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1. Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase

Author

Poutoum P. Samire, Bo Zhuang, Bertrand Légeret, Ángel Baca-Porcel, Gilles Peltier, Damien Sorigué, Alexey Aleksandrov, Frédéric Beisson, Pavel Müller, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'Optique et Biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), China Scholarship Council, ANR-18-CE11-0021,SNAPsHOTs,Dynamique structurale de l'acide gras photodécarboxylase(2018), ANR-18-CE43-0008,PHOTOALKANE,Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme(2018), and ANR-18-CE44-0002,ATMCADD,Modèles atomiques précis pour la conception de médicaments assistée par ordinateur(2018)

Subjects
Multidisciplinary, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [CHIM.CATA]Chemical Sciences/Catalysis
Abstract

Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP ( Cv FAP) can convert n -octanoic acid in vitro four times faster than n -hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a Cv FAP-based production rate over 10-fold higher for n -heptane than for n -pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that Cv FAP’s high catalytic activity on n -octanoic acid is, in part, due to an autocatalytic effect of its n -heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons.

Published
2023

2. Fatty Acid Photodecarboxylase Is an Interfacial Enzyme That Binds to Lipid-Water Interfaces to Access Its Insoluble Substrate

Author

Cyril Aselmeyer, Frédéric Carrière, Anaïs Bénarouche, Bertrand Legeret, Goetz Parsiegla, Damien Sorigué, Fred Beisson, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bioénergie et Microalgues (EBM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
congenital, hereditary, and neonatal diseases and abnormalities, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Photoenzyme, Carboxy-Lyases, Phospholipid, Palmitic Acid, Chlorella, 7. Clean energy, 01 natural sciences, Biochemistry, Micelle, 03 medical and health sciences, chemistry.chemical_compound, Biofuel, Animals, Microemulsion, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], neoplasms, Micelles, Unilamellar Liposomes, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Liposome, biology, 010405 organic chemistry, Algal Proteins, beta-Cyclodextrins, Active site, Fatty acid, Substrate (chemistry), Water, Serum Albumin, Bovine, Lipids, digestive system diseases, Hydrocarbons, 0104 chemical sciences, Kinetics, Membrane, chemistry, biology.protein, Biophysics, Biocatalysis, Cattle, Emulsions, Adsorption
Abstract

International audience; Fatty Acid Photodecarboxylase (FAP), one of the few natural photoenzymes characterized so far, is a promising biocatalyst for lipid-to-hydrocarbon conversion using light. However, the optimum supramolecular organization under which the fatty acid (FA) substrate should be presented to FAP has not been addressed. Using palmitic acid embedded in phospholipid liposomes, phospholipid-stabilized microemulsions and mixed micelles, we show that FAP displays a preference for FAs present in liposomes and at the surface of microemulsions. Adsorption kinetics onto phospholipid and galactolipid monomolecular films further suggests the ability of FAP to bind to and penetrate into membranes, with higher affinity in the presence of FAs. FAP structure reveals a potential interfacial recognition site with clusters of hydrophobic and basic residues surrounding the active site entrance. The resulting dipolar moment suggests the orientation of FAP at negatively charged interfaces. These findings provide important clues for the mode of action of FAP and the development of FAP-based bioconversion processes.

Published
2021

3. Light harnessing by Algae: from fundamental investigations to light-based biotechnologies

Author

Damien Sorigué, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bioénergie et Microalgues (EBM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
chemistry.chemical_classification, congenital, hereditary, and neonatal diseases and abnormalities, biology, Fatty acid, biology.organism_classification, Photosynthesis, digestive system diseases, Organic molecules, [SPI]Engineering Sciences [physics], Algae, chemistry, Biochemistry, 13. Climate action, neoplasms
Abstract

International audience; Light is the most abundant source of energy on earth and is used by photosynthetic organisms to drive the synthesis of organic molecules. Light also allows the catalysis of few enzymes, the photoenzymes. Among them, the fatty acid photodecarboxylase (FAP) isolated from microalgae converts fatty acids into hydrocarbons. We present here our understanding of the role of hydrocarbons produced by FAP in vivo, the catalytic mechanism of the FAP and its potential biotechnological applications.

