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Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-producing polynesiensis and G.excentricus () Kohli, G. S., Campbell, K., John, U., Smith, K. F., Fraga, S., Rhodes, L. L., & Murray, S. A. (2017). Role of Modular Polyketide Synthases in the Production of Polyether Ladder Compounds in Ciguatoxin-producing Gambierdiscus polynesiensis and G.excentricus (Dinophyceae). DOI: 10.1111/jeu.12405

Published in: The Journal of Eukaryotic Microbiology

Document Version: Peer reviewed version

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Download date:09. Sep. 2018 Rhodes This article isprotected Allrights bycopyright. reserved. doi: 10.1111/jeu.12405-4819 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen f e d c b a Gurjeet S.Kohli Ciguatoxin-producing in Role ofModular Polyketide Kohli etal.---PKS Genes in Article type:Original Article Date:03-Feb-2017Accepted Date:31-Jan-2017Revised Received Date :06-Apr-2016 DR. GURJEET S KOHLI(Orci David Keir Building,David Road,Belfast, 5AG Stranmillis UKBT9 University, Singapore 689528 CB07.06.039, POBox 123 Broa [email protected]; guruk Shauna A.Murray&Gurjeet S.Koh author:Corresponding 36390 Vigo, Spain NSW 2007, Australia Instituto Español de Instituto Oceanograf CawthronInstitute, Alfred Wegener Institute Polarandfor Marine Research, Bremerh Institute for GlobalFood Securi Plant Functional Biology and Climate Change Cluster, University Cluster, Change Biology Climate and Plant Functional AcceptedSingapore Centre for Environmental Life Sciences Engineering, N Article e , Shauna A.Murray a, b a, , Katrina Campbell 98Halifax Street East, Nelson 7010, Z New Gambierdiscus polynesiensis Gambierdiscus polynesiensis a d ID :0000-0002-4578-2355) Synthases inthe Production ofPolyether Ladder Compounds dway, NSW 2007, Australia [email protected] Tel: +61295148 ty, School Biologicalof Scienc ía, CentroOceanográfico deVig li; E-mail: shauna.murray@uts. c , Uwe John on and undergone full peer review buthasnot review undergone fullpeer on and d , Kirsty F. Smith Version of Record. PleaseVersion cite thisarticle as & G.excentricus and and es, Queen’s University Belfast, Belfast, University Queen’s es, G.excentricus ealand e o, Subida a Radio Faro 50, , Santiago Fraga Santiago , ofTechnology S edu.au anyang Technological 404; Fax: +61295144079; aven 27515, Germany (Dinophyceae) (Dinophyceae) f , Lesley L. ydney, Ultimo, Ultimo, ydney, more than 11congeners now desc arethermostableCTXs polyetChemically, andliposoluble cyclic (Skinner etal.2011). re cases CFP of rate The States, underreported (Fleming et al. worldwide, andimpacts ~50,000- contaminated with analogues toxinof Iti the (CTX). ciguatoxin CIGUATERA Fish Poisoning (CFP) is a syndrome caused by the cons Ciguatoxin, Keywords: biosynthesis pathways in . pathways in organisms. other in genes PKS/FAS known with clustering arrangement of domains on the co were found. plants in Type IPKS dehydratase andenoyl-reductase K KS, domain single encoding contigs Seventeen compound. ladder produced by one ofthese megasyn evidence to support that P-CTX-1B, P-CTX-2 and P-CTX-3 are deri are P-CTX-3 and P-CTX-2 P-CTX-1B, that support to evidence 1991). Though nostrains of present in carni toxin principle Indian Oceans) (Kohli the thefrom from Caribbean/At C-CTXs (P-CTXs Pacific, toxicity congeners can be divided into th PKS domains (forming typical typ dinoflagellates. Additionally, 24contigs ( 7, synthases (KS; This article isprotected Allrights bycopyright. reserved. Rhodes etal.2014) in culture. M 4A, CTX-4B) and et al. 19 (Chinain region Pacific Ocean the Polynesia in French 1990, Lewis andHolmes 1993, Yasumoto etal. 2000). biotransformation of CTXs produced by of that polyketide synthases (PKS) Ciguatera. Ciguatoxins are ladder polyether compounds have that Gambierdiscus Abstract G.polynesiensis

Accepted ArticleG. excentricus Gambierdiscus Gambierdiscus , a benthic , prod a benthic , dinoflagellate, G. excentricus and Type 2 (CTX-3C, M-seco-CTX-3C, 49-epiCTX-3C) P- 2(CTX-3C, M-seco-CTX-3C, Type : 8)with to sequence similarity G. polynesiensis et al. 2015aandreferences therein). In , biosynthesis, polyketide syn polyketide biosynthesis, , is important, facilitate as will approaches is it toinvestigatin Gambierdiscus : 106, 1998, Friedman etal. 2008). InP ported has increased recently b vorous fish implicated in CFP ( CFP in implicated fish vorous are involved in their productio ultiple strains of this specie and type II FAS genes were dis andtypeIIFAS ree based on types structural c e IPKSs modules) werefound. T with sequence similarity totyp with sequence similarity 500,000 people despiteannually, ribed (Kohli etal. 2015aandre ntigs and their sequence simila G. polynesiensis thases resemblesthases carb apartial and

Gambierdiscus found 264 contigs G. excentricus have been toprod have shown uces ciguatoxins that cause the uces ciguatoxins type I PKSs, as reportedtype as ot IPKSs, in : 143)andketoreductases (KR; G. polynesiensis : 3, , P-CTX-4A andP-CTX4B (Murataetal. , P-CTX-4A thase, fatty acid thase, fattyacid synthase G.polynesiensis

This differentiation of PKS and FAS encoding single domain ketoacyl s nowbeenstudied s CTX have for Murata etal. 1990, etal. Lewis the Pacific, P-CTX-1B is the 99) 1(CTX- producesType both y 60% over a 10-year period period a10-year over y 60% s common in tropical countries acific Small Island Developing Small acific n. sequencedWe transcriptomes hanges, geographical origin and and origin geographical hanges, tinguished based on the her ladder compounds, with lantic and I-CTXs from the andI-CTXs lantic e II fatty acid synthases (FAS) (FAS) e IIfatty acidsynthases rity andphylogenetic ved from the the from ved ferences therein). These a polyketide origin, indicating on backbone ofapolyether he proposed structure R, s-malonyltransacylase, being significantly significantly being umption of seafood CTXs (Chinain etal.2010,CTXs (Chinain uce P-CTX-1B, there is , first described from : 21) encoding multiple human illness her G. excentricus g toxin g toxin : This article isprotected Allrights bycopyright. reserved. li inthe channels sodium inhibit the presence of novelc indicate polynesiensis in cells with excitable membrane The proposed mechanism oftoxici characterised (Fraga etal. 2011). based ontheproduce CTX it Neuro-2a cell based assay,though Ocean Atlantic region (Fraga the from Canaryin Islands species al. 2010, Rhodes etal. 2014, production, and non-producing strains of magnetic resonance is clearly required. required. clearly is resonance magnetic characterisation ofthetoxin profile of these of species spectrometry (LCMS) techniques, profiles andstructures toxin beendeterminedCTX byliqui have (Kohli etal.2015a andreferences therein). references gastrointestinal andn This therein). various causes ce effected in potentials action spontaneous of and onset cells resu ofsodium ions, influx massive therea is channels, sodium

