Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in ...

12 Pages·2015·3.15 MB·English

Expanded Natural Product Diversity Revealed by Analysis of
Lanthipeptide-Like Gene Clusters in Actinobacteria
QiZhang,a*JamesR.Doroghazi,bXilingZhao,aMarkC.Walker,a WilfredA.vanderDonka,b
DepartmentofChemistryandHowardHughesMedicalInstitute,UniversityofIllinoisatUrbana-Champaign,Urbana,Illinois,USAa;InstituteforGenomicBiology,
UniversityofIllinoisatUrbana-Champaign,Urbana,Illinois,USAb
Lanthionine-containingpeptides(lanthipeptides)arearapidlygrowingfamilyofpolycyclicpeptidenaturalproductsbelonging
tothelargeclassofribosomallysynthesizedandposttranslationallymodifiedpeptides(RiPPs).Lanthipeptidesarewidelydis-
tributedintaxonomicallydistantspecies,andtheircurrentlyknownbiosyntheticsystemsandbiologicalactivitiesarediverse. D
Buildingontherecentnaturalproductgeneclusterfamily(GCF)project,wereportherelarge-scaleanalysisoflanthipeptide- o
w
likebiosyntheticgeneclustersfromActinobacteria.Ouranalysissuggeststhatlanthipeptidebiosyntheticpathways,andbyex-
n
trapolationthenaturalproductsthemselves,aremuchmorediversethancurrentlyappreciatedandcontainmanydifferentpost- lo
a
translationalmodifications.Furthermore,lanthioninesynthetasesaremuchmorediverseinsequenceanddomaintopology d
thancurrentlycharacterizedsystems,andtheyareusedbythebiosyntheticmachineriesfornaturalproductsotherthanlanthip- e
d
eptides.Thegeneclusterfamiliesdescribedheresignificantlyexpandthechemicaldiversityandbiosyntheticrepertoireoflan-
f
r
thionine-relatednaturalproducts.Biosynthesisofthesenovelnaturalproductslikelyinvolvesunusualandunprecedentedbio- o
m
chemistries,asillustratedbyseveralexamplesdiscussedinthisstudy.Inaddition,classIVlanthipeptidegeneclustersareshown

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nottobesilent,settingthestagetoinvestigatetheirbiologicalactivities. t
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Lanthipeptidesarearapidlygrowingfamilyofpolycyclicpep- functionalimportanceoflanthioninescaffoldsandaconvergent e
m
tides characterized by the presence of the thioether cross- evolutionprocesstoproducethem(12).Notably,productsgen-
.
linked amino acids lanthionine and methyllanthionine (1–5). eratedbythesefourclassesoflanthioninesynthetasesarenotlim- a
s
Thesecompoundsarewidelydistributedintaxonomicallydistant itedtolanthipeptides.Forexample,manylanthipeptide-likebio- m
speciesanddisplayverydiversebiologicalactivities,rangingfrom syntheticgeneclustersencodeprecursorpeptidesthathavenoCys .o
antimicrobialtoantiallodynic(5–7).Antibacteriallanthipeptides, residues,andtheirproductsarethereforenotlanthipeptides(19). rg
suchasthecommerciallyusedfoodpreservativenisin,aretermed Overthepast5years,severalstudieshavereportedbioinfor- /
o
lantibiotics(8).Lanthipeptidesaregeneratedfromaribosomally matic genome mining efforts based on the then-available ge- n

synthesized linear precursor peptide (generically termed LanA) nomes.Thesestudieshavetypicallyfocusedonanenzyme-spe- N
o
andthereforebelongtothelargeclassofnaturalproductsthatare cificquery,suchasclassILanBandLanCenzymes(20),classII v
ribosomally synthesized and posttranslationally modified pep- LanM proteins (21), or the bifunctional LanT proteins, which e
m
tides(RiPPs)(9).TheprecursorpeptideLanAconsistsofaC-ter- transportlanthipeptidesandremovetheirleadersequences(22). b
minalcorepeptidewhereallposttranslationalmodificationstake Buildingontherecentnaturalproductgeneclusterfamily(GCF) e
r
placeandanN-terminalleaderpeptidethatisimportantforpost- project(23),herewereportlarge-scaleanalysisoflanthipeptide- 1
translationalmodificationsandthatissubsequentlyremovedby likebiosyntheticgeneclustersthatisdrivenbysimilaritiesinen- 7
,

proteolysis(10,11).Theinstallationofthe(methyl)lanthionine tireclustersratherthanspecificenzymes.Wefocusedtheanalysis 2
0
thioetherbridgesisachievedbytheinitialdehydrationofSerand onlanthipeptidesfromActinobacteria,whicharerelativelyunder- 1
Thrresiduesintheprecursorpeptides,followedbystereoselective studiedcomparedtofamilymembersfromFirmicutes.Actinobac- 8