Published
2021

4. Mechanism and dynamics of fatty acid photodecarboxylase

Author

Sébastien Boutet, Catherine Berthomieu, Laura Antonucci, A. Gorel, Manuel Joffre, Marco Cammarata, A. Benachir, Sergio Carbajo, Solène L. Y. Moulin, Martin Weik, Michel Sliwa, Stephane Cuine, Yonghua Li-Beisson, Nicolas Coquelle, Didier Nurizzo, P. Samire, Jacques-Philippe Colletier, Alexey Aleksandrov, Robert L. Shoeman, Guillaume Gotthard, Antoine Royant, Marten H. Vos, Bo Zhuang, M. Hilpert, Adeline Bonvalet, Ilme Schlichting, Xavier Solinas, Martin Byrdin, Pascal Arnoux, Gilles Peltier, Pierre Legrand, F. Beisson, Klaus Brettel, R. Hienerwadel, Thomas R. M. Barends, R.B. Doak, Lutz Foucar, T. Domratcheva, Damien Sorigué, Marco Kloos, Stéphanie Blangy, Giorgio Schirò, Kyprianos Hadjidemetriou, Bertrand Légeret, Thomas J. Lane, Marie Luise Grünbein, Pavel Müller, Elisabeth Hartmann, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), European Synchroton Radiation Facility [Grenoble] (ESRF), Laboratoire d'Optique et Biosciences (LOB), École polytechnique (X)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), SLAC National Accelerator Laboratory (SLAC), Stanford University, Institut de Physique de Rennes (IPR), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Luminy Génétique et Biophysique des Plantes (LGBP), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire Avancé de Spectroscopie pour les Intéractions la Réactivité et l'Environnement - UMR 8516 (LASIRE), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Interactions Protéine Métal (IPM), Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), Microbiologie Environnementale et Moléculaire (MEM), STepLADDER (724362), European Research Council, 724362, European Research Council, SNAPsHOTs, Agence Nationale de la Recherche, Photoalkane, Agence Nationale de la Recherche, SignalBioNRJ, Agence Nationale de la Recherche, BioXFEL, Agence Nationale de la Recherche, Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-18-CE11-0021,SNAPsHOTs,Dynamique structurale de l'acide gras photodécarboxylase(2018), ANR-18-CE43-0008,PHOTOALKANE,Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme(2018), ANR-15-CE32-0004,BioXFEL,Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides(2015), European Project: 724362,STePLADDER - H2020-EU.1.1., Bioénergie et Microalgues (EBM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), ILL, Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille Institut (CLIL), Vos, Marten, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Dynamique structurale de l'acide gras photodécarboxylase - - SNAPsHOTs2018 - ANR-18-CE11-0021 - AAPG2018 - VALID, APPEL À PROJETS GÉNÉRIQUE 2018 - Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme - - PHOTOALKANE2018 - ANR-18-CE43-0008 - AAPG2018 - VALID, Caractérisation d'états intermédiaires de protéines fluorescentes en utilisant des lasers à électrons libres X et les spectroscopies UV-visible et infrarouge ultra-rapides - - BioXFEL2015 - ANR-15-CE32-0004 - AAPG2015 - VALID, and Solving The Pathway of LADDERane biosynthesis - STePLADDER - H2020-EU.1.1. - 724362 - INCOMING