fractions of (Rhodes etal.2010, Rhodes etal.2014, Kohli etal. 2015b). T belizeanus G. cou P-CTX-4A, P-CTX-4B) P-CTX-3C, (P-CTX-3B, that noCTXs shown CCMP401 has beenrepor and (Hamilton etal., 2002a, Hamilton 1989, Murata etal., 1990a, Lewis 2010). Qualitative detection ofP CTX standards available (Berda compounds of known molecular Atpresent, ave weights. is there andfract isolation the involves byLCMS of toxin Confirmation AcceptedLiposoluble extracts ofother 1).(Table has sometimes been a mismatch between results from cell-based a been determined with certainty (Table 1), and ofthose species amounts. Forthistrace reason, to lowstruggles reach the ofdetection requiredlevels forCTX Article G. belizeanus G. G. australes strain CCMP401 (Kohli etal. 2015b in the study of strains from Polynesia (Chinain the etal. ofstrains in study French have been reported tobe toxic , G. toxicus ted previously (Roeder et al. 2010). Howe Murrayetal unpub data). Gambierdiscus ongeners of CTXs and/or other p other and/or of CTXs ongeners the toxin profile of relatively let etal., 2012). Currently stat posoluble extracts theseof sp -CTX-3C inthe liposoluble fra s such as the peripheral nerve etal.,2002b). These methods , et al., 1991, Satake et al., accompanied by nuclear magnetic ty of CTXs is based on its abil based ty ofCTXs onits is G. pacificus G. polynesiensis species such as as species such and ) or inseveraldifferent st via thereceptor-binding assay G. belizeanus G. G. excentricus Gambierdiscus havenotyetbeenreported (Chinain et 1996) andradio ligand binding G. australes few species of he toxicity ofthe toxicity liposoluble he e ofthe artLCMS equipment lls (Kohli etal.2015a and ecies. Therefore, detailed ction of s, as they may be highly toxic in in toxic behighly they as may s, eurological symptoms in humans ionation ofthe CTXvarious lting in the depolarisation of the werelow compared tothatof require standards. purifiedCTX that that been have there examined, ity to activate sodium channels etal.2011), was found to cells. When CTXs binds tothe binds When cells. CTXs s toxin profile not yetbeen toxin has s d chromatography-mass olyketide compounds that ry limited supply of purified supplyry limited ofpurified ssays, and those using LCMS LCMS using andthose ssays, ver, other studies have have studies other ver, , arecently described ld be detected via LCMS LCMS ld bedetected via in resonance (Murata via LC-MS and/ornuclear viaLC-MS G. belizeanus , G. toxicus 2010). This might might This 2010). rains of of rains Gambierdiscus (Chinain etal. , G. australes G. australes G. pacificus strain strain

et al., have have

G. This article isprotected Allrights bycopyright. reserved. toxicology, chemistry, as well as includin science, fundamental their of our understanding aiding An understanding of the genetic basis of ciguatoxin synthesis w are present in asingle protein Three types of PKSs have been des et al.1999, Jenke-Kodama etal ultimately releasing thepolyketide compound from the megasynth essential for FASs. The thioeste enoylreductase (ER)and ketoredu al. 2005). Otherdomains that mod forms the core structure of FASs acyl alongwith transferasethe (A betweenacylreaction units, history (Kohli etal. 2016). The related are closely (FAS) andPKSs andhav synthases Fatty acid 1987, Leeetal. 1989, Wright et synthesis pathways indinoflag clear that cyclic polyether compounds such as CTXs are produced Based on radio labelled precursor studies, and determinations o health. public cyclic polyether production in dinoflagellates. Previous studie 2015b), this poses asi dinoflagellatesto other (Veldhuis et al.1997, Hou and 200 Lin , (32.5 Gbpfor As et al.,1999). proteins direc acts (that is, it monomer performs a specific func 2005; Khosla etal.,1999). Type IIIPKSs are self-contained ho andplants (Cane bacteria in type analogous FASs to II fashion on aseparatepeptide domain is acid derivatives), partially red 2005). Iterative typeIPKSs can analogous animals andfungito fattyacid synthesis is (Khosla 2005; Khosla etal.,1999). Type the elongation process and releases the polyketide chain (Cane reaches the atoms, last untilit thatmodule 2 carbon contains needed toperform one condensati where modules, are organised sequential each in module contains (Jenke-Kodama etal.2005 and references therein). In modular t

AcceptedGambierdiscus Article species amongs topossess appear species gnificant challenge inthe ongoing effor G. australes tly onacyl units), also called ucing PKS and non-reducing PKSs and non-reducing PKS ucing and used in a fashion re and usedin cyclic . 2005andreferences therein). ellates (Kalaitzis etal. 2010,L ketosynthase (KS) domain, whic rase (TE) domain hydrolyses the al.1996, Murata al.1998).et be further subdivided intored assisting in thedevelopment in assisting II PKSs consist multiof prote andPKSs (Cane et 1998,al. Kh which function as mono-function tion in an iterative manner wit manner iterative an in tion ify the acyl-units condeafter ctase (KR), are selectively pre on reaction and increase the le and the on reaction increase cribed so far. ty Initerative so cribed and35Gbpfor t the largest genomes knownt thelargest fr G. belizeanus G. as chalcone synthase PKSs (Ferrer like

peatedly for chain elongation, ee etal. 1986, Chou and Shimizu in complexes where complexes in catalytic each t to for the responsible find genes modimeric enzymes where each the the domain, TE terminates which of monitoring measures toprotect pe I PKS, a set of catalytic domains domains pe IPKS, asetof catalytic et al. 1999, Jenke-Kodama etal. s have therefore have s used 9, Shoguchi et 2013,al. Kohli etal. et al., 1998;Jenke-Kodama etal.,

T) and acyl andacyl proteinT) (ACP) carrier ucing PKSs (that PKSs ucing fatty produce nsation are dehydratasensation (DH), osla etal. 1999, Jenke-Kodama et ould be immensely useful in h performs the condensation et al., 1998; Jenke-Kodama etal., e a common evolutionary g molecular ecology, evolution, polyketide chain from ACP (that produce truepolyketides) ype I PKSs, ype IPKSs, domains catalytic sent/absent in PKSs, however is it structure, chemical their f hout the usehout ofacyl carrier ngth ofthe polyketide chain by by way of polyketide polyketide bywayof al proteins in an iterative ) (Kohli etal. 2015b), similar allthe cataly ase (Cane etal.1998, Khosla tic domains tic om This article isprotected Allrights bycopyright. reserved. JF303074, large ribosomal subunit Island, Spain (Fraga etal. 2011) macroalgae and turfonatidal p excentricus (21°13’24.18” S;15°43’54.94” W) Gambierdiscus polynesiensis and assembly conditions, RNA extraction Culture MATERIAL AND METHODS andnon-produ producing in synthesis polyether cyclic to linked th was toinvestigate comparison profiles compared,is toxin o is A comparative transcriptomic approach, in which transcriptomes (Stüken etal.2011, Murray etal.2015). 2015, Eichholz etal.2012). AsPK 2008, Pawlowiez etal.2014, Ryan presence ofanovel in typeIPKS the dinoflagel and indicating phylogenetically cluster with ot transcript domain per one encode minutum Symbiodinium polyketide (PKS)synthase genes. brevis transcriptomic sequencing to identify candidate genes related t AcceptedGambierdiscus oftwogeneticallytranscriptomes relatively closely related, m (Rhodes etal.2014), which likelyproduced knownCTX analogs, generated transcriptomes oftwospecies, the presence absenceor of relat of genes likely responsible forcertain pathways has used circu bee has dinoflagellates in pathways genetic of confirmation the Articlespecies, including developing knock-outs, and difficulties in a genetic successfully in difficulties genetically, characterised Due to several factors, including the lack ofdinoflagellate mo better understand thepolyketide/toxin production in todifferentiateimportant betw ostenfeldii circularisquama belizeanus (Monroe andVan Dolah 2008), (Kohli et al. 2015b), (Jaeckisch etal. 2011, Eichholz e (VGO790) was collected onMarc species, species, (Salcedo et al.2012), G. belizeanus G. (Beedessee etal.2015). In encodingdinoflagellates, contigs (CAWD212) Rarotonga, was isolated from Cook Islands ne approach to overcome this di Azadinium spinosum een thegenes encoding the twog her typeIPKS-KS ther domains, . Strain VGO790 is barcoded in ond PuntaHid in ed genes in species with aprop with species ed genes in e presence and expression of ca Recently, PKSgenes werealso Ss andFASs share a similar e (afeature typically observed in March 2013in (Rhodes et al.2 et al.2014, Kohli etal.201 , D8-D10, 771bp), (GenBankID: and G. polynesiensis Karenia mikimotoi Karenia G. australes G. excentricus t al.2012) and h 28th, 2004 asanepiphyte on algo 28º34’27’ (Meyeretal.2015), (Kohli et al.2015b). The this of aim (Pawlowiez etal.2014), Gambierdiscus (Fraga etal.2011)and (Kimura al.2015),et and successfully ally manipulating dinoflagellate del species that have been well xenically culturing dinoflagellates, 5b, Kohli etal.2016, Meyer al. et lates (Monroe and Dolah Van ost likelynon-CTX producing GenBank(GenBank ID: nzymatic domain structure it is is it structure domain nzymatic mstantial information, such as fficulty. In this study, we ensity toproduce that toxin n difficult. To date, confirmation o toxin production o toxin in efore, classified as type I PKS PKS I type as classified efore, ’N, 16º19’52’’W, Tenerife in type II PKSs) but typeII PKSs) in cing similar species. species. similar cing identified in the genome of roups of enzymes, order in to 014). and compared them to the ndidate genes that be that genes may ndidate of species with contrasting contrasting with species of HQ877874, large ribosomal . identified hundreds of Gambierdiscus Heterocapsa small filamentous G. australes, G. polynesiensis Alexandrium Alexandrium KS domains Karenia Karenia

