Multifunctional polyketide synthase genes identified by genomic survey of the symbiotic dinoflagellate, Symbiodinium minutum
Beedessee et al. BMC Genomics
Multifunctional polyketide synthase genes identified by genomic survey of the symbiotic dinoflagellate, Symbiodinium minutum
Girish Beedessee 0
Kanako Hisata 0
Michael C. Roy 1
Noriyuki Satoh 0
Eiichi Shoguchi 0
0 Marine Genomics Unit, Okinawa Institute of Science and Technology Graduate University , Onna, Okinawa 904-0495 , Japan
1 Imaging and Instrumental Analysis Section, Okinawa Institute of Science and Technology Graduate University , Onna, Okinawa 904-0495 , Japan
Background: Dinoflagellates are unicellular marine and freshwater eukaryotes. They possess large nuclear genomes (1.5-245 gigabases) and produce structurally unique and biologically active polyketide secondary metabolites. Although polyketide biosynthesis is well studied in terrestrial and freshwater organisms, only recently have dinoflagellate polyketides been investigated. Transcriptomic analyses have characterized dinoflagellate polyketide synthase genes having single domains. The Genus Symbiodinium, with a comparatively small genome, is a group of major coral symbionts, and the S. minutum nuclear genome has been decoded. Results: The present survey investigated the assembled S. minutum genome and identified 25 candidate polyketide synthase (PKS) genes that encode proteins with mono- and multifunctional domains. Predicted proteins retain functionally important amino acids in the catalytic ketosynthase (KS) domain. Molecular phylogenetic analyses of KS domains form a clade in which S. minutum domains cluster within the protist Type I PKS clade with those of other dinoflagellates and other eukaryotes. Single-domain PKS genes are likely expanded in dinoflagellate lineage. Two PKS genes of bacterial origin are found in the S. minutum genome. Interestingly, the largest enzyme is likely expressed as a hybrid non-ribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly of 10,601 amino acids, containing NRPS and PKS modules and a thioesterase (TE) domain. We also found intron-rich genes with the minimal set of catalytic domains needed to produce polyketides. Ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) along with other optional domains are present. Mapping of transcripts to the genome with the dinoflagellate-specific spliced leader sequence, supports expression of multifunctional PKS genes. Metabolite profiling of cultured S. minutum confirmed production of zooxanthellamide D, a polyhydroxy amide polyketide and other unknown polyketide secondary metabolites. Conclusion: This genomic survey demonstrates that S. minutum contains genes with the minimal set of catalytic domains needed to produce polyketides and provides evidence of the modular nature of Type I PKS, unlike monofunctional Type I PKS from other dinoflagellates. In addition, our study suggests that diversification of dinoflagellate PKS genes comprises dinoflagellate-specific PKS genes with single domains, multifunctional PKS genes with KS domains orthologous to those of other protists, and PKS genes of bacterial origin.
Gene diversification; Horizontal gene transfer; Spliced-leader trans-splicing; Polyketide synthase; Bacterial PKS; NRPS; Zooxanthellamide D; Symbiodinium minutum; Dinoflagellates; Genome-wide survey
Dinoflagellates are unicellular eukaryotes found in both
marine and freshwater environments. Some are crucial
symbionts of reef-building corals, and others sometimes
cause toxic algal blooms [
]. Dinoflagellates are rich
sources of structurally unique and bioactive secondary
metabolites and are of interest to natural product
chemists, biologists, and ecologists. These metabolites are
unique in size, structure, and potency, and many are of
polyketide origin [
Dinoflagellate toxins have been classified into three
main categories: (i) polycyclic polyethers, (ii) macrolides,
and (iii) linear polyethers [
]. The majority of these
compounds display remarkable biological activities,
including ion channel modulation, phosphatase inhibition,
hemolysis, mycotoxicity, and cytotoxicity [
possible explanation for their high potency is to
compensate for high dilution when they are released into the
water . Much is known regarding the biosynthesis of
polyketides from terrestrial and freshwater organisms;
however, only in the last decade have dinoflagellate
polyketides been investigated.