Subjects
Models, Molecular, Light, Carboxy-Lyases, Protein Conformation, Decarboxylation, Chlorella, Reaction intermediate, Flavin group, Crystallography, X-Ray, 010402 general chemistry, Photochemistry, 01 natural sciences, [PHYS] Physics [physics], Electron Transport, 03 medical and health sciences, Catalytic Domain, Alkanes, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acids, Alkyl, 030304 developmental biology, chemistry.chemical_classification, [PHYS]Physics [physics], Photons, 0303 health sciences, Multidisciplinary, Algal Proteins, Fatty Acids, Temperature, Fatty acid, Substrate (chemistry), Hydrogen Bonding, Carbon Dioxide, Chromophore, Electron transport chain, 0104 chemical sciences, Bicarbonates, Amino Acid Substitution, chemistry, 13. Climate action, Biocatalysis, Flavin-Adenine Dinucleotide, Mutant Proteins, Oxidation-Reduction
Abstract

Light makes light work of fatty acids Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally. Science , this issue p. eabd5687

Published
2021

5. Fatty acid photodecarboxylase is an ancient photoenzyme that forms hydrocarbons in the thylakoids of algae

Author

Bertrand Légeret, Damien Sorigué, Magali Floriani, Adrien Burlacot, Stéphan Cuiné, Audrey Beyly-Adriano, Stéphanie Blangy, Fred Beisson, Poutoum-Palakiyem Samire, Solène L. Y. Moulin, Gilles Peltier, Yonghua Li-Beisson, Bioénergie et Microalgues (EBM), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'écotoxicologie des radionucléides (PRP-ENV/SERIS/LECO), Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'écotoxicologie des radionucléides (IRSN/PRP-ENV/SERIS/LECO), Service de Recherche et d'Expertise sur les Risques environnementaux (IRSN/PRP-ENV/SERIS), Institut de Radioprotection et de Sûreté Nucléaire (IRSN)-Institut de Radioprotection et de Sûreté Nucléaire (IRSN), and ANR-18-CE43-0008,PHOTOALKANE,Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme(2018)

Subjects
0106 biological sciences, congenital, hereditary, and neonatal diseases and abnormalities, Genotype, Light, Carboxy-Lyases, Physiology, Chlamydomonas reinhardtii, Plant Science, Genes, Plant, Thylakoids, 01 natural sciences, 03 medical and health sciences, Algae, Gene Expression Regulation, Plant, Microalgae, Genetics, Cold acclimation, Plastid, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], neoplasms, Research Articles, 030304 developmental biology, 0303 health sciences, biology, Endosymbiosis, Chemistry, Fatty Acids, Genetic Variation, Ectocarpus, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, Photochemical Processes, biology.organism_classification, digestive system diseases, Light intensity, Biochemistry, Mutation, Nannochloropsis, 010606 plant biology & botany
Abstract

Fatty acid photodecarboxylase (FAP) is one of the few enzymes that require light for their catalytic cycle (photoenzymes). FAP was first identified in the microalga Chlorella variabilis NC64A, and belongs to an algae-specific subgroup of the glucose–methanol–choline oxidoreductase family. While the FAP from C. variabilis and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activities, their activities and physiological functions have not been studied in vivo. Furthermore, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a C. reinhardtii FAP knockout line (fap), we showed that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon of this alga. We further showed that CrFAP was predominantly membrane-associated and that >90% of 7-heptadecene was recovered in the thylakoid fraction. In the fap mutant, photosynthetic activity was not affected under standard growth conditions, but was reduced after cold acclimation when light intensity varied. A phylogenetic analysis that included sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was retained in secondary photosynthetic endosymbiosis lineages but absent from those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP photochemical activity was present in red and brown algae, and was not limited to unicellular species. These results thus indicate that FAP was conserved during the evolution of most algal lineages where photosynthesis was retained, and suggest that its function is linked to photosynthetic membranes.

