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+3344225626 F.B. conceived the original research project; F.B., S.M. and G.P. designed the experiments and analyzed the data; S.M., A.B., S.B., D.S., B.L., and A.B. performed experiments; M.F. performed the TEM study; S.M. performed phylogenetic analysis; F.B. and S.M. wrote the article with contributions from Y.L.-B. and G.P.

This project has received funding from CEA (DRF Impulsion Invention E2FAP to F.B.) and from Agence Nationale de la Recherche (PHOTOALKANE, N° ANR-18-CE43-0008-01, to G.P.). This work was also supported by the HelioBiotec platform funded by the EU, the région PACA, the French Ministry of Research, and the CEA. S.M. has received a PhD scholarship 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.

Most organisms have the ability to synthesize highly hydrophobic compounds made only of carbon and hydrogen called hydrocarbons (HCs). Many HCs are isoprenoids but others like nalkanes and their unsaturated analogues (n-alkenes) derive from fatty acids (Herman and Zhang 2016). In plants, C29-C35 n-alkanes are synthesized in epidermis from very-long-chain fatty acids and secreted onto the surface of aerial organs (Lee and Suh 2013). Plant n-alkanes are important for adaptation to the terrestrial environment because they constitute a major part of the cuticular wax layer that prevents the loss of internal water. In microorganisms, n-alka(e)nes are presumably mostly located in membranes and their function is more elusive. Roles in cell growth, cell division, photosynthesis and salt tolerance have been proposed for cyanobacterial n-alka(e)nes (Berla et al. 2015; Lea-Smith et al. 2016; Yamamori et al. 2018; Knoot and Pakrasi 2019). In eukaryotic microalgae, occurrence of n-alka(e)nes has been reported their subcellular location and physiological function remain unknown (Sorigué et al. 2016). Besides the elucidation of their biological roles, n-alkanes and n-alkenes have also attracted attention because of their biotechnological interest. Indeed, a bio-based alka(e)ne production would be highly desirable to replace part of petroleum-derived HCs in fuels, cosmetics, lubricants and as synthons in organic chemistry (Jetter and Kunst 2008).

A number of n-alka(e)ne-forming enzymes have been identified and characterized in the last decade and it is now clear that conversion of fatty acids to HCs occurs through a variety of reactions and proteins (Herman and Zhang 2016). Besides, for the same type of reaction, the biosynthetic enzymes involved are not conserved across phylogenetic groups. For instance, it has been shown in bacteria that synthesis of terminal olefins (1-alkenes) occurs through decarboxylation of a saturated long-chain fatty acid and that this reaction is catalyzed by a cytochrome P450 in Jeotgalicoccus spp. (Rude et al. 2011) and a non-heme iron oxidase in Pseudomonas (Rui et al. 2014). In the bacterium Micrococcus luteus, yet another pathway has been described, which consists in the head-to-head condensation of fatty acids to form verylong-chain n-alkenes with internal double bonds (Beller et al. 2010). In cyanobacteria, nalka(e)nes are produced by two distinct pathways which are mutually exclusive (Coates et al. 2014). The first one forms terminal olefins and involves a type I polyketide synthase that elongates and decarboxylates a Cn fatty acid to yield a Cn+1 alkene (Mendez-Perez et al. 2011). The second one is a two-step pathway with a fatty aldehyde intermediate and involves an acylACP reductase (AAR) and an aldehyde deformylating oxygenase (ADO) (Schirmer et al. 2010; Li et al. 2012). In plants, the pathway producing the very-long-chain n-alkanes of the cuticular waxes is known to require the Arabidopsis homologs CER1 and CER3 and would involve an aldehyde intermediate (Bernard et al. 2012).