b
intramolecularMichael-typeadditionofCysthiolstothenewly y
formeddehydroaminoacids(Fig.1A). g
u
Four classes of biosynthetic enzymes are known to catalyze Received24February2015 Accepted14April2015 e
lanthionineformation(2,12)(Fig.1B).ClassIlanthioninesyn- Acceptedmanuscriptpostedonline17April2015 s
t
thetasesconsistofadehydrataseandacyclasethataregenerically CitationZhangQ,DoroghaziJR,ZhaoX,WalkerMC,vanderDonkWA.2015.
termedLanBandLanC,respectively(8).ClassIIenzymesarege- Expandednaturalproductdiversityrevealedbyanalysisoflanthipeptide-like
geneclustersinActinobacteria.ApplEnvironMicrobiol81:4339–4350.
nericallynamedLanMs,whicharesinglepolypeptidescontaining
doi:10.1128/AEM.00635-15.
anN-terminaldehydratasedomainthatbearsnohomologytoany
Editor:M.A.Elliot
functionallyknownenzymesandaC-terminalLanC-likecyclase
AddresscorrespondencetoWilfredA.vanderDonk,[email protected].
domain (13, 14). Class III and class IV synthetases are trifunc-
*Presentaddress:QiZhang,DepartmentofChemistry,FudanUniversity,
tionalenzymestermedLanKC(15)andLanL(16),respectively. Shanghai,China.
TheseenzymescontainanN-terminallyasedomainandacentral Q.Z.andJ.R.D.sharethepositionoffirstauthor.
kinasedomainbutdifferintheirCtermini.LanCandtheC-ter- Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
minaldomainsofLanMandLanLcontainaconservedzinc-bind- /AEM.00635-15.
ingmotif(Cys-Cys-His/Cys)(17,18),whereastheC-terminalcy- Copyright©2015,AmericanSocietyforMicrobiology.AllRightsReserved.
clasedomainsofLanKClacktheseconservedresidues(Fig.1B). doi:10.1128/AEM.00635-15
The four distinct classes of biosynthetic machinery reflect the
July2015 Volume81 Number13 AppliedandEnvironmentalMicrobiology aem.asm.org 4339
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FIG1Biosynthesisoflanthipeptides.(A)Themechanismof(methyl)lanthionineformation.(B)Thefourcurrentlyknownclassesoflanthipeptidesynthetases. e
m
Xnrepresentsapeptidelinker.Theconservedzinc-bindingmotifsarehighlightedbythepurplelinesinthecyclasedomains.SpaB_Cisaneliminationdomain
.
presentasastand-aloneproteininthiopeptidebiosynthesisandastheC-terminaldomainofLanBenzymesinlanthipeptidebiosynthesis. a
s
m
.
teriacarryawealthofnaturalproductbiosyntheticgeneclusters mationcanbefoundathttp://www.igb.illinois.edu/labs/metcalf/gcf/lant o
r
whose products encompass highly diverse chemical structures. .html. Use of these pages requires a JavaScript-enabled web browser. g
/
This phylum has been the source and/or inspiration for the Mouseoverofageneprovidesaroughannotationandshouldbecon- o
majorityofpharmaceuticallyusefulnaturalproducts(24,25). firmedwithadditionalmethodsbeforefurtheruse.Mouseoveralsohigh- n
N
Although Actinobacteria constitute only a small proportion of lightsothergenesonthepagethatareinthesamehomologygroup.On
o
knownlanthipeptideproducers,theyencodeadiversesetofpost- click,eachgenewillopenanewtabcontainingtheaminoacidsequence v
andalinktoBLASTanalysisofthesequence.Straindesignationssuchas e
translationalmodificationsthathavenotbeenobservedforlan- m
Streptomyces species B-3253 refer to strains in the Culture Collection
thipeptidesfromotherphyla(2)(e.g.,lysinoalanineformationin b
(NRRL)oftheAgriculturalResearchService(ARS).Thegenomicinfor- e
cinnamycin [26], tryptophan chlorination and proline dihy- r
mationcanbefoundbyenteringB-3253intotheNCBIsearchfieldand
droxylationinmicrobisporicin[27,28,alsocalledNAI-107[29], 1
then searching in the Assembly or BioSample databases. Designations 7
andglycosylationinNAI-112[30]).Theuniquemodificationsin ,
suchasStreptomycesspeciesB-3253_23refertocluster23inthisstrain.
2
actinobacteriallanthipeptidebiosynthesissuggestgreatpotential
Network analysis was performed by BLASTP searches, comparing 0
forexploringunknownlanthipeptidechemicalspaceinthisphy- eachsequenceagainsteachothersequence.AVBAscriptwaswrittento 18
lum. Our analysis suggests that lanthipeptides are likely much removeallduplicatecomparisons,andtheresultwasimportedintothe b
more diverse than is currently appreciated and contain many Cytoscapesoftwarepackage(31).Thenodeswerearrangedbyusingthe y

