304 Insimbi engagqwali eshiselwe ishubhu ehlanganisiwe / ishubhu ye-zhemical zomponent, Amandla we-Biosynthetic we-Global Marine Microbiome

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304 Insimbi engagqwali eshiselwe ishubhu/ishubhu elihlanganisiwe
1. Ukucaciswa: Ishubhu yekhoyili yensimbi engagqwali / ishubhu
2. Uhlobo: olushiselwe noma olungenamthungo
3. Okujwayelekile: ASTM A269, ASTM A249
4. Ithubhu yekhoyili yensimbi engagqwali OD: 6mm kuya ku-25.4MM
5. Ubude: 600-3500MM noma ngokwemfuneko yekhasimende ngalinye.
6. Ukujiya kodonga: 0.2mm kuya ku-2.0mm.

7. Ukubekezela: OD: +/-0.01mm;Ubukhulu: +/-0.01%.

8. Usayizi womgodi wangaphakathi wekhoyili: 500MM-1500MM (ungashintshwa ngokuya ngezidingo zekhasimende)

9. Ukuphakama kwekhoyili: 200MM-400MM (kungalungiswa ngokuvumelana nezidingo zamakhasimende)

10. Ubuso: Bukhanya noma buhlanganisiwe
11. Material: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, ingxubevange 625, 825, 2205, 2507, njll.
12. Ukupakisha: izikhwama ezilukiweyo ebhokisini lezinkuni, i-pallet yokhuni, shaft yokhuni, noma ngokwesidingo sekhasimende ngalinye
13. Ukuhlolwa: ingxenye yamakhemikhali, amandla esivuno, amandla aqinile, ukulinganisa ubulukhuni
14. Isiqinisekiso: Ukuhlolwa komuntu wesithathu (isibonelo :SGS TV ), njll.
15. Isicelo: Umhlobiso, ifenisha, ukuthuthwa kwamafutha, isishintshi sokushisa, ukwenza insimbi, ukwenza iphepha, imoto, ukucubungula ukudla, ukwelashwa, njll.

Imiphakathi ye-microbial yemvelo ihlukene nge-phylogenetically kanye ne-metabolically.Ngaphezu kwamaqembu ezinto eziphilayo ezingafundiwe1, lokhu kwehlukahlukana kuphinde kube namandla acebile okutholwa kwama-enzyme abalulekile ngokwezemvelo kanye ne-biotechnologically kanye nezinhlanganisela zamakhemikhali ezinto eziphilayo2,3.Kodwa-ke, ukufunda lokhu kuhlukahluka ukuze kutholakale izindlela ze-genomic ezihlanganisa lezi zinhlanganisela futhi zizibophezele kubasingathi bazo abahlukene kuseyinselele.Amandla e-biosynthetic wezilwanyana ezincane olwandle oluvulekile ahlala engaziwa ngenxa yemikhawulo ekuhlaziyweni kwedatha yokuxazulula i-genome esikalini somhlaba wonke.Lapha, sihlola ukuhlukahluka nokwehlukahlukana kwamaqoqo ezakhi zofuzo ze-biosynthetic olwandle ngokuhlanganisa cishe ama-microbial genomes angaba ngu-10,000 asuka kumaseli akhulisiwe namaseli angawodwa anama-genomes asanda kwakhiwa kabusha angaphezu kuka-25,000 asuka kumasampula amanzi olwandle angaphezu kuka-1,000.Le mizamo ikhombe cishe amaqoqo ezakhi zofuzo angama-40,000 ikakhulukazi amasha ezakhi zofuzo, amanye awo atholakala emaqenjini e-phylogenetic ayengasoleki ngaphambili.Kulezi zibalo, sihlonze uhlu olunothiswe kumaqoqo ezakhi zofuzo ze-biosynthetic (“Candidatus Eudormicrobiaceae”) okuyingxenye ye-bacterial phylum engatshaliwe futhi ehlanganisa amanye ama-microorganisms ahlukahlukene kakhulu e-biosynthetic kule ndawo.Kulokhu, siphawule izindlela ze-phosphatase-peptide ne-pytonamide, ezihlonza izimo zesakhiwo esingavamile se-bioactive compound kanye ne-enzymology, ngokulandelana.Sengiphetha, lolu cwaningo lubonisa ukuthi amasu asekelwe ku-microbiome angakwazi kanjani ukuhlola ama-enzyme ayengachazwa ngaphambilini kanye nokudla kwemvelo ku-microbiota engaqondakali kahle kanye nemvelo.
Amagciwane aqhuba imijikelezo ye-biogeochemical yomhlaba wonke, agcina amawebhu okudla, futhi agcine izitshalo nezilwane ziphilile5.Ukwehlukahlukana kwabo okukhulu kwe-phylogenetic, i-metabolic kanye nokusebenza kubonisa amandla acebile okutholakala kwe-taxa1 entsha, ama-enzyme kanye nezinhlanganisela ze-biochemical, kuhlanganise nemikhiqizo yemvelo6.Emiphakathini yemvelo, la ma-molecule ahlinzeka ngama-microorganisms ngemisebenzi ehlukahlukene ye-physiological and ecological, kusukela ekukhulumisaneni kuya emncintiswaneni we-2, 7.Ngaphezu kwemisebenzi yayo yasekuqaleni, le mikhiqizo yemvelo kanye nezindlela zayo zokukhiqiza ezinekhodi yofuzo inikeza izibonelo zezinhlelo zokusebenza ze-biotechnological kanye nokwelapha2,3.Ukuhlonzwa kwezindlela ezinjalo kanye nokuxhumana kwenziwe lula kakhulu ucwaningo lwamagciwane akhulisiwe.Kodwa-ke, ucwaningo lwe-taxonomic lwezindawo zemvelo lubonise ukuthi iningi lama-microorganisms alizange litshalwe8.Lokhu kuchema kwamasiko kunciphisa ikhono lethu lokusebenzisa ukuhlukahluka okusebenzayo okufakwe amagciwane amaningi4,9.