Polyketides are synthesized by specific enzymes
called polyketide synthases, through a series of
condensation and reduction reactions involving at least
three protein domains. These include ketosynthase
(KS), acyl transferase (AT), and acyl carrier protein
(ACP) (PP-binding) domains. In addition, polyketide
synthesis may involve three optional domains:
ketoreductase (KR), dehydratase (DH), and enoylreductase
]. In 2008, full-length transcripts of Type
Ilike, modular PKS were sequenced from Karenia
brevis, with seven out of eight transcripts containing
single PKS domains, a feature typical of Type II PKS
]. Eichholz et al. [
] characterized five transcripts
for Type I-like, PKS-encoding KS proteins that are
expressed as monofunctional units, from the
dinoflagellates, Alexandrium ostenfeldii and Heterocapsa
triquetra. Transcriptomic analysis of the non-toxic
Heterocapsa circularisquama, revealed 61 polyketide
synthase-encoding expressed sequence tags (EST)
contigs, including one contig with two domains
]. Similar analysis revealed Type I-like
polyketide synthases in the toxic dinoflagellate,
Gambierdiscus polynesiensis, the main producer of
]. Meyer et al. [
] reported finding all
genes essential for polyketide toxin synthesis in
Azadinium spinosum, known to produce azaspiracid
toxins. Recently, Kohli et al. catalogued 162 unique
transcripts encoding complete KS domains in two
species of Gambierdiscus, which are putatively
involved in polyketide biosynthesis [
Among marine dinoflagellates, the Genus
Symbiodinium includes major coral symbionts that are also
associated with other invertebrate taxa (Porifera,
Mollusca, and Platyhelminthes) [
]. The draft genome
of S. minutum, encoding ~42,000 protein-coding genes,
has provided an opportunity for better understanding of
its PKS system . Snyder et al. [
] reported Type I
PKSs in several dinoflagellates, including Symbiodinium
sp.; however, there has been no detailed survey of genes
involved in polyketide synthesis in S. minutum. We
probed the S. minutum genome with respect to enzymes
involved in polyketide synthesis and phylogenetically
analyzed the KS domains of PKSs. We found a
nonribosomal peptide synthetase-polyketide synthase
(NRPS-PKS) hybrid and confirmed that PKSs of S.
minutum belong to the protistan Type I PKS group, along
with some unexpected sequences associated with a
Diversification of KS domain-containing genes in the S.
In total, 65 genes with ketoacyl synthase domains
(Pfam IDs: PF00109) were screened from the
predicted 41,925 genes in the S. minutum genome
BLASTP searches, we also checked S. minutum genes
similar to reported PKSs and confirmed the aligned
sequences manually. After removing sequences for
partial domains, 25 genes that encoded full KS
domains in S. minutum were selected for sequence
characterization (Table 1; see Additional file 1: Table
S1) and molecular phylogenetic analysis. Sequence
comparisons with KS domains showed that the most
similar genes are those reported from other
dinoflagellates, although several genes were unexpectedly
most similar to bacterial (Bacillus) genes. Eleven KS
domain-containing genes likely encode multifunctional
proteins with other domains related to PKS synthesis
(AT, ACP, KR, DH, and ER) (Table 1). Careful
examination of the S. minutum genome identified 25
intron-rich genes for KS sequences (Table 1) that are
expressed under standard culture conditions (see
Additional file 1: Figure S1). Only one KS gene
(symbB1.v1.2.039083.1) is likely to be more highly
expressed than genes [
] for RNA polymerase (data
not shown). Quantitative expression analysis under
different conditions will be useful for functional
predictions. An interesting feature was the presence of
tandemly aligned KS genes (symbB1.v1.2.015790.t1,
symbB1.v1.2.015788.t2 and symbB1.v1.2.015789.t1) on
scaffold 1186.1, in addition to two KS genes on
scaffold 514.1 (Table 1). Since domain combinations are
not conserved completely, duplication and/or splitting
are hypothesized as the mechanisms for these
expansions (see Fig. 1). This hypothesis is not
unreasonable, considering a recent report of dinoflagellates
possessing por (protochlorophyllide oxidoreductase)
gene duplicates [
]. Gene duplications can have
metabolic advantages and can eventually become fixed
in a population.