novel posttranslational modifications. More intriguingly, our yFiles organic layout provided with Cytoscape version 2.8.3. Sequence g
u
analysissuggeststhatinsomecaseslanthipeptidesynthetaseshave logosweregeneratedonlineusingWeblogo(32). e
beenrepurposedforproducingnaturalproductsotherthanlan- Cellcultures,samplepreparation,andMSanalysis.Dimethylsul- st
thipeptides,expandingnaturalproductdiversity. foxide(DMSO)stocksofStreptomyceskatraeISP5550werepreparedby
mixing600(cid:2)lofanS.katrae ISP5550liquidculturewithanequalvolume
MATERIALSANDMETHODS ofsterilized20%dimethylsulfoxide(DMSO).Theresultingsolutionwas
Chemicals,media,andgeneralmethods.Unlessotherwisenoted,reagents mixed,flashfrozen,andstoredat(cid:3)80°C.S.katraeISP5550wasinocu-
werepurchasedfromFisherScientific.YeastextractwaspurchasedfromBec- lated from DMSO stocks onto solid ATCC172 medium (20 g soluble
tonDickinsonandN-ZaminetypeAfromSigma-Aldrich.Acetonitrilefor starch,5gyeastextract,5gN-ZaminetypeA,1gcalciumcarbonate,15g
matrix-assistedlaserdesorptionionizationmassspectrometry(MALDIMS) agar,and1literofMilli-Qwater;afterautoclavingthemedium,sterilized
analysiswaspurchasedfromAvantorPerformanceMaterials.MALDI–time glucosewasaddedtoa1%finalconcentration)andincubatedat30°Cfor
offlight(MALDI-TOF)MSwascarriedoutonaBrukerUltrafleXtremeTOF/ 4to5days.
TOF.Reversed-phaseliquidchromatographyelectrosprayionization(ESI) ThepresenceofvenezuelinswasdetectedusingcolonyMALDI-TOF
tandemMSwascarriedoutandprocessedusingaSynaptESIquadrupole MS.Asmallportion(approximately1by1mm)ofcolonywastransferred
TOFmassspectrometrysystem(Waters). ontoaBrukerMTP384polishedsteeltargetplate.Ontopofthecolony,2
Bioinformatics. GCF analysis, BLAST searches, and phylogenetic (cid:2)lofsinapicacidmatrixwasoverlaidandallowedtoairdry.Sinapicacid
analysiswereallperformedasdescribedpreviously(23).AllGCFinfor- matrixwaspreparedbysaturatinganacetonitrile(MeCN)–Milli-Qwater
4340 aem.asm.org AppliedandEnvironmentalMicrobiology July2015 Volume81 Number13
Lanthipeptide-LikeGeneClustersinActinobacteria
(80:20) solution with sinapic acid (Sigma-Aldrich). MALDI-TOF MS 12h.Thereactionmixturewasdriedandmixedwithasolutionofacetyl
analysis of the sample was performed using a Bruker UltrafleXtreme chloride(Sigma-Aldrich)andmethanol.Followingrefluxat110°Cfor45
MALDI-TOFTOF-MSmaintainedbytheUIUCSchoolofChemicalSci- min,thereactionmixturewasdriedand3mlofdichloromethane(Sigma-
ences Mass Spectrometry Laboratory. Data acquisition was completed Aldrich)wasadded.Aftercoolingthesolutionto0°C,1mlofpentafluo-
usingBruker’sFlexControlprogram,whileFlexAnalysiswasusedfordata ropropionicanhydride(Sigma-Aldrich)wasadded,andthereactionmix-
analysis.Priortodataacquisition,theinstrumentwascalibratedusing turewasrefluxedfor15minat110°C.Thereactionmixturewasdriedand
peptidecalibrationstandardII(BrukerDaltonics,Billerica,MA)andthe storedat(cid:3)80°C,andpriortoGC-MSanalysis,thesamplewasredissolved
matrixsuppressionmodesettodeflectionmode.Thedatawerenotsub- in100(cid:2)lofmethanol.Particulateswereremovedbycentrifugationat
jectedtoprocessing. 23,700(cid:4)gfor4min.AnAgilentHewlett-Packard5973massspectrom-
UpondetectionofvenezuelinsbycolonyMALDI-TOFMSfromsolid eterequippedwithaCP-Chirasil-LVal0.12-(cid:2)m,25-mby0.25-mmcol-
culturesofS.katraeISP5550,liquidcultureswereanalyzedforthepro- umn(7-in.cage;AgilentTechnologies)wasusedtoperformtheGC-MS
ductionofvenezuelins.Asinglecolonywastransferredinto50to100ml analysis.Thesamplewasintroducedtotheinstrumentviaasplit(1:5)
ofliquidATCC172medium,preparedinthesamewayasthesolidme- injectionof2to5(cid:2)lwitha2.0-ml/minheliumgasflowrate.Theinitial
diumwiththeomissionofagar.Theliquidculturewasincubatedwith inlettemperaturewas190°C,andagradientof20°C/minfrom160°Cfor
shakingat30°Cfor12days.Supernatantfromtheliquidcultureswas 5minto180°Cfor10minwasused.Thefollowingmassspectrometer D
o
obtainedbycentrifugingat4,650(cid:4)gfor15min.Thehydrophobiccom- settingswereused:185°CfortheMSDtransferlineheater,150°Cforthe w
ponents of the supernatant were desalted by ZipTip (EMD Millipore) MS Quad analyzer, and 230°C for the ion source. Data were acquired n
accordingtothemanufacturer’sinstructions.TheZipTipcontentswere usingscanandselectedionmonitoringat365and379Da.Theidentityof lo
directlyelutedontoaBrukerMTP384polishedsteeltargetplateonaspot (methyl)lanthionineresidueswasconfirmedbyaddingauthenticmeth- ad
containing1(cid:2)lofsinapicacidmatrix.AnalysisbyMALDI-TOFMSwas yl(lanthionine)standardstothesampleandanalyzingtheresultingmix- e
performedasdescribedabove. turebyGC-MS.Themixturesofsampleandstandardwerepreparedsuch d
InpreparationforESI-MS,peptidecomponentsofthesupernatant thattherewasanapproximatelyequalcontributionofsignalarisingfrom fro
wereprecipitatedusingammoniumsulfate.A60%finalconcentrationof thesampleandstandard. m
ammoniumsulfatewasachievedwiththeadditionof30gofammonium h
sulfateto50mlofsupernatant.Thesolutionwasallowedtoincubateat RESULTS tt
p
roomtemperaturefor30min.Theresultingprecipitatewaspelletedby PhylogeneticanalysisofLanC-likeenzymes.Miningof830acti- :
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centrifugation at 22,789 (cid:4) g for 10 min, and the supernatant was de- nobacterial genomes, including 344 newly sequenced genomes /a
canted. The pellet was redissolved in Milli-Q water and subjected to obtainedfortheGCFproject(23),revealed1,163lanthipeptide- e
m
ESI-MSanalysisusinganAcquityultraperformanceliquidchromatogra- likegeneclusters,whichweregroupedintomorethan100differ- .
a
phy(UPLC)systemcoupledtoaWatersSynaptESI-QTOF.TheUPLC entfamiliesaccordingtotheirsequenceandorganizationalsimi- s
wasequippedwithanAcquityUPLCBEHC81.7-(cid:2)m,1.0-by100-mm larities(seeFig.S1inthesupplementalmaterial)(23).Strikingly, m
column.SamplewasintroducedintothecolumnviaanAcquityautoin- the GCFs that produce the known lanthipeptides produced by .o
jectorandsubjectedtoagradientof2%Bfrom0to2min,2%to98%B r
Actinobacteriamakeupaverysmallfraction,illustratingthetre- g
from2to12min,and98%Bfrom12to15min(Awas0.1%formicacid /
mendousdiversityofyet-to-be-investigatedGCFs.Tofirstinves- o
inwaterandBwas0.1%formicacidinMeCN).TheWatersMassLynx n
tigate the phylogenetic relationship of LanC-like enzymes from
V4.1programwasusedtotune,calibrate,andacquiredata.[Glu1]-Fibri- N
nopeptideBhuman(Sigma-Aldrich)wasusedasanexternalcalibrant, different gene clusters, we constructed a maximum-likelihood o
andthemassofvenezuelinwasverifiedtobewithin5ppmofitscalculated (ML) tree using enzymes from 29 large families that each are v
e
mass.Collision-induceddissociationwasachievedusingaseriesofenergy foundinatleastfivedifferentspecies(oroperationaltaxonomic m
rampsrangingfrom30to60eV. units with a clustering threshold of 99% identity in ribosomal b
e
Isolationofvenezuelins.S.katraeISP5550liquidculturesupernatant proteins [23]). This analysis makes it possible to infer a gene’s r
wassubjectedtopressure-basedsampleconcentrationratherthanammo- involvementinaGCFbasedonconservationoverevolutionary 1
niumsulfateprecipitation.Aftercentrifugation,40mlofculturesuper- time(34).GeneconservationwithinaGCFreliesontheinclusion 7,
natantwasfurtherclarifiedbyfiltrationthrougha0.22-(cid:2)mnylonmem- ofmultiplespeciesintheanalysis,asgenomediversitywithina 2
0
braneandthenloadedintoanAmiconstirredcellequippedwitha1-kDa
singleStreptomycesspeciesismuchreducedandsyntenyextends 1
nominal-molecular-mass limit regenerated cellulose disc (EMD Milli- 8
nearlytheentirelengthofthechromosome(35).Thetreeshows
pore).Theclarifiedsupernatantwasconcentratedatroomtemperature b
distinct phylogenetic distributions of enzymes from different y
fromavolumeof40to2mloverapproximately4h.Theconcentrated
supernatantwasflashfrozenandstoredat(cid:3)20°Cpriortofractionation classes,asLanCandtheC-terminaldomainsofLanM,LanL,and gu
by analytical reversed-phase high-performance liquid chromatography LanKCfallintofourdifferentclades(Fig.2).Thisobservationis e
(HPLC)usinganAgilent1260InfinityinstrumentequippedwithaPhe- consistent with our previous proposal that the four classes of st
nomenexLuna10-(cid:2)m,C (2)100-Å,250-by4.6-mmcolumn.Thein- lanthipeptide synthetases have evolved from different ancestors
18
strumentwasmanagedusingAgilentInstrument1(online)software.The (12,36).Interestingly,threecasesofnovelGCFsemergewithin
mobilephaseconsistedofA(0.1%trifluoroaceticacid[TFA][AcrosOr- an existing GCF: GCF100 branches within the LanM clade of
ganics])inwaterandB(0.1%TFAin80%MeCN–20%water),witha GCF146 (this GCF also contains a LanKC-encoding gene and
gradientof2%Bfrom0to5min,2to100%Bfrom5to50min,and100% henceappearstwiceinFig.2),GCF1andGCF29branchwithin
Bfrom50to60min.A0.5-(cid:2)laliquotofeachfractionelutingafter20min
GCF20,andGCF133andGCF140branchwithinGCF31.Below
wasspottedwithanequalvolumeofsinapicacidmatrixandanalyzedby
we highlight several families of lanthipeptide-like gene clusters
MALDI-TOFMSforthepresenceofvenezuelin.Venezuelin-containing
thataremostinterestinginthattheydeviatefromthecurrently
fractions(typicallyelutingat35to38min)wereflashfrozenanddriedin
knownlanthipeptidebiosyntheticsystemsandlikelyencodenovel
vacuo.Three1-mlinjectionsofconcentratedS.katraeISP5550superna-
tantyielded100(cid:2)gofHPLC-purified,drymaterial. andunusualbiochemistryinthebiosynthesisoflanthionine-re-
Detectionoflanthionine.HPLC-purifiedvenezuelinwasanalyzedby latednaturalproducts.
gaschromatography(GC)-MSaspreviouslydescribed(33).Inbrief,the Novelmulticomponentlanthipeptidesystems.Two-compo-
samplewasredissolvedin3mlof6MHClandheatedat110°Cfor10to nentlanthipeptidesarenormallypotentantimicrobialagentsin
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FIG2 AnMLtreeofLanCsandLanC-likedomains.Intheouterring,thefourdifferentclassesofenzymesaredepictedbythecolordefinedinthelegend, N
whereasthedifferentGCFsareshownintheinnerringwithrandomcolors.TheGCFnumbersaredefinedinreference23andcanbefoundathttp://www.igb o
v
.illinois.edu/labs/metcalf/gcf/lant.html.PKS,polyketidesynthetase;NRPS,nonribosomalpeptidesynthetase;TOMM,thiazole/oxazole-modifiedmicrocins. e
m
b
e
whichthetwoposttranslationallymodifiedpeptidesexertstrong recently characterized NpnAs (19). Like in the Npn cluster, an r