Published
2021

6. High-resolution structure and reaction cycle of fatty acid photodecarboxylase: anatomy of a crime scene

Author

Damien Sorigué, Kyprianos Hadjidemetriou, Stéphanie Blangy, Guillaume Gotthard, Pierre Legrand, Didier Nurizzo, Antoine Royant, Catherine Berthomieu, Martin Weik, Tatiana Domratcheva, Klaus Brettel, Martin H. Vos, Ilme Schlichting, Pavel Müller, Fred Beisson, and Pascal Arnoux

Subjects
Inorganic Chemistry, Structural Biology, General Materials Science, Physical and Theoretical Chemistry, Condensed Matter Physics, Biochemistry
Abstract

Fatty Acid Photodecarboxylase (FAP) is a recently discovered photoenzyme that catalyzes the conversion of fatty acids into alkane and CO2 under light, with potential importance in green chemistry applications [1]. Its mechanism was still not fully understood and partly relied on a low-resolution crystal structure obtained from crystals with a twinning default [1]. Here, we present high-resolution crystal structures of FAP obtained in the dark and after light illumination at cryogenic temperatures (Figure 1). Combined with structural, computational, and spectroscopic techniques we are now able to provide a detailed reaction cycle of FAP. The reaction mechanism starts with an electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved CO2 unexpectedly transforms in 100 ns, most likely into bicarbonate. This is orders of magnitude faster than in solution, which indicates a catalytic step. FT-IR, structural and functional studies on variants centered on two conserved active site residues (R451 and C432) showed that R451 is essential for substrate stabilization and proton transfer. Altogether this study provides a detailed characterization of this unique enzyme and reveals a striking and unanticipated mechanistic complexity [2].

Published
2021

7. Fatty acid photodecarboxylase is an ancient photoenzyme responsible for hydrocarbon formation in the thylakoid membranes of algae

Author

Gilles Peltier, Adrien Burlacot, Solène L. Y. Moulin, Stéphanie Blangy, F. Beisson, Yonghua Li-Beisson, Audrey Beyly, Damien Sorigué, Bertrand Légeret, and Magali Floriani

Subjects
Chlorella, biology, Biochemistry, Algae, Chemistry, Chlamydomonas, Cold acclimation, Chlamydomonas reinhardtii, Green algae, Ectocarpus, Plastid, biology.organism_classification
Abstract

Fatty acid photodecarboxylase (FAP) is one of the three enzymes that require light for their catalytic cycle (photoenzymes). FAP has been first identified in the green microalga Chlorella variabilis NC64A and belongs an algae-specific subgroup of the glucose-methanol-choline oxidoreductase family. While the FAP from Chlorella and its Chlamydomonas reinhardtii homolog CrFAP have demonstrated in vitro activity, their activity and physiological function have not been studied in vivo. Besides, the conservation of FAP activity beyond green microalgae remains hypothetical. Here, using a Chlamydomonas FAP knockout line (fap), we show that CrFAP is responsible for the formation of 7-heptadecene, the only hydrocarbon present in this alga. We further show that CrFAP is associated to the thylakoids and that 90% of 7-heptadecene is recovered in this cell fraction. In the fap mutant, photosynthesis activity was not affected under standard growth conditions but was reduced after cold acclimation. A phylogenetic analysis including sequences from Tara Ocean identified almost 200 putative FAPs and indicated that FAP was acquired early after primary endosymbiosis. Within Bikonta, FAP was kept in photosynthetic secondary endosymbiosis lineages but absent in those that lost the plastid. Characterization of recombinant FAPs from various algal genera (Nannochloropsis, Ectocarpus, Galdieria, Chondrus) provided experimental evidence that FAP activity is conserved in red and brown algae and is not limited to unicellular species. These results thus indicate that FAP has been conserved during evolution of most algal lineages when photosynthesis was kept and suggest that its function is linked to photosynthetic membranes.One sentence summaryFAP is present in thylakoids and conserved beyond green algae.

