John, Uwe

Kohli, Gurjeet

Murray, Shauna A.

Van Dolah, Frances M.

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Func%onal	and	evolu%onary	dis%nc%veness	of	fa2y	acid	and	polyke%de	synthesis	in	microbial	eukaryotes	Gurjeet	S.	Kohlia,b,	Uwe	Johnc,	Frances	M.	Van	Dolahd,	Shauna	A.	Murraya	aPlant	Func%onal	Biology	and	Climate	Change	Cluster,	University	of	Technology	Sydney,	Australia;	bCurrent	address:	Singapore	Centre	for	Environmental	Life	Sciences	Engineering,	Singapore;	cAlfred-Wegener-Ins%tute	Helmholtz	Center	for	Polar	and	Marine	Research,	Bremerhaven,	Germany;	dMarine	Biotoxins	Program,	Na%onal	Oceanic	and	Atmospheric	Administra%on,	Charleston,	SC	USA		CyanobacteriaRed algaeHigher plantsGreen algaeRhizariaHaptophytesCryptophytesRaphidophytesDiatomsBolidophytesPelagophytes/DictyochophytesDictyochophytesPinguiophytes/XanthophytesChrysophytes/SynurophytesDinoflagellatesVitrellaApicomplexaChromera0.2OpisthokontsAmoebozoaExcavatesGlaucophytesRed AlgaeCryptophytesHigher plantsGreen AlgaeHaptophytesRhizariaStramenopilesAlveolates250 500 750 1000 1250 1500 1750 20000500Red AlgaeCryptophytesHigher plantsGreen AlgaeHaptophytesRhizariaStramenopilesAlveolatesB. Tree of life and molecular clock analysisA. Type II FASC. Type I & II PKSs & FASsType II FASType I PKS & FASGreen Algae Type II FAS Dinoflagellate Type II FASApicomplexa Viterella & Chromera Type II FASStramenopiles Type II FASHaptophytes Type II FASFungal non-reducing Type I PKSFungal reducing Type I PKSAnimal Type I FASBacterial trans AT modular Type I PKSBacterial cis AT modular Type I PKSGreen Algae Type I PKSHaptophytes Type I PKSPelagophytes & Dictyochophytes Type I PKSDictyochophytes,  Synurophytes & Chrysophytes Type I PKSDinoflagellate Type I PKSApicomplexa & Viterella Type I PKS0.50.4Anabaena sp. Porphyridium aerugineumChondrus crispusArabidopsis thalianaCamelina sativaBrassica rapaChlamydomonas euryaleDunaliella tertiolectaPolytomella parvaTetraselmis striataPicocystis salinarumMicromonas sp. Pyramimonas parkeaeOchromonas sp. Chromulina nebulosaDinobryon sp. Chromera veliaEimeria tenellaNeospora caninumToxoplasma gondiiVitrella brassicaformisProrocentrum minimumPolerella glacialisAzadinium spinosumGymnodimium catenatumPelagodinium beiiSymbiodinium sp. Brandtodinium nutriculumScrippsiella hanganoePeridinium aciculiferumCeratium fususLingulodinium polyedraPyrodinium bahamenseGambierdiscus belizeanusAlexandrium monilatumProtoceratium reticulatumAmphidinium carteraeTogula jollaHeterocapsa rotundataVaucheria litoreaPhaeomonas parvaPinguiococcus pyrenoidosusHeterosigma akashiwoChattonella subsalsaCryptomonas curvataGeminigera cryophilaHanusia phiGuillardia thetaProteomonas sulcataRhodomonas sp. Hemiselmis anderseniiBolidomonas pacificaAulacoseira subarcticaStephanopyxis turrisRhizosolenia setigeraCoscinodiscus wailesiiAsterionellopsis glacialisGrammatophora oceanicaLicmophora paradoxaNitzschia punctataPseudo−nitzschia australisFragilariopsis