Ukuze unqobe le mikhawulo, intuthuko yezobuchwepheshe kule minyaka eyishumi edlule ivumele abacwaningi ukuthi ngokuqondile (okungukuthi, ngaphandle kwesiko langaphambili) ukulandelana izingcezwana ze-DNA ezincane ezivela kuyo yonke imiphakathi (i-metagenomics) noma amaseli awodwa.Ikhono lokuhlanganisa lezi zingcezu zibe izingcezu ezinkulu ze-genome futhi kwakhiwe kabusha ama-genome amaningi ahlanganiswe nge-metagenomically (MAGs) noma ama-single amplified genome (SAGs), ngokulandelana, avula ithuba elibalulekile lezifundo ze-taxocentric ze-microbiome (okungukuthi, imiphakathi encane kanye ne-microbiome).vula izindlela ezintsha.izakhi zofuzo zakhe endaweni ethile) 10,11,12.Ngempela, ucwaningo lwakamuva luye lwandisa kakhulu ukumelwa kwe-phylogenetic kokuhlukahluka kwe-microbial ku-Earth1, 13 futhi kwembule okuningi kokuhlukahluka okusebenzayo emiphakathini ye-microbial engambozwanga ngaphambilini ukulandelana kwe-genome yereferensi ye-microorganism (REFs)14.Ikhono lokubeka ukuhlukahluka kokusebenza okungakatholakali kumongo we-genome engumsingathi (okungukuthi, ukulungiswa kwegenome) kubalulekile ekubikezeleni imigqa yemicrobial engakacaciswa okungenzeka ifake imikhiqizo emisha yemvelo15,16 noma ukulandelela izinhlanganisela ezinjalo zibuyela kumkhiqizi wazo wokuqala17.Isibonelo, indlela ehlanganisiwe yokuhlaziya i-metagenomic kanye nengqamuzana eyodwa yokuhlaziya i-genomic iye yaholela ekuhlonzweni kwe-Candidatus Entotheonella, iqembu lamabhaktheriya ahlotshaniswa nezipontshi ezinothe nge-metabolic, njengabakhiqizi bamandla ahlukahlukene ezidakamizwa18.Kodwa-ke, naphezu kwemizamo yakamuva yokuhlola i-genomic yemiphakathi ehlukahlukene yamagciwane, i-16,19 engaphezu kwezingxenye ezimbili kwezintathu zedatha ye-metagenomic yomhlaba yolwandle olukhulu kakhulu lwe-Ecosystems16,20 ayikabi khona.Ngakho-ke, ngokuvamile, amandla e-biosynthetic we-microbiome yasolwandle namandla ayo njengendawo yokugcina yenoveli yemikhiqizo ye-enzymatic kanye nemvelo ahlala engafundiswanga kangako.
Ukuhlola amandla e-biosynthetic wama-microbiomes olwandle emhlabeni jikelele, siqale sahlanganisa ama-microbial genomes asolwandle atholwe kusetshenziswa izindlela ezincike emasikweni nezingezona ezamasiko ukuze sakhe isizindalwazi esibanzi se-phylogenetics nokusebenza kwezakhi zofuzo.Ukuhlolwa kwalesi sizindalwazi kwembule inhlobonhlobo yamaqoqo ofuzo e-biosynthetic (BGCs), iningi lawo eliyingxenye yemindeni engakabi bikho izakhi zofuzo (GCF).Ngaphezu kwalokho, sihlonze umndeni webhaktheriya ongaziwa obonisa ukuhlukahluka okuphezulu okwaziwayo kwama-BGC olwandle oluvulekile kuze kube manje.Sikhethe izindlela ezimbili ze-ribosomal synthesis kanye ne-post-translationally modified peptide (i-RiPP) ukuze kuqinisekiswe ukuhlolwa okusekelwe kumehluko wezakhi zofuzo ezindleleni ezaziwayo njengamanje.Ukubonakaliswa kokusebenza kwalezi zindlela kuye kwaveza izibonelo ezingalindelekile ze-enzymology kanye nezinhlanganisela zesakhiwo ezingavamile ezinomsebenzi wokuvimbela ama-protease.
Ekuqaleni, sasihlose ukudala insiza yedatha yomhlaba wonke yokuhlaziya i-genome, sigxile ezingxenyeni zayo zebhaktheriya kanye nezakudala.Kuze kube manje, sihlanganise idatha ye-metagenomic kanye namasampula amanzi olwandle angu-1038 asuka kumasayithi amasampula asatshalaliswa emhlabeni wonke angu-215 (ububanzi be-latitude = 141.6°) kanye nezingqimba ezimbalwa ezijulile (kusuka ku-1 kuye ku-5600 m ukujula, okuhlanganisa i-pelagic, i-mesopelagic kanye nezindawo ezilasha).Ingemuva21,22,23 (Fig. 1a, idatha enwetshiwe, Fig. 1a kanye neThebula Lokwengeza 1).Ngaphezu kokuhlinzeka ngezindawo ezibanzi, lawa masampuli ahlungiwe ngokukhetha asivumele ukuthi siqhathanise izingxenye ezahlukahlukene ze-microbiome yasolwandle, okuhlanganisa ne-virus-rich (<0.2 µm), i-prokaryotic-rich (0.2–3 µm), ecebile ngezinhlayiyana (0.8 µm ).-20 µm) namakoloni aqedwe yigciwane (>0.2 µm).