In microalgae, we have shown that C15-C17 n-alka(e)nes occur in Chlorella variabilis NC64A (named Chlorella from here on) and that they are synthesized through decarboxylation of long-chain fatty acids (Sorigué et al. 2016). A Chlorella protein with a fatty acid decarboxylase activity was then identified as a photoenzyme (Sorigué et al. 2017), a rare type of enzyme that requires light as energy (Bjorn 2015). The Chlorella protein was thus named fatty acid photodecarboxylase (FAP, E.C. 4.1.1.106). It is one of the three photoenzymes discovered so far, the two others being

DNA photolyases and light-dependent protochlorophyllide oxidoreductase. FAP belongs to a family of flavoproteins (Sorigué et al. 2017), the glucose-methanol-choline (GMC) oxidoreductases, which includes a large variety of enzymes present in prokaryotic and eukaryotic organisms (Zamocky et al. 2004). FAP activity thus represents a new type of chemistry in the GMC oxidoreductase family (Sorigué et al. 2017). Molecular phylogeny has shown that Chlorella FAP and CrFAP belong to an algal branch of GMC oxidoreductases. However, whether FAP activity is conserved in other algal lineages beyond green algae and whether FAP is indeed responsible for n-alka(e)ne formation in vivo remains to be demonstrated. Besides, the subcellular location and role of FAP in the algal cells have not yet been investigated.

In this work, we isolate and characterize in Chlamydomonas reinhardtii (hereafter named Chlamydomonas) an insertional mutant deficient in FAP (fap mutant strain). We show that FAP is indeed responsible for the formation of 7-heptadecene, the only HC present in this alga. In addition, we provide evidence for a thylakoid localization of Chlamydomonas FAP and its alkene product. We also show that although growth and photosynthesis are not affected in the knockout under laboratory conditions, but photosynthetic efficiency is impacted under cold when light intensity varies. Finally, we build a large molecular phylogeny of GMC oxidoreductases based on TARA Ocean data and identify almost 200 new putative FAP sequences across algal lineages. Experimental evidence is provided that FAP photochemical activity is conserved in red and brown algae and is not limited to unicellular species but is also present in macroalgae.

Chlamydomonas has been previously shown to produce 7-heptadecene (C17:1-alkene) from cis-vaccenic acid (Sorigué et al., 2016) and to have a FAP homolog that can also perform photodecarboxylation of fatty acids in vitro (Sorigué et al., 2017). Although it seemed likely that FAP proteins are indeed responsible for the synthesis of alka(e)nes produced by Chlamydomonas and Chlorella, the possibility that the alkenes are formed in vivo by another enzyme could not be ruled out. In order to address this issue and investigate the biological role of FAP, a Chlamydomonas strain mutated for FAP was isolated from the Chlamydomonas library project (CliP)(Li et al., 2016). This strain showed complete absence of FAP protein (Fig. 1) and was named fap from here on. The only HC in Chlamydomonas i.e. 7-heptadecene could not be detected in the fap mutant (Fig. 1). After performing nuclear complementation using the genomic FAP gene under the promotor PsaD, 4 independent transformants with different expression levels of Chlamydomonas FAP (CrFAP) were isolated (named Cp 1 to 4). In these complemented strains, production of 7-heptadecene was clearly related to FAP amount. These results thus demonstrated that CrFAP is indeed responsible for alkene formation in vivo in Chlamydomonas.

Molecular phylogeny of GMC oxidoreductases has previously shown that CrFAP and the FAP from Chlorella variabilis NC64A (CvFAP) are present in a branch containing only sequences from algae (Sorigué et al., 2017). The term <algae= is used here in the classical sense of photosynthetic organisms that have chlorophyll a as their primary photosynthetic pigment and lack a sterile covering of cells around the reproductive cells (Lee, 2008). To investigate whether FAP activity has been conserved in other algal groups than green algae, genes encoding putative FAPs from selected algal lineages were cloned and expressed in E. coli and the bacterial HC content was analyzed. Considering the basal position of red algae, we decided to explore FAP activity in Rhodophytes selecting the microalga Galdieria sulphuraria and the macroalga Chondrus crispus. For algae deriving from secondary endosymbiosis, we also chose the microalga Nannochloropsis gaditana and the macroalga Ectocarpus silicosus. E. coli strains expressing the various FAPs all produced a range of n-alkanes and n-alkenes with different chain lengths (C15 to C17) in various proportions (Fig. 2 and Supplemental Fig. S1). These results thus demonstrate that FAP activity is present in red algae, and has been conserved in algae with secondary plastids and is not limited to unicellular algae.

