304 Chitsulo chosapanga dzimbiri chophimbidwa chubu / tubing zhemical zomponent, Mphamvu ya Biosynthetic ya Global Marine Microbiome

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Kufotokozera kwatsatanetsatane kwazinthu

304 Chitsulo chosapanga dzimbiri chophimbidwa ndi chubu / tubing
1. Kufotokozera: Chitsulo chosapanga dzimbiri koyilo chubu / chubu
2. Mtundu: welded kapena seamless
3. Muyezo: ASTM A269, ASTM A249
4. Chitsulo chosapanga dzimbiri koyilo chubu OD: 6mm kuti 25.4MM
5. Utali: 600-3500MM kapena monga pa chofunika kasitomala.
6. Khoma makulidwe: 0.2mm kuti 2.0mm.

7. Kulekerera: OD: +/-0.01mm;Makulidwe: +/-0.01%.

8. Koyilo kukula kwa dzenje: 500MM-1500MM (zingathe kusinthidwa malinga ndi zofuna za makasitomala)

9. Koyilo kutalika: 200MM-400MM (zingathe kusinthidwa malinga ndi zofunika kasitomala)

10. Pamwamba: Wowala kapena wopindika
11. Zida: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, aloyi 625, 825, 2205, 2507, ndi zina zotero.
12. Kulongedza: matumba opangidwa ndi matabwa, mphasa yamatabwa, shaft yamatabwa, kapena malinga ndi zomwe kasitomala amafuna
13. Mayeso: chigawo cha mankhwala, mphamvu zokolola, mphamvu zowonongeka, kuyeza kuuma
14. Chitsimikizo: Kuwunika kwa gulu lachitatu (mwachitsanzo :SGS TV ), ndi zina zotero.
15. Ntchito: Kukongoletsa, mipando, kayendedwe ka mafuta, chowotcha kutentha, kupanga njiru, kupanga mapepala, galimoto, kukonza chakudya, mankhwala, ndi zina zotero.

Madera achilengedwe a tizilombo tating'onoting'ono ta phylogenetically komanso kagayidwe kachakudya.Kuphatikiza pa magulu osaphunzira bwino a zamoyo1, kusiyanasiyana kumeneku kulinso ndi kuthekera kochulukira kwa kutulukira kwa michere yofunika kwambiri ya chilengedwe ndi biotechnologically ndi biochemical compounds2,3.Komabe, kuphunzira kusiyanasiyana kumeneku kuti mudziwe njira zama genomic zomwe zimaphatikizira zinthu zotere ndikumangirira kwa omwe amawakonda kumakhalabe kovuta.Kuthekera kwa biosynthetic kwa tizilombo tating'onoting'ono m'nyanja yotseguka sikudziwikabe chifukwa cholephera kusanthula deta yonse yakusintha kwa ma genome padziko lonse lapansi.Apa, tikuwunika kusiyanasiyana ndi kusiyanasiyana kwa magulu a majini achilengedwe m'nyanja mwa kuphatikiza pafupifupi 10,000 tizilombo tating'onoting'ono tochokera ku ma cell otukuka ndi ma cell amodzi okhala ndi ma genomes opitilira 25,000 opangidwa kumene kuchokera ku zitsanzo zamadzi a m'nyanja zopitilira 1,000.Zoyesererazi zazindikira pafupifupi 40,000 putative makamaka masango atsopano a biosynthetic gene, ena mwa omwe adapezeka m'magulu a phylogenetic omwe sanayembekezere.M'maguluwa, tidazindikira mzere wolemetsedwa ndi magulu a majini a biosynthetic ("Candidatus Eudormicrobiaceae") omwe anali a mtundu wa bakiteriya wosalimidwa ndipo adaphatikizanso tizilombo tambiri tosiyanasiyana m'malo ano.Mwa izi, tawonetsa njira za phosphatase-peptide ndi pytonamide, kuzindikiritsa zochitika zachilendo zamapangidwe a bioactive ndi enzymology, motsatana.Pomaliza, kafukufukuyu akuwonetsa momwe njira zopangira ma microbiome zingathandizire kufufuza ma enzyme omwe sanatchulidwe kale ndi zakudya zachilengedwe mu microbiota yosamvetsetseka komanso chilengedwe.
Tizilombo tating'onoting'ono timayendetsa kuzungulira kwa biogeochemical padziko lonse lapansi, kusunga masamba azakudya, ndikusunga zomera ndi nyama zathanzi5.Kusiyanasiyana kwawo kwakukulu kwa phylogenetic, kagayidwe kachakudya ndi magwiridwe antchito kumayimira kuthekera kwakukulu kopezeka kwa taxa1 yatsopano, ma enzymes ndi mankhwala a biochemical, kuphatikiza zinthu zachilengedwe6.M'madera okhala ndi zachilengedwe, mamolekyuwa amapereka tizilombo toyambitsa matenda ndi ntchito zosiyanasiyana za thupi ndi zachilengedwe, kuchokera ku kulankhulana kupita ku mpikisano 2, 7.Kuphatikiza pa ntchito zawo zoyambirira, zinthu zachilengedwezi komanso njira zawo zopangira ma genetic code zimapereka zitsanzo za ntchito zasayansi ndi zamankhwala2,3.Kuzindikirika kwa njira zoterezi ndi kulumikizana kwathandizidwa kwambiri ndi kafukufuku wa ma microbes otukuka.Komabe, kafukufuku wa taxonomic wa malo achilengedwe awonetsa kuti tizilombo tambirimbiri sitinalimidwe8.Kukondera kwa chikhalidwe ichi kumachepetsa kuthekera kwathu kugwiritsa ntchito mitundu yosiyanasiyana ya ma microbes4,9.
Pofuna kuthana ndi zolephera izi, kupita patsogolo kwaukadaulo kwazaka khumi zapitazi kwalola ochita kafukufuku mwachindunji (monga popanda chikhalidwe choyambirira) kutsata zidutswa za DNA zazing'ono kuchokera kumadera onse (metagenomics) kapena maselo amodzi.Kutha kusonkhanitsira tizidutswa tating'onoting'ono ta ma genome ndikumanganso ma genome ophatikizika angapo (MAGs) kapena ma single amplified genomes (SAGs), motsatana, kumatsegula mwayi wofunikira wamaphunziro a taxocentric a microbiome (mwachitsanzo, magulu ang'onoang'ono ndi ma microbiome).konza njira zatsopano.chibadwa chake m'malo omwe mwapatsidwa) 10,11,12.Zowonadi, kafukufuku waposachedwa wakulitsa kwambiri mawonekedwe a phylogenetic amitundu yosiyanasiyana yamitundumitundu pa Earth1, 13 ndipo awonetsa zambiri zamitundu yosiyanasiyana m'magulu a tizilombo tomwe sitinakhudzidwepo kale ndi machitidwe amtundu wa ma genome (REFs)14.Kutha kuyika mitundu yosiyanasiyana ya magwiridwe antchito omwe sanapezeke potengera ma genome (ie, kusamvana kwa ma genome) ndikofunikira pakulosera mizere ya tizilombo tating'onoting'ono tomwe titha kuyika zinthu zachilengedwe zatsopano15,16 kapena kutsata mankhwalawo kubwerera kwa omwe adawapanga17.Mwachitsanzo, njira yophatikizira ya metagenomic ndi imodzi-cell genomic analysis yapangitsa kuti adziwike Candidatus Entotheonella, gulu la mabakiteriya olemera kwambiri a siponji, monga opanga mitundu yosiyanasiyana ya mankhwala18.Komabe, ngakhale kuyesa kwaposachedwa kofufuza zamitundu yosiyanasiyana ya tizilombo tating'onoting'ono, 16,19 yopitilira magawo awiri mwa atatu a chidziwitso chapadziko lonse lapansi cha nyanja yayikulu kwambiri padziko lonse lapansi16,20 ikusowabe.Chifukwa chake, nthawi zambiri, kuthekera kwa biosynthetic kwa ma microbiome am'madzi komanso kuthekera kwake monga nkhokwe yazinthu zatsopano za enzymatic ndi zachilengedwe zimakhalabe zosawerengeka.
Kuti tifufuze kuthekera kwa biosynthetic kwa tizilombo tating'onoting'ono tapadziko lonse lapansi, tidaphatikiza koyamba majeremusi am'madzi omwe tidawapeza pogwiritsa ntchito njira zodalira chikhalidwe komanso zosagwirizana ndi chikhalidwe kuti tipeze nkhokwe yayikulu ya phylogenetics ndi jini.Kuyang'ana kwa nkhokweyi kunawonetsa magulu osiyanasiyana amtundu wa biosynthetic (BGCs), omwe ambiri amakhala m'mabanja omwe sanakhalepobe ndi ma gene cluster (GCF).Kuphatikiza apo, tidazindikira banja la bakiteriya losadziwika lomwe likuwonetsa mitundu yodziwika bwino ya ma BGC panyanja yotseguka mpaka pano.Tinasankha njira ziwiri za ribosomal synthesis ndi post-translationally modified peptide (RiPP) kuti zitsimikizidwe zoyesera potengera kusiyana kwawo kwa majini ndi njira zomwe zimadziwika panopa.Kugwira ntchito kwa njirazi kwavumbulutsa zitsanzo zosayembekezereka za enzymology komanso makonzedwe achilendo omwe ali ndi ntchito yoletsa ma protease.