synergisticbiologicalactivity(37).Todate,isolatedtwo-compo- oxidoreductasecontainingthecharacteristicNAD-bindingRoss- 1
7
nent lantibiotics have been confined to Firmicutes, but several mannfoldisalsoencodedinthegenecluster.Apossiblerolefor ,

familiesoflanthipeptidegeneclustersintheActinobacterialikely thisoxidoreductaseistoproduceD-aminoacidsbystereoselective 20
encodetwo-componentlanthipeptides.Averyrecentstudyalso reductionofthedehydroaminoacidsresultingfromLanMcatal- 1
8
suggestedthepresenceoftwo-componentlanthipeptidesinthree ysis(45–47).Thesefindingssetthestageforinvestigationwhether
b
Streptomyces strains (22). Among the most interesting class is thematureproductsoftheLanMandLanKCsubstratesindeedform y

GCF146(70members),whichiscurrentlythesecond-largestlan- a novel functional two-component lanthipeptide system in which g
u
thipeptideGCF(seeFig.S1inthesupplementalmaterial).Each onepeptidedoesnotcontainlanthionine/labioninstructures. e
memberencodesaLanMandaLanKC,andthecorepeptidesof GCF30 (12 members) consists of gene clusters appearing in s
t
the putative LanKC substrates have a strictly conserved CxSxxS two different types of organization, both of which encode two
motif(Fig.3A).PreviousstudieshaveshownthatLanKCproteins LanMs and two LanA substrates (Fig. 3B). Both LanAs have a
canturnSxxSxxxCmotifsintoalabioninscaffoldbyinitialMi- Gly-Gly/Alamotifcharacteristicforleaderpeptideremovaland
chaeladditionoftheC-terminalCystoacentraldehydroalanine multipleCysandSer/Thrresidues(seeFig.S2inthesupplemental
(formedfromSer)andsubsequentadditionoftheresultingeno- material).Therefore,theproductsofthesegeneclustersarelikely
late onto the N-terminal dehydroalanine of the motif (38–42). similartowell-knowntwo-componentlantibioticssuchashalo-
TheCxSxxSmotifinGCF146couldbeavariationofthelabionin- duracinandlichenicidinproducedbybacilli(20,37,48–51).One
forming sequence. Another interesting finding is that a radical GCF30 member from Streptomyces viridochromogenes was also
SAM protein containing a canonic CxxxCxxC motif (43, 44) is notedinarecentgenomeminingstudyfocusingonLanTanalogs
strictly conserved in these gene clusters, which may catalyze an (22).
unprecedented posttranslational modification in lanthipeptide GCF46 (6 members) consists of gene clusters encoding two
maturation.TheputativeLanMsubstratesinGCF146,however, LanAsandtwoLanKCenzymes(Fig.3C).ThetwoLanKCsshare
donotcontaincysteineresidues(Fig.3A)andaresimilartothe highsequencesimilarities(about40%identity)witheachother,
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FIG3GCFsthatlikelyproducetwo-componentlanthipeptides.(A)ArepresentativegeneclusterofGCF146andthesequencelogosoftheprecursorpeptide b
substrates.ThepredictedLanKCsubstrate/enzymepairisshowninblue,andthepredictedLanMsubstrate/enzymepairisshowninred.Thestrictlyconserved e
CxSxxSmotifoftheputativeLanKCsubstrateandtheGG/AleaderpeptidecleavagesiteoftheputativeLanMsubstratearehighlightedbyredboxes.(B)A r
1
representativegeneclusterofGCF30thatlikelyencodesahaloduracin-likesystem.Thegeneclustersappearintwodifferenttypes,andhomologousenzymesare 7
linkedbybluedashedlines.(C)ArepresentativegeneclusterofGCF46thatmayencodeclassIIItwo-componentlanthipeptides.(D)Arepresentativegenecluster ,