To provide a wider picture of the occurrence and evolution of putative FAP photoenzymes within algal groups and to increase the reservoir of FAPs for future biotechnological purposes, a large phylogenetic analysis of GMC oxidoreductases sequences was conducted. We used

GMC oxidoreductases retrieved from public databases, from sequenced algal genomes (BlabyHaas and Merchant, 2019) and from the Tara Ocean project (de Vargas et al., 2015). Tara data gave a unique opportunity to enlarge the FAP dataset with marine algal species that may not be easy to grow under laboratory conditions and whose genome has not been sequenced. Protein sequences sharing between 50 and 33% of homology with the sequence of Chlorella variabilis FAP were retrieved using Basic Local Alignment Search Tool (BLAST) (Supplemental table S1 and table S2). Over 500 GMC oxidoreductases were thus identified in the algal genomes and in the TARA dataset using annotations of the reconstructed genomes done by the Tara group. Additional GMC oxidoreductases selected from public databases were from various taxa including the three different kingdoms.

Molecular phylogeny confirmed that all the sequences of the FAP clade belong to algal species (Fig. 3, Supplemental Fig. S2). Sequences from plants as well as other streptophytes (including charophytes) did not group with algal FAPs. Absence of FAP in charophytes indicated early loss of FAP function in streptophytes. No putative FAP sequences could be found in cyanobacteria although this group is highly represented in TARA data (de Vargas et al. 2015). Phylogeny within the FAP branch indicated that red algae (rhodophytes) sequences were the most basal. Interestingly, FAP sequences from secondary endosymbiosis-derived species appeared to be more closely related to FAPs of green algae (chlorophytes) than red algae. Overall, the new putative FAPs that could be identified in algae were present in a variety of algal groups, including stramenopiles (heterokonts), haptophytes and dinophytes. Logo sequence of FAPs compared to other GMC oxidoreductases exhibited conserved patterns (Supplemental Fig. S3), including residues specific to FAP and thought to play a role in the catalysis such as C432 and R451 of CvFAP (Sorigué et al. 2017). Most eukaryotic algae harbored one putative FAP and no other GMC oxidoreductase, but a few algae showed no FAP and/or several non-FAP GMC oxidoreductases (Fig. 4). Indeed, no putative FAP could be found in the sequenced algal genomes of the glaucocystophyte Cyanophora paradoxa, the Mamiellophyceae Ostreoccocus, Micromonas and Bathycoccus, the diatom Thalassiosira pseudonana. Conversely, only a few algal sequences could be found in other branches of the GMC oxidoreductase family. Existence in the diatom Thalassiosira pseudonana of a GMC oxidoreductase grouping with bacterial choline dehydrogenase was supported by one sequence from the sequenced genome (Tps-GMC) and one sequence from Tara (48230190). A Tara sequence annotated as a Pelagomonas protein (5166790) also turned out not to be located on the FAP clade. The cryptophyte Guillardia theta had 3 different GMC oxidoreductases in 3 different branches but none of them grouped with FAPs. Ulva mutabilis had 11 predicted GMCs, but only one was in the FAP clade. The ten other members of this multigene family of Ulva appeared to form a group close to plant GMC oxidoreductases. Although exceptions similar to these ones probably exist in algal diversity, the general picture appears to be that most algae have one GMC oxidoreductase, which groups with CrFAP and CvFAP in the phylogenetic tree.

Chlamydomonas FAP and most of its alkene products are found in the thylakoid fraction FAP is predicted to be addressed to the chloroplast by Predalgo (Tardif et al., 2012), a software dedicated to the analysis of subcellular targeting sequences in green algae. This is consistent with the finding that CrFAP was found in a set of 996 proteins proposed to be chloroplastic in Chlamydomonas (Terashima et al., 2011). A broader study of putative targeting peptides using Predalgo (Tardif et al., 2012) and ASAFind algorithms (Gruber et al., 2015) indicated that FAPs from various green and red algae were largely predicted to be chloroplastic (Supplemental Fig. S4). In algae with secondary plastids (i.e. containing 3 or 4 membranes), the presence of a signal peptide was consistent with a targeting to the ER or chloroplast ER (CER) membrane. Analysis performed using ASAFind, a prediction tool designed to recognize CER targeting motifs in signal peptides, indicated that such a motif was present in Ectocarpus silicosis and Nannochloropsis gaditana. Taken together, these results suggest that FAP homologs are very likely to be localized to chloroplasts in green algae, in red algae and also in at least some of the algae that acquired plastids through secondary endosymbiosis.

To consolidate these observations, subcellular fractionation of Chlamydomonas cells was performed (Fig. 5A). Thylakoid membranes were isolated from whole cells using a sucrose gradient. Co-purification with thylakoids was followed by D1 protein (PsbA) from PSII core complex, a thylakoid membrane protein. The fact that the phosphoribulokinase (PRK) control from stroma could barely be detected in our thylakoid fraction, indicated the presence of little amount of intact chloroplasts or cells. It is thus clear that CrFAP is present in the chloroplast of Chlamydomonas and at least partially bound to the thylakoids. When analyzing the percentage of 7-heptadecene in total fatty acids in whole cells versus purified thylakoid membranes, a slight but significant enrichment in alkene was found in the thylakoids (Fig. 5B). Using the fatty acid C16:1(3t) as a marker of the thylakoid lipids, it could be estimated that the enrichment in 7heptadecene corresponds in fact to the localization of >90% of this compound to thylakoids (Fig. 5C). These results therefore demonstrate that part of the FAP and the vast majority of the FAP product are associated to the thylakoid membranes of Chlamydomonas.

The lack of FAP and HCs in chloroplasts of C. reinhardtii did not result in any obvious differences in the overall organization of cells or chloroplast as seen by transmission electron microscopy (TEM) (Supplemental Fig. S5). To try to gather clues on FAP function, FAP transcriptomic data publicly available were mined. Transcriptomic data from Zones et al., 2015 shows that FAP has a similar expression pattern as those genes encoding proteins of the photosynthetic apparatus (Supplemental Fig. S6). In order to determine whether FAP product varied with time, we monitored total fatty acids and 7-heptadecene content during a day-night cycle in synchronized Chlamydomonas cells. While total fatty acid content per cell increased during the day and was divided by two during cell division at the beginning of night (Fig. 6A), a constant level of 7-heptadecene representing 0.45% of total fatty acids was found most of the time (Fig. 6B). A significant peak (0.7%) was observed before cell division, which decreased during mitosis. This result therefore indicates that the extra-amount of HCs synthesized before cell division must be somehow lost or metabolized during cell division.

Fatty acid and membrane lipid compositions are altered in the fap mutant Since 7-heptadecene content varied during cell cycle and may thus play a role in cell division, growth of the WT and fap strains were analyzed. Growth at 25 °C was compared in photoautotrophic conditions (mineral medium (MM)) and in mixotrophic conditions (Trisacetate-phosphate medium (TAP)). No difference between WT and fap could be detected neither in growth rates nor in cell volumes under these conditions (Supplemental Fig. S7). In addition, no difference between WT and fap strains could be observed when cells were grown under various concentrations of sodium chloride (Supplemental Fig. S8). Although the lack of HCs had no effect on growth, fatty acid profile showed some differences in C16:1(9), C18:1(9), C18:3(9-12-15) (Supplemental Fig. S9). Synchronized cells also showed no growth differences between WT and fap strains but exhibited differences in the dynamics of some fatty acid species (Supplemental Fig. S10). Changes in fatty acid profiles prompted us to perform a lipidomic analysis by UPLC-MS/MS. Interestingly, it revealed that a limited set of lipid molecular species were significantly different between WT and fap and that were all plastidial lipids belonging to the galactolipid classes digalactosyldiacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG) (Fig. 7 and Supplemental Fig. S11). The decrease in the relative content of these galactolipid species appeared to be fully restored by complementation in the case of DGDG but not of MGDG (Fig. 7). Taken together, these results show that the lack of 7-heptadecene in the fap mutant causes a change in thylakoid lipid composition, which is evidenced by the decrease of the relative content in at least 3 galactolipid species belonging to the DGDG class.

FAP is not strongly associated to photosynthetic complexes and lack of HCs has no effect on their organization In cyanobacteria a role of HCs in photosynthesis has been suggested (Berla et al., 2015) but is controversial (Lea-Smith et al., 2016). In C. reinhardtii, there was no difference in the 77K chlorophyll fluorescence spectrum between WT, complementant and fap mutant, which indicated that no major changes in antenna distribution around photosystems (Supplemental complementant and fap strains grown under standard laboratory conditions (Supplemental Fig. S12B). Membrane inlet mass spectrometry (MIMS) experiments conducted to quantify O2 exchange showed no difference in respiration and photosynthesis rates between the two genotypes (Supplemental Fig. S12C). Native electrophoresis of proteins from purified thylakoids and FAP immunodetection revealed that FAP could only be detected at an apparent molecular size of the monomeric FAP (Fig. 8), indicating no strong association to proteins of photosynthetic complexes. Besides, no difference in organization of photosynthetic complexes between WT and fap could be seen on the native protein electrophoresis.

Photosynthesis is affected under light and cold stress in the fap mutant Lack of HCs in the fap strain did not cause changes in the photosynthesis activity under standard growth conditions. However, since significant modifications in the composition of membrane lipids could be detected, we explored harsher conditions to challenge further photosynthetic membranes. We chose to investigate chilling temperatures because cold is well-known to affect both membrane physical properties and photosynthesis. Using multicultivator in turbidostat mode, we first stabilized cultures at 25 °C under medium light (200 µmol photons m22 s21), electron transfer rate (ETR) showed no difference (Fig. 9A). When cooling down the culture to 15 °C and after 3 days of acclimation, both ETR and 77 K chlorophyll fluorescence spectra still showed no differences (Fig. 9B,C) (Fig. 9C). After one day at a lower light intensity (50 µmol photons m22s21), the maximal PSII yield was equal for all the strains but ETR was lower for the mutant when measured at high light intensities (Fig. 9D). Interestingly, longer acclimation to this condition (3 days) led to the disappearance of this phenotype.

In order to provide support for a possible link between HCs and cold acclimation, 7heptadecene content was quantified under various growth temperatures. Relative HC content in cells clearly increased under cold conditions (Fig. 10). As expected, an increase in the relative content in polyunsaturated species occurred upon cold treatment (Supplemental Fig. S13), but no difference in the dynamics of fatty acid remodeling was observed between WT and fap strains.

Here, we report the isolation and characterization of an insertional Chlamydomonas mutant deficient in FAP and we perform a phylogenetic and functional analyses of algal homologs. We show FAP and the vast majority of its 7-heptadecene product are associated to thylakoid membranes. It is also shown that the FAP gene is present in most algal lineages and encodes a functional fatty acid photodecarboxylase in some species of red algae, of secondary algae as well as in some macroalgae. By studying a FAP knock-out Chlamydomonas mutant, we provide evidence that lack of hydrocarbons is correlated with small changes in galactolipid composition but has no impact on photosynthesis and growth in Chlamydomonas under standard culture conditions. However, in the absence of hydrocarbons generated by FAP, the photosynthetic activity is transitorily affected during cold acclimation. The possible significance of these results for algal physiology as well as FAP function and evolution are discussed below.

Based on the characterization of a fap mutant, we first show that FAP is responsible for the synthesis of all fatty acid-derived HCs found in Chlamydomonas cells (Fig. 1). Our result clearly demonstrates that the fatty acid photodecarboxylase activity measured in vitro for CrFAP (Sorigué et al., 2017) is not a promiscuous secondary activity and indeed corresponds to a genuine biological activity, namely the light-driven synthesis of 7-heptadecene from cisvaccenic acid (Fig. 11). Also, the fap knockout line shows that no other enzyme is able to synthesize 7-heptadecene in Chlamydomonas. Besides, the fact that HC production was found to be correlated with the quantity of FAP present in complemented lines, indicated that FAP is a limiting factor for 7-heptadecene production in vivo. Thus, the putative lipase activity that must be acting upstream of FAP to generate the free cis-vaccenic acid is not limiting in the pathway.

Based on subcellular fractionation and anti-FAP antibodies, we show here that FAP is able to associate to thylakoid membranes (Fig. 5). This result is consistent with the predicted plastid localization and with the fact that thylakoid membranes harbored 90% of the 7-heptadecene product. Presence of plastid transit peptides in FAPs seems to be a general rule in green and red algae (primary plastids) and is also predicted for some secondary endosymbiosis-derived algae (Supplemental Fig. S4).