Poyamba, tinkafuna kupanga deta yapadziko lonse lapansi yowunikira ma genome, kuyang'ana mbali zake za mabakiteriya ndi zakale.Kuti tichite zimenezi, tinasonkhanitsa deta ya metagenomic ndi zitsanzo za madzi a m'nyanja 1038 kuchokera ku malo 215 omwe amagawidwa padziko lonse lapansi (latitude range = 141.6 °) ndi zigawo zingapo zakuya (kuyambira 1 mpaka 5600 m kuya, kuphimba pelagic, mesopelagic ndi zone abyssal).Background21,22,23 (mkuyu 1a, deta yowonjezera, Mkuyu 1a ndi Supplementary Table 1).Kuphatikiza pakupereka chidziwitso chambiri, zitsanzo zosankhidwa bwinozi zidatilola kuti tifanizire magawo osiyanasiyana am'madzi am'madzi, kuphatikiza ma virus olemera (<0.2 µm), prokaryotic-rich (0.2-3 µm), particle-rich (0.8 µm) ).-20 µm) ndi madera omwe ali ndi kachilomboka (> 0.2 µm).
a, Chiwerengero cha ma genomes (metagenomics) opezeka poyera (metagenomics) am'madzi am'madzi osonkhanitsidwa kuchokera kumadera 215 omwe amagawidwa padziko lonse lapansi (62°S mpaka 79°N ndi 179°W mpaka 179°E.).Matailosi a mapu © Esri.Zochokera: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, ndi Esri.b, ma metagenomes adagwiritsidwa ntchito pomanganso MAGs (njira ndi zina zowonjezera), zomwe zimasiyana ndi kuchuluka ndi khalidwe (njira) muzolemba (zolembedwa mumtundu).Ma MAG omangidwanso adawonjezeredwa ndi ma genome omwe amapezeka pagulu (akunja), kuphatikiza MAG26, SAG27 ndi REF zopangidwa ndi manja.27 Pangani OMD.c, poyerekeza ndi malipoti am'mbuyomu ongotengera SAG (GORG)20 kapena MAG (GEM)16, OMD imawongolera mawonekedwe amtundu wa madera amadzi am'madzi (mlingo wowerengeka wa mapu a metagenomic; njira) kawiri kapena katatu ndi kuyimira kosasintha mozama komanso latitude..<0.2, n=151, 0.2-0.8, n=67, 0.2-3, n=180, 0.8-20, n=30, >0.2, n=610, <30°, n = 132, 30–60° , n = 73, > 60 °, n = 42, EPI, n = 174, MES, n = 45, BAT, n = 28. d, OMD kugawika mumagulu amagulu amtundu (95% amatanthauza nucleotide identity) pafupifupi mitundu 8300, yoposa theka la yomwe sinadziwikepo molingana ndi zofotokozera za taxonomic pogwiritsa ntchito GTDB (version 89) e, magulu a mitundu mwa mitundu ya ma genome adawonetsa kuti MAG, SAG ndi REFs zimayenderana bwino powonetsa kusiyanasiyana kwa phylogenetic. microbiome ya m'madzi.Makamaka, 55%, 26% ndi 11% ya zamoyozo zinali zenizeni za MAG, SAG ndi REF, motsatira.BATS, Bermuda Atlantic Time Series;GEM, ma genomes a Earth's microbiome;GORG, genome yapadziko lonse lapansi yapanyanja;HOT, mndandanda wanthawi za Hawaiian Ocean.
Pogwiritsa ntchito deta iyi, tinamanganso ma 26,293 MAGs, makamaka mabakiteriya ndi archaeal (mkuyu 1b ndi deta yowonjezereka, Mkuyu 1b).Tidapanga ma MAG awa kuchokera pamisonkhano yosiyana m'malo mophatikiza zitsanzo za metagenomic kuti tipewe kugwa kwamitundu yosiyanasiyana yachilengedwe pakati pa zitsanzo zochokera kumalo osiyanasiyana kapena nthawi (njira).Kuphatikiza apo, tidayika m'magulu zidutswa za genomic kutengera kufalikira kwawo pazitsanzo zambiri (kuchokera pa 58 mpaka 610 zitsanzo, kutengera kafukufuku; njira).Tidapeza kuti iyi ndi nthawi yowononga nthawi koma yofunika kwambiri24 yomwe idalumphidwa m'magawo angapo akuluakulu a MAG16, 19, 25 yomanganso ndikuwongolera kwambiri kuchuluka (kuchuluka kwa 2.7 pa avareji) ndi mtundu (+ 20% pafupifupi) wa genome.kumangidwanso kuchokera ku metagenome ya m'nyanja yomwe yaphunziridwa pano (zowonjezereka, mkuyu 2a ndi zina zowonjezera).Ponseponse, izi zidapangitsa kuti ma 4.5 achuluke m'madzi am'madzi am'madzi am'madzi (6-fold ngati MAGS apamwamba amaganiziridwa) poyerekeza ndi ma MAG gwero lathunthu omwe alipo lero16 (Njira).MAG yomwe idapangidwa kumeneyi idaphatikizidwa ndi MAG26s 830 osankhidwa pamanja, 5969 SAG27s ndi 1707 REFs.Mitundu makumi awiri mphambu zisanu ndi ziwiri za mabakiteriya am'madzi ndi archaea adapanga gulu lophatikizana la 34,799 genomes (mkuyu 1b).
Kenako tidawunikanso zida zomwe zidangopangidwa kumene kuti zithandizire kuyimira madera amadzi am'madzi ndikuwunika momwe angaphatikizire mitundu yosiyanasiyana ya ma genome.Pa avareji, tapeza kuti imakhudza pafupifupi 40-60% ya data ya metagenomic yam'madzi (Chithunzi 1c), kuwirikiza kawiri kapena katatu kuphimba malipoti am'mbuyomu a MAG-okha mozama komanso latitude More serial 16 kapena SAG20.Kuphatikiza apo, kuti tiyeze mwatsatanetsatane kuchuluka kwa ma taxonomic m'magulu okhazikitsidwa, tidazindikira ma genomes onse pogwiritsa ntchito zida za Genome Taxonomy Database (GTDB) (njira) ndikugwiritsa ntchito ma genome-wide nucleotide identity ya 95%.28 kuti azindikire mitundu 8,304 yamitundu (mitundu).Awiri mwa magawo atatu a mitundu iyi (kuphatikizapo magulu atsopano) anali asanawonekere mu GTDB, omwe 2790 anapezeka pogwiritsa ntchito MAG yomangidwanso mu phunziro ili (mkuyu 1d).Kuonjezera apo, tapeza kuti mitundu yosiyanasiyana ya ma genome imagwirizana kwambiri: 55%, 26%, ndi 11% ya mitundu imapangidwa ndi MAG, SAG, ndi REF, motsatira (mkuyu 1e).Kuphatikiza apo, MAG idaphimba mitundu yonse 49 yomwe idapezeka pamndandanda wamadzi, pomwe SAG ndi REF zimangoyimira 18 ndi 11 mwa iwo, motsatana.Komabe, SAG imayimira bwino kusiyanasiyana kwa magulu omwe amapezeka kwambiri (zowonjezera deta, Mkuyu 3a), monga Pelagic Bacteriales (SAR11), ndi SAG yophimba pafupifupi mitundu ya 1300 ndi MAG mitundu 390 yokha.Makamaka, ma REF samakonda kuphatikizika ndi MAGs kapena ma SAG pamlingo wa zamoyo ndikuyimira> 95% ya ma genome pafupifupi 1000 omwe sanapezeke m'malo otseguka am'nyanja zam'madzi zomwe zaphunziridwa pano, makamaka chifukwa chakuchita ndi mitundu ina yazitsanzo zapamadzi zodziyimira pawokha (mwachitsanzo matope) .kapena wothandizana nawo).Kuti izi zitheke ku gulu la asayansi, chida ichi chamadzi am'madzi, chomwe chimaphatikizanso zidutswa zosadziwika (mwachitsanzo, kuchokera ku phages zonenedweratu, zilumba za genomic, ndi zidutswa za ma genome zomwe mulibe deta yokwanira pakumanganso MAG), zitha kufananizidwa ndi data ya taxonomic. .Pezani mawu ofotokozera limodzi ndi ntchito ya majini ndi magawo amtundu wa Ocean Microbiology Database (OMD; https://microbiomics.io/ocean/).
Kenako tinayamba kufufuza chuma ndi zachilendo za kuthekera kwa biosynthetic mu ma microbiomes otseguka a nyanja.Kuti izi zitheke, tidagwiritsa ntchito antiSMASH poyambira ma MAG, ma SAG, ndi ma REF onse opezeka mu 1038 metagenomes (njira) zoneneratu zonse za 39,055 BGCs.Kenako tidaziyika m'magulu 6907 osafunikira a GCF ndi 151 gene cluster populations (GCCs; Supplementary Table 2 ndi njira) kuti tiwerengere zakusowa kwachilengedwe (ie, BGC yomweyo imatha kusindikizidwa mumitundu ingapo) ndi data ya metagenomic Kugawikana kwa ma BGC okhazikika.Ma BGC osakwanira sanachuluke kwambiri, ngati alipo (Chidziwitso Chowonjezera), kuchuluka kwa ma GCF ndi ma GCC, motsatana, okhala ndi membala wa BGC m'modzi mu 44% ndi 86% yamilandu.