G.

excentricus BLASTx analysis was performed at an E-value cutoff of 10 Transcriptome Analysis (NNNNs) of gaps 100bpand/or containing that more werenot anal and theassembly wasa using default software setting 7.0 Version Workbench Genomics CLC using into contigs assembled CD-HIT out onwebserver carried was andcomparison clustering have afull spliced leader (all first/last the A,orG)” within (D=T, “DCCGTAGCCATTTTGGCTCAAG detected using ChloroP (Emanuels a totalof of1,064,432,504 cDNA librarywas sequenced using San Diego, CA),generating atotalof135,064,950 100 bppaired analysed anyfurther. Identifica HMMPanther, Gene3D, Phobius, C HMMSmart, HMMTigr, ProfileScan, which uses the BlasProDom, followingBLAST2GO databases: FPrint 2015b). This article isprotected Allrights bycopyright. reserved. intensity 60 μmol m (Fraga etal.2011). Strains were subunit, D1-D3, 822bp) and (GenB PKS/NRPS and FAS genes using Pfa using genes andFAS PKS/NRPS and FAS genes. Sequences identif residues and amino acid of for conserved fu used identification sequences from HMMER, (Puntaetal.Pfam 2012) and (Marchle CDD for HMMused the databases beenprovidedhave supplementary in sequences from other dinoflagella developed using sequences identified via text searching the ann usingwere alsoidentified etal.2011), i HMMER(Finn which in annotations (ketosynthase, polyketide PKS, synthase, ketoreduct 2007) was also used. Initial sear transcriptomes, the Eukaryoticof the Core Genes Mapping approa theannotationimprove of the tr using default software settings (Götz etal.2008)BLAST2GO andK Accepted Article andlibrary extraction RNA prepa

The The ) reads mapped assembly. During during theassembly, scaffoldin G. excentricus -2 s -1 lso validated using read mapping. Anycon ; 12:12 light:dark cycle) in in f/2 medium (Guillard in f/2medium and cycle) in ; 12:12light:dark Ryther cDNAlibrary was sequenced ona 22 bp)/partial spliced leader ( leader spliced bp)/partial 22 s andatotalof 883,662,391 ( were used toperformwere used an mapping tion oftransit peptide targete 100 bppaired-end reads. Raw grown at25 °C under cool whi ch for PKS/FAS genes was carri anscriptomes, InterProScan anal ied by HMMER/BLASTx where nofu byHMMER/BLASTx where ied ration was carried ration descr outas oils (Mitchell etal. 2014).To a tes for each enzyme investigat son etal.1999). Contigs conta ank ID:JF303065, large ribosom twolanes ofHiSeq2000 (Illumi yoto Encyclopedia Geneyoto of and m and CDDm could we beobtained, HAMAP, PatterScan, SuperFamily, -9 G. polynesiensis against the of nr database GenBank. against 11-21 bp,) sequence. Sequence half laneof HiSeq2000 (Illumi d towards chloroplast was was chloroplast towards d reads were quality filtered and nctional prediction of PKS/NRPS of PKS/NRPS prediction nctional te fluorescent (light light n-house HMM weredatabases -end reads. The ed out by text searching the ed out the bytextsearching otations andalreadyknown nalyse the comprehensiveness comprehensiveness the nalyse ibed previously (Kohli et al. al. previously et (Kohli ibed ed in this study. Alignments ining the sequence ases, FAS). PKS/FAS genes ysis was carried out ysis in d annotation To oftranscripts. tigs lesstigs than 300bpoflength ch (CEGMA)ch tool(Parraetal. (Huang etal.2010). na, San Diego, CA), generating generating CA), Diego, na, San Genomes (KEGG) analysis Genomes (KEGG) analysis al subunit, D1-D3, 822bp) ysed anyysed further. file 1. After obtaining the (CLC bio, Cambridge, MA) Scan, HMMPIR, HMMPfam, 100bpwere considered to r-Bauer et al.2014)were re discarded and not SignalPHMM, TMHMM, nctional predictions of ) and 118,044,360 ( g was performed performed g was G. polynesiensis 1962). na, na, G.

unique and this indicates that these contigs are not andthis contigs unique isofo indicates these just that different sequence similarity thresholds) and 90.1- different thresholds) sequence99.3% similarity co of therefore wekeptall contigs in did differencesequence notaffect the high numbers contig b in contigs, respectively, had great analysis of et al.2014, Ryanetal.2014, K For For 57.9% the of contigs lacked similarity to any sequences in the transcript candidates. The revealedtranscript 98.6-99.9% analysis that of within and between both the tran 95% and98% a sequence similarity range forother studies protist the in nr databa anysequences to lacked similarity contigs the 20.9% the of contigs had an annota This article isprotected Allrights bycopyright. reserved. SRR3348983 ( Raw datawas submitted totheNCB GETK00000000) under Bioproject numbers PRJNA317708 ( transcriptome shotgun assembly archive ( wi (combined sequences assembled were submitted toGenbank under resulted in77,393 (Mean length Comprehensive transcriptomic libraries of analysisTranscriptomic AND RESULTS DISCUSSION heterogeneity with 1000 bootstraps. was carried outinRAxML Version andtrimmedinspected to they the ensure spanned re coding same software (Kearse et al. 2012) wereused for sequence alignments (Katoh MAFFT et al.2002) and/or Phylogenetic Analysis ( 10 2014). For content 61.6%) contigs > 300 bp, (Shoguchi etal.2013, Pawlowiez (62.3 %for G. polynesiensis

Accepted-9 Article , 24.7%of thecontigs had ananno G. polynesiensis G. excentricus G. excentricus, G. excentricus G. excentricus ). it was determined using BLASTx analysis that atane-value cut that analysis was it BLASTx using determined and it was determined using BLASTx BLASTx determined itwas using 61.6%forand ) andSRR3358210 ( G. polynesiensis ohli etal.2015b, Meyer etal. (Keeling etal.2014) butparti er than 20xcoverage 2). er than (Table the CD-HIT analyses. cluster a : 1148,GC content: 62.3%) and1 etal.2014, Kohli etal.2015 scriptomes to identify potentia respectively. The GC contento accession numbers KX395751 - KX 7.0 (Stamatakis 2006) using GA Clustal etal.199 W (Thompson ted match, 16.1 % hadanon-a I sequence read archive under th partialth PKS/FAS sequences) w t nucleotide level) toidentif tated match, 17.4 %had anon G. polynesiensis G. excentricus G. excentricus transcriptomes transcriptomes showed that 72.1% and88.4% of G. polynesiensis GETL00000000; ) as compared toother) as eukaryotes and analysis that atane-value cut 2015). Read mapping/coverage cular for dinoflagellates (Pawlowiez G. excentricus rms from the same gene in the gene in from the same rms b, Meyer etal.2015, Ryanetal. G. polynesiensis nalysis was performed (at 90%, nr database of GenBank (Table 2). nr database of GenBank(Table Excluding lower the coverage se of GenBank. This is of the in is GenBank.This se etween both species and contigs in in contigs ntigs in ). Full-length PKS and FAS genes genes andFAS ). PKS Full-length y highly similar sequences sequences similar y highly f the transcriptomes was high l isoforms and positive false . Alignments were manually 15,780 (Mean length: 924.5,GC nnotated and63%match of 4) aligners within Geneious® MMA and LG models ofrateMMA andLGmodels accession numbers gion. Phylogenetic analysis -annotated match and and match -annotated ere submitted tothe NCBI 395902 andtherest ofthe G. polynesiensis G. polynesiensis ) and PRJNA317942 G. excentricus were analysed, and -off of 10 were (at -off of

