Transcriptomic analysis of polyketide synthases in a highly ciguatoxic dinoflagellate, Gambierdiscus polynesiensis and low toxicity Gambierdiscus pacificus, from French Polynesia

Abstract

Marine dinoflagellates produce a diversity of polyketide toxins that are accumulated in marine food webs and are responsible for a variety of seafood poisonings. Reef-associated dinoflagellates of the genus Gambierdiscus produce toxins responsible for ciguatera poisoning (CP), which causes over 50,000 cases of illness annually worldwide. The biosynthetic machinery for dinoflagellate polyketides remains poorly understood. Recent transcriptomic and genomic sequencing projects have revealed the presence of Type I modular polyketide synthases in dinoflagellates, as well as a plethora of single domain transcripts with Type I sequence homology. The current transcriptome analysis compares polyketide synthase (PKS) gene transcripts expressed in two species of Gambierdiscus from French Polynesia: a highly toxic ciguatoxin producer, G. polynesiensis, versus a non-ciguatoxic species G. pacificus, each assembled from approximately 180 million Illumina 125 nt reads using Trinity, and compares their PKS content with previously published data from other Gambierdiscus species and more distantly related dinoflagellates. Both modular and single-domain PKS transcripts were present. Single domain β-ketoacyl synthase (KS) transcripts were highly amplified in both species (98 in G. polynesiensis, 99 in G. pacificus), with smaller numbers of standalone acyl transferase (AT), ketoacyl reductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) domains. G. polynesiensis expressed both a larger number of multidomain PKSs, and larger numbers of modules per transcript, than the non-ciguatoxic G. pacificus. The largest PKS transcript in G. polynesiensis encoded a 10,516 aa, 7 module protein, predicted to synthesize part of the polyether backbone. Transcripts and gene models representing portions of this PKS are present in other species, suggesting that its function may be performed in those species by multiple interacting proteins. This study contributes to the building consensus that dinoflagellates utilize a combination of Type I modular and single domain PKS proteins, in an as yet undefined manner, to synthesize polyketides.

Fig 1. Phylogenetic analysis of G. polynesiensis and G. pacificus KS domains.

The alignment consisted of 227 KS domains from G. polynesiensis TB92 and G. pacificus MUR4 and from prokaryotic and eukaryotic type I and type II PKS and FAS. Analysis was carried out by PhyML using the LG model of rate heterogeneity and 100 bootstraps. Only bootstrap values >50% are displayed.

Fig 2. Phylogenetic analysis of KS domains extracted from modular PKS and NRPS/PKS.

The alignment consisted of 116 sequences, including all modular KS from this study and previously published Gambierdiscus spp., K. brevis, and cis- and trans-AT prokaryotic and eukaryotic type I PKS and FAS. Type II PKS and FAS served as outgroups. Analysis carried out by PhyML using the LG model of rate heterogeneity and 100 bootstraps. Only bootstrap values >50% are displayed. Sequence logos of the active site are shown for each major clade.

Fig 3. Modular PKSs sharing domain architecture and sequence homology with the 7-module PKS found in G. polynesiensis.

The starting KS of each module is in red.