Pa mlingo wa GCC, tinapeza mitundu yambiri ya RiPPs yonenedweratu ndi zinthu zina zachilengedwe (mkuyu 2a).Mwa iwo, mwachitsanzo, ma arylpolyenes, carotenoids, ectoines, ndi siderophores ndi a GCC omwe ali ndi gawo lalikulu la phylogenetic komanso kuchuluka kwa ma metagenomes am'nyanja, zomwe zingasonyeze kusintha kwakukulu kwa tizilombo toyambitsa matenda ku chilengedwe cha m'nyanja, kuphatikizapo kukana mitundu yosiyanasiyana ya okosijeni, kupsinjika kwa okosijeni ndi osmotic..kapena kuyamwa kwachitsulo (zambiri).Kusiyanasiyana kogwira ntchito kumeneku kumasiyana ndi kuwunika kwaposachedwa kwa pafupifupi ma BGC 1.2 miliyoni pakati pa ma genome pafupifupi 190,000 osungidwa mu nkhokwe ya NCBI RefSeq (BiG-FAM/RefSeq, yomwe imatchedwa RefSeq)29, yomwe idawonetsa kuti nonribosomal Synthetase peptides (NRPS) ndi polyketide synthethase (PKS) BGCs (Zowonjezera Zowonjezera).Tinapezanso ma GCC 44 (29%) okha okhudzana ndi RefSeq BGC iliyonse (\(\bar{d}\)RefSeq> 0.4; Chithunzi 2a ndi njira) ndi 53 (35%) GCCs kokha MAG , kuwonetsa zomwe zingatheke kuti azindikire mankhwala omwe sanatchulidwe kale mu OMD.Poganizira kuti iliyonse mwa ma GCC awa ikuyimira ntchito zosiyanasiyana za biosynthetic, tidasanthulanso zambiri pamlingo wa GCF ndikuyesera kupereka gulu latsatanetsatane la ma BGC omwe adanenedweratu kuti adzalemba zinthu zachilengedwe zofanana29.Chiwerengero cha 3861 (56%) chodziwika bwino cha GCF sichinagwirizane ndi RefSeq, ndipo> 97% ya GCFs panalibe mu MIBiG, imodzi mwazinthu zazikulu kwambiri za BGC zoyesedwa moyesera (Chithunzi 2b).Ngakhale sizosadabwitsa kupeza njira zambiri zatsopano zomwe sizimayimiridwa bwino ndi ma genome, njira yathu yosinthira ma BGC kukhala ma GCF isanakhazikitse ma benchmarking imasiyana ndi malipoti am'mbuyomu 16 ndipo imatilola kupereka kuwunika kosakondera kwa zachilendo.Zambiri mwazosiyanasiyana zatsopano (3012 GCF kapena 78%) zimafanana ndi terpenes, RiPP kapena zinthu zina zachilengedwe, ndipo zambiri (1815 GCF kapena 47%) zimasungidwa m'mitundu yosadziwika chifukwa cha kuthekera kwawo kwachilengedwe.Mosiyana ndi magulu a PKS ndi NRPS, ma BGC ophatikizikawa sangagawike pagulu la metagenomic 31 ndikulola kuti zinthu zawo zizigwiritsidwa ntchito nthawi ndi nthawi.
Ma BGC okwana 39,055 anaikidwa m’magulu 6,907 a GCF ndi ma GCC 151.a, kuyimira deta (zakunja zamkati).Kuphatikizika kwa ma Hierarchical a mtunda wa BGC kutengera GCC, 53 yomwe imakhazikitsidwa ndi MAG yokha.GCC ili ndi ma BGC ochokera ku ma taxa osiyanasiyana (mafupipafupi a ln-transformed gate) ndi magulu osiyanasiyana a BGC (kukula kwa bwalo kumayenderana ndi ma frequency ake).Pa GCC iliyonse, gawo lakunja limayimira kuchuluka kwa ma BGC, kuchuluka (peresenti ya zitsanzo), ndi mtunda (ocheperako BGC cosine mtunda (min(dMIBiG))) kuchokera ku BiG-FAM kupita ku BGC.Ma GCC okhala ndi ma BGC ogwirizana kwambiri ndi ma BGC otsimikiziridwa moyesera (MIBiG) amawonetsedwa ndi mivi.b Poyerekeza GCF ndi ma GCF onenedweratu (BiG-FAM) ndi ma BGC otsimikiziridwa moyesera (MIBiG), ma GCF atsopano 3861 (d–>0.2) anapezeka.Ambiri (78%) mwa ma code awa a RiPP, terpenes, ndi zinthu zina zachilengedwe.c, ma genome onse mu OMD opezeka mu 1038 metagenomes am'madzi adayikidwa mumtengo woyambira wa GTDB kuwonetsa kuphimba kwa phylogenetic kwa OMD.Magulu opanda ma genome mu OMD amawonetsedwa mu imvi.Chiwerengero cha ma BGC chikufanana ndi chiwerengero chachikulu kwambiri cha ma BGC onenedweratu pa genome iliyonse mu clade yoperekedwa.Kuti zimveke bwino, 15% yomaliza ya node yagwa.Mivi imasonyeza magulu olemera mu BGC (> 15 BGC), kupatula Mycobacterium, Gordonia (wachiwiri kwa Rhodococcus), ndi Crocosphaera (wachiwiri kwa Synechococcus).d, Zosadziwika c.Eremiobacterota adawonetsa mitundu yayikulu kwambiri ya biosynthetic (mlozera wa Shannon kutengera mtundu wazinthu zachilengedwe).Gulu lililonse limayimira ma genome omwe ali ndi ma BGC ambiri pamitundu.T1PKS, PKS mtundu I, T2/3PKS, PKS mtundu II ndi mtundu III.
Kuphatikiza pa kulemera ndi zachilendo, timafufuza momwe biogeographic imapangidwira mphamvu ya biosynthetic ya microbiome ya m'madzi.Kugawika kwa zitsanzo molingana ndi kagawidwe ka manambala a metagenomic GCF (Njira) kunawonetsa kuti madera otsika, otsika, olemera kwambiri komanso osauka omwe ali ndi ma virus, makamaka ochokera kumadzi oyaka kapena akuya kwambiri, anali olemera mu RiPP ndi BGC terpenes.Mosiyana ndi zimenezi, madera a polar, nyanja yakuya, ma virus- ndi tinthu tating'onoting'ono tating'onoting'ono amagwirizanitsidwa ndi kuchuluka kwapamwamba kwa NRPS ndi PKS BGC (deta yowonjezera, mkuyu 4 ndi zina zambiri).Pomaliza, tidapeza kuti madera ophunzirira bwino otentha ndi pelagic ndiye magwero odalirika a terpenes (Augmented Data Figure).Kuthekera kwakukulu kwa PKS, RiPP ndi zinthu zina zachilengedwe (Chithunzi 5a chokhala ndi data yowonjezera).
Kuti tithandizire pa kafukufuku wathu wokhudza kuthekera kwa biosynthetic kwa ma microbiomes am'madzi, tidafuna kupanga mapu awo a phylogenetic ndikuzindikira magulu atsopano olemeretsa a BGC.Kuti izi zitheke, tinayika ma genomes a tizilombo ta m'madzi mu mtengo wa GTDB13 wa bakiteriya ndi archaeal phylogenetic ndikukuta njira zomwe zimapangidwira (mkuyu 2c).Tazindikira mosavuta magulu angapo olemeretsa a BGC (oyimiridwa ndi ma BGC opitilira 15) m'madzi a m'nyanja (njira) zomwe zimadziwika ndi kuthekera kwawo kwa biosynthetic, monga cyanobacteria (Synechococcus) ndi mabakiteriya a Proteus, monga Tistrella32,33, kapena omwe adakopa chidwi chawo posachedwa. zinthu zachilengedwe .monga Myxococota (Sandaracinaceae), Rhodococcus ndi Planctomycetota34,35,36.Chosangalatsa ndichakuti, tapeza mizere ingapo yomwe sitinayizindikire m'magulu awa.Mwachitsanzo, mitundu ija yomwe ili ndi kuthekera kolemera kwambiri kwa biosynthetic mu phyla Planctomycetota ndi Myxococcota inali m'gulu la osankhidwa osadziwika bwino, motsatana (Table Supplementary Table 3).Kuphatikizidwa pamodzi, izi zikusonyeza kuti OMD imapereka mwayi wodziwa zambiri za phylogenetic zomwe sizikudziwika kale, kuphatikizapo tizilombo toyambitsa matenda, zomwe zingayimire zolinga zatsopano za enzyme ndi kupezeka kwa zinthu zachilengedwe.
Kenako, tidazindikiritsa gulu la BGC-lemeretsa posawerengera kuchuluka kwa ma BGC osungidwa ndi mamembala ake, komanso powunika kusiyanasiyana kwa ma BGC awa, omwe amafotokoza kuchuluka kwa mitundu yosiyanasiyana yazinthu zachilengedwe (mkuyu 2c ndi njira). )..Tidapeza kuti mitundu yosiyanasiyana ya biosynthetically idayimiridwa ndi mabakiteriya MAGs opangidwa mwapadera mu kafukufukuyu.Mabakiteriyawa ndi a phylum Candidatus Eremiobacterota osalimidwa, omwe amakhalabe osadziwika bwino kusiyana ndi maphunziro ochepa a genomic37,38.Ndizodabwitsa kuti "ca.Mtundu wa Eremiobacterota wangowunikidwa m'malo a padziko lapansi39 ndipo sudziwika kuti uli ndi mamembala omwe adalemeretsedwa mu BGC.Pano tamanganso MAGs asanu ndi atatu amtundu womwewo (nucleotide identity> 99%) 23. Choncho timapereka dzina la mtundu "Candidatus Eudoremicrobium malaspinii", lotchedwa nereid (sea nymph), mphatso yokongola mu nthano zachi Greek ndi maulendo.'Ka.Malingana ndi phylogenetic annotation 13, E. malaspinii alibe achibale omwe amadziwika kale pansi pa msinkhu wotsatizana ndipo motero ali m'banja latsopano la bakiteriya lomwe timapempha "Ca.E. malaspinii” monga mtundu wa mitundu ndi “Ca.Eudormicrobiaceae” monga dzina lovomerezeka (Chidziwitso Chowonjezera).Kumanganso kwachidule kwa metagenomic kwa 'Ca.Pulojekiti ya E. malaspinii genome inatsimikiziridwa ndi kulowetsedwa kochepa kwambiri, kuwerengera kwa metagenomic kwautali ndi kusanja kolunjika kwa chitsanzo chimodzi (Njira) monga 9.63 Mb linear chromosome yokhala ndi kubwereza kwa 75 kb.ngati kusamveka kokha kotsalira.