-9 , G. excentricus This article isprotected Allrights bycopyright. reserved. investigated via CEGMA polynesiensis 78 %(361of 458)and74.6 %(342 (CEGMA)mapping approach (Parraetal.2007), and th found that To further assess the completeness ofthe transcriptomes, weus (Table S2). synthesis andhistidine synthesis lysine acid-Glutamine synthesis, Cysteine-Methionine synthesis, valine threonien synthesis, arginine-proline synthesis, Alanine-Aspart dTDP anddTTP),dTMP, tyrosine-phenylalanine-tryptopham synthes ofdCDP, dU dCTP, CTP synthesis andCDP; of UDP,UTP, synthesis 10 datasets. respective To thecompare twotranscriptomes aBLASTn indicating that the two species thatthe twospecies indicating % ofthe match to the the 18S ribosomal 18S the phylogeny of In the species used as a database. 63-75% that had ofthe positive i contigs query species matches datasets of comparisons were carried BLASTn o (Figure Similar S1). species Gambierdiscus polyedrum 2015), reported ofthe of or studies As in genomes transcriptomes transcriptome, 6869 (6% of thetra and Dol (Lidie (SL) Van sequence partial (defined as >11bp within 100bp of ends of contig) dinof of dGDP and dGTP), pyrimidine nucleotide synthesis (Uridine mon synthesis of AMP, ADP, ATP, synthesis of dADP, dATP; synthesis tricaroxalic acid cycle, purine nucleotide synthesis (Inosine m for C-3 carbon oxidative cycle, ( polynesiensis proteins (H2A, H2B,coding H3,H Gambierdiscus G. excentricus

Accepted Article -20 ) was performed,) was where G. excentricus Symbiodinium minutum Symbiodinium minutum G. polynesiensis (RoyandMorse 2012), G. excentricus , respectively. This is comparab G. excentricus (Table S1). Transcriptomes of contigs. The analysis r species occur on much longer branch lengths as longeras occurcompared species onmuch to branch lengths : 159of167 and et (Kohli species 2015b),al. contigs afullsuiteencoding of transcriptome, 8890 (11.5% of t analysis todate(Ryanetal. 2014,Kohl sequences that matched the , G. belieanus transcriptomic database. Howeve G. polynesiensis phosphorylation, pentose phosph G. polynesiensis are genetically quite distant t are quite genetically distant (Bayer etal.2012, Sh Gambierdiscus ah 2007),ah atthe 5’endandSim 4) werefound the in transcipto Azadinium spinosum nscriptome) contigs hada ful of 458) ofthe core eukaryoti evealed that66.8 %of , G. australes G. excentricus le to other dinoflagellate tran contigs wereused as aquery against adatabase of : 161 of167) theessent and and Alexandrium oguchi etal. 2013), G. excentricus he transcriptome) contigs had G. polynesiensis (Meyeretal.2015)and two and G. polynesiensis Symbiodinium kawagutii o each other. This is also evident in in also evident is This other. o each r, the of percentage similarity onophosphate synthesis; G. polynesiensis lagellate-specific spliced leader leader spliced lagellate-specific i et al.2015b, Meyer etal.2015). ic acid-Asparginine-Glutamic n the transcriptome ofn thetranscriptome the species (Figure which in S1), species -leucine-isoleucine synthesis, ed the core eukaryotic genes ilarly, in the l orpartial SL sequence. of GMP, GDP, GTP and synthesis of GMP, GDP, andsynthesis GTP ate pathway, glycolysis, c genes for mes ofmes ut among the transcriptomic transcriptomic the ut among DP and dUTP; synthesis of DP anddUTP; synthesis e transcriptomes contained transcriptome was 70-90%, transcriptome ophosphate synthesis, ophosphate synthesis, analysis (e-value analysis cut-off of is, serine-glycine- is, . The analysis revealed Lingulodinium Lingulodinium ial enzymes required G. excentricus essential histone- essential scriptomes scriptomes contigs had apositive had contigs G.excentricus Alexandrium G. polynesiensis also encoded 95% a full or (Lin etal. (Lin and and

93.6 G.

G. This article isprotected Allrights bycopyright. reserved. typ in enzyme involved seven the hydroxyacyl-ACP dehydratase (DH- (AT-FabD), ACP trans3-ketoacyl s-malonyltransacylase FabH), ACP Fatty acidand polyketide biosynthesis in Murray etal.2015). Gambierdiscus Peridiniales (except broadly follows the trend of din velia Chromera type IIFAS enzymes (KASIII-FabH In ordertounderstand the evolu the process of fatty acid synthesis appears to take place in th (Table S3). These features indic the contigs encoding type IIFAS in type II FASs) in al. 2016), each transcript encode are likely involved in fatty acid synthesis. As seen in other d transcriptomes of We found genes encoding 3-ketoac genera) (Kohli etal., 2016). dinoflag reported many in were of plants, that resembling genes plants (White etal., 2005; Bro and functional characterisation fatty acid synthesis occurs (McFadden, 1999 and references ther each polypeptide nuclearseparate geneencodes that targeted is multi-protein complexes (Jenke-Kodama et al. 2005 and reference on domain catalytic each carry which FASs, II type have Plants Fatty acidsynthesis Accepted evolutionarily clade, closely is that rela sister it indicating (Figure2).W clade monophyletic found in elonga during reaction the out condensation carries which FabF, type IIFAS in lineages green algae 1) phototrophic (Figure as such plants and that and 71typeIketosynthase domain Article Gambierdiscus Gambierdiscus Gambierdiscus group together within the grouptogether the within clade, and was anout-group, was as used and d Gambierdiscus G. australes, G. belizeanus, G.excentricusandpolynesiensis G. australes, G.belizeanus, 3-ketoacyl ACP synthase II with other genes typeII 3-ketoacylcluster synthase FA ACP Azadinium . Transit peptides targetedtowa . Transit . Unlike other. Unlike protists, only on wn etal.,2010andreferences t ) and Suessiales form monophylet form Suessiales ) and ate that the genes are theate that genes fullyfu oflagellate evolution, inwhich of all the FAS domainof all FAS catalytic the enzymes (Table S3). Residues c . Phylogenetic analysis of32ty tionary history of these genes, d individual FASenzyme/domain e IIFASwereconfirmed all in ithin the dinoflagellate clade, , AT-FabD, DH-FabZ, ERER-FabI, yl ACP synthase I, II&III(KA s from prokaryotic andeukaryo from prokaryotic s FabZ), andenoyl-ACP reductase Gambierdiscus inoflagellates formed adistinc rds therds chloroplast werefound ted to e type of gene family encoding e typeofgenefamily encoding inoflagellates and (Kohliprotists et

e chloroplast. nctional, andthat wholeorpart of herein). Recently, these type II FAS typeIIherein). FAS Recently, these the orders Gonyaulacales, s has been s carried higher has outin the evolution genes these of separate polypeptides that form a concatenated phylogeny of 5 pe II 3-ketoacyl ACP synthase I pe II synthase 3-ketoacyl ACP four species of of species four , indicating the presence of presence , the a indicating SI-FabB, KASII-FabF, KASIII- omprising the omprising foractive sites Gambierdiscus ellates and other protists (116 towards the chloroplast, where tion ofthefatty ein). Gene knockout studies studies knockout Gene ein). ic clades. All fourspecies clades. of ic s therein). a In therein). plants, s (afeature observed typically Alexandrium KR- FabG) was carried carried KR- FabG)was out. tic PKS and FASs, revealed reductase FabG), 3- (KR- (ER-FabI) the in (Table S3) that S3) (Table t well-supported (Orretal.2012, Gambierdiscus formed the formed acid chain, was S genes from in 70%of KASII- I al. 2015). synthas peptide nonribosomal al. 2015b).However, recently at transcriptomes ofabacterial co (encoding multiple PKS domains) within bacterialdomains the typeIPKS clade during phylogenet previous studies, duetothe lac None ofthese transcripts encode PKS domains per transcript were also found in transcriptsIn additiontothe en transcritpomes also encoded the of the each domain in KS asingle encoding transcripts conserved ExExGYLGcentrally moti domain pertranscript (Table 3).Dinoflagellate transcripts enc these contigs encoding KS domains in domains, they were classified as cluste phylogenetically but PKSs) II type observed in typically 2014). Asthese contigs coding KS (Kohli et al.2015b)partial) in domains and22KS (Kroken etal.2003, John etal. partial and7full KRdomains wer transcriptomes of werestudy, domains encodingIn this KS contigs found tobehig This article isprotected Allrights bycopyright. reserved. have to occurred likely functions also are novel for selection Accepted bacterial homology,recent that well as multiple toconsider as need toconsi pathways attempting toidentify genes inv saxitoxin synthesis into dinoflagellates species (Stüken et al. novel introduc the for responsible likely be to found been have relatively recent bacterial orig that dinoflagellates ca indicate was also foundNRPS in on phylogenetic analysis (Bach dinoflagellate despite origin, t ArticleS4). In encoded 86full and20partial KS australes primary focus of this study. In domains KS areanessentia Since Polyketide biosynthesis G. polynesiensis G. polynesiensis and