Bayesian inference and maximum likelihood analysis
of the 25 KS sequences were carried out with acyl carrier
protein synthase (ACPS) and Type II PKS sequences as
outgroups to understand relationships of KS sequences
from S. minutum compared with those of other
dinoflagellates. After alignment and trimming, a sequence of
Fig. 1 (See legend on next page.)
Apicomplexa PKS and FAS
PKS and FAS
Type II PKS
(See figure on previous page.)
Fig. 1 A molecular phylogenetic tree of Type I KS domains from prokaryotic and eukaryotic PKS and FAS, analyzed by Bayesian inference, reveals the
diversification of the KS domain gene family. Symbiodinium minutum possesses genes belonging to three major groups within this gene family. Type II
PKS and acyl carrier protein synthases (ACPS) were used as outgroups. Numbers at nodes indicate posterior probabilities. Details regarding S. minutum
sequences are provided in Table 1. Red stars indicate S. minutum proteins with single PKS-related domains. Green circles indicate S. minutum proteins with
multiple PKS-related domains. Dinoflagellate KSs (yellow) are classified as a well-supported group within the protistan Type I PKS clade.
235 amino acids (aa) was used for analysis. Molecular
phylogenetic analyses significantly placed most
sequences with those of other dinoflagellate Type I PKSs
(Fig. 1). Bayesian inference clearly demonstrated that 22
S. minutum proteins and other protistan proteins were
included in that clade (Fig. 1). There was strong
Bayesian support (posterior probability: 1.00) for a
‘protistan’ clade of Type I PKS sequences comprising of
apicomplexan, dinoflagellate, chlorophyte, and haptophyte
sequences (Fig. 1). These major groups formed two
separate sub-groups within the protistan clade. One
subgroup with all existing sequences from A. ostenfeldii, A.
spinosum, G. polynesiensis, and H. triquetra, included 10
proteins of S. minutum with one PKS-related domain
(KS). The second sub-group, with
chlorophyte/haptophyte proteins, contained four S. minutum sequences
with multiple PKS-related domains. The third
subgroup, with bacterial proteins, contained two S.
minutum proteins (symbB1.v1.2.027671 and symbB1.
v1.2.036002). Interestingly, the sequences were more
closely related to cyanobacterial KS sequences than to
other eukaryote sequences. A similar pattern has been
reported in K. brevis, in which some sequences grouped
with cyanobacterial proteins [
]. Dinoflagellate proteins
were not found in clades of animal fatty acid synthase
(FAS) and fungal PKS. It is worth mentioning that
NRPS-PKS hybrid (symbB1.v1.2.012436.t1) was in a
clade with PKS and FAS of chlorophyta/haptophyta
(Emiliania huxleyi, Ostreococcus tauri, Chlamydomonas
reinhardii, Ostreococcus lucimarinus and Micromonas
pusilla), which encode the largest PKS protein in S.
minutum. Maximum likelihood analysis provided additional
support for a “protistan clade” containing Type I PKS
sequences (Additional file 1: Figure S2).
To explore the possibility of spliced leader
transsplicing from a large transcriptome to single-domain,
protein-coding transcriptomes, we mapped
transcriptome data from the TSS (transcription start site). The
mapping of SL (spliced leader)-removed TSS (red lines
in Figure S1) showed that each of three gene models
(symbB1.v1.2.000535 on scaffold 31, symbB1.v1.2.020241
on scaffold 1693, symbB1.v1.2.028834 on scaffold 3094)
predicts two transcripts by SL trans-splicing (red arrows
in Figure S1). They are genes with single PKS-related
domains (Table 1; Fig. 1). Therefore, proteins with
multiple PKS-related domains are likely to be expressed in
cultured S. minutum.