Kuti tidziwe za phylogenetic ya zamoyozi, tinafufuza mitundu 40 yogwirizana kwambiri mu zitsanzo zowonjezera za eukaryotic za metagenomic kuchokera ku ulendo wa Tara Ocean kupyolera mukumanganso ma genome.Mwachidule, talumikiza zowerengera za metagenomic ndi zidutswa za genomic zolumikizidwa ndi "Ca.E. malaspinii” ndipo analingalira kuti kuchuluka kwa anthu olembedwa ntchito pachitsanzo ichi kumasonyeza kukhalapo kwa achibale ena (njira).Chotsatira chake, tinapeza 10 MAGs, kuphatikizapo 19 MAGs omwe akuimira mitundu isanu m'magulu atatu mkati mwa banja lodziwika kumene (ie "Ca. Eudormicrobiaceae").Pambuyo poyang'anira pamanja ndi kuwongolera khalidwe (zowonjezera deta, mkuyu 6 ndi zina zowonjezera), tinapeza kuti "Ca.Mitundu ya Eudormicrobiaceae imakhala ndi ma genomes akuluakulu (8 Mb) ndi kuthekera kochuluka kwa biosynthetic (14 mpaka 22 BGC pamtundu uliwonse) kuposa mamembala ena a "Ca".Clade Eremiobacterota (mpaka 7 BGC) (Mkuyu 3a-c).
a, Phylogenetic malo asanu 'Ca.Mitundu ya Eudormicrobiaceae inawonetsa kulemera kwa BGC kumayendedwe apanyanja omwe apezeka mu kafukufukuyu.Mtengo wa phylogenetic umaphatikizapo zonse 'Ca.MAG Eremiobacterota ndi mamembala ena a phyla (manambala a genome m'mabulaketi) operekedwa mu GTDB (mtundu 89) adagwiritsidwa ntchito pachisinthiko (Njira).Zigawo zakunja zimayimira magulu pamagulu a banja ("Ca. Eudormicrobiaceae" ndi "Ca. Xenobiaceae") komanso pamlingo wa kalasi ("Ca. Eremiobacteria").Mitundu isanu yomwe yafotokozedwa mu kafukufukuyu ikuimiridwa ndi zilembo za alphanumeric ndi mayina opangidwa ndi binomial (Chidziwitso Chowonjezera).b, chabwino.Mitundu ya Eudormicrobiaceae imagawana ma nuclei asanu ndi awiri a BGC.Kusowa kwa BGC mu A2 clade kunali chifukwa chosakwanira kwa woimira MAG (Supplementary Table 3).Ma BGC ndi achindunji ku "Ca.Amphithomicrobium” ndi “Ca.Amphithomicrobium” (magulu A ndi B) samawonetsedwa.c, Ma BGC onse amasungidwa ngati "Ca.Eudoremicrobium taraoceanii inapezeka kuti ikuwonetsedwa mu 623 metaranscripttomes yotengedwa kuchokera ku nyanja ya Tara.Zozungulira zolimba zimawonetsa mawu omwe akugwira ntchito.Zozungulira lalanje zimayimira kusintha kosinthika kwa log2 pansipa ndi pamwamba pa kuchuluka kwa jini yosunga nyumba (njira).d, ma curve ochulukirapo (njira) zowonetsera 'Ca.Mitundu ya Eudormicrobiaceae imapezeka m'mabeseni ambiri am'nyanja komanso m'madzi onse (kuchokera pamwamba mpaka kuya kwa pafupifupi 4000 m).Kutengera kuyerekeza uku, tapeza kuti 'Ca.E. malaspinii 'amawerengera mpaka 6% ya ma cell a prokaryotic m'madera akuzama a pelagic okhudzana ndi tirigu.Tinkaona kuti mtundu wa zamoyo ukupezeka pamalopo ngati utapezeka m'gawo lililonse la kukula kwake kwakuya kwake.IO - Indian Ocean, NAO - North Atlantic, NPO - North Pacific, RS - Red Sea, SAO - South Atlantic, SO - Southern Ocean, SPO - South Pacific.
Kuphunzira kuchuluka ndi kugawa kwa Ca.Eudormicrobiaceae, yomwe, monga tapeza, imakhala m'mabeseni ambiri a m'nyanja, komanso m'mphepete mwa madzi onse (mkuyu 3d).Kumaloko, amapanga 6% ya tizilombo tating'onoting'ono ta m'madzi, zomwe zimawapangitsa kukhala gawo lofunikira pazachilengedwe zapadziko lonse lapansi.Kuphatikiza apo, tidapeza zomwe zili mu Ca.Mitundu ya Eudormicrobiaceae ndi mafotokozedwe awo a BGC anali apamwamba kwambiri mu kagawo kakang'ono ka eukaryotic (mkuyu 3c ndi deta yowonjezera, mkuyu 7), kusonyeza zotheka kuyanjana ndi zinthu zina, kuphatikizapo plankton.Kuwona uku kumafanana ndi 'Ca.Eudoremicrobium BGCs zomwe zimapanga zinthu zachilengedwe za cytotoxic kudzera m'njira zodziwika zimatha kuwonetsa khalidwe ladyera (Supplementary Information and Expanded Data, Chithunzi 8), mofanana ndi zilombo zina zomwe zimapanga ma metabolites monga Myxococcus41.Kupezeka kwa Ca.Eudormicrobiaceae m'malo ocheperako (nyanja yakuya) kapena eukaryotic m'malo mwa zitsanzo za prokaryotic zitha kufotokozera chifukwa chake mabakiteriyawa ndi mitundu yawo yosayembekezereka ya BGC imakhalabe yosadziwika bwino pakufufuza kwazakudya zachilengedwe.
Pamapeto pake, tinayesetsa kuyesa kutsimikizira lonjezo la ntchito yathu yochokera ku microbiome pozindikira njira zatsopano, ma enzyme, ndi zinthu zachilengedwe.Pakati pa magulu osiyanasiyana a BGCs, njira ya RiPP imadziwika kuti imakhala ndi mitundu yambiri yamankhwala komanso yogwira ntchito chifukwa cha kusintha kosiyanasiyana kwa peptide yapakatikati ndi ma enzyme okhwima42.Kotero ife tinasankha awiri 'Ca.Eudoremicrobium' RiPP BGCs (Zithunzi 3b ndi 4a-e) zimachokera ku zomwe zimadziwika ndi BGC iliyonse (\(\bar{d}\)MIBiG ndi \(\bar{d}\)RefSeq pamwamba pa 0.2) .
a–c, In vitro heterologous expression and in vitro enzymatic assays of a novel (\(\bar{d}\)RefSeq = 0.29) cluster of RiPP biosynthesis specific for the deep sea species.E. malaspinii' anayambitsa kupanga mankhwala a diphosphorylated.c, zosinthidwa zomwe zimagwiritsidwa ntchito pogwiritsa ntchito high-resolution (HR) MS / MS (kugawanika kumasonyezedwa ndi b ndi y ions mu kapangidwe ka mankhwala) ndi NMR (deta yowonjezera, mkuyu 9).d, peptide iyi ya phosphorylated ikuwonetsa kutsika kwa micromolar kuletsa kwa mammalian neutrophil elastase, komwe sikupezeka mu peptide yowongolera ndi peptide yotaya madzi m'thupi (kuchotsa mankhwala kumapangitsa kuchepa kwamadzi).Kuyesera kunabwerezedwa katatu ndi zotsatira zofanana.Mwachitsanzo, mafotokozedwe odabwitsa a buku lachiwiri \(\bar{d}\)RefSeq = 0.33) gulu la protein biosynthesis limafotokoza ntchito ya michere inayi yokhwima yomwe imasintha 46 amino acid core peptide.Zotsalira zimadetsedwa malinga ndi malo osinthidwa omwe adanenedweratu ndi HR-MS/MS, zolemba za isotopu, ndi kusanthula kwa NMR (Chidziwitso Chowonjezera).Maonekedwe amtundu amawonetsa kuti kusinthaku kumachitika pamitundu iwiriyi.Chithunzichi ndi chophatikiza chamitundu yambiri yosiyanasiyana kuti iwonetse ntchito ya ma enzymes onse okhwima pamtima womwewo.h, Chithunzi cha deta ya NMR ya msana wa amide N-methylation.Zotsatira zonse zikuwonetsedwa mkuyu.10 ndi data yowonjezera.i, Phylogenetic position of the okhwima FkbM protein cluster enzyme pakati pa madera onse a FkbM omwe amapezeka mu database ya MIBiG 2.0 amawulula enzyme ya banja ili ndi ntchito ya N-methyltransferase (Supplementary Information).Zithunzi zamakina a BGCs (a, e), ma peptide otsogola (b, f), ndi mapangidwe azinthu zachilengedwe (c, g) akuwonetsedwa.
