Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals

Abstract

Marine ecosystems are responsible for virtually all production of omega-3 (ω3) long-chain polyunsaturated fatty acids (PUFA), which are essential nutrients for vertebrates. Current consensus is that marine microbes account for this production, given their possession of key enzymes including methyl-end (or "ωx") desaturases. ωx desaturases have also been described in a small number of invertebrate animals, but their precise distribution has not been systematically explored. This study identifies 121 ωx desaturase sequences from 80 species within the Cnidaria, Rotifera, Mollusca, Annelida, and Arthropoda. Horizontal gene transfer has contributed to this hitherto unknown widespread distribution. Functional characterization of animal ωx desaturases provides evidence that multiple invertebrates have the ability to produce ω3 PUFA de novo and further biosynthesize ω3 long-chain PUFA. This finding represents a fundamental revision in our understanding of ω3 long-chain PUFA production in global food webs, by revealing that numerous widespread and abundant invertebrates have the endogenous capacity to make significant contributions beyond that coming from marine microbes.

Fig. 1. Distribution of ωx desaturases.

(A) A condensed phylogenetic tree depicting relationships among ωx desaturases from Cyanobacteria (8) and Eukaryota (279). Metazoan phyla (represented with 121 sequences) are named in the tree and assigned with colors for the following taxa: Nematoda (green), Cnidaria (blue), Arthropoda (purple), and Lophotrochozoa (orange). Nonmetazoan clades were collapsed and named according to their general taxonomic composition. “Miscellaneous Clade A” contains representatives of Amoeboza, Apusozoa, Haptophyceae, Heterolobosea, Nucleariidae, SAR, Chlorophyta, Rhodophyta, Cryptophyta, and the insect L. migratoria. Representatives of Excavata, Amoebozoa, Fungi, SAR, and the hexapod S. viridis compose “Miscellaneous Clade B.” The phylogenetic tree presented corresponds to the 80% rule consensus tree estimated in MrBayes. Colored dots on the nodes represent supporting values of 80% or above for MrBayes and PhyloBayes analyses (blue) or for all three (MrBayes, PhyloBayes, and RAxML) analyses (red). (B) Summary of taxonomic groups highlighting metazoans in which ωx desaturases have been identified in this study. Number of species is indicated in brackets. A list of all genomes and transcriptomes searched, including those in which no ωx desaturase genes were found, can be accessed as described in the Data and materials availability section.

Fig. 2. HGT accounts for the presence of ωx desaturase in some animal genomes.

(A) Multiple copies of ωx desaturase–like sequences scattered among several loci of the B. tabaci genome. The ωx desaturase–like sequences are found in five different scaffolds and are flanked by animal genes. * indicates sequences that did not pass the filter (probability between 0.50 and 0.70). (B) Intron-exon structures in the ωx desaturase genes from selected metazoan species. The arrows indicate aligned coding sequences (CDS) extracted from the genomic assembly of each species. The positions of introns are indicated as solid lines within the arrows. The dotted line indicates shared intron-exon boundaries. All cnidarian genes and several genes from Annelida and Mollusca have an “intronless” organization. The CDS of the three ωx desaturase genes from A. vaga (Rotifera) share the same overall structure consisting of eight exons. In addition, all genes from three Heterobranchia species have the same structure with six exons in the CDS. Variable gene structures were found in Nematoda, Annelida, and Arthropoda. The arrow colors are consistent with those used to represent clades in Fig. 1.

Fig. 3. Functional characterization of metazoan ωx desaturases.

(A) The de novo production of ω3 PUFA requires both ω6 (Δ12) (blue arrow) and ω3 (Δ15) desaturases (red arrow). Both LA (18:2ω6) and ALA (18:3ω3) can be subsequently modified through the ω6 and ω3 long-chain PUFA biosynthesis pathways that proceed separately, at least in vertebrates (9), or can be interconnected by ω3 desaturases with Δ15, Δ17, or Δ19 activities (green arrows). (B) Chromatograms of FA methyl esters (FAME) from yeast expressing ωx desaturases from A. vaga (MF448339) (top) and A. millepora (KY658237) (middle) show Δ12/Δ15 and Δ12 activities, respectively. Further ωx desaturases with Δ12 activity were characterized from P. vulgata, P. dumerilii, and L. salmonis (Table 1). (C) Chromatograms of FAME from yeast expressing the ωx desaturase from P. vulgata (KY658241) and grown with supplemented ω6 substrates including 18:2ω6 (left), 20:4ω6 (middle), and 22:4ω6 (right) show the resulting ω3 products 18:3ω3, 20:5ω3, and 22:5ω3, respectively. Similar functions obtained for ωx desaturases from A. millepora, A. vaga, P. dumerilii, and L. salmonis are shown in Table 2. Molecular structures and diagnostic mass ions of 4,4-dimethyloxazoline derivatives from desaturation products are shown in figs. S3 and S4.