Genome–Scale Metabolic Networks Shed Light on the Carotenoid Biosynthesis Pathway in the Brown Algae Saccharina japonica and Cladosiphon okamuranus

Understanding growth mechanisms in brown algae is a current scientific and economic challenge that can benefit from the modeling of their metabolic networks. The sequencing of the genomes of Saccharina japonica and Cladosiphon okamuranus has provided the necessary data for the reconstruction of Genome–Scale Metabolic Networks (GSMNs). The same in silico method deployed for the GSMN reconstruction of Ectocarpus siliculosus to investigate the metabolic capabilities of these two algae, was used. Integrating metabolic profiling data from the literature, we provided functional GSMNs composed of an average of 2230 metabolites and 3370 reactions. Based on these GSMNs and previously published work, we propose a model for the biosynthetic pathways of the main carotenoids in these two algae. We highlight, on the one hand, the reactions and enzymes that have been preserved through evolution and, on the other hand, the specificities related to brown algae. Our data further indicate that, if abscisic acid is produced by Saccharina japonica, its biosynthesis pathway seems to be different in its final steps from that described in land plants. Thus, our work illustrates the potential of GSMNs reconstructions for formalizing hypotheses that can be further tested using targeted biochemical approaches.


Contents
: Data from the annotation of algal protein-coding genes. Figure S1: Source of the S. japonica (a) and C. okamuranus (b) annotations. Upset plots represent the distribution of the three types of annotation data: EC numbers, gene ontology terms (GOT) and protein domains (PFAM) for all predicted coding sequences.
In order to provide a first source of 'annotation-based' metabolic reactions, EC numbers, Gene ontology terms (GOT), and protein domains (PFAM) were sought among the predicted coding sequences of S. japonica and C. okamuranus. 60% (10,826 out of 17,898) and 69% (10,422 out of 15,077) of them were annotated with at least one of these three types of annotations and 22% of them are annotated by all three data types. These annotations allowed the generation of GSMNs composed respectively of 4,316 and 4,003 genes, 1,237 and 1,205 pathways, 2,560 and 2,448 enzymatic reactions and 2,920 and 2,872 metabolites for S. japonica and C. okamuranus.  Metabolites present but not targeted by gap-filling due to their encoding (type: compound class)

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Fucoidan C7H14O7S Fucoidans [5] L-fucose C6H12O5 L-fucoses [2] Uronic acid C6H10O7 Uronates [2] Figure S2.   Figure S4: Maximum likelihood tree of the lycopene cyclase family. All the sequences provided with an accession number came from the NCBI database. The sequences presented on the various trees were selected based either on homology searches with the sequences identified QUERY (black arrow) or based on publications. When available, the functional annotation of the sequences were added to the sequence names. Stramenopile sequences (brown algae and diatoms) are shown in brown, red algae sequences in red, green algae sequences in green and Arabidopsis thaliana sequences in blue. Only bootstrap values greater than or equal to 70 are displayed. Tree were rooted by midpoint.
In terrestrial plants, neoxanthin is the last product of carotenoid synthesis and therefore the precursor of new oxygenated carotenoids. The enzyme responsible for this transformation, Nsy, belongs to the lycopene cyclase family characterized by the presence of the protein domain of the same name (PF05834). This carotenoid cyclase paralog has apparently been redesigned from the β-lycopene cyclase from terrestrial plants [1,2]. Homology search based on the Nsy sequence of A. thaliana on brown algae predicted protein sequences and the search for the protein domain in annotation files revealed a single candidate, identified as lycopene β-cyclase. The results obtained for red algae C. merolae, diatom P. tricornutum and green algae C. reinhardtii and V. carteri are similar to those obtained in [3]. Figure S5: Maximum likelihood tree of the NCED family. All the sequences provided with an accession number came from the NCBI database. The sequences presented on the various trees were selected based either on homology searches with the sequences identified QUERY (black arrow) or based on publications. When available, the functional annotation of the sequences were added to the sequence names. Stramenopile sequences (brown algae and diatoms) are shown in brown, red algae sequences in red, green algae sequences in green and Arabidopsis thaliana sequences in blue. Only bootstrap values greater than or equal to 70 are displayed. Tree were rooted by midpoint.
The first oxidoreductase involved in ABA synthesis belongs to the carotenoid oxygenase family (CCO) and it is composed of a single protein domain: retinal pigment epithelial membrane protein (PF03055). In plants, this family is divided into two subfamilies, carotenoid cleavage dioxygenase (CCD) and the NCED [1][2][3][4]. There seems to be a common origin between A. thaliana CCDs and the various groups of algae, but the NCED family seems to have evolved only in embryophytes [5,6]. It would therefore appear that the sequences of P. tricornutum, N. gaditana, G. sulphuraria and C. merolae annotated NCED are incorrect. Nevertheless, homology search allows us to target a set of sequences belonging to the CCDs family. Phylogenetically, for each of the seaweeds, three groups of CCDs are detected and they could be involved in the synthesis of apocarotenoids such as retinal or carotenoid-derived volatiles [6]. The first is composed of a single sequence for S. japonica and three paralogues for C. okamuranus, the second of three paralogues per organism and the third include a single sequence per organism. For instance, in terrestrial plants, CCD1 is involved in the preliminary steps of beta-ionone synthesis whereas CCD7 and CCD8 are implicated in strigolactone synthesis [6,7]. 22 The third oxidoreductase involved in ABA synthesis belongs to the aldehyde oxygenase (AO) family [1]. AO is a subfamily of xanthine oxidase (XO) which also includes xanthine dehydrogenase (XDH). The AOs have evolved independently twice from XDH paralogues [2], which explains why these sequences have a similar structure and organization in protein domains (six domains in total: 2Fe-2S iron-sulfur cluster binding domain PF00111, [2Fe-2S] binding domain PF01799, FAD binding domain in molybdopterin dehydrogenase PF00941, CO dehydrogenase flavoprotein C-terminal domain PF03450, Aldehyde oxidase and xanthine dehydrogenase, a/b hammerhead domain PF01315, Molybdopterin-binding domain of aldehyde dehydrogenase PF02738). No AOs were detected in the brown and red algae analysed, suggesting a gene loss of in those species. On the other hand, the homology search, confirmed by the annotation files, allowed us to identify the presence of XDH (involved in the purine degradation pathways) in these two types of algae.   [1]. Among the 14 types of SDR found in brown, none of them are related to the type sought. ABA2 is consequently an enzyme specific to terrestrial plants.
Among the 14 types of SDR: 8 types are shared between S. japonica and C. okamuranus, 2 are specific to S. japonica and 4 are specific to C. okamuranus. Within the algal genomes 52 (S. japonica) and 54 sequences (C. okamuranus) have been identified as potential SDRs, 10 and 13 of these sequences (~22%) could be annotated with an SDR number. For the other sequences only the type (essentially U or C) or otherwise the corresponding protein domain could be found (only concerns 2 sequences of C. okamuranus). According to homology research carried out within the 13 algae proteomes, it would appear that SDR17C and SDR152C are shared between green, red and brown algae, SDR65C between certain brown and green algae and that SDR87D is only found in Cladosiphon okamuranus and Ectocarpus siliculosus proteomes.  27 VDE is one of the two enzymes involved in the first xanthophyll cycle. It is composed of a single domain: VDE lipocalin domain (PF07137). As expected, three out-paralogs of VDE are found in brown algae proteomes, no copies of VDE gene are found in the red algae proteomes and the presence of a VDL appears to be a specificity of Phaeophyceae.