kerguelensisCylindrotheca closteriumDurinskia balticaGlenodinium foliaceumKryptoperidinium foliaceumAmphora coffeaeformisEntomoneis sp. Amphiprora sp. Synedropsis rectaThalassionema nitzschioidesThalassiothrix antarcticaHelicotheca tamensisDitylum brightwelliiChaetoceros affinisDactyliosolen fragilissimusEucampia antarcticaMinutocellus polymorphusExtubocellulus spiniferOdontella auritaTriceratium dubiumStriatella unipunctataAttheya septentrionalisSkeletonema menzeliiDetonula confervaceaThalassiosira rotulaCyclotella meneghinianaProboscia alata Corethron pennatumLeptocylindrus danicusAureococcus anophagefferensPelagomonas calceolataAureoumbra lagunensisChrysocystis fragilisFlorenciella parvulaDictyocha speculumPseudopedinella elasticaChrysoculter rhomboideusExanthemachrysis gayraliaePavlova lutheriPhaeocystis antarcticaKarenia brevisKarlodinium micrumChrysochromulina rotalisScyphosphaera apsteiniiEmiliania huxleyiIsochrysis sp. Gephyrocapsa oceanicaPleurochrysis carteraeCoccolithus pelagicusCalcidiscus leptoporusImantonia sp. Prymnesium parvumPartenskyella glossopodiaGymnochlora sp Lotharella amoebiformisChlorarachnion reptansBigelowiella natansCyanothece sp. Pleurocapsa minorMicrocystis aeruginosaDiatomsCyanobacteriaRhodophytesPlantsRhizaria HaptophytesOther AlveolatesApicomplexaCryptophytesOther StramenopilesChlorophytesDinoflagellatesHaptophytesDinoflagellatesDinoflagellatesOther Stramenopiles0 30 60 90 120 150Filamoeba nolandiNeoparamoeba aestuarinaMayorella spAcanthoeca-like sp.Neobodo designisEutreptiella gymnasticaGloeochaete witrockianaCyanoptyche gloeocystisTetraselmis striataPyramimonas parkeaeMicromonas spPicocystis salinarumPolytomella parvaDunaliella tertiolectaChlamydomonas euryalePalpitomonas bilixRhodomonas spHemiselmis spp (2)Guillardia thetaGeminigera cryophilaCryptomonas curvataScyphosphaera apsteiniiPrymnesium parvumPleurochrysis carteraePhaeocystis cordataPhaeocystis antarcticaPavlova spp (3)Isochrysis galbanaImantonia spGephyrocapsa oceanicaExanthemachrysis gayraliaeEmiliania huxleyiCoccolithus pelagicus ssp braarudiChrysoculter rhomboideusChrysochromulina polylepisCalcidiscus leptoporusBigelowiella spp (2)Elphidium margaritaceumAmmonia spPartenskyella glossopodiaLotharella spp (3)Gymnochlora spChlorarachnion reptansVaucheria litoreaPhaeomonas parvaHeterosigma akashiwoChattonella subsalsaChromulina nebulosaParaphysomonas bandaiensisSpumella elongataParaphysomonas vestitaPelagomonas calceolataAureoumbra lagunensisAureococcus anophagefferensOchromonas spDinobryon spRhizochromulina marinaPinguiococcus pyrenoidosusFlorenciella spFlorenciella parvulaDictyocha speculumChrysocystis fragilisPseudopedinella elasticaThalassiosira spp (5)Thalassionema nitzschioidesStephanopyxis turrisSkeletonema spp (2)Rhizosolenia setigeraPseudo-nitzschia australisProboscia alataOdontella auritaNitzschia punctataMinutocellus polymorphusLicmophora paradoxaLeptocylindrus danicusFragilariopsis kerguelensisEucampia antarcticaDitylum