Domoic acid biosynthesis in the red alga Chondria armata suggests a complex evolutionary history for toxin production

Abstract

Domoic acid (DA), the causative agent of amnesic shellfish poisoning, is produced by select organisms within two distantly related algal clades: planktonic diatoms and red macroalgae. The biosynthetic pathway to isodomoic acid A was recently solved in the harmful algal bloom-forming diatom Pseudonitzschia multiseries, establishing the genetic basis for the global production of this potent neurotoxin. Herein, we sequenced the 507-Mb genome of Chondria armata, the red macroalgal seaweed from which DA was first isolated in the 1950s, identifying several copies of the red algal DA (rad) biosynthetic gene cluster. The rad genes are organized similarly to the diatom DA biosynthesis cluster in terms of gene synteny, including a cytochrome P450 (CYP450) enzyme critical to DA production that is notably absent in red algae that produce the simpler kainoid neurochemical, kainic acid. The biochemical characterization of the N-prenyltransferase (RadA) and kainoid synthase (RadC) enzymes support a slightly altered DA biosynthetic model in C. armata via the congener isodomoic acid B, with RadC behaving more like the homologous diatom enzyme despite higher amino acid similarity to red algal kainic acid synthesis enzymes. A phylogenetic analysis of the rad genes suggests unique origins for the red macroalgal and diatom genes in their respective hosts, with native eukaryotic CYP450 neofunctionalization combining with the horizontal gene transfer of N-prenyltransferases and kainoid synthases to establish DA production within the algal lineages.

Keywords: biosynthetic gene cluster; genomics; natural products; neurotoxin; seaweed.

Fig. 1.

Kainoid biosynthetic genes and producing organisms. (A) DA-producing diatom P. multiseries (Bacillariophyta) and KA-producing red algae D. simplex and P. palmata with DA-producing red alga C. armata (Rhodophyta), two significantly divergent phyla of kainoid-producing algae. (B) Structure of DA. (C) Proposed DA and KA biosynthetic pathways showing diagnostic kainoid biosynthetic enzymes in teal performing the two key enzymatic transformations forming the core kainoid pyrrolidine scaffold. Verified activities and major products from this work and previous studies (17, 18) shown with solid arrows.

Fig. 2.

Visualization of syntenic comparisons between dab, rad, and kab gene clusters. Blue background highlights diatom sequences, and red background highlights red algal sequences. Cladogram connects clusters based on taxonomic relationships of organisms, not to scale with respect to evolutionary time. Intensity of shading between genes is relative to the similarity of gene pairs by amino acid sequence percent identity in agreement with discrete measures of sequence percent identity as labeled. Retro transposable elements are abbreviated as RT elements.

Fig. 3.

Activities and predicted structures of the known kainoid synthase enzymes. (A) Comparison of combined extracted ion chromatogram profiles for cNGG substrate (m/z 312.1 ± 0.2) and isodomoic acid (m/z 310.1 ± 0.2) from RadC1, RadC2, DabC, and KabC assays and C. armata extract. Relative intensity of extracted ions is shown, and all substrates and observed DA isomers are compared to purified standards (SI Appendix, Fig. S5). (B) AlphaFold2 predicted models for RadC1, DabC, and KabC. (C) Modeled pocket volume for RadC1 and KabC.

Fig. 4.

Phylogenetic analysis of (A) kainoid synthase and (B) coclustered DA CYP450 enzymes (yellow highlights, yellow circle denotes key branch points). The maximum likelihood trees were built using representative sequences from key taxonomic groups (SI Appendix, Tables S5 and S6). Kainoid synthase enzymes form their own distinct branch, independent of taxonomic origin, while the DA CYP450s are nested within their respective taxonomic groups.