ofGCF140thatmayencodethreeprecursorpeptidesandaLanKC.Theputativefunctionsofthegeneproductsareshownbycolors.Thelogosoftheprecursor 2
0
peptidesinGCF30,GCF46,andGCF140areshowninFig.S2toS4,respectively,inthesupplementalmaterial.Theboundariesofeachgeneclusterarepredicted 1
basedontheconservedgeneswithineachGCFandarenotclearlydefined.RS,radicalSAM;OR,oxidoreductase. 8

b
y

g
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andthetwoLanAsaresimilarbutnotidentical(seeFig.S3inthe Cysresidueinthecorepeptideregion,whereasonepeptidehasa e
supplementalmaterial).BothLanAshavehighlyconservedCxx- Cysresidueintheleaderpeptide(seeFig.S4inthesupplemental s
t
SxxSmotifs,suggestingapotentialfirstclassIIItwo-component material).Itremainstobedeterminedwhethertheseputativepre-
lanthipeptide.AradicalSAMproteinisalsoencodedinthegene cursor peptides are substrates of the corresponding LanKC en-
cluster,whosefunctionremainstobedetermined.Notably,the zyme.
two LanAs in the gene clusters from GCF46 are significantly Lanthionine synthetases associated with polyketide and
shorterthanotherLanAsandspanonlyabout30aminoacids(see nonribosomalpeptidebiosynthesis.Thethird-largestlanthipep-
Fig.S3inthesupplementalmaterial).Theseshortprecursorpep- tidefamilyintheinvestigatedgenomecollectionisGCF133(48
tides are possibly truncated LanAs, which have recently been members)(seeFig.S1inthesupplementalmaterial),whichrep-
found in several lanthipeptide biosynthetic systems and were resentsaveryunusualbiosyntheticcluster.Eachmemberofthis
showntobegenuinesubstrates(19). family encodes a LanKC and a series of polyketide synthetase
Another intriguing lanthipeptide biosynthetic system was (PKS)-likeenzymes,includinganacylcarrierprotein(ACP),two
foundinGCF140(29members),whosegeneclusterseachencode 3-oxoacyl-ACP synthases (KS), a malonyltransferase (MT), a
threeputativeprecursorpeptides,aLanKC,andasetofhypothet- short-chaindehydrogenase/reductase(SDR)likelytobeaketore-
icalenzymes(Fig.3D).Notably,allthreeprecursorpeptideslacka ductase(KR),andaputativeenoylcoenzymeA(enoyl-CoA)hy-
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FIG4AssociationofgenesencodinglanthipeptidesynthetaseswithPKSandNRPSgenes.(A)ArepresentativegeneclusterofGCF133thatlikelyencodes a
lanthipeptidesynthetase-PKShybridsystems.(B)ArepresentativegeneclusterofGCF26thatlikelyencodeslanthipeptidesynthetase-NRPShybridsystems,and d
alogodepictingtheprecursorpeptidesequences.Theemptysitesintheleaderpeptideregionareaconsequenceofhighsequencedivergence(i.e.,noconserved e
d
residues).ThesequencealignmentofGCF26precursorpeptidesisshowninFig.S5inthesupplementalmaterial.TheGGleaderpeptidecleavagesiteandthe
f
conservedCysresidueintheputativecoreregionarehighlightedbyaredboxandaredasterisk,respectively.Theputativefunctionsofgeneproductsareshown r
o
bycolors.MT,malonyltransferase;ACP,acylcarrierprotein;KS,ketosynthase;KR,ketoreductase;DH,dehydratase/enoyl-CoAhydratase;AL,ATP-dependent m
ligase;RNR,ribonucleotidereductase-like(theseRNR-likeproteinsappeartocontaintheligandsforadinuclearmetalclusterbutnotatyrosineforradical
h
formation);sLanB,smallLanB.TheboundariesofeachgeneclusterarepredictedbasedontheconservedgeneswithineachGCFandarenotclearlydefined.
t
t
p
:
/
/
a
e
dratase(Fig.4A).TheseenzymesarereminiscentofatypeIIPKS suggestthepossibilityofcrosstalkbetweenribosomalandnonri-
m
system,which,unlikemodularPKSs,arecomplexesofmonofunc- bosomalpeptidenaturalproducts,whichhasrecentlyalsobeen .
a
tionalenzymes(52).Alanthipeptideprecursorpeptidecouldnot reportedinanothercontextforpheganomycinbiosynthesis(55). s
m
befoundinthecluster,buttheLanKCislikelytobeinvolvedin Interestingly,theLanMenzymesencodedinGCF26alllackthe
.
thebiosynthesisoftheproductofGCF133,asitsencodinggeneis normallyhighlyconservedzincligandsthatareimportantforcy- o
r
embeddedinthemiddleofthecluster(Fig.4A).AP450enzymeis clization(seeFig.S6andTableS1inthesupplementalmaterial). g
/
alsoencodedinthegenecluster,whichsharessignificantsimilar- Ofthe211LanMenzymesencodedinthegenomesinvestigated o
n
ity(37%identityand53%similarity)withEryF,whichisinvolved here, 196 have the canonical conserved CC(H/C) zinc-binding