Presence of a hybrid NRPS-PKS gene in S. minutum
One gene model of 10,601 amino acids was identified as
a hybrid NRPS-PKS, based on PFAM domain analysis
and confirmed by anti-SMASH (antibiotics & Secondary
Metabolite Analysis Shell) [
]. It was composed of eight
modules, three NRPSs, and five PKSs (Fig. 2a). The first
NRPS is followed by three PKS modules, which contain
at least a KS domain with different domain
combinations. A second NRPS with condensation (C) and
adenylation (A) is also present, followed by an additional
NRPS-like assembly (C-HxxPF-A). Thioesterase (TE)
domains are usually located in the final NRPS module
where they catalyze product release [
]. Substrates of
adenylation (A) domains in the NRPS module can be
predicted based on residues in the binding pocket [
The reported A domains in NRPS proteins are
responsible for recruiting amino acids into the final product. In
this case, the first A domain has the sequence
DLFNLSLI while the second A domain has DVWxFSLI.
In silico methods were used to infer a hypothetical
metabolite produced by the hybrid NRPS-PKS gene. Based
on antiSMASH server prediction, the hybrid cluster
could result in a natural product consisting of a core
scaffold made from two amino acids (cysteine and
serine) (Fig. 2b). I-TASSER [
] 3D protein structure
prediction showed that the AT domain at the start of
the cluster resembled an acyl-carrier-protein
malonyltransferase (data not shown) and may provide malonyl
groups for polyketide biosynthesis.
Surveys of other PKS domains and conserved N-terminal sequences of S. minutum KS
BLAST screening of the S. minutum genome and
comparisons of domain structure resulted in identification of
candidates for all domains involved in polyketide
synthesis: KS, AT, ACP (PP), KR, DH, and ER. First, we
examined whether functionally important amino acid residues
required for enzymatic activity (cysteine, histidine, and
lysine) are present near the DTACSS-motif of S.
minutum KS enzymes. We found that most sequences (21/
25) contain these residues (see Additional file 2: Figure
S3). Other domains can also be identified by comparing
sequences and signature motifs: HxxxGxxxx with two
active residues (histidine and proline) in DH [
LxHxxxGGVG in ER with important residues (leucine,
histidine, valine, and glycine) [
], GxGxxGxxxA in KR
with three glycine and one alanine as essential residues
] and the signature motif, GHSLG, in AT domains
(see Additional file 2: Figure S4). The phylogenetic
relationship of the PKS N-terminal region among
dinoflagellates is shown in a maximum likelihood phylogeny (see
Additional file 2: Figure S5). The tree reflects the same
dinoflagellate resolution found in the KS-based
phylogeny (Fig. 1) with one main sub-clade consisting of A.
spinosum, A. ostenfeldii, H. triqueta, and K. brevis, one
sub-clade comprising only one S. minutum and two K.
brevis sequences, and a third clade consisting of three S.
minutum sequences. Alignment of N-terminal regions
revealed several conserved amino acid positions,
including the highly conserved ExExGYLG in most
dinoflagellates (see Additional file 2: Figure S5). In Symbiodinium,
only one of the 25 sequences contained the signature
GYLG and three other variants, DYLG, EYLG, and
GYMG. Many variations were found in other sequences
at the same position showing the diversified nature of
the N-terminus in S. minutum (see Additional file 2:
Identification of ZAD-D in cultured S. minutum by
ZAD (zooxanthellamide D) was identified based on
high-resolution mass data (Table 2). A NanoLC-MS
(positive ion) profile of the methanol extract of S. minutum
showed ions at m/z 1072.60 (10.6 min) and 1050.57 for the
[M + Na]+ and [M + H]+, respectively (Additional file 3:
Figure S6). The ammonium adduct [M + NH4]+ at m/z
1067.59 (10.6 min) was also observed. It should be noted
that other polyhydroxy molecules were also observed in
the crude methanol extract, but none of them
corresponded to other reported zooxanthella polyhydroxy
molecules (Table 3).