brightwelliiDactyliosolen fragilissimusCylindrotheca closteriumCyclotella meneghinianaCoscinodiscus wailesiiCorethron pennatumChaetoceros spp (4)Asterionellopsis glacialisAmphora coffeaeformisAmphiprora sp.Bolidomonas spp (2)Thraustochytrium spSchizochytrium aggregatumAurantiochytrium limacinumAplanochytrium stocchinoiAplanochytrium spCafeteria spp (2)Bicosoecid spStrombidium  inclinatumPseudokeronopsis spPlatyophrya  macrostomaLitonotus pictusFavella spp (2)Condylostoma magnumClimacostomum virensAnophryoides haemophilaChromera veliaVitrella brassicaformisOxyrrhis-marinaTogula jollaSymbiodinium sp.Scrippsiella hangoeiPyrodinium bahamenseProtoceratium reticulatumProrocentrum minimumPolarella glacialisPeridinium aciculiferumPelagodinium beiiLingulodinium polyerdaKruptoperidinium foliaceumKarlodinium micrumKarenia brevisHeterocapsa triquestraGymnodinium catenatumGonyaulax spiniferaGlenodinium foliaceumGambierdiscus australesDurinskia balticaCeratium fususBrandtodinium nutriculumAzadinium spinosumAmphidinium carteraeAlexandrium pacificumAmoebozoaExcavatesOpisthokontsGlaucophytesDinoflagellatesOxyrrhisVitrellaChromeraCiliatesBicosoecidsLabyrinthulids ThraustochytridsBolidophytesBacillariophytesDictyochophytesSynurophytesPelagophytesChrysophytesRaphidophytesPinguiophytesXanthophytesHaplosporidiaForaminiferaChlorarachniophytesHaptophytesCryptophytesPalpitomonasChlorophyceansTrebouxiophytesPrasinophytesAlveolatesGreenHaptophytesRhizariaStramenopilesAlgaeNumber of PKS (KS domains) genes                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                GonyaucalesGymnodinialesPeridinialesSuessialesProrocentralesOthers (Outgroup)Karenia brevisGambierdiscus sppLingulodinium polyedraAzadinium spinosumAmphidinium carteraeKarlodinium micrumOther generaOuter ringInner ring0.1Clade IClade IIClade IIIFigure	1:	Type	II	Fa.y	acid	synthase	genes	are	present	in	all	phototrophic	lineages			Introduc7on	Fa2y	acid	synthesis	(FAS)	is	an	essen%al	metabolic	process.	Despite	its	signiﬁcance,	li2le	is	known	about	this	process	in	pro%sts,	which	comprise	~90%	of	eukaryo%c	lineages.	Elaborate	polyke%de	compounds	are	produced	by	some	pro%sts,	in	par%cular,	the	toxins	released	during	harmful	algal	blooms	(HABs).	While	FAS	is	a	primary	metabolic	process,	other	polyke%de	compounds	are	secondary	metabolites	involved	in	processes	such	as	preda%on	defense.	Surprisingly,	some	pro%st	studies	suggest	that	the	two	processes	are	catalyzed	by	the	same	enzymes.			RNA	Extrac%on	&	Illumina	RNASeq	sequencing	PKS	&	FAS	gene	hun%ng	via	BLAST	and	Hmmer	Assembly	via	CLCBio	or	ABySS	Phylogene%c	Analysis	RAxML	V8.0	(GAMMA,	1000	bootstraps)	Methods	Transcriptomic	libraries	(213	strains/152	genera)	from	the	Marine	Microbial	Eukaryo%c	Sequencing	Project	(MMETSP)	were	screened	for	FAS	and	PKS	genes	using	the	following	key	steps:	Aims	1.  Iden%fy	the	gene%c	basis	of	FAS	and	polyke%de	synthesis	(PKS)	in	the	major	pro%st	lineages		2.  