This gene wasfound toencode di G. belizeanus G. G. excentricus transcriptome, encodedcontigs 73fulland70 partialKS domain der both genes of clear dinoflagellate or G. polynesiensis ( G australes he fact that clustered it withi 2008), thesearch for genes en in, likely through processes su processes through likely in, Gambierdiscus e (PKS-NRPS) was was in found e (PKS-NRPS) n express genes incorporated in k of a dinoflagellate specific varoff etal. 2015). Atranscript ntaminant of the strain, as the ofthe as ntaminant strain, and coding single PKS (KS) domains olved indinoflagellate seconda type I PKSs (Pawlowiez etal. (Pawlowiez typeIPKSs ranscript encoding ahybridpo d adinoflagellate specific SL l andhighly conserved domain, domains and7full KRdomains conserved N-terminal motif ExEx domains, encoded one domain p e found (Table 3, Table S4). S were considered tohave likely f at the N-terminal region (Jo f atthe N-terminal region G. polynesiensis G. excentricus : 90fulland 12 partial; transcriptome (contig 19937, (contig transcriptome Table3). results These noflagellate anda SL specific , other studies have reported KS domains from G. excentricus G. polynesiensis and and . In In . G. polynesiensis G. excentricus G. polynesiensis Amphidinium carterae n the bacterial typen the clade based bacterial IPKS SL and clustering of these KS andclustering ofthese SL 2011). This indicates that studies and ch as lateral as genech transfer, which (Murray etal. 2015). coding this domain was the domain was this coding strains werenot axenic (Kohli et sequence. 5’endof In the at the red with otherred type with IPKS-KS encoding partial hybrid PKS- 2014, Kohli etal. 2015b). G. belizeanus G. gene duplications, loss and oding single KS domains have a to the genome to the of apparently imilar toother dinoflagellates, tion of some of the genes fortion ofsome genes the ry metabolite biosynthetic igin and those with relatively lyketide synthase – lyketide synthase , 24 contigs encoding encoding , 24multiple contigs hly numerous and diverse in the the in diverse and numerous hly ic analysis, these contigs thesecontigs analysis, ic hn etal., 2012). Eighteen involved in PKS biosynthesis biosynthesis PKS in involved werefound (Table 3, Table been partof the strain TB-92 strain (Pawlowiezetal. G. polynesiensis G. polynesiensis er transcript (afeature GYLG. transcriptome, contigs contigs transcriptome, and and also encoded one polyA tail, indicating its G. excentricus : 74 fulland40 (Bachvaroff et (Table 3). (Table s and 1

G. Van Dolah 2008, Eichholz al. et dinoflagellates formed adistinc sequences (Figure 3). Within the protistan clade, transcripts e sequences with bootstraptype IPKS and high separatesupport f position of transcripts). Transcripts encoding single KS domain (b contaminants bacterial domains potential KS andidentify the belizeanus of these genes may be involved i leveland88-93 acid amino %sim KS andA,whichdomains clademonofunctional C in were 98-99% expression of monofunctional KS ( butonlyputative-MTX-3 MTX-1, Rhodes etal.2010). P-CT P-CTX-3C, produce P-CTX-3B, belizeanus G. This article isprotected Allrights bycopyright. reserved. species of appears to produce both CTXs a G. australes of KSdomains from chlorophytes, (Figuredomains 3),the protista tothe Inaddition PKSs. sub-clad Chlorophytes, apicomplexa andha hybrids werePKS-NRPS contigs notbedeterminedcould ifthese domains fromKS all four Amphidinium, Heterocapsa AT) from bacteri (cis type IPKS formeddomains separate six (Figure 3).Clade clades Dconsiste one ormore active residuessite with alltheir (Figure3). residuesdomains active intact site (Clade A,BandC),as reported dinoflagellate ofclade transcripts encoding KS domains, single Phylogenetic analysis ofall the The discovery of sequences encod com specific for genes responsible these producing biosynthesis toxin/polyketide the study profile of of strains specific (Satake etal. 1993)andgambier a range ofother polyketide comp polyether/polyketide compoun strains. This compound might be a new congener of CTX/MTX or an (Length: 30289 bp;Openreadfra AcceptedG. polynesiensis be ofdinoflagellate origin Article Gambierdiscus (Kohli et al. 2015b) 3), (Table and : putative MTX-3), and the strains used in this study andthe this MTX-3), used strains :putative in encoded a partial typical typeI encodedathat of typical PKS consisted modular partial G.belizeanus G. polynesiensis produce differenttypes produce of toxi Gambierdiscus , opensnewavenues of investig and produce MTXs (Table 1,

previously (Kohli etal. 2016). KS domainsKS found in study this 2012, Pawlowiez et 2014,al. Ko

d. Other ofspecies/strains t well-supported clade as seen a and the recently discovered h discovered recently the and a nd MTXs (Fraga et al. 2011). Desp G. polynesiensis n clade also consisted ofwell- 3).KS (Figure domains intact n theproduction of a polyketid es consisting of dinoflagellat oxide (Watanabe et al.2013). T ounds such as gambieric acids ( me: 30141 bp;GC content: 57.9 domains in the four species. We ilar atthe nucleotide level (F Rhodes et al. 2014). Based on t on Based 2014). al. et Rhodes apicomplexa and (F apicomplexa haptophytes ing typical typical modularing typeIPKSs ptophytes are alsoknowntopos X-4A, P-CTX-4B using LC-MS anal LC-MS using P-CTX-4B X-4A, is a potent of producer however is CTXs, does not produce species, species, thehowever transcripts as were incomplete it was performed toexamine thee (Figure 3).Clade Dalso c G. australes Gambierdiscus ns, no differences were found i found were differences no ns, ation (Table 3).Contig 50464 fou Gambierdiscus KS domains nothave KS clade Bdid in AandC Clade consisted of KS e monofunctional and multiple KS andmultiple monofunctional e supported and distinct sub-clades supportedsub-clades anddistinct ncoding single KS domains from from domains KS single ncoding from contigs containing multiple , and from in previous studies (Monroe and igure 3). It is possible that some they formed three sub-clades ybrid PKS-NRPS found in hli etal. 2016). Within the s groupeds protistan with other e compound produced by all d of KS domains from: modular modular from: domains KS of d herefore is it imperative to ased on the phylogenetic pounds. oxicity assays, rom other prokaryotic PKS Nagai et al.1992), gambierol

: MTX-1 and MTX-3; :MTX-1 putative or typical typeIPKSs. or typical in in observed 12 of groups have notbeenhave foundto %). KS domains%). KS encoded in ite the fact that this group of similar to each other at sess typical modular type I igure 3). y other type of y other ysis (Kohli etal. 2015b, Gambierdiscus, in order todetermine the ontained multiple other are knowntoproduce G. australes volutionary history of 6 modules modules 6 G. excentricus which may which and n the n the G. nd in nd in

processes these s that is afterthe occur might carbonbackbone polyether ladders during polyketi epoxidases/monooxygenases and 4different types epoxideof hydr and elucidated from adinoflagellate of polyether the biosynthesis associatedwith geneclusters No one species (Kohli etal.,2016). encoding single enzymes/domains; often exhibiting high sequence retain PKSs the some modular geneclusterwhy structure andoth for polyketide biosynthesis. Different evolutionary and functio polynesiensis sequences that encoded epoxidase hydrolases (Figure 5). In support ofthe above proposed biosynt epoxidation and polyepoxide cyclisation carried out via PKSs, e back the carbon which in pathway Rein andSnyder2006) and MTX-1 ( 2003). Similar totheproposed bi (Leadlay cluster gene biosynthesis the monensin in encoded also polyepoxide cyclisation that mig produced is backbone via modulara typeIPKS andunde assembly type IPKSs(Leadlay etal. 2001, polyether compounds, structurally enzymes/domencode all the which multimodular PKS as 50464such dinoflagellates in contig that p different polyketides. However, this study has also revealed th single PKS enzymes/domains might work iteratively in multiple P domains/enzymes (Mon been attributed to asecondary se In dinoflagellates, the origin of transcripts encoding single t sequence AT-DH-ER-KR-ACP-TE; similarity: 81.8%atamin encodes: 84.2% ataminoKR-ACP-TE; level)and sequence similarity: acid 85.2%similarity: atamino level), acid were alsofound in polyether ladder compound (Figure 4).Sequences resembling PKS has produced resemblesbeen bythis proposed,which aparti last the in megas module is that the it which indicates domain, This article isprotected Allrights bycopyright. reserved. Accepted Article the within clustered contig this G. polynesiensis G. polynesiensis . In toanearliertheo contrast G. excentricus (Table S5). These sequences encoded 13 different types of roe andVanDolah 2008; Eichholz etal.201 protistan clade (Figure protistan 3).Th todate. Monensin and nanchang Sun etal.2003). In monensin ht be performed by epoxidases a (contig 6057; DH-ER-KR-ACP-TE;sequence encodes: (contig osynthesis pathway forbreveto paration of typical type Imul de synthesis (Shimizu 2003), t 2003), (Shimizu synthesis de bone is polyketideproducedbone is via s/monooxygenases andepoxides/monooxygenases hy closest to polyether ladders, ains in a sequential manner (op manner a in sequential ains Kohli etal.2015b), wepropos G. australes ry stipulating epoxidation and epoxidation ry stipulating (contig 20703; (contig encodes: ACP-KS-AT-DH-ER- ype I PKS catalytic ype IPKS catalytic domains has e last module consisted of TE s biosynthesis, the carbon carbon the biosynthesis, s ynthase. Astructure that could be e presence typeI oftypical timodular PKS into single single into PKS timodular nal constraints might explain explain might nal constraints G. polynesiensis ladder compounds have been poxidases and epoxide ynthesised by PKSs. byPKSs. ynthesised and bymodular produced are hesis pathway we also found pathway we also hesis he hypothesis here proposes ossess large gene clusters largegeneclusters ossess G. belizeanus G. mycin are non-ladder non-ladder are mycin xins (BTX) (Leexins etal.1989, etal.2001, Oliynyk etal. nd epoxidehydro ers are separated into genes al carbon backbone of a of backbone al carbon KS pathwaysKS to produce e a possible CTX biosynthesis biosynthesis, followed by diversity within KS genes KS genes of within diversity rgoes epoxidation and olases in en reading frame)required 2). This indicates that these o acid level). drolases in cyclisation of G. excentricus (contig 11718; contig 50464 G. excentricus lases that are and G.

This article isprotected Allrights bycopyright. reserved. the genecatalogues of In conclusion, CONCLUSION proposed biosynthesis of ciguatoxin. Sequencing of the Prof. thank Naresh oftheKumar We University of New South Wale ACKNOWLEDGMENTS responsible for the majority of totheare crucial formation group major of biot ofthis marine producing strains. The that be polyketide may synthesis and polyketide biosynthesis in we ofPKS genes, diversity prese libraries of full transcripts described from a single genus of associated with23 regulatory pathways, thegene catalogues pro amongst the most comprehensive yet found in a dinoflagellate. I Zentrum fürZentrum Polar- undMeeresfors program research of Alfred-W PACES the was provided bythe U.J. Research Council (FT120100704, of Ramaciotti centre genomicsfor atUniversity of SouthNew Wa Russell and Tonia forMangs performing sequencin transcriptomic In Fritz Lipmann forAgeResearch, Leibniz-Institute out atthe Bachvaroff, R., T. Williams, E LITERATURE CITED Australian mobility grant and Bayer, T.,Aranda, M., Sunagawa, AcceptedBeedessee, G.,Hisata, K.,Roy, M Article Harmful Algae. module PKS/NRPS found synthase genes identified by genomic survey of the symbiotic di the dinoflagellate symbionts Voolstra, C.R.&Medina, M. 2012. Symbiodinium minutum Symbiodinium

Wellington, New Zealand. Zealand. New Wellington, results presentedresults here are a ste ., Jagus, R.&Place, A.R.(2015 BMBF PT/DLRIBFKZ 01DR14006. the marine biotoxin biotoxin the marine related sea Gambierdiscus. . . C., Satoh, N.&Shoguchi, E. BMC Genomics, nt a clear distinctionnt aclear between S., Yum, L. K., DeSalvo, M.K. associated with toxin with associated producti in dinoflagellates. DP120103199) for funding SM and chung and Austra and chung ofreef-building corals. G. polynesiensis Symbiodinium We identified numerous genesWe identified relatedto 16:1. The Conference 16th International on and lian Academylian o p towards recognising the genes the recognising p towards G. excentricus transcriptomes: genome insights into genomeinsights transcriptomes: G. excentricus ) A noncryptic noncanonical multi- noncanonical ) Anoncryptic 2015. Multifunctional polyketide dinoflagellate. In addition tothevast PLoS ONE, , Lindquist, E., Coffroth, M.A., oxins, that is currently is that oxins, stitute Jena.We Dr.Helena thank food poisonings worldwide. worldwide. poisonings food genes responsible for fatty acid on in these twociguatoxin- n addition tothegenes vide the most exhaustive les. We thank theAustralian egener-Institute Helmholtz- g of g of s (Australia)s forhelpwith the f Science DAAD German- transcriptome was transcriptome carried GK. Financial support to 7:e35269. presented hereare G. polynesiensis noflagellate, noflagellate, courtesy courtesy which which Emanuelsson, O., Nielsen, H. & Eichholz, K., Beszteri, B.& John, U.2012. Putative monofuncti Chou, H.N.& Shimizu, Y. 1987.B Chinain, M., Darius, H.T., Ung, This article isprotected Allrights bycopyright. reserved. Faust, M.A.1995. Observation of M.,Faust, Chinain, A.&Pauil M. Cane, D.E., Walsh, C. T. &Kho Brown, A.P.,Slabas, A.R.& Raffe AcceptedFraga, S.&Rodríguez, F.2014. Genus Fleming, L. E., Baden, D.G., Be Finn, R.D.,Clements, J.& Eddy, Article polynesiensis Growth and toxin production in the the production in andtoxin ciguatera-causingGrowth dinoflagel predicting chloroplast transit peptides chloroplast transit cleavage and their site predicting adinoflagellate-specificunits: feature? 109:2184-2185. of dicarboxylic andinvolvement chain possible carbon backbone polynesiensis species of issues in epidemiology outreach. and in community issues widely differing sites, including two new species. permutations, and mutations. Photosynthesis pathways, structure and organization. In: Wada, H., Murata, N. benthic dinoflagellate. benthic description of with Oceanographic Commission ofUNESCO.245-248. (eds.) Harmful T. M. L.&Wyatt, Algae searching. Gambierdiscus Nucleic AcidsRes., , sp. nov. (Dinophyceae) in culture. . Springer. Dordrecht, The Netherlands, 11-34 Gambierdiscus silvae sla, C. 1998. Harnessing thebio J. Phycol., an, J.A.,Weisman, R.& Blythe A., Cruchet, P.,Wang, Z., Pon Protist, Von Heijne, G.1999. ChloroP, a lac, 1999.S. Morphology and m S. R.2011. HMMER web server: rty, J.B. 2010. Fatty acid biorty, J.B.2010. Fattyacid (Dinophyceae): iosynthesis of brevetoxins. Ev sand-dwelling toxic Dinoflage 39:W29-W37. 165:839-853. Gambierdiscus Science, 35:1282-1296. . 282:63-8. Xunta deGalicia and Intergovernmental PLoS ONE, Toxicon, sp. nov., a new potentially toxic epiphytic nov., anew sp. toxic potentially G. pacificus in the Canary Islands (NE Atlantic Ocean) Islands (NE Canary Atlantic the in 56:739-750. 7:e48624. J. Phycol., In ton, D.,Laurent, D. & Pauillac, S. 2010. synthsis in plants-metabolic plants-metabolic in synthsis : Reguera, B.,Blanco, J.,Fernandez, , D.G.1998. Seafood diseases: toxin , sp.nov., synthetic code: combinations, onal type Ipolyketide synthase neural network-based method for idence fortheidence ofthe origin mixed olecular analyses ofthree toxic interactive sequence similarity llates (Dinophyceae) from 31:996-1003. s. G. australes 9eds), Protein Sci., late acids. Gambierdiscus Lipids in J. Am. Chem. Soc., Am.Chem. J. , sp. nov., and 8:978-984. G.

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Wright, J.L. C., Hu, T., McLach Y Zhang, J., Zheng, S.W., White, Yasumoto, T.,Igarashi, T., Legr This article isprotected Allrights bycopyright. reserved. Watanabe, R., Uchida, H., Suzuki Thompson, J.D., Higgins, D.G.& The, H.V.2009. HocSinh Tao Hai Sun, Y., Zhou, X., Dong, H., Tu, Stüken, A.,Orr, R.J.S.,Kell Stamatakis, A.2006. RAxML-VI-HP Skinner, M.P., Brewer, T. D., Jo AcceptedVeldhuis, M. J.W.,Cucci, T. L. Article spectroscopy. Structural elucidation of ciguatoxin congeners by fast-atom bom evidence foran unusual carbon deletion process. of apolyketideconfirmation proof pathway, of a baeyer−villige biosynthesis. 10303. from thefrom dinoflagellate Y., Inoue, A.&Yasumoto, T. 201 Phycol., Trang.Nha Trang, Nha Nguyen, Vinh Oceanography, Nanchangmycin. Ionophore from 6:e20096. of nuclear-encoded genes forthe thousands of taxa and mixed models. 5:e1416. Poisoning in the Pacific phytoplankton using two new fluorochromes: taxonomic andecolog taxonomic two fluorochromes: new using phytoplankton and weight gap penalties choice. matrix progressive multiple sequence alignment through sequence weight Streptomyces nanchangensis Streptomyces 33:527-541. Annu. Rev.Biochem J. Am. Chem. Soc., Am.Chem. J. mann, R.,Murray, S.A., Neilan, & Sieracki, M.E.1997. Cellul lan, J.L., Needham, J.& Walte G., Wang, M., Wang, B.&Deng, .M. &Rock,C.O.2005. The stru hnstone, R., Fleming, L. E.& and, A.M.,Cruchet, P., Chinai Islands (1998 to2008). Gambierdiscus toxicus Gibson, J.1994. W T. Gibson, CLUSTAL , T., Matsushima, R., Nagae, M. Roi Co SongVo DayVung Bien C: maximum likelihood-based maximum C: phy Chem. Biol., Chem. 122:4988-4989. 3. Gambieroxide, anovelepoxy ., 74:791-831. neurotoxin saxitoxin in dinofl in saxitoxin neurotoxin NS3226 Encoding Biosynthesis of the Encoding Polyether NS3226 Biosynthesis Bioinformatics, 10:431-441. Nucleic Acids Res., GTP2 strain. PLoS Neglected tropical diseases, B.A.&Jakobsen, K J. Am. Chem. Soc., Am.Chem. J. ar DNA content of ar DNA content marine r, J. A. 1996. Biosynthesis r, J.A.1996. Biosynthesis of DTX-4: Lewis, R.J.2011. Ciguatera Fish n, M.,Fujita, T. &Naoki, H.2000. Z. 2003.AComplete GeneCluster 22:2688-2690. ctural biology of type II fatty acid oftypeIIfattyacid ctural biology , Toyohara, Y., Satake, M.,Oshima, : improving the sensitivity of sensitivity the : improving Ven Bo Viet Institute Ven BoViet Nam. of 22:4673-4680. Tetrahedron, logenetic analyses with agellates. r oxidation step, and polyether compound bardment tandem mass 118:8757-8758. ing, position-specific . S. 2011.Discovery ical Implications. 69:10299- PLoS ONE,

J. This article isprotected Allrights bycopyright. reserved. hydroxyacyl-ACP dehydr ACP ketoacyl III,synthase s-malonyltransacylase, trans3-ketoac 2:Concatenated Figure phylogeny bootstraps. GAMMA was inferred RAxMLusing model rate tree. Analysis in of 1:Phylogenetic Figure LEGENDS FIGURE Figure 5: Proposed 5:Proposed Figure of P mechanism reductase; thioestrase. TE: acyl protein; acylKR: ketoreductase; AT: ACP: transfer carrier the megansynthase has been proposed. Following abbreviations we of resembling a carbonbackbone compound ladd polyether partial contig_50464 in representat 4:Aschematic Figure rate heterogeneity and 1000 bootstraps. 3:Phylogenetic Figure and 1000bootstraps. andthecharacters phylogeny RAxML, inferred using was GAMMAmo and animal bacter fungi, plants, dinoflagellates, apicomplexa, chromera, The alignment consisted of 531 ch IIandtypeIketosynth synthase Accepted cyclisation to form ciguatoxin. produced(preciguatoxin) bypoly other prokaryotic and eukaryotic consisted of573 characters, 478 eukaryotic typeI&IIpolyketide Article Chromera velia Chromera G. polynesiensis G. polynesiensis analysis showing a clear distinction bet distinction aclear showing analysis analysis of ketoacyl ketoacyl domai of analysis (KS) synthase which was used as anoutgroup. atase and enoyl-ACP reductase) from 18 di ia showing the position ofeach the position showing ia ase domains of polyketide synth transcriptome. Biosynthetic pa synthases (PKS) and fatty aci synthases sequences encoding KSdomain f taxa. Analysis was inferred us ketide biosynthesis undergoes e ion of type I polyketide polyketide I synth type of ion aracters and103 representativ of five enzymes involved in ty acific-ciguatoxin (P-CTX) prod (P-CTX) acific-ciguatoxin Vitrella , stramenopiles, haptophytes, green algae, The alignment consisted of 1431 d synthases. The alignment ase; DH:dehydratase; ER:enoyl ween type II3-ketoacyl ACP major group in the phylogenetic ase complex encoded by thway andastructure ofa ing RAxML,GAMMAmodeling of ns from prokaryotic from and ns yl ACP reductase,yl ACP 3- ases andfatty ases acid synthases. pe II fatty acid synthesis (3- synthesis pe IIfatty acid e sequences from rom 10dinoflagellates and30 uction. Polyene Polyene uction. poxidation and epoxide poxidation andepoxide heterogeneity and1000 er that could beproduced by re used: KS: ketosynthase; re ketosynthase; used:KS: noflagellates and one other del of rate heterogeneity

enzymes. presence denote the ofS “polyA” excentricus sequences respectively. Table S4: Polyketide synthase genes found in and Supplementary file 1: Alignments 1:Alignments file Supplementary identified in Table S5: Sequence properties of Table S3: A list of typeIIFAS found S3:Alist genes in Table out of167 and polynesiensis and Table S2: Description of sequences from S2:Description ofsequences Table This article isprotected Allrights bycopyright. reserved. Accepted Article genecatalogue ofthe of allthe S1:Alist sequen Table using RAxML, GTRCAT model ofr Phylogeneti S1: Figure SUPPORTING INFORMATION G. polynesiensis Alexandrium and G. polynesiensis encoding essential enzymes for various metabolic pathways ( pathways metabolic various for enzymes essential encoding G. polynesiensis G. polynesiensis species. The alignment consiste . G. excentrucus c analysis of18s analysis ribosomal rDNAc from var and : 161outof 167 enzyme transcriptomes. Sequences names with a prefix “sl” and/or Sequences with a prefix names “sl” transcriptomes. ces encoding the histone prot histone encoding the ces G. excentricus pliced leader and a polyA tails the transcripts encoding epoxi ate heterogeneity and1000bootst used to developfigures 1, 2, and G.polynesiensis Gambierdiscus excentricus Gambierdiscus australes Gambierdiscus australes . d of 1822 characters. Analysis s werepresent). . eins (H2A,H2B, H3, H4)found in 3 and HMM databases for various t the 5’and3’ end ofthe dases and epoxide hydrolases ious and raps. , G. belizeanus G. , Gambierdiscus G. belizeanus Gambierdiscus G. excentricus was was inferred , G. excentricus , , G. Fukuyoa : 159