N
inerythromycinbiosynthesis(53).Inaddition,theGCFencodes ligandset.Ofthe15thatdonot,12areencodedinclusterswith
o
anenzymesharingsignificantsimilaritiestononhemedinuclear NRPSgenes(GCF26andGCF98)(seeTableS1inthesupplemen- v
e
FeorFe/Mnenzymesrelatedtothesmallsubunitofribonucle- talmaterial).ThesefindingssuggestthattheseLanMenzymesare m
otidereductase(RNR).Itiscurrentlytooearlytoproposepoten- usedonlyfordehydrationprocessesandnotforcyclization. b
e
tialproductsofGCF133.Regardless,thebiosynthesisalmostcer- ClusterscontainingLanCbutnotLanBenzymes.Dehydra- r

tainlyinvolvesunprecedentedandintriguingbiochemistry. tionofSer/ThrresiduescatalyzedbyLanBsisaprerequisitefor 1
7
WehaverecentlyshownthatthenisindehydrataseNisBcon- LanC-catalyzed cyclizations (Fig. 1A). However, LanC-like en- ,

sistsofanN-terminaldomainthatutilizesglutamyl-tRNAtoglu- zymesarewidespreadineukaryotes,whereasthecorresponding 2
0
tamylatethesidechainsofSerandThrresiduesthataretargeted LanBhomologsarenotpresent(12,36).Themoleculardetailsof 1
8
for dehydration and a small C-terminal domain (SpaB_C do- thefunctionsofeukaryoticLanC-likeenzymesremainlargelyelu-
b
main)thatisrequiredforglutamateeliminationtoformthede- sive (56–61). In prokaryotes, orphan LanCs have also been ob- y

hydroamino acids (54). Notably, a set of small LanBs (sLanBs) servedsporadically,whichcouldpossiblybeevolutionaryvestiges g
u
lacking the elimination domain are apparently associated with (12,21).Inouranalysis,wenotedthatGCF125,thefourth-largest e
nonribosomal peptide synthetases (NRPSs), possibly adding lanthipeptide family in the investigated Actinobacteria genome s
t
aminoacidstothegrowingpeptideinatRNA-dependentmanner collection,with36members(seeFig.S1inthesupplementalma-
(54).WhilethemajorityofthesesLanBsresideontheirownpoly- terial), encodes LanCs but not LanBs (Fig. 5A). Putative LanA
peptide chain, the NRPS GCF348 (23) contains a gene cluster substratesarealsoencodedinthegenecluster,whicharehighly
whereinthesLanBresidesinamoduleofanNRPSalongwithan conservedinsequenceandcontainmultipleCysandSer/Thrres-
adenylationdomainandapeptidylcarrierprotein,providingfur- idues(Fig.5A).Anenzymefromthecupinfamily,alargefamilyof
ther support for integration of sLanB into the assembly line of enzymeswithhighlydiversefunctions(62),isalsopresentinthese
certainNRPSbiosyntheticpathways. geneclusters.Interestingly,ahypotheticalproteinsharingweak
Wealsonotedauniquefamily,GCF26(8members),thaten- similaritywithLanMenzymes(BLASTEvalueof(cid:5)10(cid:3)6withthe
codesansLanB,aLanM,andanNRPS(Fig.4B).AputativeLanA TIGR03897familyconsistingofLanMs)isalsoencoded(Fig.5A),
substrateisalsoencodedintheseclusters,whichhasaconserved suggestinganovellanthipeptidebiosyntheticsystem.ThisLanM-
Gly-GlyproteolyticleaderpeptideremovalsiteandmultipleSer/ likestand-aloneenzymeislikelyadehydrataseandpossiblyasur-
ThrresiduesbutonlyoneCysinthepredictedcorepeptide(Fig. rogateforthecanonicalLanBenzymesusuallyfoundinconjunc-
4B;seeFig.S5inthesupplementalmaterial).Theseobservations tion with LanC proteins. A stand-alone dehydratase domain
4344 aem.asm.org AppliedandEnvironmentalMicrobiology July2015 Volume81 Number13
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FIG5PutativeLanC-containinglanthipeptidesystemswithoutLanBenzymes.(A)Arepresentativegeneclusterandtheprecursorpeptidesequencelogoof tp
GCF125.(B)ArepresentativegeneclusterandtheprecursorpeptidesequencelogoofGCF114.Theputativefunctionsofgeneproductsareshownbycolors.The :/
/
boundariesofeachgeneclusterarepredictedbasedontheconservedgeneswithineachGCFandarenotclearlydefined. a
e
m
.
a
relatedtoLanMproteinsisalsofoundinPoyE,whichisinvolved dehydrogenaseisflavinmononucleotidedependent,andrelated s
m
in polytheonamide biosynthesis (63), although the LanM-like enzymescatalyzetheconversionofazolinestoazoles(64,72).The
.
proteins in GCF125 are more divergent from canonical LanM twoTOMMenzymesandLanMinGCF127areencodedtogether o
r
proteins than PoyE. The very high density of Ser, Cys, and Gly with a P450 enzyme, a putative precursor substrate, and three g
/
residuesinthesubstratepeptides(Fig.5A)isreminiscentofthia- transporterproteins. o
n
zole/oxazole-modified microcins (TOMMs) (64), but no other PossibleO-methylationinclassIlanthipeptidebiosynthesis.

N
RiPPbiosyntheticgeneswerefoundinthegeneclusters. Areoccurringobservationinouranalysis,onethatwasalsomade
o
AnothernotablefamilywhosemembersencodeLanCswith- previously with a smaller genome set (21), is that many class I v
e
outthecorrespondingLanBsisGCF114(9members)(Fig.5B). lanthipeptidebiosyntheticgeneclustershaveaproteinhomolo- m
Nootherlanthipeptidesynthetase-likeenzymesarefoundinthis gous to L-isoaspartate O-methyltransferase. These gene cluster b
e
family.Onceagain,theputativeLanAsencodedinthesegeneclus- families include GCF1, GCF20, GCF27, GCF29, GCF44, and r

terscontainmultipleGly,Ser,andCysresidues(Fig.5B),present- GCF124(Fig.7A;seeFig.S7inthesupplementalmaterial).The 1
7
ingapotentiallyveryinterestingnaturalproductbiosynthesissys- LanAprecursorpeptidesfromeachfamilyaredistinctfromoth- ,

temforfutureinvestigations. ers,buttheyallcontainatleastoneAspresidueinthecoreregion 2
0
Orchestration of lanthipeptide and TOMM biosynthesis. that is conserved in each GCF (see Fig. S7 in the supplemental 1
8
ThiopeptidesareaclassofRiPPscharacterizedbyasix-member material).WhethertheseAspresiduesarerelatedtomethyltrans-
b
nitrogen-containing heterocycle that is believed to be formed ferremainstobeinvestigated.Althoughmethyltransferasesindif- y

