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

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

Spectroscopie infrarouge pompe-sonde de la picoseconde à la microseconde dans une photoenzyme

National audienceNous avons développé une méthode de spectroscopie pompe sonde permettant la mesure de spectres différentiels sur des échelles de temps allant de la sub-picoseconde à la milliseconde, méthode qui s’implémente facilement sur deux lasers femtosecondes amplifiés pré-existants sans besoin d’asservir les oscillateurs [1-3]. Cette méthode dénommée AD-ASOPS pour Arbitrary Detuning Asynchronous Optical Sampling, a été appliquée à une photoenzyme récemment découverte [4], Fatty Acid Photodecarboxylase (FAP). La mesure des spectres infrarouges pompe-sonde a mis en évidence la dynamique de formation du CO2 au cours d’un processus complexe qui transforme un acide gras en hydrocarbure, et permis d’élucider une partie du mécanisme à l’œuvre dans la biomolécule [5].[1] L. Antonucci, A. Bonvalet, X. Solinas, L. Daniault, M. Joffre, Opt. Express 23, 27931 (2015), https://doi.org/10.1364/OE.23.027931 [2] X. Solinas, L. Antonucci, A. Bonvalet, M. Joffre, Opt. Express 25, 17811 (2017), https://doi.org/10.1364/OE.25.017811[3] L. Antonucci, X. Solinas, A. Bonvalet, M. Joffre, Opt. Express 28, 18251 (2020), https://doi.org/10.1364/OE.393887[4] D. Sorigué et al., Science 357, 903 (2017), https://doi.org/10.1126/science.aan6349[5] D. Sorigué et al., Science 372, 6538 (2021), https://doi.org/10.1126/science.abd568

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

International audienceFatty 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

Catalytic mechanism of fatty acid photodecarboxylase: on the detection and stability of the initial carbonyloxy radical intermediate

International audienceIn fatty acid photodecarboxylase (FAP), light‐induced formation of the primary radical product RCOO$^●$ from fatty acid RCOO– occurs in 300 ps, upon which CO2 is released quasi‐immediately. Based on the hypothesis that aliphatic RCOO$^●$ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6‑dichlorobenzoyloxy radical (DCB$^●$), much longer‐lived linear RCOO$^●$ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB$^●$decarboxylation dynamics and spectral properties and show that in contrast to DCB$^●$, aliphatic RCOO$^●$ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time‐resolved infrared spectroscopy experiment confirms very rapid, <<300 ps RCOO$^●$ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP

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

International audienceUse 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

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

International audienceOngoing 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

Microalgae synthesize hydrocarbons from long-chain fatty acids via a light-dependent pathway

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

International audienceFatty 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

An algal photoenzyme converts fatty acids to hydrocarbons

International audienceAlthough many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga $Chlorella\ variabilis$ NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or-alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid–binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons