The ability to synthesize and secrete hydrophobic compounds is believed to have been a pivotal event in the evolution of land plants from their aquatic green algal ancestors. The key to biosynthesis of plant surface alkanes is a heterodimeric complex consisting of two homologous membrane-bound proteins, ECERIFERUM 3 (CER3) and ECERIFERUM 1 (CER1), which bear distinct enzyme activities. A single homolog of CER1 and CER3, referred to as CER1/3, has long been identified in some algae. However, it has remained unknown whether CER1/3 exhibits CER1 and/or CER3 activity or another ancestral activity. Here we investigate the function of CER1/3 by using CRISPR-Cas9-mediated knockout mutants in the early-branching chlorophyte Ostreococcus tauri and by yeast heterologous expression. Genome mining shows that in green algal genomes the presence of CER1/3 is correlated with the absence of fatty acid photodecarboxylase. Knockouts provide evidence that CER1/3 is necessary for synthesizing a C21:6 alkene in Ostreococcus. Yeast expression experiments demonstrate that algal CER1/3 are bifunctional enzymes with aldehyde- and hydrocarbon-forming domains, corresponding to CER3 and CER1 activities, respectively. These findings support the idea that the land plant alkane-forming CER1/CER3 complex evolved from a bifunctional hydrocarbon-forming CER1/3 enzyme found in some of the earliest-diverging green algal lineages.

Across the tree of life, diverse organisms synthesize hydrocarbons from fatty acids using various enzymes. In plants and insects, hydrocarbons are typically excreted (eg waxes, pheromones), whereas in algae, they seem to be mostly intracellular and membrane-associated. To date, all studied organisms have been found to possess only one hydrocarbon-forming pathway. In most green algae, unesterified fatty acids are converted into hydrocarbons by the algal-specific photoenzyme fatty acid photodecarboxylase. Other green algae lacking the fatty acid photodecarboxylase are able to synthesize hydrocarbons via the ECERIFERUM 1/3 (CER1/3) protein. Here, using heterologous expression in E. coli, we show that 6 fatty acid photodecarboxylase homologs belonging to diverse groups of brown algae (Phaeophyceae) are functional. We also demonstrate that Ectocarpus species 7 and Saccharina latissima produce 2 intracellular hydrocarbons during vegetative growth: n-pentadecane (15:0 hydrocarbon) and n-heneicosahexaene (21:6 hydrocarbon), which is distinct from the n-heneicosahexaene isomer found in some green algae. Through genome editing, we show that 15:0 hydrocarbon, likely localized in chloroplast membranes, is synthesized via a fatty acid photodecarboxylase-dependent pathway in Ectocarpus, while the 21:6 hydrocarbon is produced through an unidentified alternative pathway. Our results support the hypothesis that fatty acid photodecarboxylase plays a conserved biological role in brown algae. Furthermore, this work provides the first example of a group of organisms harboring a second, distinct hydrocarbon-forming pathway, which may fulfill a unique biological function.

In plants, very-long-chain (VLC) alkanes (C25-C35) are secreted onto the epidermal surface of aerial organs, often forming the primary component of the waterproof cuticular wax layer. This secretion plays a vital role in preventing desiccation in terrestrial environments. The key to VLC alkane biosynthesis in plants lies in the complex formed by two homologous membrane-bound proteins, ECERIFERUM 3 (CER3) and ECERIFERUM 1 (CER1). This complex transforms an acyl-CoA substrate into an aldehyde intermediate, which is then converted into an alkane. The ability to synthesize and secrete alkanes is believed to have been a pivotal event in the evolution of land plants from their green algal ancestors. Interestingly, a single homolog of CER1 and CER3, known as CER1/3, has been identified in certain algae. However, the functionality of this protein remains to be investigated. In this study, we present the functional characterization in yeast of CER1/3 proteins from various algal species belonging to the green lineage. We demonstrate that CER1/3 proteins alone can efficiently mediate hydrocarbon biosynthesis. Furthermore, we demonstrate that point mutations in conserved motifs in the N- or C-terminal domains of CER1/3 lead to impaired hydrocarbon biosynthesis. Additionally, we show that coexpressing plant CER3 with algal CER1/3 results in longer alkanes being formed in yeast. Together, these findings support the hypothesis that the alkane-forming CER1/CER3 complex found in land plants evolved from a green algal CER1/3 bifunctional enzyme through a process of gene duplication followed by protein specialization. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-18-CE43-0008, ANR-24-CE20-3101-01

The photoenzyme fatty acid photodecarboxylase (FAP) has emerged as a promising catalyst for the redox-neutral biological production of hydrocarbons. Previous studies have shown that FAP can efficiently convert medium-chain fatty acids such as n-octanoic acid into hydrocarbons, outperforming its natural long-chain fatty acid substrates (C16-C18). Such observation expands the potential applications of FAP to include solvents and jet fuels. However, the limited availability of natural sources of n-octanoic acid poses a challenge to the industrial implementation of n-heptane bioproduction. This study investigates the hydrocarbon synthesis capacity of an E. coli strain that expresses FAP and produces n-octanoic acid, the precursor to n-heptane, via a specific octanoyl-ACP thioesterase. Several FAPs and thioesterases were tested. A blue light-inducible promoter ensured high expression of both enzymes, eliminating the need for chemical inducers. Fusion of FAP with thioredoxin increased n-heptane production 12-fold. Using a co-cultivation strategy, where one strain produces n-octanoic acid and another strain converts it to n-heptane, increased hydrocarbon production 14-fold compared to co-expressing FAP and thioesterase. Co-cultures operated in batch mode in 100-mL photobioreactors enabled the recovery of >90%-pure n-heptane, yielding 272 mg·L-1 over 56 h. This work lays the foundation for the development of an industrial bioproduction of n-heptane.

Summary The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl‐CoA carboxylase (ACCase). As the rate‐limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α‐carboxyltransferase (Crα‐CT) subunit of the ACCase by yeast two‐hybrid protein–protein interaction assay. Three independent CRISPR‐Cas9 mediated knockout mutants for CrCTI1 each produced an ‘enhanced oil’ phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα‐CT. However, mutating all six predicted phosphorylation sites within Crα‐CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β‐oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as waterproof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, their importance for photosynthetic membranes has remained elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid composition and photosynthesis using the cyanobacterium Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that drastic changes in the glycerolipid contents occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. These changes are associated with a higher susceptibility of photosynthesis and growth to high light in alka(e)ne-deficient strains. All these phenotypes are reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. Therefore, alkenes, despite their low abundance, are an essential component of the lipid composition of membranes. The profound remodeling of lipid composition that results from their absence suggests that they play an important role in one or more membrane properties in cyanobacteria. Moreover, the lipid compensatory mechanism observed is not sufficient to restore normal functioning of the photosynthetic membranes, particularly under high-light intensity. We conclude that alka(e)nes play a crucial role in maintaining the lipid homeostasis of thylakoid membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities.

In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOO⋅ from fatty acid RCOO − occurs in 300 ps, upon which CO 2 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.

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the α/β hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl- N,N,N -trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells.Significant statement Lipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative α/β- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl- N,N,N -trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.### Competing Interest StatementThe authors have declared no competing interest.

The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix, composed of polysaccharides and glycoproteins, must be constantly remodeled throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the dual-specificity tyrosine phosphorylation-regulated kinase (DYRKP1) family present in all eukaryotes regulates cell wall degradation after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases. Without DYRKP1, daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the dual-specificity tyrosine phosphorylation-regulated kinase complementation lines show normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked downregulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in matrix metalloproteinases retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that dual-specificity tyrosine phosphorylation-regulated kinase, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae. The DYRKP1 kinase induces the expression of matrix metalloproteinases involved in the degradation of the parental cell wall, allowing prompt hatching of daughter cells after cell division.

Fatty Acid Photodecarboxylase (FAP) has emerged as a promising catalyst for the biological production of long-chain hydrocarbons. We have recently shown that purified FAP or FAP-expressing bacteria can efficiently convert octanoic acid into heptane, thus extending the potential applications of FAP to medium-chain hydrocarbons (i.e., solvent- or kerosene-type). The scarcity of natural sources of octanoic acid presents a challenge however. Here, we explore the heptane production capacity of a FAP-expressing E. coli strain engineered to biosynthesize octanoic acid via a specific thioesterase. Various FAPs and C8-specific thioesterases were tested. A blue-light-inducible promoter was used to avoid chemical inducers. We found that the expression of FAP fused with TrxA resulted in a 10-fold increase in heptane production. Coexpression of Cuphea hookeriana thioesterase and Chlorella variabilis FAP achieved the highest heptane titer (12.5 mg.L-1). Scale-up experiments in 100 mL photobioreactors allowed a constant production of heptane over two days (22 mg.L-1.day-1). ![Figure][1] Highlights ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Using model microalga Chlamydomonas, we show that ABHD1, an α/β-hydrolase domain-containing protein, is localized to the LD surface and stimulates LD formation through two actions: one enzymatic and one structural. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed a higher content of lyso-derivatives of betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 increased LD abundance and boosted TAG content. Purified recombinant ABHD1 hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine. In vitro droplet-embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells.

Alka(e)nes are produced by many living organisms and exhibit diverse physiological roles, reflecting a high functional versatility. Alka(e)nes serve as water proof wax in plants, communicating pheromones for insects, and microbial signaling molecules in some bacteria. Although alka(e)nes have been found in cyanobacteria and algal chloroplasts, a possible role in photosynthesis and chloroplast function remains elusive. In this study, we investigated the consequences of the absence of alka(e)nes on membrane lipid remodeling and photosynthesis using the cyanobacteria Synechocystis PCC6803 as a model organism. By following the dynamics of membrane lipids and the photosynthetic performance in strains defected and altered in alka(e)ne biosynthesis, we show that a profound remodeling of the membrane lipidome and carotenoid content occur in the absence of alka(e)nes, including a decrease in the membrane carotenoid content, a decrease in some digalactosyldiacylglycerol (DGDG) species and a parallel increase in monogalactosyldiacylglycerol (MGDG) species. Under high light, this effect is accompanied in alka(e)ne deficient strains by a higher susceptibility of photosynthesis and growth, the effect being reversed by expressing an algal photoenzyme producing alka(e)nes from fatty acids. We conclude that alka(e)nes play a crucial role in maintaining lipid homeostasis of photosynthetic membranes, thereby contributing to the proper functioning of photosynthesis, particularly under elevated light intensities.Significance statement We used cyanobacteria as a model organism to explore the role of alka(e)nes related to photosynthesis. Our findings reveal that the absence of alka(e)nes induces alterations in the composition of membrane lipids and carotenoid content, resulting in an increased susceptibility of photosynthesis. By introducing a fatty acid photodecarboxylase to produce alkanes, we could reverse these effects, highlighting the critical role of alka(e)nes in maintaining lipid balance in photosynthetic membranes and ensuring efficient photosynthesis. Uncovering the physiological role of alka(e)nes provides insights to a better understanding of the widespread presence of genes encoding alka(e)nes-synthesizing enzymes in cyanobacteria and microalgae, organisms of major ecological and evolutionary importance in the global CO2 assimilation.### Competing Interest StatementThe authors have declared no competing interest.

Abstract New regulatory functions in plant development and environmental stress responses have recently emerged for a number of apocarotenoids produced by enzymatic or nonenzymatic oxidation of carotenoids. β-Cyclocitric acid (β-CCA) is one such compound derived from β-carotene, which triggers defense mechanisms leading to a marked enhancement of plant tolerance to drought stress. We show here that this response is associated with an inhibition of root growth affecting both root cell elongation and division. Remarkably, β-CCA selectively induced cell cycle inhibitors of the SIAMESE-RELATED (SMR) family, especially SMR5, in root tip cells. Overexpression of the SMR5 gene in Arabidopsis induced molecular and physiological changes that mimicked in large part the effects of β-CCA. In particular, the SMR5 overexpressors exhibited an inhibition of root development and a marked increase in drought tolerance which is not related to stomatal closure. SMR5 up-regulation induced changes in gene expression that strongly overlapped with the β-CCA–induced transcriptomic changes. Both β-CCA and SMR5 led to a down-regulation of many cell cycle activators (cyclins, cyclin-dependent kinases) and a concomitant up-regulation of genes related to water deprivation, cellular detoxification, and biosynthesis of lipid biopolymers such as suberin and lignin. This was correlated with an accumulation of suberin lipid polyesters in the roots and a decrease in nonstomatal leaf transpiration. Taken together, our results identify the β-CCA–inducible and drought-inducible SMR5 gene as a key component of a stress-signaling pathway that reorients root metabolism from growth to multiple defense mechanisms leading to drought tolerance.

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 (CvFAP) 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 CvFAP-based production rate over 10-fold higher for n-heptane than for n-pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that CvFAP’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.

New regulatory functions in plant development and environmental stress responses have recently emerged for a number of apocarotenoids produced by enzymatic or non-enzymatic oxidation of carotenoids. β-cyclocitric acid (β-CCA) is one such compound derived from β-carotene which triggers defense mechanisms leading to a marked enhancement of plant tolerance to drought stress. We show here that this response is associated with an inhibition of root growth affecting both root cell elongation and division. Remarkably, β-CCA selectively induced cell cycle inhibitors of the SIAMESE-RELATED (SMR) family, especially SMR5, in root tip cells. Overexpression of the SMR5 gene in Arabidopsis induced molecular and physiological changes that mimicked in large part the effects of β-CCA. In particular, the SMR5 overexpressors exhibited an inhibition of root development and a marked increase in drought tolerance which is not related to stomatal closure. SMR5 up-regulation induced changes in gene expression that strongly overlapped with the β-CCA-induced transcriptomic changes. Both β-CCA and SMR5 led to a down-regulation of many cell cycle activators (cyclins, cyclin-dependent kinases) and a concomitant up-regulation of genes related to water deprivation, cellular detoxification and biosynthesis of lipid biopolymers such as suberin and lignin. This was correlated with an accumulation of suberin lipid polyesters in the roots and a decrease in non-stomatal leaf transpiration. Taken together, our results identify the β-CCA-and drought-inducible SMR5 gene as a key component of a stress signaling pathway that reorients root metabolism from growth to multiple defense mechanisms leading to drought tolerance.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase–peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare , we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.

Fatty Acid Photodecarboxylase a 2 Its mechanism in the we reaction cycle of FAP. The reaction mechanism with an transfer from the fatty to a

Contrary to animals, little is known in plants about enzymes able to produce fatty acid epoxides. In our attempt to find and characterize a new fatty acid epoxygenase in Arabidopsis thaliana, data mining brought our attention on CYP77B1. Modification of the N-terminus was necessary to get enzymatic activity after heterologous expression in yeast. The common plant fatty acid C18:2 was converted into the diol 12,13-dihydroxy-octadec-cis-9-enoic acid when incubated with microsomes of yeast expressing modified CYP77B1 and AtEH1, a soluble epoxide hydrolase. This diol originated from the hydrolysis by AtEH1 of the epoxide 12,13-epoxy-octadec-cis-9-enoic acid produced by CYP77B1. A spatio-temporal study of CYP77B1 expression performed with RT-qPCR revealed the highest level of transcripts in flower bud while, in open flower, the enzyme was mainly present in pistil. CYP77B1 promoter-driven GUS expression confirmed reporter activities in pistil and also in stamens and petals. In silico co-regulation data led us to hypothesize that CYP77B1 could be involved in cutin synthesis but when flower cutin of loss-of-function mutants cyp77b1 was analyzed, no difference was found compared to cutin of wild type plants. Phylogenetic analysis showed that CYP77B1 is strictly conserved in flowering plants, suggesting a specific function in this lineage.