The KS domain is the most conserved domain of Type I
PKS proteins and has divergent homologs, which permit
comparative phylogenetic analysis of PKSs [
phylogenetic trees resolved previously reported clades
11, 14, 29
]. Addition of novel sequences from S.
minutum to the KS phylogenetic dataset provided evidence
for three groups of dinoflagellate KS during PKS gene
evolution. Other protist groups that diverged earlier also
retain their KS evolutionary signatures and remain
within well-supported clades, as shown by our analysis.
A smaller clade comprising only S. minutum and K.
brevis sequences showed alterations in their active sites (see
Additional File 2: Figure S5). This topology may indicate
a history of early gene duplication within the
As in PKSs, NRPSs also produce diverse secondary
metabolites and have modular organizations with each
module assuming specific functions. Formation of hybrid
systems or clusters has been reported in bacteria [
Table 2 High-resolution MS of the target molecule, ZAD-D45
[M + H]+
Obs. Mass (m/z) Theo. Mass Delta (mmu) Formula
1050.5657 1050.5632 2.46
[M + NH4]+ 1067.5922
Fungal and bacterial NRPS and PKS have gained attention
in recent years, mainly due to their complex evolutionary
]. Lawrence et al. [
] provided evidence
for horizontal gene transfer (HGT) of the hybrid
NRPSPKS gene from a putative bacterial donor in the
Burkholderiales and suggested a HGT early in the history of the
fungal Phylum Ascomycota. Bushley and Turgeon [
identified NPS genes encoding NRPS and NRPS-like
proteins in fungal genomes and suggested mechanisms for
this modular architecture. In Aspergillus spp., genes
involved in secondary metabolite biosynthesis tend to be
located in subtelomeric regions, which may contribute to
their rapid evolution [
]. Gene transfer from
cyanobacteria to dinoflagellates has been suggested by
LópezLegentil et al.  and this could explain the grouping of
two S. minutum sequences with cyanobacterial sequences.
The hybrid gene symbB1.v1.2.012436.t1 shares features
with chlorophyte/haptophyte sequences used in our
analysis. A data mining study found a surprisingly high
number of hybrid NRPS-PKS gene clusters across three
domains of life and this might be a consequence of
longterm convergence between NRPS and PKS [
survey showed that the S. minutum genome encodes only
one NRPS domain-containing gene. Hybrid NRPS-PKSs
have been reported in other dinoflagellates (K. brevis [
and H. circularisquama [
]). Dinoflagellate genomes are
punctuated with a high number of simple and complex
repeats and well known for frequent recombination events
]. Additionally, these genomes contain genes in
high copy numbers, an indication of frequent gene
duplication events during dinoflagellate evolution .
Shoguchi et al. [
] predicted that a total of 17,703 genes
of S. minutum might have originated by gene duplication.
It will be interesting to determine what types of natural
products are synthesized by this NRPS-PKS gene and
what role they do play in S. minutum.
Eichholz et al. [
] speculated that the N-terminus is
related to the monofunctional nature of KS domains and
may play a role in structural rearrangements, substrate
docking, or protein-protein interactions. The N-termini
of PKS multi-enzymes contain regularities in amino acid
sequences. Recent studies have highlighted the potential
role of these regions as “linkers” and their interactions
with linker regions at the C-termini of PKS
]. A low degree of conservation was
noted within the N-terminal ExExGYLG signature
sequence of Symbiodinium KS sequences. The GYLG
conserved sequence has also been reported in G.
polynesiensis, along with several variants (DYLG, HYLG,
YYLG, GLLG and ALLG) . Alteration of this
signature has also been reported in A. spinosum (AFLG) [
Eichholz et al. [
] found that PKS domains are
expressed as monofunctional units and that this feature
may be unique to dinoflagellates. However, a transcript
containing more than one domain has been reported in
which one EST contig encoding two Type I PKS
domains was found in the transcriptome of H.
circularisquama, raising the possibility that there may be
multimodular PKS genes in dinoflagellates [
]. One feature
suggesting this possibility is the presence of the ACP (PP
arm), upstream of the KS domain. Such a group could
serve as a swinging arm to present substrates to catalytic
sites on PKS. Modular Type I PKS proteins have been
reported in a closely related apicomplexan
(Cryptosporidium parvum) and in a haptophyte (Emiliana huxleyi)
with several different enzymatic domains arranged in
distinct modules [
]. Given that there is partial or
complete absence of the conserved GYLG sequence in
the N-terminus and that ACP precedes the KS domains,
our genome-wide survey provides evidence for
multifunctional PKS genes in the Symbiodinium genome
along with monofunctional units.
ZAD-D is a linear polyhydroxylated polyketide and
has been reported from Symbiodinium strain JCUCS-1
]. It is related to amphidinols isolated from the
dinoflagellate, Amphidinium sp. [
]. This molecule is a
polyhydroxy amide consisting of a C22-acid moiety and a
C32-amine moiety; it furnishes three tetrahydropyran
rings and six isolated butadiene chromophores. Apart
from ZAD-D, other unknown polyhydroxy molecules
were found in the methanol extract, and characterization
of these unknown compounds could be interesting (see
Additional File 3 Figure S6). Other natural products
have been isolated from Symbiodinium sp. that displayed
significant biological activities (Table 3). Hybrid
NRPSPKS systems are capable of incorporating both amino
acids and short carboxylic acids into final products,
eventually leading to greater chemical structural
diversity. It is not yet known what type of natural products
are synthesized by the hybrid NRPS-PKS reported here;
further work is needed to characterize its end products
as well as products of other PKSs in order to determine
their role in S. minutum.
We demonstrate that three structural types of enzymes
for polyketide synthesis, single-domain PKS,
multidomain PKS, and NRPS-PKS hybrid, are present in the
dinoflagellate, S. minutum. Based on the ketosynthase
domain, dinoflagellate PKSs can be evolutionarily
classified into three groups. It is not yet clear why S. minutum
possesses a polyketide biosynthetic pathway and how
these multifunctional PKS proteins have evolved.
Genomic characterization of dinoflagellate PKS genes will
likely provide insights for combinatorial biosynthesis of
polyketides with wide range of applications. Due to large
and complex dinoflagellate genomes, it is more difficult
to perform comprehensive analyses; however, cultured S.
minutum, which has a comparatively small genome,
might provide further insights into these phenomena.
Symbiodinium minutum, originally provided by Dr. Mary
Alice Coffroth, of the University of New York, Buffalo,
was cultured in autoclaved, artificial seawater containing
1X Guillard’s (F/2) marine-water enrichment solution
(Sigma-Aldrich: G0154), plus three antibiotics, ampicillin
(100 μg/mL), kanamycin (50 μg/mL), and streptomycin
(50 μg/mL) [
]. Mass culturing was performed in a 2 L
final volume by inoculating, and sampling at stationary
phase one month later. A 12 h light/dark regime at 25 °C
was maintained with a TOMY incubation chamber
Transcriptome sequences of S. minutum were obtained in
our previous study [
]. RNAseq reads have been deposited
at DDBJ Sequence Read Archive (http://trace.ddbj.nig.
ac.jp/dra/index_e.html) [DRR003865-DRR003871]. Reads
from the Transcription start site (TSS) library have been
also deposited at the DDBJ Sequence Read Archive
[DRR023220-DRR023221]. Spliced leader (SL) sequences
(DCCGTAGCCATTTTGGCTCAAG ) (D = T, A, or G)
were removed from single TSS reads (99-bp). SL sequences
were deleted to yield 77-bp reads that were then used for
mapping onto the genome [
]. Reads were mapped onto
S. minutum genome version 1 [DDBJ/EMBL/GenBank:
DF239013-DF260911 (scaffolds)], using TopHat with
default parameters [
]. SAMtools software was used for
visualization of read coverage [
Amino acid sequences of KS domains were obtained
from NCBI Genbank with additional sequences from
Eichholz et al [
]. Type I and II PKS and FAS
sequences, representing 39 different taxa, were used for
Bayesian inference and maximum likelihood analysis.
These represent major clades from prokaryote, fungal,
animal, apicomplexan, haptophyte, and chlorophyte
PKSs. Data included KS sequences from other
dinoflagellates (K. brevis, A. ostenfeldii, H. triqueta, G.
polynesiensis, and A. spinosum). The genome browser,
] was accessed in order to retrieve
PKS sequences. Multiple amino acid alignment was
performed with the MUSCLE algorithm [
] included in
MEGA 6 [
]. A maximum likelihood phylogenetic tree
was generated with MEGA 6 using a Le-Gasquel amino
acid replacement matrix with 1000 bootstraps. Bayesian
inference was conducted with MrBayes v.3.2 [
the same replacement model and run for four million
generations and four chains until the posterior
probability approached 0.01. Statistics and trees were
summarized using a burn-in of 25 % of the data [
were edited using Figtree
Active sites of KS, AT, DH, ER, and KR domains and the Nterminus of the KS domain
In order to investigate whether active sites of major
enzyme motifs were conserved, dinoflagellate sequences
from NCBI were aligned with reference sequences from
A. ostenfeldii, A. spinosum, H. triquetra, and K. brevis.
For the N-termini of dinoflagellate KS sequences, other
regions were separated from the KS domain and
searched against the NCBI and PFAM databases [
Multiple alignments and phylogenies of the truncated
sequences were calculated as described above for the KS
NRPS-PKS gene cluster
The antiSMASH server [
] was used to identify
nonribosomal peptide synthetase (NRPS) and polyketide
synthase (PKS) domains. FASTA format protein
sequences were used as input. Results were compared and
further annotation was conducted using PFAM database
]. NRPSpredictor2 was used to identify binding
specificity of the A domains in the NRPS modules [
ITASSER was used to identify the AT domain associated
with the PKS module [
Polyol extraction from S. minutum culture
Cultured cells were collected by centrifugation (9,000 g
and 14,000 g, 10 min, 10 °C). After discarding the
supernatant, the cell pellet was extracted with methanol (three
times) at room temperature. Methanol (100 μL) was
added to the biomass (37 mg, wet weight) followed by
vortexing (1 min), sonication (10 min), and
centrifugation (14,000 g, 10 min, 10 °C) to yield a methanol
extract. The resulting clear solution was transferred to a
new tube. By adding methanol (100 μL) to the residue, a
second methanol extraction was carried out in the same
fashion. The clear second methanol extract was
combined with the first and stored at –30 °C. Additional
methanol (100 μL) was added to the residue, vortexed
(1 min), and kept overnight at room temperature. After
centrifugation, the third methanol extract was pooled
with the previous extracts (total 300 μL), and designated
as the crude extract. To remove lipophilic materials, an
aliquot (50 μL) of the crude extract was suspended in
50 μL water-methanol (90:10) containing 0.5 % formic
acid. The suspension was vortexed (30 sec) and
centrifuged (14,000 g, 10 min, 10 °C) to give a clean solution.
The clean solution was transferred into a new tube
(stock solution) and the insoluble part was discarded.
The stock solution was kept at –30 °C before
NanoLCMS analysis or immediately analyzed after dilution.
NanoLC-MS analysis of the Symbiodinium methanol extract
A Thermo Scientific hydride (LTQ Orbitrap) mass
spectrometer was used for MS data collection. The mass
spectrometer was equipped with an HPLC (Paradigm
MS4, Michrom Bioresources Inc.), an auto-sampler
(HTC PAL, CTC Analytics) and a nanoelectrospray ion
source (NSI). High-resolution MS spectra were acquired
at 60,000 resolution in FTMS mode (Orbitrap), full mass
range m/z 400–2,000 Da with 200 °C capillary
temperature, 1.9 kV spray voltage in positive ion mode.
The lipid-depleted crude extract (stock solution) was
diluted 1:50 by adding water-methanol (50:50)
containing 0.25 % formic acid and separated on a capillary
ODS column (50 × 0.18 mm, 3 μm, C18, Supelco). A
20min gradient was used for polyol separation (10 % B for
0.0–2.0 min, 10–100 % B for 2.0–10.0 min, hold 100 %
B for 10.0–15.0 min, equilibration 10 % B for 15.1–
20.0 min; where solvent A is water:acetonitrile 98:2
and solvent B is water:acetonitrile 2:98, both
containing 0.1 % formic acid; flow rate 2.0 μL/min, injection
2.0 μL loop).