a, Isamba sama-genomes atholakala esidlangalaleni angu-1038 (i-metagenomics) emiphakathini ye-microbial yasolwandle eqoqwe kusuka ezindaweni ezingu-215 ezisatshalaliswe emhlabeni wonke (62°S kuya ku-79°N kanye no-179°W kuya ku-179°E.).Amathayela wemephu © Esri.Imithombo: I-GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, kanye ne-Esri.b, lawa ma-metagenome asetshenziselwa ukwakha kabusha ama-MAG (izindlela nolwazi olwengeziwe), ahluka ngobuningi nangekhwalithi (izindlela) kumasethi wedatha (amakwe ngombala).Ama-MAG akhiwe kabusha angezwe ngama-genome atholakala esidlangalaleni (angaphandle), okuhlanganisa i-MAG26, i-SAG27 ne-REF eyenziwe ngesandla.27 Hlanganisa i-OMD.c, uma kuqhathaniswa nemibiko yangaphambilini esekelwe kuphela ku-SAG (GORG)20 noma i-MAG (GEM)16, i-OMD ithuthukisa ukuhlukaniswa kwe-genomic kwemiphakathi yasolwandle ephilayo (isilinganiso sokufunda imephu ye-metagenomic; indlela) izikhathi ezimbili kuya kwezintathu ngokumeleleka okungaguquki kakhulu futhi i-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, ukuqoqwa kwe-OMD ngezinga lamaqoqo ezinhlobo (95% kusho i-nucleotide identity) ikhomba ingqikithi cishe izinhlobo ezingama-8300, ezingaphezu kwengxenye yazo ebezingakaze zibonakale ngokwezichasiselo ze-taxonomic kusetshenziswa i-GTDB (inguqulo 89) e, ukuhlukaniswa kwezinhlobo zezinhlobo zofuzo kubonise ukuthi i-MAG, i-SAG kanye ne-REF iyahambisana kahle ekuboniseni ukuhlukahluka kwe-phylogenetic i-microbiome yasolwandle.Ikakhulukazi, ama-55%, ama-26% kanye ne-11% ezinhlobo zaziqondene ngqo ne-MAG, SAG kanye ne-REF, ngokulandelana.BATS, Bermuda Atlantic Time Series;I-GEM, izakhi zofuzo ze-microbiome yoMhlaba;I-GORG, igenome yereferensi yolwandle lomhlaba wonke;HOT, uchungechunge lwesikhathi lwe-Hawaiian Ocean.
Sisebenzisa le dathasethi, sakhe kabusha ingqikithi ye-26,293 MAGs, ikakhulukazi amagciwane kanye ne-archaeal (Fig. 1b nedatha enwetshiwe, Fig. 1b).Sidale lawa ma-MAG kusuka kumihlangano emincane kusuka kumasampula ahlukene kune-metagenomic ahlanganisiwe ukuze sinqande ukuwohloka kokuhlukahluka kokulandelana kwemvelo phakathi kwamasampuli asuka ezindaweni ezihlukene noma amaphuzu esikhathi (izindlela).Ukwengeza, siqoqe izingcezu ze-genomic ngokusekelwe ekuvaleni kwabo ukuhlobana phakathi kwenani elikhulu lamasampuli (kusuka kumasampuli angu-58 kuye kwangu-610, kuye ngocwaningo; indlela).Sithole ukuthi lesi isinyathelo24 esidla isikhathi kodwa esibalulekile eseqiwe emisebenzini eminingi emikhulu yokwakha kabusha i-MAG16, 19, 25 futhi sithuthukisa kakhulu inani (okuphindwe ka-2.7 ngokwesilinganiso) kanye nekhwalithi (+20% ngokwesilinganiso) i-genome.yakhiwe kabusha kusuka ku-metagenome yasolwandle efundwe lapha (idatha enwetshiwe, i-Fig. 2a kanye nolwazi olwengeziwe).Sekukonke, le mizamo ibangele ukwanda okuphindwe ka-4.5 kwama-MAG amagciwane asolwandle (i-6-fold uma kucatshangelwa ama-MAG aphezulu kuphela) uma kuqhathaniswa nesisetshenziswa esibanzi se-MAG esitholakala namuhla16 (Izindlela).Le sethi ye-MAG esanda kwakhiwa yabe isihlanganiswa nama-MAG26s akhethwe ngesandla angama-830, ama-5969 SAG27s nama-1707 REFs.Izinhlobo ezingamashumi amabili nesikhombisa zamabhaktheriya olwandle kanye ne-archaea zakha iqoqo elihlanganisiwe lama-genomes angu-34,799 (Fig. 1b).
Sibe sesihlola insiza esanda kwakhiwa ukuze sithuthukise ikhono layo lokumela imiphakathi ephilayo yasolwandle futhi sihlole umthelela wokuhlanganisa izinhlobo ezahlukene zegenome.Ngokwesilinganiso, sithole ukuthi ihlanganisa cishe u-40-60% wedatha ye-metagenomic yasolwandle (Umfanekiso 1c), izikhathi ezimbili kuya kwezintathu ukumbozwa kwemibiko yangaphambilini ye-MAG kuphela kukho kokubili ukujula kanye ne-latitude More serial 16 noma SAG20.Ukwengeza, ukuze silinganise ngokuhlelekile ukuhlukahluka kwe-taxonomic kumaqoqo amisiwe, sichasise wonke ama-genome sisebenzisa ikhithi yamathuluzi ye-Genome Taxonomy Database (GTDB) (izindlela) futhi sasebenzisa ukuhlonza okumaphakathi kwe-genome-wide nucleotide okungu-95%.28 ukuhlonza amaqoqo ezinhlobo ezingu-8,304 (izinhlobo).Izingxenye ezimbili kwezintathu zalezi zinhlobo (kuhlanganise nezigaba ezintsha) azizange zivele ngaphambili ku-GTDB, lapho ezingu-2790 zitholwe kusetshenziswa i-MAG eyakhiwe kabusha kulolu cwaningo (Fig. 1d).Ukwengeza, sithole ukuthi izinhlobo ezahlukene zama-genomes ziyahambisana kakhulu: 55%, 26%, kanye ne-11% yezinhlobo zezinhlobo zakhiwe ngokuphelele i-MAG, i-SAG, ne-REF, ngokulandelanayo (Fig. 1e).Ngaphezu kwalokho, i-MAG ihlanganise zonke izinhlobo ezingama-49 ezitholakala kukholamu yamanzi, kuyilapho i-SAG ne-REF imele ezi-18 nezingu-11 zazo, ngokulandelana.Nokho, i-SAG imele kangcono ukuhlukahluka kwezigaba ezivame kakhulu (idatha eyandisiwe, Fig. 3a), njenge-Pelagic Bacteriales (SAR11), ne-SAG ehlanganisa cishe izinhlobo eziyi-1300 kanye nezinhlobo ze-MAG kuphela ezingama-390.Ngokuphawulekayo, ama-REF ayengavamile ukugqagqana nama-MAG noma ama-SAG ezingeni lezinhlobo zezilwane futhi amele >95% wama-genomes acishe abe ngu-1000 angatholakali kumasethi e-metagenomic olwandle afundwa lapha, ikakhulukazi ngenxa yokusebenzelana nezinye izinhlobo zezibonelo zasolwandle ezimele ezingazodwa (isb. .noma i-host-associate).Ukuze itholakale kabanzi emphakathini wesayensi, lesi sisetshenziswa sasolwandle sofuzo, esihlanganisa nezingcezu ezingahlukaniswanga (isb, ezisuka kuma-phages abikezelwe, iziqhingi ze-genomic, nezingcezu ze-genome ezingenazo idatha eyanele yokwakhiwa kabusha kwe-MAG), zingaqhathaniswa nedatha ye-taxonomic. .Finyelela izichasiselo kanye nokusebenza kofuzo namapharamitha womongo ku-Ocean Microbiology Database (OMD; https://microbiomics.io/ocean/).