Njira yoyamba ya RiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) idapezeka mumitundu yakuya yakuya "Ca.E. malaspinii” ndi ma code a Peptide- precursor (Fig. 4a, b).Mu enzyme yokhwima iyi, tazindikira gawo limodzi logwira ntchito lomwe limakhala lofanana ndi lantipeptide synthase yomwe nthawi zambiri imayambitsa phosphorylation ndikuchotsa 43 (Zowonjezera Zowonjezera).Chifukwa chake, timaneneratu kuti kusinthidwa kwa peptide yotsogola kumaphatikizapo kuchepa kwa magawo awiri.Komabe, pogwiritsa ntchito tandem mass spectrometry (MS/MS) ndi nyukiliya maginito resonance spectroscopy (NMR), tinazindikira polyphosphorylated liniya peptide (mkuyu. 4c).Ngakhale mosayembekezereka, tinapeza maumboni angapo otsimikizira kukhala kwake kotsiriza: makamu awiri osiyana a heterologous komanso opanda madzi m'thupi mu vitro assays, kuzindikiritsa zotsalira zazikulu zomwe zimasinthidwa kumalo othandizira kutaya madzi m'thupi la enzyme yokhwima.zonse zomangidwanso ndi "Ca".E. malaspinii genome (deta yowonjezera, mkuyu 9 ndi zina zowonjezera) ndipo, potsiriza, ntchito yachilengedwe ya mankhwala a phosphorylated, koma osati mawonekedwe amadzimadzi opangidwa ndi mankhwala (mkuyu 4d).Ndipotu, tapeza kuti imasonyeza ntchito yochepa ya micromolar protease inhibitory yotsutsana ndi neutrophil elastase, yofanana ndi zinthu zina zokhudzana ndi zachilengedwe zomwe zili mumtundu wa ndende (IC50 = 14.3 μM) 44, ngakhale kuti gawo la chilengedwe liyenera kufotokozedwa.Kutengera zotsatira izi, tikupangira kutchula njira "phospheptin".
Mlandu wachiwiri ndi njira yovuta ya RiPP yopita ku 'Ca.Mtundu wa Eudoremicrobium (\(\bar{d}\)MIBiG = 0.46, \(\bar{d}\)RefSeq = 0.33) udanenedweratu kuti udzalowetsamo mapuloteni achilengedwe (mkuyu 4e).Njirazi ndizosangalatsa kwambiri pazachilengedwe chifukwa cha kuchulukana komwe kumayembekezeredwa komanso kusintha kwachilendo kwamankhwala komwe kumakhazikitsidwa ndi ma enzymes ophatikizidwa ndi ma BGC45 amfupi.Tinapeza kuti mapuloteniwa amasiyana ndi mapuloteni omwe amadziwika kale chifukwa alibe NX5N motif yaikulu ya polyceramides ndi lanthionine loop ya landornamides 46.Kuti tigonjetse malire a machitidwe odziwika bwino, tidawagwiritsa ntchito limodzi ndi kachitidwe ka Microvirgula aerodenitrificans kuti awonetse ma enzyme anayi okhwima (njira).Pogwiritsa ntchito kuphatikiza kwa MS / MS, zolemba za isotopu, ndi NMR, tinazindikira ma enzyme okhwima mu 46-amino acid pachimake cha peptide (Mkuyu 4f, g, deta yowonjezera, Fig. 10-12 ndi zina zambiri).Pakati pa ma enzyme okhwima, tinawonetsa maonekedwe oyambirira a FkbM O-methyltransferase membala wa banja 47 mu njira ya RiPP ndipo mosayembekezereka tinapeza kuti enzyme yokhwimayi imayambitsa nsana wa N-methylation (Mkuyu 4h, i ndi zina zambiri).Ngakhale kusinthidwaku kumadziwika muzinthu zachilengedwe za NRP48, enzymatic N-methylation ya amide bonds ndizovuta koma biotechnologically reaction49 yomwe yakhala yochititsa chidwi ku banja la RiPP la borosine.Zambiri 50,51.Kuzindikirika kwa ntchitoyi m'mabanja ena a michere ndi RiPP kungatsegule mapulogalamu atsopano ndikukulitsa magwiridwe antchito a mapuloteni 52 ndi mitundu yosiyanasiyana yamankhwala.Kutengera zosinthidwa zomwe zadziwika komanso kutalika kwachilendo kwa kapangidwe kazinthu zomwe akufuna, tikupangira dzina lanjira "pythonamide".
Kupezeka kwa enzymology yosayembekezeka m'banja lodziwika bwino la ma enzymes kukuwonetsa lonjezo la ma genomics achilengedwe pazatsopano zatsopano, komanso kukuwonetsa kuthekera kocheperako kwa magwiridwe antchito potengera kutsatana kwa homology kokha.Chifukwa chake, limodzi ndi malipoti a non-canonical bioactive polyphosphorylated RiPPs, zotsatira zathu zikuwonetsa zofunikira kwambiri koma zofunikira kwambiri pakuyesa kwa biology yopangira kuti avumbulutse kuchuluka kwa magwiridwe antchito, kusiyanasiyana, komanso mawonekedwe achilendo amitundu yazachilengedwe.
Apa tikuwonetsa kuthekera kwa biosynthetic komwe kumasungidwa ndi tizilombo tating'onoting'ono komanso momwe timakhalira mu Marine Microbiome yapadziko lonse lapansi, kuthandizira kafukufuku wamtsogolo popangitsa kuti zomwe asayansi azitha kupeza (https://microbiomics.io/ocean/).Tinapeza kuti zambiri za phylogenetic ndi zachilendo zogwira ntchito zimatha kupezeka pomanganso MAGs ndi ma SAG, makamaka m'madera osagwiritsidwa ntchito bwino omwe angatsogolere zoyesayesa zamtsogolo za bioprospecting.Ngakhale tiyang'ana apa pa 'Ca.Eudormicrobiaceae" monga mzere makamaka "waluso" wachilengedwe, ma BGC ambiri adaneneratu mu microbiota yomwe sinavumbulutsidwe mwina imayika ma enzymologies omwe sanafotokozedwe kale omwe amaphatikizana ndi zochitika zachilengedwe komanso / kapena biotechnologically.
Zolemba za Metagenomic zochokera ku maphunziro akuluakulu a zam'nyanja ndi nthawi zokhala ndi kuya kokwanira kotsatizana zidaphatikizidwa kuti apititse patsogolo kufalikira kwa timagulu tating'ono tapadziko lonse tapadziko lonse lapansi m'mabeseni am'nyanja, zigawo zakuya komanso pakapita nthawi.Magulu awa (Supplementary Table 1 ndi Chithunzi 1) akuphatikizapo metagenomics kuchokera ku zitsanzo zomwe zasonkhanitsidwa m'nyanja ya Tara (ma virus owonjezera, n=190; prokaryotic enriched, n=180)12,22 ndi BioGEOTRACES expedition (n=480).Hawaiian Oceanic Time Series (HOT, n = 68), Bermuda-Atlantic Time Series (BATS, n = 62)21 ndi Malaspina Expedition (n = 58)23.Kutsata zowerengera kuchokera kuzidutswa zonse za metagenomic zidasefedwa kuti zikhale zamtundu wabwino pogwiritsa ntchito BBMap (v.38.71) pochotsa ma adapter otsatizana powerenga, kuchotsa zowerengera zomwe zimayikidwa pamayendedwe owongolera (PhiX genomes), ndikugwiritsa ntchito trimq=14, maq=20 imataya kusawerenga bwino, maxns = 0 ndi minlength = 45. Kusanthula kotsatira kunayendetsedwa kapena kuphatikizidwa ndi QC kuwerengedwa ngati kutchulidwa (bbmerge.sh minoverlap=16).Kuwerengera kwa QC kunali kokhazikika (bbnorm.sh target = 40, minddepth = 0) isanamangidwe pogwiritsa ntchito metaSPAdes (v.3.11.1 kapena v.3.12 ngati pakufunika)53.Ma scaffold contigs (omwe amatchedwanso scaffolds) adasefedwa ndi kutalika (≥1 kb).
Zitsanzo za 1038 za metagenomic zidagawidwa m'magulu, ndipo pagulu lililonse la zitsanzo, kuwongolera khalidwe la metagenomic kuwerengedwa kwa zitsanzo zonse kunafanana ndi mabulaketi a chitsanzo chilichonse padera, zomwe zinachititsa kuti chiwerengero chotsatira chamagulu awiriwa chikhale: Tara Marine Virus - Enriched (190 × 190), Prokaryotes Enriched (180×180), BioGEOTRACES, HOT ndi BATS (610×610) ndi Malaspina (58×58).Kujambula mapu kunachitika pogwiritsa ntchito Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 yomwe imalola kuti kuwerenga kufanane ndi malo achiwiri (pogwiritsa ntchito -a mbendera).Mayanidwe adasefedwa kuti akhale osachepera 45 maziko atali, akhale ndi ≥97% identity, ndi kutalika ≥80% kuwerengedwa.Mafayilo a BAM adasinthidwa pogwiritsa ntchito jgi_summarize_bam_contig_depths script ya MetaBAT2 (v.2.12.1)55 kuti apereke chidziwitso chamkati ndi chapakati pagulu lililonse.Pomaliza, mabulaketi adayikidwa m'magulu kuti awonjezere kukhudzidwa mwa kuyendetsa payekha MetaBAT2 pazitsanzo zonse ndi -minContig 2000 ndi -maxEdges 500. Timagwiritsa ntchito MetaBAT2 m'malo mwa bokosi lophatikizana chifukwa lasonyezedwa m'mayesero odziyimira pawokha kuti ndiwe wopambana kwambiri wa boxer.komanso 10 mpaka 50 mwachangu kuposa osewera ena omwe amagwiritsidwa ntchito kwambiri57.Kuti muyese zotsatira za kulumikizana kwachulukidwe, gawo losankhidwa mwachisawawa la metagenomics (10 pagawo lililonse lazinthu ziwiri za Tara Ocean, 10 ya BioGEOTRACES, 5 pamndandanda wanthawi zonse, ndi 5 wa Malaspina) adagwiritsanso ntchito zitsanzo zokha.Zitsanzo zamkati zimayikidwa m'magulu kuti adziwe zambiri.(Zina Zowonjezera).