Published
2020

8. Continuous photoproduction of hydrocarbon drop-in fuel by microbial cell factories

Author

Yonghua Li-Beisson, Gilles Peltier, Stéphanie Blangy, Adrien Burlacot, Damien Sorigué, Fred Beisson, Pascaline Auroy, Bertrand Légeret, Solène L. Y. Moulin, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Programme CEA DRF Impulsion Invention, Alcasun, HelioBiotec platform funded by the EU, the région PACA, the French Ministry of Research, and the CEA, ANR-18-CE43-0008,PHOTOALKANE,Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme(2018), Burlacot, Adrien, APPEL À PROJETS GÉNÉRIQUE 2018 - Production biosourcée d'hydrocarbures basée sur une nouvelle photoenzyme - - PHOTOALKANE2018 - ANR-18-CE43-0008 - AAPG2018 - VALID, Bioénergie et Microalgues (EBM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Light, lcsh:Medicine, 01 natural sciences, 7. Clean energy, Continuous production, Article, 03 medical and health sciences, Key point, Thioesterase, Continuous release, Escherichia coli, Microalgae, lcsh:Science, chemistry.chemical_classification, Multidisciplinary, Drop (liquid), lcsh:R, Fatty Acids, Fatty acid, Pulp and paper industry, Hydrocarbons, Enzymes, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 030104 developmental biology, Hydrocarbon, chemistry, Biofuels, lcsh:Q, Volatility (chemistry), Metabolic engineering, 010606 plant biology & botany
Abstract

Use of microbes to produce liquid transportation fuels is not yet economically viable. A key point to reduce production costs is the design a cell factory that combines the continuous production of drop-in fuel molecules with the ability to recover products from the cell culture at low cost. Medium-chain hydrocarbons seem ideal targets because they can be produced from abundant fatty acids and, due to their volatility, can be easily collected in gas phase. However, pathways used to produce hydrocarbons from fatty acids require two steps, low efficient enzymes and/or complex electron donors. Recently, a new hydrocarbon-forming route involving a single enzyme called fatty acid photodecarboxylase (FAP) was discovered in microalgae. Here, we show that in illuminated E. coli cultures coexpression of FAP and a medium-chain fatty acid thioesterase results in continuous release of volatile hydrocarbons. Maximum hydrocarbon productivity was reached under low/medium light while higher irradiance resulted in decreased amounts of FAP. It was also found that the production rate of hydrocarbons was constant for at least 5 days and that 30% of total hydrocarbons could be collected in the gas phase of the culture. This work thus demonstrates that the photochemistry of the FAP can be harnessed to design a simple cell factory that continuously produces hydrocarbons easy to recover and in pure form.

Published
2019

9. An algal photoenzyme converts fatty acids to hydrocarbons

Author

Bertrand Légeret, Pavel Müller, Yonghua Li-Beisson, Yohann Couté, David Pignol, Fred Beisson, Gilles Peltier, Damien Sorigué, Emmanuelle Billon, Stéphanie Blangy, Didier Nurizzo, Pascal Arnoux, Pierre Richaud, Solène L. Y. Moulin, Klaus Brettel, Stéphan Cuiné, Sabine Brugière, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Synthèse et étude de systèmes à intêret biologique (SEESIB), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Centre National de la Recherche Scientifique (CNRS), Troubles cognitifs dégénératifs et vasculaires (DN2M), Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-INSERM, Bioénergie et Microalgues (EBM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Etude de la dynamique des protéomes (EDyP), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), European Synchrotron Radiation Facility (ESRF), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Microbiologie Environnementale et Moléculaire (MEM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ANR-13-JSV5-0005,MUsCA,Ingénierie métabolique d'une microalgue verte en vue de la production d'alcanes à chaine moyenne(2013), ANR-11-IDEX-0001,Amidex,INITIATIVE D'EXCELLENCE AIX MARSEILLE UNIVERSITE(2011), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Biologie végétale et microbiologie environnementale - UMR7265 ( BVME ), Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Aix Marseille Université ( AMU ), Laboratoire de Biologie à Grande Échelle ( BGE - UMR S1038 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Grenoble Alpes [Saint Martin d'Hères]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), European Synchrotron Radiation Facility ( ESRF ), Biologie et Biotechnologie des Cyanobactéries ( B2CYA ), Département Microbiologie ( Dpt Microbio ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), and Environnement, Bioénergie, Microalgues et Plantes (EBMP)

