial acyl carrier protein Δ9-desaturase, a key enzyme in fatty acid modification and analyzed the impact of overexpression and knock out of this gene on glycerolipid metabolism. This revealed a previously unknown role of this soluble desaturase in EPA synthesis and production of triacylglycerol. This study provides further insight into the distinctive nature of lipid metabolism in the marine diatom P. tricornutum and suggests additional approaches for tailoring oil composition in microalgae.

Smith R ，Jouhet J ，Gandini C ，Nekrasov V ，Marechal E ，Napier JA ，Sayanova O ... -  《-》

Proteomes reveal the lipid metabolic network in the complex plastid of Phaeodactylum tricornutum.

Phaeodactylum tricornutum plastid is surrounded by four membranes, and its protein composition and function remain mysterious. In this study, the P. tricornutum plastid-enriched fraction was obtained and 2850 proteins were identified, including 92 plastid-encoded proteins, through label-free quantitative proteomic technology. Among them, 839 nuclear-encoded proteins were further determined to be plastidial proteins based on the BLAST alignments within Plant Proteome DataBase and subcellular localization prediction, in spite of the strong contamination by mitochondria-encoded proteins and putative plasma membrane proteins. According to our proteomic data, we reconstructed the metabolic pathways and highlighted the hybrid nature of this diatom plastid. Triacylglycerol (TAG) hydrolysis and glycolysis, as well as photosynthesis, glycan metabolism, and tocopherol and triterpene biosynthesis, occur in the plastid. In addition, the synthesis of long-chain acyl-CoAs, elongation, and desaturation of fatty acids (FAs), and synthesis of lipids including TAG are confined in the four-layered-membrane plastid based on the proteomic and GFP-fusion localization data. The whole process of generation of docosahexaenoic acid (22:6) from palmitic acid (16:0), via elongation and desaturation of FAs, occurs in the chloroplast endoplasmic reticulum membrane, the outermost membrane of the plastid. Desaturation that generates 16:4 from 16:0 occurs in the plastid stroma and outer envelope membrane. Quantitative analysis of glycerolipids between whole cells and isolated plastids shows similar composition, and the FA profile of TAG was not different. This study shows that the diatom plastid combines functions usually separated in photosynthetic eukaryotes, and differs from green alga and plant chloroplasts by undertaking the whole process of lipid biosynthesis.

Huang T ，Pan Y ，Maréchal E ，Hu H ... -  《-》