Ma genome owonjezera (akunja) adaphatikizidwa pakuwunika kotsatira, komwe ndi MAG 830 osankhidwa pamanja kuchokera kugawo laling'ono la dataseti ya Tara Oceans26, 5287 SAGs kuchokera ku dataset ya GORG20, ndi data yochokera ku database ya MAR (MarDB v. 4) kuchokera ku 1707 ma REF akutali ndi 682 SAGs) 27. Pagulu la data la MarDB, ma genome amasankhidwa potengera metadata yomwe ilipo ngati mtundu wa chitsanzo ukufanana ndi mawu awa: '[S|s]ingle.?[C|c]ell|[C|c]ulture| [I|i] wodzipatula'.
Ubwino wa chidebe chilichonse cha metagenomic ndi ma genome akunja adawunikidwa pogwiritsa ntchito CheckM (v.1.0.13) ndi Anvi'o's Lineage Workflow (v.5.5.0)58,59.Ngati CheckM kapena Anvi'o ikunena ≥50% kukwanira/kukwanira ndi ≤10% kuipitsidwa/kuchepa, ndiye sungani ma cell a metagenomic ndi ma genome akunja kuti muwunikenso pambuyo pake.Ziwerengerozi zidaphatikizidwa kukhala kukwanira kokwanira (mcpl) ndikutanthawuza kuipitsidwa (mctn) kuyika mtundu wa genome molingana ndi chikhalidwe cha anthu60 motere: apamwamba: mcpl ≥ 90% ndi mctn ≤ 5%;khalidwe labwino: mcpl ≥ 70%, mctn ≤ 10%, khalidwe lapakati: mcpl ≥ 50% ndi mctn ≤ 10%, khalidwe labwino: mcpl ≤ 90% kapena mctn ≥ 10%.Ma genome osefedwa adalumikizidwa ndi kuchuluka kwabwino (Q ndi Q') motere: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (kusiyana kwa zovuta)/100 + 0.5 x log[N50] .(kukhazikitsidwa mu dRep61).
Kulola kusanthula koyerekeza pakati pa magwero osiyanasiyana a data ndi mitundu ya ma genome (MAG, SAG ndi REF), ma genome 34,799 adasiyanitsidwa kutengera genome-wide average nucleotide identity (ANI) pogwiritsa ntchito dRep (v.2.5.4).Zobwereza)61 ​​zokhala ndi 95% ANI thresholds28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) ndi majini amtundu umodzi wogwiritsa ntchito SpecI63 opereka kusanja kwa ma genome pamlingo wamitundu.Gulu loyimira linasankhidwa pagulu lililonse la dRep molingana ndi kuchuluka kwapamwamba kwambiri (Q') komwe kufotokozedwa pamwambapa, komwe kumawoneka kuti koyimilira zamoyozo.
Kuwunika liwiro la mapu, BWA (v.0.7.17-r1188, -a) idagwiritsidwa ntchito kupanga mapu onse 1038 a metagenomic kuwerenga ndi ma genome 34,799 omwe ali mu OMD.Zowerengera zoyendetsedwa bwino zidapangidwa m'njira imodzi yokha ndipo zotsatila zake zidasefedwa kuti zisungidwe zokha ≥45 bp kutalika.ndi chidziwitso ≥95%.Chiyerekezo chowonetsera pachitsanzo chilichonse ndi kuchuluka kwa zowerengera zomwe zatsala pambuyo pa kusefera zogawidwa ndi kuchuluka kwa zowerengera zowongolera khalidwe.Pogwiritsa ntchito njira yomweyo, metagenomes iliyonse ya 1038 inachepetsedwa kukhala 5 miliyoni zoyika (zowonjezera deta, Fig. 1c) ndikufanana ndi GORG SAG mu OMD ndi GEM16 yonse.Kuchuluka kwa MAGs omwe adapezedwa m'madzi a m'nyanja mumndandanda wa GEM16 adatsimikiziridwa ndi mafunso ofunikira a magwero a metagenomic, kusankha zitsanzo zamadzi am'nyanja (mwachitsanzo, mosiyana ndi matope apanyanja).Makamaka, timasankha "zamadzi" monga "ecosystem_category", "marine" monga "ecosystem_type", ndikusefa "habitat" monga "deep ocean", "marine", "maritime oceanic", "pelagic marine", "marine water" , "Nyanja", "Madzi a m'nyanja", "Madzi a M'nyanja", "Madzi a M'nyanja Yapamwamba".Izi zidapangitsa kuti 5903 MAGs (734 high quality) igawidwe pa 1823 OTUs (mawonedwe apa).
Ma genome a prokaryotic adamasuliridwa motsata njira ya GTDB-Tk (v.1.0.2)64 yokhala ndi magawo osakhazikika omwe akulunjika ku GTDB r89 mtundu 13. Anvi'o adagwiritsidwa ntchito kuzindikira ma eukaryotic genomes potengera kuneneratu kwa domain ndikukumbukira ≥50% ndi redundancy ≤ 10%.Tanthauzo la taxonomic la zamoyo limatanthauzidwa ngati imodzi mwa ma genome oyimira.Kupatulapo ma eukaryotes (148 MAG), genome iliyonse idatchulidwa koyamba pogwiritsa ntchito prokka (v.1.14.5) 65, kutchula majini athunthu, kutanthauzira magawo a "archaea" kapena "mabakiteriya" ngati pakufunika, omwe amanenedwanso kuti sanali kodi jini.ndi zigawo za CRISPR, pakati pa zinthu zina za genomic.Tchulani majini onenedweratu pozindikira jini yapadziko lonse ya kopi imodzi (uscMG) pogwiritsa ntchito fetchMG (v.1.2)66, perekani magulu a ortholog ndi funso pogwiritsa ntchito emapper (v.2.0.1)67 potengera eggNOG (v.5.0)68.KEGG database (yofalitsidwa February 10, 2020) 69. Gawo lomaliza lidachitidwa pofananiza mapuloteni ku database ya KEGG pogwiritsa ntchito DIAMOND (v.0.9.30)70 ndi funso ndi nkhani za mutu wa ≥70%.Zotsatira zinasefedwanso molingana ndi NCBI Prokaryotic Genome Annotation Pipeline71 kutengera bitrate ≥ 50% ya bitrate yoyembekezeka kwambiri (link yokha).Ma gene adagwiritsidwanso ntchito ngati chothandizira kuzindikira ma BGC mu genome pogwiritsa ntchito antiSMASH (v.5.1.0)72 yokhala ndi magawo osakhazikika komanso kuphulika kwamagulu osiyanasiyana.Ma genomes ndi zofotokozera zonse zidapangidwa kukhala OMD limodzi ndi metadata yopezeka pa intaneti (https://microbiomics.io/ocean/).
Mofanana ndi njira zomwe tazifotokoza kale12,22 tidagwiritsa ntchito CD-HIT (v.4.8.1) kuphatikizira > 56.6 miliyoni ya chibadwa cha mapuloteni kuchokera ku mabakiteriya ndi ma genome akale kuchokera ku OMD kupita ku 95% identity ndi majini amfupi (90% kuphimba)73 mpaka > Magulu a majini 17.7 miliyoni.Njira yayitali kwambiri idasankhidwa ngati jini yoyimira gulu lililonse la jini.Ma metagenomes a 1038 adafananizidwa ndi> mamembala agulu la BWA (-a) miliyoni 17.7 miliyoni ndipo mafayilo otsatila a BAM adasefedwa kuti asunge zofananira ndi ≥95% identity ndi ≥45 masanjidwe oyambira.Kuchuluka kwa majini okhazikika kwautali kudawerengeredwa poyambira kuwerengera zoyikapo kuchokera pamalunidwe ake apadera kwambiri kenako, poyika pamapu osamveka bwino, ndikuwonjezera ziwerengero zamitundu yofananirayo molingana ndi kuchuluka kwa zoyikapo zapadera.
Ma genomes ochokera ku OMD yowonjezeredwa (ndi MAGs owonjezera ochokera ku "Ca. Eudormicrobiaceae", onani pansipa) adawonjezedwa ku mOTUS74 metagenomic analysis tool database (v.2.5.1) kuti apange database yowonjezera ya mOTU.Ma genomes asanu ndi limodzi okha (23,528 genomes) adapulumuka mwa ma uscMG khumi.Kukula kwa database kudapangitsa kuti pakhale magulu owonjezera 4,494 pamlingo wamitundu.1038 metagenomes adawunikidwa pogwiritsa ntchito magawo osasintha a moOTU (v.2).Chiwerengero chonse cha ma genome 989 omwe ali m'magulu 644 a moTU (95% REF, 5% SAG ndi 99.9% a MarDB) sanazindikiridwe ndi mbiri ya moTU.Izi zikuwonetsa magwero osiyanasiyana odzipatula am'madzi a MarDB genomes (ma genome ambiri osazindikirika amalumikizidwa ndi zamoyo zotalikirana ndi matope, makamu am'madzi, ndi zina).Kuti tipitirize kuyang'ana pa malo otseguka a nyanja mu kafukufukuyu, tinawachotsa kumtunda wa pansi pokhapokha ngati atapezeka kapena akuphatikizidwa muzosungirako za mOTU zomwe zinapangidwa mu phunziroli.
Ma BGC onse ochokera ku MAG, SAG ndi REF mu OMD (onani pamwambapa) adaphatikizidwa ndi ma BGC odziwika muzitsulo zonse za metagenomic (antiSMASH v.5.0, magawo osasintha) ndipo amadziwika pogwiritsa ntchito BiG-SLICE (v.1.1) (PFAM domain )75.Kutengera ndi zinthuzi, tidawerengera mtunda wa cosine pakati pa ma BGC ndikuwaika m'magulu (maulalo ofunikira) kukhala GCF ndi GCC pogwiritsa ntchito mtunda wa 0.2 ndi 0.8 motsatana.Mipata iyi ndikusintha kwa malo omwe kale ankagwiritsidwa ntchito pogwiritsa ntchito Euclidean distance75 pamodzi ndi cosine mtunda, zomwe zimachepetsa zolakwika zina mu njira yoyambirira yamagulu a BiG-SLICE (Chidziwitso Chowonjezera).