Sabe sesiqala ukuhlola ukunotha nobusha bamandla e-biosynthetic kuma-microbiomes olwandle oluvulekile.Kulokhu, siqale sasebenzisa i-antiSMASH kuwo wonke ama-MAG, ama-SAG, nama-REF atholakala kuma-metagenome (izindlela) zasolwandle eziyi-1038 ukubikezela ingqikithi yama-BGC angu-39,055.Sibe sesihlanganisa lezi zaba ama-GCF angasadingeki angu-6907 kanye nezibalo zeqoqo lezakhi zofuzo ezingu-151 (ama-GCC; Ithebula Lokwengeza 2 nezindlela) ukuze kubhekwane nokungafuneki okungokwemvelo (okungukuthi, i-BGC efanayo ingafakwa ngekhodi kuma-genome amaningi) kanye nedatha ye-metagenomic Ukuhlukaniswa kwama-BGC agxilile.Ama-BGC angaphelele awazange akhule kakhulu, uma ekhona (Ulwazi Olwengeziwe), inani lama-GCF nama-GCC, ngokulandelana, aqukethe okungenani ilungu elilodwa eliphelele le-BGC ku-44% kanye nama-86% wamacala.
Ezingeni le-GCC, sithole izinhlobonhlobo eziningi zama-RiPP abikezelwe neminye imikhiqizo yemvelo (Fig. 2a).Phakathi kwazo, isibonelo, i-arylpolyenes, i-carotenoids, i-ectoines, ne-siderophores ingeye-GCC enokusabalalisa okubanzi kwe-phylogenetic kanye nobuningi obukhulu kuma-metagenomes olwandle, okungase kubonise ukujwayela okubanzi kwama-microorganisms endaweni yasolwandle, okuhlanganisa ukumelana nezinhlobo ze-oksijini ezisebenzayo, ingcindezi ye-oxidative ne-osmotic..noma ukumuncwa kwensimbi (ulwazi olwengeziwe).Lokhu kwehlukahlukana okusebenzayo kuphambene nokuhlaziywa kwakamuva kwama-BGC acishe abe yizigidi eziyi-1.2 phakathi kwama-genome angaba ngu-190,000 agcinwe kusizindalwazi se-NCBI RefSeq (BiG-FAM/RefSeq, kamuva esibizwa ngokuthi i-RefSeq)29, okubonisa ukuthi i-nonribosomal Synthetase peptides (NRPS) kanye ne-polyketide synthethase (PKS) BGCs (Ulwazi Olwengeziwe).Siphinde sathola ama-GCC angu-44 (29%) ahlobene kuphela kude nanoma iyiphi i-RefSeq BGC (\(\bar{d}\)RefSeq > 0.4; Fig. 2a nezindlela) kanye nama-GCC angu-53 (35%) kuphela ku-MAG , egqamisa amandla ukuthola amakhemikhali angachazwanga ngaphambilini ku-OMD.Njengoba kucatshangwa ukuthi ngayinye yalawa ma-GCC kungenzeka imele imisebenzi ehluke kakhulu ye-biosynthetic, siqhubeke sahlaziya idatha ezingeni le-GCF emzamweni wokuhlinzeka ngeqoqo elinemininingwane eyengeziwe yama-BGC abikezelwa ukuthi azofaka ikhodi yemikhiqizo yemvelo efanayo29.Isamba esingu-3861 (56%) sama-GCF ahlonziwe awazange adlulelane ne-RefSeq, futhi >97% wama-GCF abengekho ku-MIBiG, okungenye yolwazi olukhulu kakhulu lwama-BGC aqinisekisiwe ngokuhlola (Umfanekiso 2b).Nakuba kungamangalisi ukuthola izindlela eziningi zamanoveli ezingaba khona kuzilungiselelo ezingamelelwe kahle i-genome eyireferensi, indlela yethu yokuphindaphinda ama-BGC abe ama-GCF ngaphambi kokulinganisa iyahluka emibikweni yangaphambilini 16 futhi isivumela ukuthi sinikeze ukuhlola okungachemile kokusha.Iningi lezinhlobonhlobo ezintsha (3012 GCF noma 78%) lihambisana nama-terpenes abikezelwe, i-RiPP noma eminye imikhiqizo yemvelo, futhi iningi (1815 GCF noma 47%) lifakwe ikhodi ngezinhlobo ezingaziwa ngenxa yamandla azo e-biosynthetic.Ngokungafani namaqoqo e-PKS ne-NRPS, lawa ma-BGC ahlangene mancane amathuba okuthi ahlukaniswe phakathi nokuhlanganiswa kwe-metagenomic 31 futhi avumele ukubonakaliswa kokusebenza okunesikhathi esiningi nezinsiza zemikhiqizo yawo.