fromcycloadditionofdehydroalanineresidues(65).Inaddition, ferent gene cluster families share significant similarities, they g
u
thiopeptidestypicallycontainazoleheterocycles.Thedehydroala- mostly separate into different clusters (Fig. 7B), suggesting that e
ninesinthiopeptidesareformedbyaLanB-likeenzymeandthe eachfamilyofenzymesmayhaveevolvedseparately.Interestingly, s
t
azoles by a cyclodehydratase/dehydrogenase pair (66, 67). thephylogenyofLanCisdifferentfromtheclusteringofthemeth-
GCF127 (8 members; currently limited to Kitasatospora ) is a yltransferases.Forexample,theLanCsfromGCF1,GCF29,and
uniquefamilythatalsocombineslanthipeptideandTOMMbio- GCF124aremorecloselyrelatedtoeachotherandareonasepa-
syntheticmachinerybutinamannerdifferentfromthatforthe ratebranchfromGCF27(Fig.2),butthemethyltransferasesfrom
thiopeptides.ThisfamilyencodesaLanMandapairofTOMM GCF27andGCF124areverycloselyrelatedandseparateonlywith
biosyntheticenzymes,includingacyclodehydrataseandadehy- verystringentcutoff(Fig.7B),suggestingthattheLanCandmeth-
drogenase.Notably,basedonsequence,theLanMhasafunctional yltransferaseenzymesmaynothavecoevolved.Thisfindingisin
dehydratasedomainbuttheC-terminaldomainisnothomolo- contrasttotheobservationthat,exceptforrarecases,LanCand
goustothecanonicalLanC-likecyclizationdomain.Theputative LanBenzymesappeartohavecoevolved(12).Anotablefinding
cyclodehydratase is a fusion protein containing an N-terminal forGCF20andGCF29isthatastand-aloneSpaB_Cprotein,sim-
dockingdomainandaC-terminalYcaOdomain(68),anorgani- ilartotheeliminationproteininthiopeptidebiosynthesis(54),is
zationsimilartothatofthecyclodehydratasesinvolvedinthio- encodedinthegenecluster(Fig.7A);thisSpaB_Cproteinlikely
peptide and cyanobactin biosynthesis (Fig. 6 ) (64, 66–71). The involvesnovelchemistry.
July2015 Volume81 Number13 AppliedandEnvironmentalMicrobiology aem.asm.org 4345
Zhangetal.
FIG6 ArepresentativegeneclusterofGCF127thatlikelyencodeslanthipeptide-TOMMhybridsystems.Theputativefunctionsofgeneproductsareshownby D
colors.TheboundariesofeachgeneclusterarepredictedbasedontheconservedgeneswithineachGCFandarenotclearlydefined.CD,cyclodehydratase;DHG, o
w
dehydrogenase.
n
lo
a
d
Venezuelins produced by Actinobacteria. Although they TOFMS).ThreeStreptomycesstrains(S.katraeISP5550,S.laven-
e
share similar enzyme structure and organization, class IV LanL dulaesubsp.lavendulaeNRRLB-2508,andStreptomycessp.strain d

enzymesaresignificantlyoutnumberedbytheclassIIILanKCsin NRRLB-2375)producedvenezuelinsindetectableamounts(Fig. fr
o
Actinobacteria(Fig.2).Venezuelin,previouslyobtainedonlyviain 8C;seeFig.S8inthesupplementalmaterial).Venezuelinsofdif- m
vitro modification of the precursor peptide VenA by VenL fol- ferentlengthsweredetected,similartotheobservationswithclass
h
lowedbyproteolyticremovaloftheleaderpeptide(16),represents IIIlanthipeptides(38,39,42,73).Thevariationinlengthsofthe tt
p
theonlyclassIVlanthipeptidecharacterizedsofar.However,pro- observedvenezuelinscorrespondedtothepresenceorabsenceof :
/
/
ductionofvenezuelinwasdetectedneitherinthewild-typestrain Phe28andAla29(numberingbasedonVenAsequence)(Fig.8B; a
e
Streptomycesvenezuelaenorafterattemptedexpressionofthegene seeFig.S8inthesupplementalmaterial),presumablyarisingfrom m
clusterinStreptomyceslividans,raisingthequestionwhetherven- stepwiseremovalof(partof)theleaderpeptides.Thevenezuelin .
a
ezuelinisagenuinenaturallanthipeptide.Inourcurrentanalysis, fromS.katraeISP5550,whichcontainedAla29,wasfurtherana- s
m
wenotedthatGCF147(33members)(seeFig.S1inthesupple- lyzedbyelectrosprayionization(ESI)-MS-MSanalysis(Fig.8D).
.
mentalmaterial)consistsmostlyofvenezuelin-likegeneclusters Thelackofanyfragmentationshowedthatthepeptidelikelycon- o
r
(Fig.8A),andtheputativeprecursorpeptidesequencesarehighly sistsofoverlapping(methyl)lanthioninerings,consistentwiththe g
/

conserved (Fig. 8B), suggesting the wide occurrence of venezu- previouslyproposedringtopologyofvenezuelinsbasedonexten- o
n
elin-likelanthipeptidegeneclusters(althoughitwasfirstdiscov- sivemutagenesisstudies(16).Additionalverificationthatvenezu-
N
ered in S. venezuelae, here we collectively designate all these elinfromS.katraeISP5550isindeedalanthipeptidewasprovided
o
similar putative lanthipeptides venezuelins). The conserved byhydrolysisofthepeptideandderivatizationoftheconstituent v
e
genesinGCF147includeaLanAgene,aLanLgene,andtwoABC aminoacidsasmethylestersandpentafluoropropionamides.Chi- m
transportergenes(Fig.8A),indicatingthat(methyl)lanthionine ralgaschromatography(GC)-MSanalysisofthederivatizedven- b
e
formation is likely the only posttranslational modification on ezuelin residues revealed the presence of (methyl)lanthionine, r