Infer	the	constraints	and	processes	in	the	evolu%onary	history	of	pro%st	FAS	and	PKS	genes	Type	II	FAS	genes	recently	transferred	from	the	plas7d	to	the	nucleus	of	its	ter7ary	hosts	(in	some	cases,	e.g.	haptophytes	&	diatoms,	then	to	their	dinoﬂagellate	hosts)		Figure	2:	Compara7ve	evolu7on	of	fa.y	acid	and	polyke7de	synthase.		A.	Concatenated	phylogeny	of	type	II	FAS	genes.	B.	Dated	molecular	clock	phylogeny	of	the	eukaryotes,	showing	absolute	%me	scale	(million	years)1.	C.	Phylogeny	of	3-ketoacyl	ACP	synthase	II	(FAS)	and	type	I	ketosynthase	(PKS)	domains.	Solid	circles	indicate	bootstrap	values	above	≥	90.	Results	•  Six	key	type	II	FAS	enzyme	are	present	in	all	phototrophic	lineages	(Figure	1).	•  Evidence	supports	type	II	FAS	genes	being	nuclear	encoded,	and	ini%al	fa2y	acid	synthesis	steps	occur	in	the	chloroplast	(transit	pep%des;	polyA	tail,	dinoﬂagellate	spliced	leader;	genes	encoded	in	E.	huxleyi	genome).	•  Enormous	diversity	of	PKS	genes	are	found	in	selected	lineages	of	pro%sts	(Figs	3	&	4).	Clear	dis7nc7on	between	FAS	and	PKS	genes	in	pro7sts	Conclusions:	•  FAS	is	essen%al	for	survival.	Thus,	pro%sts	likely	retained	genes	in	the	nucleus	due	to	strong	selec%ve	pressure;		•  Large	PKS	gene	diversity	in	dinoﬂagellates,	suggests	mul%ple	gene	duplica%on	events,	domain	shuﬄing	and	losses;	•  Relaxed	selec%on	pressure	may	have	acted	on	the	evolu%on	of	PKS	genes	acquired	or	lost	based	on	func%onality;	•  Diﬀeren%a%on	of	PKS	and	FAS	will	facilitate	inves%ga%on	of	harmful	algal	toxin	biosynthesis	pathways	in	pro%sts.	Figure	3:	Abundance	of	expressed	type	I	PKS	ketosynthase	domains	among	pro7st	lineages	PKS	gene	family	is	highly	expanded	in	dinoﬂagellates	and	haptophytes	Dinoﬂagellates	type	I	PKS	ketosynthase	domains	form	three	dis7nct	clades,	but	the	pa.ern	of	distribu7on	is	not	related	to	the	species	phylogeny	and/or	the	chemical	structures	these	organisms	produce	Acknowledgements:	Australian	Research	Council	grants	(FT120100704,	DP120103199)	to	SM;	Australian	Academy	of	Science	Germany-Australia	mobility	call	2013-2014	and	BMBF	PT/DLR	IB	FKZ	01DR14006	awarded	to	SM	and	UJ;PACES	research	program	of	AWI	to	UJ;	Na%onal	Oceanic	and	Atmospheric	Administra%on	NCCOS	project	02E00025	to	FV;	SCELSE	travel	grant	to	GSK		Figure	4:	Polyke7de	synthase	gene	phylogeny	in	dinoﬂagellates:	Phylogene%c	analysis	of	1633	type	I	PKS	ketosynthase	domains.	Evolu7on	of	type	II	FAS	genes	broadly	follows	the	evolu7on	pa.ern	of	the	organism	Contact	Gurjeet	Singh	Kohli	gurjeetkohli@ntu.edu.sg	gurukohli@gmail.com	Ph:	+65	8381	7647	References:	1Parfrey	LW,	Lahr	DJG,	Knoll	AH,	Katz	LA.	(2011).	Proc	Natl	Acad	Sci	108:	13624-13629			

Functional and evolutionary distinctiveness of fatty acid and polyketide synthesis in microbial eukaryotes

https://epic.awi.de/id/eprint/48764/1/ISME_2016_poster_GSK_SL_uts_FVD.pdf

Functional and evolutionary distinctiveness of fatty acid and polyketide synthesis in microbial eukaryotes

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