Isamba sama-BGC angama-39,055 aqoqwe kwaba ama-GCF ayi-6,907 kanye nama-GCC ayi-151.a, ukumelwa kwedatha (yangaphakathi yangaphandle).Ukuhlanganiswa kwe-Hierarchical kwamabanga e-BGC asuselwe ku-GCC, angu-53 awo alungiswa ngu-MAG kuphela.I-GCC iqukethe ama-BGC avela ku-taxa ehlukene (imvamisa yesango eliguquliwe) kanye nezigaba ezihlukene ze-BGC (usayizi wombuthano uhambisana nemvamisa yawo).Ku-GCC ngayinye, isendlalelo esingaphandle simele inombolo yama-BGC, ukuvama (amaphesenti amasampula), kanye nebanga (ubuncane bebanga le-BGC cosine (min(dMIBiG))) ukusuka ku-BiG-FAM ukuya ku-BGC.Ama-GCC anama-BGC ahlobene eduze nama-BGC aqinisekisiwe (MIBiG) agqanyiswe ngemicibisholo.b Ukuqhathanisa i-GCF nama-BGC abikezelwe (i-BiG-FAM) kanye nama-BGC aqinisekisiwe (MIBiG), angu-3861 (d–>0.2) ama-GCF amasha atholakele.Iningi (78%) lale khodi ye-RiPP, i-terpenes, neminye imikhiqizo yemvelo ebekayo.c, wonke ama-genome ku-OMD atholakala kuma-metagenome olwandle angu-1038 afakwa esihlahleni sesisekelo se-GTDB ukukhombisa ukumbozwa kwe-phylogenetic ye-OMD.Ama-clades angenawo ama-genomes ku-OMD aboniswa ngokumpunga.Inombolo yama-BGC ihambisana nenani elikhulu kunawo wonke lama-BGC abikezelwe ngofuzo ngalunye esigabeni esithile.Ukuze kucace, i-15% yokugcina yama-node iwile.Imicibisholo ibonisa izigaba ezinothe nge-BGC (>15 BGC), ngaphandle kwe-Mycobacterium, Gordonia (yesibili kuphela ku-Rhodococcus), ne-Crocosphaera (yesibili kuphela ku-Synechococcus).d, Okungaziwa c.I-Eremiobacterota ibonise ukuhlukahluka kwe-biosynthetic okuphezulu kakhulu (inkomba ye-Shannon esekelwe ohlotsheni lomkhiqizo wemvelo).Ibhendi ngayinye imele i-genome enama-BGC amaningi ohlotsheni.T1PKS, PKS uhlobo I, T2/3PKS, PKS uhlobo II kanye nohlobo III.
Ngaphezu kokunotha nobusha, sihlola ukwakheka kwe-biogeographic yamandla e-biosynthetic we-microbiome yasolwandle.Ukuqoqwa kwamasampula ngesilinganiso sokusatshalaliswa kwenombolo yekhophi ye-metagenomic GCF (Izindlela) kubonise ukuthi imiphakathi ene-latitude ephansi, indawo engaphezulu, enothile nge-prokaryotic kanye nempofu ngegciwane, ikakhulukazi emanzini angaphezulu noma ajulile akhanya ilanga, yayicebile nge-RiPP kanye ne-BGC terpenes.Ngokuphambene, imiphakathi ye-polar, deep-sea, virus- kanye nezinhlayiyana yayihlotshaniswa nenani eliphakeme le-NRPS ne-PKS BGC (idatha eyandisiwe, i-Fig. 4 nolwazi olwengeziwe).Ekugcineni, sithole ukuthi imiphakathi yasezindaweni ezishisayo kanye ne-pelagic efundwe kahle iyimithombo ethembisayo yama-terpenes amasha (Umfanekiso Wedatha Ongeziwe).Amandla aphezulu we-PKS, i-RiPP neminye imikhiqizo yemvelo (Umfanekiso 5a onedatha enwetshiwe).
Ukuze sigcwalise ucwaningo lwethu lwamandla e-biosynthetic wama-microbiomes olwandle, sasihlose ukwenza imephu ukusatshalaliswa kwe-phylogenetic nokuhlonza izigaba ezintsha ezinothiswe yi-BGC.Kuze kube manje, sibeke ama-genomes amagciwane olwandle esihlahleni esijwayelekile se-GTDB13 sebhaktheriya kanye ne-archaeal phylogenetic futhi sembesa izindlela ezifaka ama-biosynthetic azihlanganisayo (Fig. 2c).Sithole kalula izigaba ezimbalwa ezithuthukisiwe ze-BGC (emelwe ama-BGC angaphezu kuka-15) kumasampula amanzi olwandle (izindlela) ezaziwa ngamandla awo e-biosynthetic, njenge-cyanobacteria (Synechococcus) kanye namagciwane e-Proteus, afana ne-Tistrella32,33, noma asanda kuheha ukunaka kwawo. imikhiqizo yemvelo .njenge-Myxococota (i-Sandaracinaceae), i-Rhodococcus ne-Planctomycetota34,35,36.Kuyathakazelisa ukuthi sithole izinhlu ezimbalwa ebezingahloliwe ngaphambili kulezi zigaba.Isibonelo, lezo zinhlobo zezilwane ezinamandla acebe kakhulu e-biosynthetic ku-phyla Planctomycetota kanye ne-Myxococcota kwakungezama-oda wekhandidethi kanye nesizukulwane, ngokulandelana (Ithebula Lokwengeza 3).Kuhlanganiswe ndawonye, ​​lokhu kuphakamisa ukuthi i-OMD inikeza ukufinyelela olwazini lwe-phylogenetic olungaziwa ngaphambili, okubandakanya ama-microorganisms, angase amele okuhloswe okusha kwe-enzyme nokutholakala komkhiqizo wemvelo.
Okulandelayo, siphawule isigaba esinothiswe yi-BGC ngokungabali kuphela inani eliphezulu lama-BGC afakwe ngekhodi amalungu awo, kodwa futhi ngokuhlola ukuhlukahluka kwalawa ma-BGC, okuchaza ukuvama kwezinhlobo ezahlukene zemikhiqizo yekhandidethi yemvelo (Fig. 2c nezindlela )..Sithole ukuthi izinhlobo ezihlukahlukene kakhulu ze-biosynthetically zimelelwe ama-MAG ebhaktheriya aklanywe ngokukhethekile kulolu cwaningo.Lawa mabhaktheriya ayingxenye ye-phylum Candidatus Eremiobacterota engatshalwanga, ehlala ingahlolisiswanga ngaphandle kwezifundo ezimbalwa ze-genomic37,38.Kuyaphawuleka ukuthi “ca.Uhlobo lwe-Eremiobacterota luhlaziywe kuphela endaweni39 yasemhlabeni futhi akwaziwa ukuthi luhlanganisa noma yimaphi amalungu anothiswe ku-BGC.Lapha siye sakha kabusha ama-MAG ayisishiyagalombili ezinhlobo ezifanayo (i-nucleotide identity> 99%) 23. Ngakho-ke siphakamisa igama lezinhlobo elithi "Candidatus Eudoremicrobium malaspinii", eliqanjwe nge-nereid (i-nymph yasolwandle), isipho esihle emlandweni wamaGreki kanye nohambo.'Ka.Ngokwesichasiselo se-phylogenetic 13, i-E. malaspinii ayinazo izihlobo ezaziwayo ngaphambili ezingaphansi kwezinga lokulandelana futhi ngaleyo ndlela ingeyomndeni omusha webhaktheriya esiphakamisa ukuthi “Ca.E. malaspinii” njengohlobo lwezinhlobo kanye “Ca.I-Eudormicrobiaceae” njengegama elisemthethweni (Ulwazi Olwengeziwe).Ukwakhiwa kabusha kwe-metagenomic kafushane kwe-'Ca.Iphrojekthi ye-genome ye-E. malaspinii yaqinisekiswa okokufaka okuphansi kakhulu, ukulandelana kwe-metagenomic efundiwe isikhathi eside kanye nokuhlanganisa okuhlosiwe kwesampula eyodwa (Izindlela) njengekhromozomu yomugqa engu-9.63 Mb ene-75 kb yokuphindaphinda.njengokuwukuphela kokungacaci kahle okusele.
Ukuze sithole umongo we-phylogenetic walolu hlobo, sifune izinhlobo zezinhlobo ezisondelene ezingu-40 kumasampula e-metagenomic engeziwe e-eukaryotic-enothiswe ohambweni lwase-Tara Ocean ngokwakhiwa kabusha kwe-genome okuhlosiwe.Kafushane, sixhume ukufundwa kwe-metagenomic nezingcezu ze-genomic ezihlobene ne-“Ca.E. malaspinii” futhi kucatshangelwe ukuthi izinga lokuqashwa elinyukile kuleli sampula libonisa ukuba khona kwezinye izihlobo (izindlela).Ngenxa yalokho, sithole ama-MAG angu-10, inhlanganisela ye-19 MAGs emele izinhlobo ezinhlanu ezizukulwaneni ezintathu phakathi komndeni osanda kuchazwa (okungukuthi "Ca. Eudormicrobiaceae").Ngemva kokuhlola mathupha kanye nokulawula ikhwalithi (idatha enwetshiwe, Fig. 6 nolwazi olwengeziwe), sithole ukuthi “Ca.Izinhlobo ze-Eudormicrobiaceae zethula ama-genome amakhulu (8 Mb) namandla acebile e-biosynthetic (14 kuya ku-22 BGC ngohlobo ngalunye) kunamanye amalungu e-“Ca”.Clade Eremiobacterota (kufika ku-7 BGC) (Fig. 3a–c).
a, Izikhundla ze-Phylogenetic ze-'Ca ezinhlanu.Izinhlobo ze-Eudormicrobiaceae zibonise ukunotha kwe-BGC okuqondene nemigqa yasolwandle ekhonjwe kulolu cwaningo.Isihlahla se-phylogenetic sihlanganisa yonke i-'Ca.I-MAG Eremiobacterota kanye namalungu enye i-phyla (izinombolo zofuzo kubakaki) anikezwe ku-GTDB (inguqulo 89) asetshenziselwa isizinda sokuziphendukela kwemvelo (Izindlela).Izendlalelo ezingaphandle zimele izigaba ezingeni lomndeni (“Ca. Eudormicrobiaceae” kanye “neCa. Xenobiaceae”) kanye nezinga lekilasi (“Ca. Eremiobacteria”).Izinhlobo ezinhlanu ezichazwe kulolu cwaningo zimelelwe amakhodi ezinhlamvu nezinombolo namagama ahlongozwayo we-binomial (Ulwazi Olwengeziwe).b, kuhle.Izinhlobo ze-Eudormicrobiaceae zihlanganyela ama-nuclei ayisikhombisa avamile e-BGC.Ukungabikho kwe-BGC ku-clade ye-A2 kwakungenxa yokungapheleli kwe-MA emele (Ithebula Lokwengeza 3).Ama-BGC aqondene ngqo ne-“Ca.Amphithomicrobium” kanye “Ca.I-Amphithomicrobium” (izigatshana A no-B) aziboniswa.c, Wonke ama-BGC afakwe ikhodi njengokuthi “Ca.I-Eudoremicrobium taraoceanii itholwe ivezwe kuma-metaranscripttomes angama-623 athathwe olwandle lwaseTara.Imibuthano eqinile ibonisa ukuloba okusebenzayo.Imibuthano ewolintshi isho izinguquko ezigoqekayo eziguquliwe ze-log2 ngezansi nangaphezulu kwezinga lokuvezwa kofuzo lokugcinwa kwendlu (izindlela).d, amajika obuningi obuhlobene (izindlela) ezibonisa 'Ca.Izinhlobo ze-Eudormicrobiaceae zisabalele ezindaweni eziningi zolwandle kanye nakuwo wonke amakholomu amanzi (kusuka phezulu kuya ekujuleni okungamamitha angama-4000).Ngokusekelwe kulezi zilinganiso, sithole ukuthi 'Ca.I-E. malaspinii' ifinyelela ku-6% wamaseli e-prokaryotic emiphakathini ehlobene nokusanhlamvu kwe-deep-sea pelagic.Sicabangele uhlobo oluthile olukhona endaweni uma lutholakala kunoma iyiphi ingxenye yobukhulu bongqimba obunikeziwe bokujula.IO – Indian Ocean, NAO – North Atlantic, NPO – North Pacific, RS – Red Sea, SAO – South Atlantic, SO – Southern Ocean, SPO – South Pacific.