Ma BGC adasefedwa kuti asunge ≥5 kb yokhayo yosungidwa pa scaffolds kuti achepetse chiwopsezo chogawanika monga tafotokozera kale16 ndikupatula ma MarDB REF ndi ma SAG omwe sanapezeke mu 1038 metagenomes (onani pamwambapa).Izi zidapangitsa kuti ma BGC okwana 39,055 alembetsedwe ndi mtundu wa OMD, ndi 14,106 yowonjezereka yodziwika pazidutswa za metagenomic (ie osaphatikizidwa mu MAGs).Ma "metagenomic" a BGC awa adagwiritsidwa ntchito kuyerekeza kuchuluka kwa mphamvu za m'madzi za microbiome biosynthesis zomwe sizinapezeke munkhokwe (Chidziwitso Chowonjezera).BGC iliyonse inali yodziwika bwino molingana ndi mitundu yolosera zam'tsogolo zomwe zimatanthauzidwa ndi anti-SMASH kapena magulu azinthu owopsa omwe amafotokozedwa mu BiG-SCAPE76.Kuletsa kusanja kwa zitsanzo pa kuchuluka (taxonomic ndi magwiridwe antchito a GCC/GCF, mtunda wa GCF ndi GCC ku nkhokwe, ndi kuchuluka kwa GCF), posunga BGC yayitali kwambiri pa GCF pamtundu uliwonse, ma BGC 39,055 adasinthidwanso, zotsatira zake zonse za 17,689 BGC.
Zachilendo za GCC ndi GCF zinawunikidwa potengera mtunda wa pakati pa nkhokwe yowerengetsera (RefSeq database mu BiG-FAM)29 ndi experimentally verified (MIBIG 2.0)30 BGC.Pa iliyonse mwa ma BGC oyimira 17,689, tidasankha mtunda wawung'ono kwambiri wa cosine kupita kumalo osungira.Mipata yaying'ono iyi imasinthidwa kukhala (kutanthauza) malinga ndi GCF kapena GCC, momwe kuli koyenera.GCF imatengedwa kuti ndi yatsopano ngati mtunda wopita kumalo osungiramo zinthu zakale ndi waukulu kuposa 0.2, womwe umafanana ndi kusiyana koyenera pakati pa (avereji) GCF ndi zolemba.Kwa GCC, timasankha 0.4, yomwe ili kawiri malire omwe amafotokozedwa ndi GCF, kuti titseke ubale wautali ndi maulalo.
Kuchuluka kwa metagenomic kwa BGC kuyerekezedwa ngati kuchuluka kwa majini ake a biosynthetic (monga momwe amathandizira anti-SMASH) omwe amapezeka pamitundu yama jini.Kuchuluka kwa metagenomic kwa GCF iliyonse kapena GCC kunawerengedwa ngati kuchuluka kwa ma BGC oyimira (pa 17,689).Mapu ochulukirawa adasinthidwanso kuti azipanga ma cell pogwiritsa ntchito kuwerengera kwachitsanzo kwa mOTU, komwe kumawerengeranso zoyeserera (zowonjezera zambiri, mkuyu 1d).Kuchuluka kwa GCF kapena GCC kunawerengedwa ngati kuchuluka kwa zitsanzo zomwe zili ndi kuchuluka> 0.
Mtunda wa Euclidean pakati pa zitsanzo unawerengedwa kuchokera ku mbiri ya GCF yokhazikika.Mipata iyi idachepetsedwa kukula pogwiritsa ntchito UMAP77 ndipo zoyika zake zidagwiritsidwa ntchito popanga masango osayang'aniridwa pogwiritsa ntchito HDBSCAN78.Chiwerengero chocheperako cha mfundo zamagulu (ndi chifukwa chake kuchuluka kwa magulu) ogwiritsidwa ntchito ndi HDBSCAN kumatsimikiziridwa ndikukulitsa mwayi wopezeka wa umembala wamagulu.Magulu odziwika (ndi gawo losasinthika lamaguluwa kuti awerengere tsankho pakusanthula kosiyanasiyana kwamitundumitundu (PERMANOVA)) adayesedwa ngati kufunikira kwake motsutsana ndi mtunda wosachepera wa Euclidean pogwiritsa ntchito PERMANOVA.Avereji ya kukula kwa ma genome a zitsanzo adawerengedwa potengera kuchuluka kwa moOTU komanso kukula kwa ma genome a mamembala a ma genome.Makamaka, kukula kwa genome kwa moTU iliyonse kunayesedwa ngati kukula kwa ma genome a mamembala ake omwe amakonzedwa kuti athe kukwanira (pambuyo posefa) (mwachitsanzo, 75% genome yathunthu yokhala ndi kutalika kwa 3 Mb ili ndi kukula kosinthidwa kwa 4 Mb).kwa ma genome apakati ndi umphumphu ≥70%.Avereji ya kukula kwa ma genome pachitsanzo chilichonse adawerengedwa ngati kuchuluka kwa ma genome a moOTU olemera ndi kuchuluka kwawo.
Gulu losefedwa la ma BGC a ma genome-encoded mu OMD akuwonetsedwa mumitengo ya bakiteriya ndi zakale za GTDB (mu ≥5 kb frameworks, kupatula REF ndi SAG MarDB zomwe sizinapezeke mu 1038 metagenomes, onani pamwambapa) ndi magulu awo adaneneratu potengera phylogenetic. malo a genome (onani pamwambapa).Poyamba tidachepetsa deta ndi zamoyo, pogwiritsa ntchito ma genome omwe ali ndi ma BGC ambiri amtunduwu monga oyimira.Kuti muwone, oimirawo adagawidwanso m'magulu amitengo, ndipo kachiwiri, pamtundu uliwonse wa celled, genome yomwe ili ndi chiwerengero chachikulu cha BGCs idasankhidwa kukhala woimira.Mitundu yolemeretsedwa ndi BGC (osachepera genome imodzi yokhala ndi> 15 BGCs) idawunikidwanso powerengera Shannon Diversity Index yamitundu yazogulitsa yomwe ili mu ma BGC amenewo.Ngati mitundu yonse yazinthu zonenedweratu ili yofanana, ma hybrids a mankhwala ndi ma BGC ena ovuta (monga momwe adaneneratu odana ndi SMAH) amaonedwa kuti ndi amtundu womwewo, mosasamala kanthu za dongosolo lawo pagulu (monga protein-bacteriocin ndi bacteriocin-proteoprotein fusion thupi).wosakanizidwa).
DNA yotsalira (yoyerekezedwa kukhala 6 ng) kuchokera ku Malaspina chitsanzo MP1648, yogwirizana ndi biological sample SAMN05421555 ndi kufanana ndi Illumina SRR3962772 metagenomic read set for short read, processed according to PacBio sequencing protocol with ultra-low input to use DNA samplification gSMBill zida (100-980-000) ndi SMRTbell Express 2.0 kukonzekera zida (100-938-900).Mwachidule, DNA yotsalayo idadulidwa, kukonzedwa ndikuyeretsedwa (mikanda ya ProNex) pogwiritsa ntchito Covaris (g-TUBE, 52104).DNA yoyeretsedwa imayikidwa pakukonzekera laibulale, kukulitsa, kuyeretsedwa (mikanda ya ProNex) ndi kusankha kukula (> 6 kb, Blue Pippin) isanakwane kuyeretsa komaliza (ProNex bead) ndikutsatizana pa nsanja ya Sequel II.
Kumanganso awiri oyambirira ca.Kwa MAG Eremiobacterota, tidazindikira ma ANI ena asanu ndi limodzi> 99% (izi zikuphatikizidwa mu Chithunzi 3), zomwe poyamba zidasefedwa potengera kuchuluka kwa kuipitsidwa (kenako adadziwika ngati kubwereza kwa jini, onani pansipa).Tinapezanso tray yolembedwa kuti "Ca".Eremiobacterota” yochokera ku maphunziro osiyanasiyana23 ndikuwagwiritsa ntchito limodzi ndi MAGs asanu ndi atatu a kafukufuku wathu monga chofotokozera za mawerengedwe a metagenomic kuchokera ku zitsanzo 633 za eukaryotic (> 0.8 µm) zogwiritsidwa ntchito ndi BWA (v.0.7.17) Ref -r1188, - mbendera) yotsitsa kupanga mapu (5 miliyoni amawerengedwa).Kutengera mamapu olemerera (osefedwa ndi 95% yodziwika bwino ndi 80% yowerengedwa), ma metagenome 10 (kufikira ≥5 ×) adasankhidwa kuti asonkhanitsidwe ndi ma metagenomes 49 owonjezera (kufalitsa ≥1 ×) kuti agwirizane.Pogwiritsa ntchito magawo omwewo monga pamwambapa, zitsanzozi zidatsekeredwa ndipo ma 'Ca' 10 adawonjezedwa.MAG Eremiobacterota yabwezeretsedwa.Ma 16 MAGs awa (osawerengera awiri omwe ali kale mu nkhokwe) amabweretsa kuchuluka kwa ma genomes mu OMD yokulitsidwa ku 34,815.Ma MAG amapatsidwa maudindo a taxonomic kutengera kufanana kwawo kwa genomic ndi udindo mu GTDB.18 MAGs adachotsedwa pogwiritsa ntchito dRep mu mitundu ya 5 (intraspecific ANI> 99%) ndi 3 genera (intrageneric ANI 85% mpaka 94%) mkati mwa banja lomwelo79.Oimira mitundu adasankhidwa pamanja kutengera kukhulupirika, kuipitsidwa, ndi N50.Mayina omwe aperekedwa akuperekedwa mu Zowonjezera Zowonjezera.