Considering that HCs are hydrophobic compounds, it is not surprising that in Chlamydomonas the 7-heptadecene is mostly located where it is produced. In a work on cyanobacterial mutants devoid of fatty acid-derived HCs, it has been suggested that HCs are located in membranes and may play a role in cell division (Lea-Smith et al., 2016). In the proposed cyanobacterial model, integration of HCs into the lipid bilayer would be responsible of membrane flexibility and curvature. HCs may play a similar role in thylakoid membranes of green algae. The fact that the percentage of 7-heptadecene in the total fatty acids stays rather stable during a 16-hour day, indicates that HC production follows lipid production during cell growth, except just before mitosis (Fig. 6B). In addition, the ratio of HCs to FAMEs decreased at the beginning of night, when cells are dividing, indicating that some HCs are lost during cell division. A simple mechanism which could explain HC loss during cell division involves enrichment in HCs at breaking points of plastidial membranes before cell division, exclusion from these membranes during division and loss to the gas phase of the culture due to HC volatility (Supplemental Fig. 6B).

HCs might thus impact local flexibility of algal plastidial membranes and participate in lipid membrane remodeling during cell division. However, under standard culture conditions, the presence of HCs is apparently not critical for chloroplast structure (Supplemental Fig. 5), cell size and cell division rate (Supplemental Fig. 7). A role of cyanobacterial HCs in resistance to salt stress has also been suggested (Yamamori et al., 2018). In Chlamydomonas, contrary to what has been shown in cyanobacteria, no difference could be detected in growth under increasing salt concentrations (Supplemental Fig. 8). One could thus hypothesize that even if HCs are produced in chloroplast and accumulated in thylakoids, their function might be different from that in cyanobacteria. It is also possible that laboratory culture conditions used for Chlamydomonas (this study) are far from natural growth conditions where HCs may be necessary. Alternatively, compensation mechanism for HC loss may operate differently in Chlamydomonas and in cyanobacteria. In Chlamydomonas, part of this mechanism may involve changes in membrane lipid composition. Interestingly, lipidomic analysis under standard growth conditions unraveled specific changes in DGDG molecular species (Fig. 7) but no other significant differences in other class of lipids (Supplemental Fig. S11). Taken together, these results suggest that in Chlamydomonas HCs play no crucial role in cell division and growth under standard conditions. Cells may adapt to a lack of HCs by some changes in the composition of membranes, which could specifically involve some DGDG galactolipids. Alternatively, or in addition to this proposed effect on properties of the membrane lipid phase, it cannot be ruled out that 7-heptadecene may act locally to disrupt or enhance some specific protein-protein interactions, or may play a yet to be defined role, such as acting as a signaling molecule or its precursor (Fig. 11).

The fact that the FAP gene expression that follows that of photosynthesis genes in day-night cycles, the likely localization of FAP in plastids of green and red algae as well as in some secondary algae, and the localization of part of FAP and almost all its alkene product in Chlamydomonas thylakoids point toward a role of FAPs in the photosynthetic function of algal cells. This idea is strongly reinforced by the conservation of the FAP-encoding gene in many eukaryotic algae but not in non-photosynthetic protists (Fig. 3 and Fig. 4) and in Polytomella, an algae that has kept some of its plastidial function but lost photosynthesis (Smith and Lee 2014). As standard culture conditions did not allow to reveal any photosynthesis phenotype in Chlamydomonas fap mutant (Supplemental Fig. S12), more challenging conditions involving colder temperatures and variations in light intensity were tested. These experiments have revealed a difference between WT and fap mutant in the photosynthesis activity measured under high light during acclimation to cold (Fig. 9). Interestingly, colder temperatures are correlated with increased HCs (Fig. 10) while fatty acid profiles follow the same trend in WT and fap strains (Fig. S13). Taken together, these observations indicate that adaptations in membrane lipid composition compensate partly for the loss of HCs in standard growth conditions but not in harsher conditions such as cold temperatures.

According to molecular phylogeny (Fig. 3, Fig. 4), FAP proteins appear to be specific to algae and highly conserved in many algae species. A noticeable exception is the Mamiellophyceae class of the green algae. Algae is a common denomination that gathers photosynthetic eukaryotes which mainly live in aquatic environments. This polyphyletic group includes organisms derived from a first endosymbiosis as well as organisms derived from a secondary or even tertiary endosymbiosis. However, a functional FAP can be found in chlorophytes (green algae), rhodophytes (Chondrus and Galdieria) and stramenopiles (in the phaeophyceae Ectocarpus and the Eustigmatophyceae Nannochloropsis) as proven by heterologous expression in E. coli of the corresponding identified FAPs (Fig. 2). FAP activity was therefore conserved during secondary endosymbiotic event(s) that gave rise to the red lineage. Moreover, FAP activity is not specific to the unicellular state as FAPs were also functional in the pluricellular algae (macroalgae) Ectocarpus silicosus and Chondrus crispus. Considering homology of sequences, FAP function is thus expected to be present in most algal phyla, including haptophytes and dinophytes (dinoflagellates). Importantly, some aminoacid residues that are likely to be involved in fatty acid substrate stabilization or photocatalysis, such as CvFAP Arg451 or Cys432 (Sorigué et al., 2017) are strictly conserved in the 198 putative FAPs (Supplemental Fig. S3). This observation reinforces the idea that all the putative FAPs identified in this work have the ability to photo-produce HCs from fatty acids.

FAP neofunctionalization from GMC oxidoreductases may have occurred early during evolution of algae, almost concomitantly with the very first endosymbiosis shared by green and red algae. No GMC could be found in glaucophytes, which may indicate that this event has occurred after separation of glaucophytes from red and green algae. However it should be noted that so far only one complete glaucophyte genome is available. Absence of FAP in charophytes indicates early loss of FAP function in streptophytes. Phylogeny points out that FAPs from secondary endosymbiosis lineages are more closely related to core chlorophytes than rhodophytes. FAP could thus be one of the genes that was inherited from green algae by horizontal gene transfer (Moustafa et al., 2009).

Concerning the conservation of FAP activity, it should be noted that the FAPs selected for heterologous expression produced various HCs profiles (Fig. 2). For example, Chondrus FAP showed high specificity for C18:1 fatty acid producing 95% of C17:1 alkene, while Ectocarpus FAP produced 70% of C15:0 alkane. This indicates that the algal biodiversity contains FAPs which are more selective or more active on shorter chain fatty acids than FAPs of Chlorella and Chlamydomonas. FAPs with different properties may be useful for biotechnological application aiming to enhance the production of short chain volatile HCs by microbial cell factories (Moulin et al., 2019).

In conclusion, the results presented here show that FAP activity is conserved beyond green microalgae and identify a big reservoir of FAPs that may be useful for biotechnological applications. It also provide some important clues for future studies aiming at unravelling the exact role of the FAP photoenzyme in eukaryotic algae.

The fap mutant and its corresponding wild-type strain of C. reinhardtii were ordered from the CLiP library (Li et al. 2016). Upon reception, strains were plated on Tris-acetate-phosphate (TAP) medium and streaked to allow formation of single colonies. For each strain, after 1-week growth in the dark, three single-clone derived colonies were randomly chosen for characterization. Wild type strains are CC-4533 cw15 mt- for mating type minus and CC-5155 cw15 mt+ (Jonikas CMJ030 F5 backcross strain) [isolate E8] for mating type plus. Mutant LMJ.RY0402.226794 was used in this study, which is predicted to harbor a first insertional cassette in coding sequence of Cre12.g514200 encoding FAP. A second insertion in the line LMJ.RY0402.226794 was predicted in Cre14.g628702. To remove this side mutation we backcrossed the mutant strain to CC-5155. Analysis of one full tetrad showed 2 progeny strains resistant to paromomycin which were mutated in FAP gene. The region of Cre14.g628702 was amplified by PCR and sequenced for the 4 progeny strains of the tetrad. No insertion was actually found, therefore a potential insertion at this locus was ruled out and the initial prediction by the CLip project was not accurate and this could happen due to the strain mixing during handling of large number of clones (Li et al. 2016). Work on mutant strains was conducted on one parental isolated strain with the mutation from LMJ.RY0402.226794 and the 2 mutants of the full tetrad from the backcross with CC-5155. This 3 strains are thereafter named fap-1, fap-2, fap-3 respectively. Wild type (WT) strains were parental strain CC5155 (WT-1) and single colony-derived lines of background strain CC4533 (WT-2, WT-3).For liquid culture experiments, cells were grown in 24 deep well plates of 25 mL under 100 µmol photons m-2 s1 with constant shaking at 25 °C. Cells were grown in TAP or minimal medium (MM) (Hoober 1989) for mixotrophic and autotrophic conditions, respectively. Cell growth was followed using a cell counter Multisizer (Beckman Coulter). For day night cycle experiment, cells were cultivated autotrophically in 1L-photobiorectors in turbidostat mode (Dang et al. 2014; Sorigue et al. 2016) (OD880nm at 0.4) under 16 hours of light (40 µmol photons m-2 s-1) 8 hours of dark at 25 °C. For photosynthesis analysis, cells were grown autotrophically in 80 mL photobioreactors (multicultivator, Photon Systems Instruments) with turbidostat module (OD680nm at 0.8). Conditions were 25 °C, medium light (200 µmol photons m-2 s-1), or 15 °C medium light or 15 °C low light (50 µmol photons m-2 s-1). All cultures were done under ambient air.