Ukufunda ngobuningi nokusatshalaliswa kweCa.I-Eudormicrobiaceae, okuyinto, njengoba sitholile, ihamba phambili ezitsheni eziningi zasolwandle, kanye nakuyo yonke ikholomu yamanzi (Fig. 3d).Endaweni, benza u-6% wezilwane zasolwandle, okubenza babe yingxenye ebalulekile ye-microbiome yasolwandle yomhlaba wonke.Ngaphezu kwalokho, sithole okuqukethwe okuhlobene kwe-Ca.Izinhlobo ze-Eudormicrobiaceae kanye namazinga azo okuvezwa kwe-BGC ayephezulu kungxenyana ethuthukisiwe ye-eukaryotic (Fig. 3c nedatha enwetshiwe, Fig. 7), ebonisa ukusebenzelana okungaba khona nezinhlayiyana, okuhlanganisa i-plankton.Lokhu okubonayo kufana nokuthi 'Ca.Ama-Eudoremicrobium BGC akhiqiza imikhiqizo yemvelo eyi-cytotoxic ngokusebenzisa izindlela ezaziwayo angase abonise impatho ehlaselayo (Ulwazi Olwengeziwe kanye Nedatha Enwetshiwe, Umfanekiso 8), efana nezinye izilwane ezidla ezinye ezikhiqiza ngokukhethekile ama-metabolite njenge-Myxococcus41.Ukutholwa kweCa.I-Eudormicrobiaceae etholakala kancane (ekujuleni kolwandle) noma amasampula e-eukaryotic kunamasampula e-prokaryotic ingase ichaze ukuthi kungani la magciwane kanye nokuhlukahluka kwawo okungalindelekile kwe-BGC kuhlala kungacacile kumongo wocwaningo lokudla kwemvelo.
Ekugcineni, safuna ukuqinisekisa ngokuhlola isithembiso somsebenzi wethu osuselwe ku-microbiome ekutholeni izindlela ezintsha, ama-enzyme, nemikhiqizo yemvelo.Phakathi kwezigaba ezihlukene zama-BGC, indlela ye-RiPP yaziwa ngokuhlanganisa amakhemikhali anothile kanye nokuhlukahluka okusebenzayo ngenxa yokuguqulwa okuhlukahlukene kwangemuva kokuhumusha kwe-peptide ewumongo ngama-enzyme asekhulile42.Ngakho-ke sikhethe ama-'Ca amabili.I-Eudoremicrobium' RiPP BGCs (Izibalo 3b kanye ne-4a-e) zisekelwe ngokufanayo nanoma iyiphi i-BGC eyaziwayo (\(\bar{d}\)MIBiG kanye \(\bar{d}\)RefSeq ngenhla 0.2) .
a–c, In vitro heterologous expression kanye ne-in vitro enzymatic assays yenoveli (\(\bar{d}\)RefSeq = 0.29) iqoqo le-RiPP biosynthesis eqondene nezinhlobo ze-deep Sea.I-E. malaspinii' iholele ekukhiqizweni kwemikhiqizo ye-diphosphorylated.c, ukuguqulwa okuhlonzwe kusetshenziswa i-high-resolution (HR) MS/MS (ukuhlukaniswa okuboniswa yi-b kanye ne-y ions esakhiweni samakhemikhali) kanye ne-NMR (idatha eyandisiwe, Fig. 9).d, le peptide ye-phosphorylated ibonisa ukuvinjelwa okuphansi kwe-micromolar ye-mammalian neutrophil elastase, engatholakali ku-peptide yokulawula kanye ne-peptide eqeda amanzi emzimbeni (ukususwa kwamakhemikhali okubangelwa ukuphelelwa amandla kwamanzi).Ukuhlolwa kuphindwe kathathu ngemiphumela efanayo.Isibonelo, ukuvezwa kwe-heterologous yenoveli yesibili \(\bar{d}\)RefSeq = 0.33) iqoqo le-protein biosynthesis licacisa umsebenzi wama-enzyme amane avuthiwe aguqula i-46 amino acid core peptide.Izinsalela zinamabala ngokuya ngendawo yokuguqulwa ebikezelwe i-HR-MS/MS, ukulebula kwe-isotope, nokuhlaziywa kwe-NMR (Ulwazi Olwengeziwe).Umbala odeshi ubonisa ukuthi ukuguqulwa kwenzeka kunoma iyiphi yalezi zinsalela ezimbili.Isibalo siyinhlanganisela yezakhi eziningi ezihlukene ukuze kuboniswe umsebenzi wawo wonke ama-enzyme avuthiwe ku-nucleus efanayo.h, Umdwebo wedatha ye-NMR ye-backbone amide N-methylation.Imiphumela egcwele iboniswa ku-fig.10 ngedatha enwetshiwe.i, Isikhundla se-Phylogenetic se-enzyme evuthiwe ye-protein ye-FkbM phakathi kwazo zonke izizinda ze-FkbM ezitholakala ku-database ye-MIBiG 2.0 yembula i-enzyme yalo mndeni ngomsebenzi we-N-methyltransferase (Ulwazi Olwengeziwe).Imidwebo eyisikimu ye-BGCs (a, e), izakhiwo ze-peptide ezandulela (b, f), kanye nezakhiwo zamakhemikhali ezibekayo zemikhiqizo yemvelo (c, g) ziyaboniswa.