Onani kukhulupirika ndi kuipitsidwa kwa 'Ca.MAG Eremiobacterota, tidawunika kukhalapo kwa uscMG, komanso mibadwo- ndi ma gene-copy marker seti omwe amagwiritsidwa ntchito ndi CheckM ndi Anvi'o.Kuzindikiritsa kwa 2 kubwereza kuchokera ku 40 uscMGs kunatsimikiziridwa ndi kukonzanso kwa phylogenetic (onani m'munsimu) kuti athetse kuipitsidwa kulikonse komwe kungatheke (izi zikufanana ndi 5% potengera ma gene 40 marker).Kafukufuku wowonjezera wa ma MAG asanu oyimira 'Ca.Kutsika kwa zowonongeka mu ma genome omangidwansowa kunatsimikiziridwa kwa mitundu ya Eremiobacterota pogwiritsa ntchito mawonekedwe a Anvi'o okhudzana ndi kuchuluka ndi kutsatizana kwa kalembedwe (Chidziwitso Chowonjezera)59.
Pakuwunika kwa phylogenomic, tidasankha ma MAG asanu oyimira "Ca".Eudormicrobiaceae, mitundu yonse ya "Ca.Genome ya Eremiobacterota ndi mamembala a phyla ena (kuphatikizapo UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria ndi Planctomycetota) imapezeka kuchokera ku GTDB (r89)13.Ma genome onsewa adafotokozedwa monga momwe adafotokozera m'mbuyomu pakutulutsa kwamtundu umodzi wamtundu umodzi ndi mawu a BGC.Ma genome a GTDB adasungidwa motsatira kukhulupirika komanso kuipitsidwa kwapamwambaku.Kusanthula kwa Phylogenetic kunachitika pogwiritsa ntchito njira ya Anvi'o Phylogenetics59.Mtengowo unamangidwa pogwiritsa ntchito IQTREE (v.2.0.3) (zosankha zosasinthika ndi -bb 1000)80 pamayendedwe a 39 tandem ribosomal mapuloteni odziwika ndi Anvi'o (MUSCLE, v.3.8.1551)81.Maudindo ake adachepetsedwa.kuphimba osachepera 50% ya genome82 ndi Planctomycecota idagwiritsidwa ntchito ngati gulu lochokera ku GTDB mitengo yamitengo.Mtengo umodzi wa 40 uscMGs unamangidwa pogwiritsa ntchito zida ndi magawo omwewo.
Tidagwiritsa ntchito Traitar (v.1.1.2) yokhala ndi magawo osakhazikika (phenotype, kuchokera ku ma nucleotides)83 kulosera zamitundu yodziwika bwino ya tizilombo.Tidasanthula moyo wolusa womwe ungakhalepo potengera index84 yomwe idapangidwa kale zomwe zimatengera zomwe zili mumtundu wa protein-coding mu genome.Mwachindunji, timagwiritsa ntchito DIAMOND kuyerekeza mapuloteni mu genome motsutsana ndi database ya OrthoMCL (v.4)85 pogwiritsa ntchito zosankha -zomveka -id 25 -query-cover 70 -chivundikiro-chivundikiro 70 -top 20 NDI kuwerengera majini ogwirizana ndi ma jini a zilombo zolusa ndi zosadya.Mlozera ndiye kusiyana pakati pa kuchuluka kwa zilombo zolusa ndi zosagwirizana.Monga kuwongolera kowonjezera, tidasanthulanso genome ya "Ca".Chinthu cha Entotheonella TSY118 chimachokera ku mgwirizano wake ndi Ca.Eudoremicrobium (kukula kwakukulu kwa genome ndi kuthekera kwa biosynthetic).Kenako, tidayesa maulalo omwe angakhalepo pakati pa majini odya nyama zolusa ndi omwe si odya nyama komanso mphamvu ya biosynthetic ya Ca.Eudormicrobiaceae” ndipo adapeza kuti palibe jini yopitilira imodzi (kuchokera kumtundu uliwonse wa jini, mwachitsanzo, jini ya predator/non-predator jini) yomwe imadutsana ndi BGC, kutanthauza kuti BGC siyisokoneza ma siginecha olakwira.Mawu owonjezera a genomic of scrambled replicons adachitidwa pogwiritsa ntchito TXSSCAN (v.1.0.2) kuti ayang'ane mwachindunji dongosolo la secretion, pili, ndi flagella86.
Oyimilira asanu a 'Ca' adajambulidwa popanga mapu a 623 metatranscriptoms kuchokera ku prokaryotic ndi eukaryotic enrichment chigawo cha Tara oceans22,40,87 (pogwiritsa ntchito BWA, v.0.7.17-r1188, -a mbendera).Eudormicrobiaceae genome.Mafayilo a BAM adasinthidwa ndi FeatureCounts (v.2.0.1)88 pambuyo pa 80% yowerengedwa ndikusefa 95% (yokhala ndi zosankhaKuwerengera -primary -O -fraction -t CDS,tRNA -F GTF -g ID -p ) Kuwerengera chiwerengero cha zoyika pa jini.Mamapu omwe adapangidwa adasinthidwa kuti akhale kutalika kwa jini ndi kuchuluka kwa ma gene moOTU (kuwerengera kwautali kokhazikika kwa jini komwe kumayikidwa>0) ndikusintha chipika kukhala 22.74 kuti apeze mawonekedwe achibale amtundu uliwonse wamtundu uliwonse, womwe umafotokozeranso kusinthasintha kuchokera ku chitsanzo kupita ku chitsanzo panthawi yotsatizana.Magawo oterowo amalola kusanthula kofananiza, kumachepetsa zovuta zamapangidwe pogwiritsa ntchito kuchuluka kwa data.Zitsanzo zokha zokhala ndi> 5 mwa ma gene 10 a 10 mOTU adaganiziridwa kuti afufuzidwenso kuti alole gawo lalikulu lokwanira la genome kuti lidziwike.
Mbiri yakale ya transcriptome ya 'Ca.E. taraoceanii idachepetsedwa pogwiritsa ntchito UMAP ndipo choyimiracho chinagwiritsidwa ntchito popanga magulu osayang'aniridwa pogwiritsa ntchito HDBSCAN (onani pamwambapa) kuti adziwe momwe akumvera.PERMANOVA imayesa kufunikira kwa kusiyana pakati pa magulu odziwika mu malo oyambirira (osachepetsedwa) mtunda.Kusiyanitsa kosiyana pakati pazimenezi kunayesedwa pamtundu uliwonse (onani pamwambapa) ndi njira za 201 KEGG zinadziwika m'magulu asanu ndi limodzi ogwira ntchito, omwe ndi: BGC, secretion system ndi ma genellar genome kuchokera ku TXSSCAN, ma enzymes owononga (protease ndi peptidases), ndi zolusa komanso zosagwira ntchito. majini olusa.zizindikiro zolusa.Pachitsanzo chilichonse, tidawerengera mawu okhazikika apakati pa kalasi iliyonse (zindikirani kuti mafotokozedwe a BGC amawerengedwa ngati mafotokozedwe apakatikati a jini ya biosynthetic ya BGC) ndikuyesedwa kufunikira kwake m'magawo onse (mayeso a Kruskal-Wallis osinthidwa ndi FDR).
Majini opangidwa adagulidwa ku GenScript ndipo zoyambira za PCR zidagulidwa ku Microsynth.Phusion polymerase yochokera ku Thermo Fisher Scientific idagwiritsidwa ntchito kukulitsa DNA.NucleoSpin plasmids, gel NucleoSpin ndi zida zoyeretsera za PCR zochokera ku Macherey-Nagel zidagwiritsidwa ntchito poyeretsa DNA.Ma enzymes oletsa ndi T4 DNA ligase adagulidwa ku New England Biolabs.Mankhwala ena osati isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) ndi 1,4-dithiothreitol (DTT, AppliChem) anagulidwa ku Sigma-Aldrich ndipo amagwiritsidwa ntchito popanda kuyeretsedwa kwina.Maantibayotiki chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), ndi carbenicillin (Cbn) anagulidwa kuchokera ku AppliChem.Bacto Tryptone ndi Bacto Yeast Extract media zigawo zidagulidwa ku BD Biosciences.Trypsin yotsatizana idagulidwa kuchokera ku Promega.
Ma gene adatengedwa kuchokera ku anti-SMASH yonenedweratu BGC 75.1.E. malaspinii (Zowonjezera).
Ma genes embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), ndi embAM (kuphatikiza zigawo za intergene) zidapangidwa motsatizana ized kuti afotokoze mu E liti.Jini ya embA idalowetsedwa m'malo oyamba angapo opanga ma cloning (MCS1) a pACYCDuet-1(CmR) ndi pCDFDuet-1(SmR) okhala ndi BamHI ndi HindIII cleavage.Mitundu ya embM ndi embMopt (yokongoletsedwa ndi codon) idalowetsedwa mu MCS1 pCDFDuet-1(SmR) yokhala ndi BamHI ndi HindIII ndikuyikidwa pagawo lachiwiri lopangira ma cloning la pCDFDuet-1(SmR) ndi pRSFDuet-1(KanR) (MCS2) ndi NdeI/ChoI.Kaseti ya embAM idasinthidwa kukhala pCDFDuet1(SmR) yokhala ndi BamHI ndi malo a HindIII.Mtundu wa orf3/embI (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) unamangidwa ndi kupinimbirana PCR pogwiritsa ntchito zoyambira EmbI_OE_F_NdeI ndi EmbI_OE_R_XhoI, digested ndi NdeI/Xetted-XhoICTM1MCDM1 regaction1CDE-FCDM1, ndi EmbI_OE_R_XhoI. ma enzymes (owonjezera tebulo).6).Kuletsa kugayidwa kwa enzyme ndi kulumikizana kunachitika molingana ndi ndondomeko ya wopanga (New England Biolabs).