Construct for complementation of knockout strain for FAP gene was carried out using pSLHyg vector containing an AphVII cassette conferring hygromycin resistance (Supplemental Fig. S14). This vector allowing nuclear transformation was kindly provided by Pr. Steven Ball (University of Lille, France). WT copy of the FAP gene was obtained by PCR of WT genomic DNA using primers Cr-F and Cr-R (This and all other primer sequences were shown in Supplemental Table S3). It was cloned into TOPO-XL vector. pSL-Hyg vector and FAP gene were digested with EcoRV and SpeI and ligated. Then, the vector was linearized with PvuI and was electroporated into the fap strains. Level of complementation was verified by immunoblot to assess quantity of protein and by transmethylation of whole cells to assess quantity of HCs.

Cells (10–15 mL) were harvested by centrifugation at 3,000 g for 2 min. Pellets were then frozen in liquid nitrogen and stored at -80 °C until use. Pellets were resuspended in 400 mL 1% (w) SDS and then 1.6 mL acetone precooled to -20 °C was added. After overnight incubation at -20 °C, samples were centrifuged (14,000 rpm, 10 min, 4 °C). Supernatant was removed and used for chlorophyll quantification using SAFAS UVmc spectrophotometer (SAFAS). Pellets were resuspended to 1 mg chlorophyll mL-1 in LDS in the presence of NuPAGE reducing agent (ThermoFischer) and loaded on 10% (w/v) PAGE Bis-Tris SDS gel. To load equal protein amounts for immunoblot analysis, protein contents were estimated by Coomassie Brilliant Blue staining of the gel using an Odyssey IR Imager (LICOR). After gel electrophoresis, proteins were transferred to nitrocellulose membranes for 75 min at 10 V using a semi-dry set up. Membranes were blocked in TBST, milk 5% (w/v) overnight at 4 °C then incubated at room temperature in the presence of the following antibodies: anti-Cyt f, anti-AtpB, anti-PsaD, antiPsbA, anti-LHCSR3 (Agrisera), or anti-FAP (see below). After 2 hour incubation, primary antibody was removed by rinsing 3 times in TBST, and a peroxidase-coupled secondary antibody was added for at least 1 h. Luminescence was detected with a Gbox imaging system (Syngene).

Codon-optimized synthetic gene encoding C. reinhardtii FAP (Sorigué et al. 2017) was cloned into the pLIC7 expression vector, allowing the production of a recombinant FAP fused to TEVcleavable His-tagged Escherichia coli thioredoxin. Production was performed in the E. coli BL21 Star (DE3) strain initially grown at 37 °C in TB medium. Induction was initiated at an OD600nm of 0.8 by adding 0.5 mM isopropyl b-D-thiogalactoside (IPTG), cultures were then grown at 20 °C. Following overnight incubation, cells were centrifuged and protein was purified as described previously (Sorigué et al. 2017). Purity of the purified protein was controlled on SDS-PAGE and it was brought to a final concentration of 2 mg mL-1 using an Amicon-Ultra device (Millipore). Polyclonal antibodies against FAP were raised in rabbits (ProteoGenix, Schiltigheim, France).

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