LanA core peptides in this family. We screened for venezuelin supportingthedesignationofvenezuelinasalanthipeptide(Fig. 1
7
production in strains from GCF147 using matrix-assisted laser 8E).The(methyl)lanthioninederivativesallhadtheDLconfigu- ,
2
desorptionionization–timeofflightmassspectrometry(MALDI- ration.Ouranalysisvalidatesthatvenezuelinsaregenuinenatural 0
1
8

b
y

g
u
e
s
t
FIG7O-MethyltransferasesinclassIlanthipeptidebiosynthesis.(A)RepresentativegeneclustersofGCFsthatencodeclassIlanthipeptidesystemswitha
putativeO-methyltransferase.(B)SequencesimilaritynetworkanalysisoftheputativeO-methyltransferasesinpanelA.
4346 aem.asm.org AppliedandEnvironmentalMicrobiology July2015 Volume81 Number13
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p
:
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a
e
m
.
a
s
m
.
o
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g
/

o
n

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o
v
e
m
b
e
FIG8ProductionofvenezuelinsinActinobacteria.(A)RepresentativegeneclustersofGCF147.(B)AlogodepictingtheGCF147precursorpeptidesequences. r
(C)MALDI-TOFMSanalysisofvenezuelinproducedbyStreptomyceskatraeISP5550.MALDI-TOFMSanalysisofvenezuelinsproducedbyStreptomyces 1
lavendulaesubsp.lavendulaeNRRLB-2508andStreptomycessp.NRRLB-2375areshowninFig.S8inthesupplementalmaterial.(D)ESI-MS-MSanalysisof 7
,
venezuelinproducedbyS.katraeISP5550.Thelackoffragmentationisconsistentwithaglobular,overlappingringtopology.(E)ChiralGC-MSanalysisof
2
hydrolyzedandderivatizedvenezuelinresidues,whichrevealedthepresenceofDL-lanthionineandDL-methyllanthionine.Tracei,sampleshowingthepresence 0
ofDL-lanthionine;traceii,samplespikedwithDL-lanthioninestandard;traceiii,sampleshowingthepresenceofDL-methyllanthionine;traceiv,samplespiked 1
8
withDL-methyllanthioninestandard.
b
y

g
u
products,andbasedonthefrequencyoftheirbiosyntheticgenes thusfar.Analysisofjust17selectedGCFsthatareamongthemost e
(seeFig.S1inthesupplementalmaterial),theymaybeproduced abundantGCFsalreadyrevealednovelposttranslationalmodify- s
t
by many Actinobacteria. This observation also further supports ingenzymesthatappeartohavebeenrecruitedforlanthipeptide
theprevioussuggestionthatidentificationoftheproductsofsilent biosynthesis, as well as several intriguing systems that illustrate
clusters may be accomplished by screening several strains that evenmorecrosstalkbetweendifferentRiPPsystemsthanrecog-
containtheGCF(23). nizedatpresent(9).Theanalysisalsoshowsthattheclassification
systemcurrentlyinusemayneedtobeupdated,sinceseveralof
DISCUSSION theGCFsdonotfallclearlyinthefourclassesastheyarecurrently
Lanthipeptidesareanintriguingclassofnaturalproductsnotonly defined.Forinstance,GCF146appearstobeahybridofclassII
because of their potential medicinal values but also because of andIIIlanthipeptidegeneclusters,andGCFs26and125contain
theirwideoccurrenceandsuitabilityforgenomeminingandhet- genesencodingenzymesthatwouldcurrentlydefinethemasclass
erologousexpressionstudies.AnextensiveanalysisofActinobac- IorII.Inaddition,theanalysessuggestcrosstalkbetweenlanthi-
teriagenomesbuildingontherecentgeneclusterfamilyproject peptidebiosyntheticenzymesandPKSandNRPSsystems.
shows that the chemical space of lanthipeptide-related natural WeemphasizethatinadditiontotheseinterestingGCFsthat
productsisfarbeyondwhattheresearchcommunityhastapped containatleast5members,averylargenumberofsmallerGCFsas
July2015 Volume81 Number13 AppliedandEnvironmentalMicrobiology aem.asm.org 4347
Zhangetal.
wellassingletonclusterswereidentifiedinActinobacteria(seeFig. DJ,DawsonM,DittmannE,DonadioS,DorresteinPC,EntianK-D,
S1 in the supplemental material) (23), which suggests an even Fischbach MA, Garavelli JS, Göransson U, Gruber CW, Haft DH,
HemscheidtTK,HertweckC,HillC,HorswillAR,JasparsM,KellyWL,
largerdiversitythanthatdiscussedhere.Infact,withtheexcep-
KlinmanJP,KuipersOP,LinkAJ,LiuW,MarahielMA,MitchellDA,
tionoftheclassIIIlanthipeptidessuchasthemorphogenSapB
MollGN,MooreBS,MüllerR,NairSK,NesIF,NorrisGE,OliveraBM,
and various peptins (both encoded within GCF31, the largest Onaka H, Patchett ML, Piel J, Reaney MJT, Rebuffat S, et al. 2013.
GCF),theknownlanthipeptidesfromActinobacteria,suchasmi- Ribosomallysynthesizedandpost-translationallymodifiedpeptidenatu-
crobisporicinandactagardine,arefoundinsuchsmallGCFsor ralproducts:overviewandrecommendationsforauniversalnomencla-
ture.NatProdRep30:108–160.http://dx.doi.org/10.1039/C2NP20085F.
singletonclusters(seeFig.S1inthesupplementalmaterial),fur-
10. OmanTJ,vanderDonkWA.2010.Followtheleader:theuseofleader
therhighlightingthatalargeamountofstructuralandfunctional peptidestoguidenaturalproductbiosynthesis.NatChemBiol6:9–18.
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andquiteafewhaveanti-allodynic/antinociceptiveactivity(38, 2004.Lacticin481:invitroreconstitutionoflantibioticsynthetaseactivity. d

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ThisworkwassupportedbytheNationalInstitutesofHealth(GMPO1 /science.1121422. r
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GM077596 to W.A.V.D.D. and GM R01 058822 to W.A.V.D.D.). The 18. TangW,vanderDonkWA.2012.Structuralcharacterizationoffour /

BrukerUltrafleXtremeMALDI-TOF/TOFmassspectrometerwaspur- prochlorosins:anovelclassoflantipeptidesproducedbyplanktonicma- o
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chasedinpartwithagrantfromtheNationalInstitutesofHealth(S10 rine cyanobacteria. Biochemistry 51:4271–4279. http://dx.doi.org/10
RR027109).J.R.D.wasfundedthroughanInstituteforGenomicBiology .1021/bi300255s. N
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fellowship,andM.C.W.issupportedby1F32GM0112284-01(NIH). 19. ZhangQ,YangX,WangH,vanderDonkWA.2014.Highdivergence v
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