This article isprotected Allrights bycopyright. reserved. Acceptedbelizeanus G. G. australes Article excentricus G. CTX Various MTX polynesiensis G. CTX LC-MS MTX assays Geographical Species of 1:Geographic distribution andtoxicity Table 2010) al. et (Litaker Caribbean Barthelemy Island- unpubl. Data),St. Australia (murray 2011), Queensland, Pakistan (Muniretal. et al.2011), (Leaw Becerril 2004),Malaysia BecerrilAlmazán and Caribbean (Hernández- 2009) ,Mexican al. et (Litaker Florida Belize (Faust1995), 2014) and Rodríguez Canary Islands (Fraga (Munir etal.2011), al. 2009),Pakistan et (Litaker Hawaii USA (Rhodes etal.2010), Islands2013), Cook (Nishimura etal. Japan (Chinain etal.1999), Polynesia French et al.2015) pers. cominNascimento 2015), Oman(Saburova al. et (Nascimento al. 2011),Brazil Canary Islands et (Fraga al. 2014) Cook Islands (Rhodes et Vietnam (The2009), 2011), NhaTrang- Pakistan (Muniretal. (Chinain etal.1999), Polynesia French distribution 2011) al. et (Fraga positive NCBA- 2010) (Chinain etal. RBA- positive et al.2014), 1999, Rhodes (Chinain etal. MBA-positive Toxicity 2010) (Chinain etal. RBA- positive 2010) (Chinain etal. positive 2010), RBA- Rhodes etal. al. 2013, Nishimura et 1999, (Chinain etal. MBA-positive Gambierdiscus 2011) al. et (Fraga positive NCBA- 2014) Rhodes etal. al. 1999, (Chinain et positive MBA- al. 2013) (Holland et positive HELA- 2010) Rhodes etal. al. 2013, Nishimura et al. 1999, (Chinain et positive MBA- positive , HELA- species used in this study. used in this species N/K al. 2014) Rhodes et 2010, al. et (Chinain Yes al. 2015b) al. 2015b) (Kohli et N/D al. 2015b) Kohli et al. 2014, Rhodes et al. 2010, (Rhodes et N/D 2014) al. et (Rhodes detected only MTX3 2015b) al. (Kohli et detected only MTX3 2014) al. et (Rhodes detected MTX3 MTX and (percentage) 300-6418 contigs Total numberof 872 10* 69x-993x* 200-295 > 10,000x 26712 1000x-10000x 300-7993 57449 100x-1000x 300-15192 8902 50x-100x 300-30289 8363 20x-50x 300-29981 12367 5x-20x 1105 1x-5x 300-8556 311-1756 (percentage) 300-722 contigs 43 Total numberof 1* 16x* > 10,000x 245* 300-5417 487 1000x-10000x - 1* - 25122 100x-1000x 1* 300-7279 14409 50x-100x 300-7013 15779 20x-50x 17443 5x-20x 300-7378 4109 1x-5x 300-7742 300-1596 No. Coverage of Annotated

This article isprotected Allrights bycopyright. reserved. Accepted Article e-value cut-off of 10 Table 2: Coverage and annotation statistics of 77393 245-7742 contigs 115780 200-30289 -9 -9 was applied during analysis. BLASTx Gambierdiscus polynesiensis polynesiensis Gambierdiscus abedsu xetiu excentricusGambierdiscus (962.7) (962.7) (811.1) (505.9) (355.2) (1148) (807.2) (885.1) (1182.8) (1441) (1393.9) (814.2) (415.1) (mean) Length (924.5) (924.5) (245.8)* (695.8) (972.6) (1017.4) 21 11 22 10 251 146 90 12790 7395 4937 36 7113 4431 2865 38 8825 4058 2896 28 12349 2736 2358 12 3493 343 273 match (20.9%) (20.9%) 24192 - 10* - 389 10* 326 157 13740 7484 5488 46 34627 13042 9780 101 6273 1304 1325 10 6483 931 949 5 10365 1064 938 1007 41 57 (24.7%) 19131 G. excentricus LSxaayi PKS BLASTx analysis and match match Annotated Non- (16.1%) (16.1%) 18704 (17.4%) 13430 G. polynesiensis G. match match No (63%) (63%) 72884 (57.9%) 44832 gene catalogue. An sequences 172 115

thioestrase;reductase; TE: A:Ade ketoreductase; acyl ACP: acyl protein; AT: transferase;carrier from Kohli etal., 2015. Followi 156K -C 61.1 11718 63.2 13526 AT(Partial)-DH-ER-KR-ACP- 94589 KS-ACP 45222 ACP-KS G. belizeanus KS-(Partial)-AT-TE-KR 66.9 This article isprotected Allrights bycopyright. reserved. Accepted G. australes Organism G. belizeanus G. australes polynesiensis G. excentricus G. ArticleOrganism Gambierdiscus 3: Totalnumber of Table d (PKS)polyketide synthase associated sequences* PKS below 300bp

transcriptomes. Results for 47 SAP 58.5 65.1 38797 ACP-KS-AT-KR-ACP 24973 KS-ACP 70 SA 65.9 67.6 63.7 105365 ACP-KS(Partial) 64398 ACP-KS(Partial) 47701 KS-AT 45392 KS(Partial)-DH-KR-ER-ACP- / R 58.6/62.2 60.0/60.3 20703 ACP-KS-AT-DH-ER-KR- 64.3 10913 KS-KR-DH-ER-ACP 58.8/65.8 60.4/59.1 Name Sequence 58.5/66.8 59.9/60.9 6/2 KR 74/40 KS 59.8 7/4 KR 60.4/62.7 90/12 KS 7/1 KR 73/70 KS 7/0 KR 86/20 KS domain (Full/Partial) Transcripts of Total number Sequences with multiple PKS domains ng abbreviations were used: KS: Sequences with single PKS domains nylation domain of non-riboso TE TE(Partial) noigDmis GC content (%) ACP-TE Encoding Domains (%) AverageGCcontent Domain Encoding Gambierdiscus australes # DH:dehydratase; ER:enoyl ketosynthase; KR: mal peptide synthase. omains found in four and 60.1 63.1 59.0 (Full/Partial) domain G. belizeanus weretaken This article isprotected Allrights bycopyright. reserved. # non-ribosomal peptide polyketide synthase synthases *Hybrid 144687 141304 KR-ACP-KS(Partial) 139846 55.5 (Partial)KS-A 55.5 ACP-KS(Partial) 138240 136511 (Partial)KS-KR-ACP- 135859 (Partial)KS-D 118767 ER-KR-ACP-KS(Partial) 117253 A-ACP-KS(Partial)* 114998 65.7 KR(Partial)-ACP 55.9 66.8 107384 68.3 ACP-KS_DH 100672 63.4 KR-ACP-KS 93355 KR-ACP-KS 88992 DH-KR-ACP-KS-KR 64449 64.0 ACP-KS(Partial) 65.4 64.3 ACP-KS(Partial) 50464 38791 Partial-KS(1)-DH-ER-KR- KS(Partial)-KR-DH-ER-PP 35740 21774 KS(Partial)-AT-TE- 63.8 65.5 20287 PP-KS(Partial) 19937 (Partial)KS-D polynesiensis TE-A-ACP-KS(Partial)* G. 60.3 6057 64.4 9137 DH-ER-KR-ACP-TE 60.4 KS-ACP excentricus G. SequencesAccepted similar Article

48436 KS(Partial)-AT-TE-KR 48436 KS(Partial)-AT-TE-KR 68.2 1250 KS(Partial)-AT-DH-KR 1250 KS(Partial)-AT-DH-KR 58.4 to G.polynesiensis KS(Partial) TE KS(7)-AT-DH-ER-KR-ACP- KR-ACP-KS(6)-KR-ACP- ER-KR-ACP-ACP-KS(5)-DH- KS(3)-KR-ACP-KS(4)-DH- ACP-KS(2)-DH-KR-ACP- KR(Partial) contig 50464 H-KR(Partial) 65.4 H-KR(Partial) T(Partial) 56.2 T(Partial) 65.4 H(Partial) -KS(Partial) 68.1 -KS(Partial) 57.9% 67.4 59.2 This article isprotected Allrights bycopyright. reserved. Accepted Article

This article isprotected Allrights bycopyright. reserved. Accepted Article

This article isprotected Allrights bycopyright. reserved. Accepted Article

This article isprotected Allrights bycopyright. reserved. Accepted Article

This article isprotected Allrights bycopyright. reserved. Accepted Article