Subjects
0301 basic medicine, Photosynthetic reaction centre, Light, Carboxy-Lyases, Decarboxylation, [SDV]Life Sciences [q-bio], Chlorella, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Alkenes, 010402 general chemistry, 01 natural sciences, Cofactor, 03 medical and health sciences, chemistry.chemical_compound, Oxidoreductase, Alkanes, Phylogeny, ComputingMilieux_MISCELLANEOUS, Plant Proteins, chemistry.chemical_classification, Flavin adenine dinucleotide, Multidisciplinary, biology, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, Fatty Acids, Fatty acid, Lipid metabolism, Lipid Metabolism, Photochemical Processes, 0104 chemical sciences, 030104 developmental biology, Enzyme, chemistry, Biochemistry, Biocatalysis, Flavin-Adenine Dinucleotide, biology.protein, Oxidoreductases
Abstract

Algal enzyme driven by blue light Microalgae make hydrocarbons. In searching for the enzyme responsible, Sorigué et al. found a glucose-methanolcholine oxidoreductase (see the Perspective by Scrutton). Expression of the enzyme in Escherichia coli showed that hydrocarbon production requires visible light. In fact, the enzyme requires a constant input of blue photons to carry out its catalytic reaction. A long hydrophobic tunnel in the enzyme stabilizes the fatty acid substrates in proximity to the flavin adenine dinucleotide cofactor. Science , this issue p. 903 ; see also p. 872

Published
2017

10. Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

Author

Damien Sorigué, Yonghua Li-Beisson, Pablo Morales, Reinhard Jetter, Bertrand Legeret, Fred Beisson, Geneviève Guedeney, Gilles Peltier, Boris Mirabella, Stéphan Cuiné, Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Botany [Vancouver] (UBC Botany), University of British Columbia (UBC), Bioénergie et Microalgues (EBM), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Synthèse et étude de systèmes à intêret biologique (SEESIB), Centre National de la Recherche Scientifique (CNRS)-Université Blaise Pascal - Clermont-Ferrand 2 (UBP), APHIS, International Services, United States Department of Agriculture, Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements Extrêmes (LEMIRE), Biologie et Biotechnologie des Cyanobactéries (B2CYA), Département Microbiologie (Dpt Microbio), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and Dept Bot

Subjects
0301 basic medicine, Chloroplasts, Light, Physiology, [SDV]Life Sciences [q-bio], Chlorophyceae, Chlamydomonas reinhardtii, Oleic Acids, Plant Science, Chlorella, Alkenes, 03 medical and health sciences, chemistry.chemical_compound, Biosynthesis, Alkanes, Genetics, Microalgae, Phaeodactylum tricornutum, Biomass, ComputingMilieux_MISCELLANEOUS, Diatoms, biology, Chlamydomonas, Fatty Acids, food and beverages, Articles, biology.organism_classification, Hydrocarbons, Biosynthetic Pathways, Chloroplast, 030104 developmental biology, chemistry, Biochemistry, Biofuels, lipids (amino acids, peptides, and proteins), Nannochloropsis, Stearic Acids
Abstract

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D31-16:0 (palmitic) acid yielded D31-18:0 (stearic) acid, D29-18:1 (oleic and cis-vaccenic) acids, and D29-heptadecene. These findings showed that loss of the carboxyl group of a C18 monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C21 hexaene, without detectable C15-C19 hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C16 and C18 fatty acids into alka(e)nes by a new, light-dependent pathway.

Published
2016

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