Availability of supporting data
The genome and transcriptome data of Symbiodinium
minutum are available at http://marinegenomics.oist.jp/
Additional file 1: Table S1. Predicted domains from transcriptome
contigs Figure S1. Expression of KS domain-containing genes on
scaffolds of S. minutum. Read coverages of RNAseq (gray line) on KS
domain-containing genes (surrounded by green) show expression in
our standard cultured conditions. In addition, the SL sequence containing
reads (red line) from transcription start site (TSS) library suggest large
multifunctional genes are expressed as a transcript that is not trans-spliced.
Red arrows show trans-spliced sites, located internally in KS
domaincontaining genes. Figure S2. Molecular phylogenetic tree of Type I
and Type II KS domains from prokaryotic and eukaryotic PKS and
FAS, analyzed by maximum likelihood. Type II KS and acyl carrier protein
synthases (ACPS) were used as outgroups. Bootstrap values ≥ 50 % are
marked at appropriate nodes. Details regarding S. minutum sequences are
provided in Table 1. (PDF 9386 kb)
Additional file 2: Figure S3. (A) Alignment of KS domains with those of
other dinoflagellates and animal PKSs and FASs. Asterisks indicate conserved
amino acids required for catalytic activity. (B) Additional Symbiodinium and
K. brevis sequences that are consistent with previous reports. Figure S4.
Alignments of motifs within AT, ACP, KR, ER, and DH domains. Active sites
within the motifs are boxed with dashed lines. Figure S5. (A) Multiple
alignment of the truncated, conserved, N-termini of dinoflagellate KSs (B)
Additional Symbiodinium sequences with the divergent signatures. Lower
panel: Maximum likelihood tree of the N-termini computed with 1000
bootstrap replicates. Bootstrap values ≥ 50 % are marked at appropriate
nodes. (PDF 2705 kb)
Additional file 3: Figure S6. (A) NanoLC-MS (positive ion) profile of the
methanol extract of Symbiodinium minutum. Top Chromatogram, Center
Top: Extract ion (m/z 1072.60, 10.6 min), Center bottom: (m/z 1050.57,
10.6 min), Bottom: MS spectrum. (B) MS spectrum (positive ion) of
the methanol extract (expanded). (PDF 145 kb)
ACP: Acyl carrier protein; ACPS: Acyl carrier protein synthase;
antiSMASH: Antibiotics and Secondary Metabolite Analysis Shell; AT: Acyl
transferase; DH: Dehydratase; ER: Enoylreductase; EST: Expressed sequence
tags; FAS: Fatty acid synthase; HPLC: High-performance liquid
chromatography; I-TASSER: Iterative Threading ASSEmbly Refinement;
KR: Ketoreductase; KS: Ketosynthase; MT: Methyltransferase; NRPS-PKS:
Nonribosomal peptide synthetase-polyketide synthase; PKS: Polyketide synthase;
The authors declare that they have no competing interests.
GB, ES, and MR wrote the manuscript. GB, KH, and ES analyzed the genomic
data. MR performed mass sample preparation, mass data acquisition, and
data interpretation. NS and ES designed and led the research program. All
authors read and approved the manuscript.
We thank Steven D. Aird for editing the manuscript. We also thank Sutada
Mungpakdee for the useful comments from the preliminary surveys for PKS
genes. The authors are grateful to Dr. Mary Alice Coffroth for providing the S.
minutum isolate. The authors acknowledge Drs. Yutaka Suzuki and Sumio
Sugano for TSS sequencing and the OIST sequencing section for RNAseq
sequencing. This work was supported in part by Grants-in-Aids from MEXT
(No. 25440182, 221S0002) and by generous support of Okinawa Institute of
Science and Technology Graduate University to the Marine Genomics Unit.
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