Indlela yokuqala ye-RiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) yatholakala kuphela ezinhlotsheni ezijulile zolwandle "Ca.E. malaspinii” kanye namakhodi e-Peptide- precursor (Fig. 4a, b).Kule enzyme evuthiwe, sihlonze isizinda esisodwa esisebenzayo esine-homologous esizindeni sokuphelelwa amanzi emzimbeni se-lantipeptide synthase esivame ukuvuselela i-phosphorylation kanye nokususwa okulandelayo kwe-43 (Ulwazi Olwengeziwe).Ngakho-ke, sibikezela ukuthi ukuguqulwa kwe-peptide eyandulelayo kuhilela ukuphelelwa amandla kwamanzi okuzinyathelo ezimbili.Nokho, sisebenzisa i-tandem mass spectrometry (MS/MS) kanye ne-nuclear magnetic resonance spectroscopy (NMR), sihlonze i-polyphosphorylated linear peptide (Fig. 4c).Nakuba kungalindelekile, sithole imigqa eminingana yobufakazi obusekela ukuba kwayo umkhiqizo wokugcina: ababungazi ababili abahlukene be-heterologous futhi akukho ukuhlolwa kokuphelelwa amandla kwamanzi ku-in vitro, ukuhlonza izinsalela ezibalulekile eziguquliwe endaweni ye-catalytic dehydration ye-enzyme evuthiwe.konke kwakhiwe kabusha ngokuthi “Ca”.I-E. malaspinii genome (idatha eyandisiwe, i-Fig. 9 kanye nolwazi olwengeziwe) futhi, ekugcineni, umsebenzi wezinto eziphilayo zomkhiqizo we-phosphorylated, kodwa hhayi ifomu elikhishwe ngamakhemikhali (Fig. 4d).Eqinisweni, sithole ukuthi ibonisa umsebenzi ophansi we-micromolar protease inhibitory ngokumelene ne-neutrophil elastase, uma kuqhathaniswa neminye imikhiqizo yemvelo ehlobene ebangeni lokuhlushwa (IC50 = 14.3 μM) 44, naphezu kokuthi indima yezemvelo isazocaciswa.Ngokusekelwe kule miphumela, siphakamisa ukuqamba indlela "phospheptin".
Icala lesibili indlela ye-RiPP eyinkimbinkimbi eqonde ku-'Ca.I-genus Eudoremicrobium (\(\bar{d}\)MIBiG = 0.46, \(\bar{d}\)RefSeq = 0.33) yabikezelwa ukuthi izofaka imikhiqizo yamaprotheni yemvelo (Fig. 4e).Lezi zindlela zinentshisekelo ethile ye-biotechnology ngenxa yokuminyana okulindelekile kanye nenhlobonhlobo yezinguquko zamakhemikhali ezingajwayelekile ezisungulwe ama-enzyme afakwe ikhodi ama-BGCs45 amafushane kakhulu.Sithole ukuthi le phrotheni ihlukile kumaprotheni anezimpawu zangaphambili ngoba ayinakho kokubili i-NX5N motif eyinhloko ye-polyceramides kanye ne-lanthionine loop ye-landornamides 46.Ukuze sinqobe imikhawulo yamaphethini avamile wokukhuluma nge-heterologous, sawasebenzisa kanye nesistimu ye-Microvirgula aerodenitrificans yangokwezifiso ukuze ibonise ama-enzyme ezindlela ezine ezivuthiwe (izindlela).Sisebenzisa inhlanganisela ye-MS/MS, ilebula ye-isotope, ne-NMR, sithole lawa ma-enzyme avuthiwe ku-46-amino acid core we-peptide (Umfanekiso we-4f, g, idatha eyandisiwe, ama-Figs. 10-12 nolwazi olwengeziwe).Phakathi kwama-enzyme avuthiwe, sibonise ukubonakala kokuqala kwelungu lomndeni le-FkbM O-methyltransferase i-47 endleleni ye-RiPP futhi sathola ngokungalindelekile ukuthi le enzyme evuthiwe ingenisa umgogodla we-N-methylation (Fig. 4h, i kanye nolwazi olwengeziwe).Nakuba lokhu kuguqulwa kwaziwa emikhiqizweni ye-NRP48 yemvelo, i-enzymatic N-methylation yamabhondi e-amide iwukusabela okuyinkimbinkimbi kodwa okubalulekile ngokwe-biotechnologically49 osekunentshisekelo kuze kube manje emndenini we-RiPP wama-borosine.Ukucaciswa okungu-50,51.Ukuhlonzwa kwalo msebenzi kweminye imindeni yama-enzyme kanye ne-RiPP kungase kuvule izinhlelo zokusebenza ezintsha futhi kwandise ukuhlukahluka okusebenzayo kwamaprotheni 52 kanye nokuhlukahluka kwawo kwamakhemikhali.Ngokusekelwe ekulungisweni okuhlonziwe kanye nobude obungavamile besakhiwo somkhiqizo ohlongozwayo, siphakamisa igama lendlela elithi “pythonamide”.
Ukutholwa kwe-enzymology engalindelekile emndenini onezimpawu ezisebenzayo zama-enzyme kukhombisa isithembiso se-genomics yezemvelo kokutholwa okusha, futhi kukhombisa umthamo olinganiselwe wokuqagela kokusebenza okusekelwe ku-homology yokulandelana kuphela.Ngakho, kanye nemibiko ye-non-canonical bioactive polyphosphorylated RiPPs, imiphumela yethu ibonisa inani elikhulu kakhulu lezinsiza kodwa elibucayi emizamweni yokwenziwa yebhayoloji yokudalula ngokugcwele ukunotha okusebenzayo, ukuhlukahluka, kanye nezakhiwo ezingajwayelekile zezinhlanganisela ze-biochemical.
Lapha sibonisa ububanzi bamandla e-biosynthetic afakwe ama-microbes kanye nomxholo wawo we-genomic ku-microbiome yasolwandle yomhlaba wonke, kusiza ucwaningo lwesikhathi esizayo ngokwenza insiza ewumphumela itholakale emphakathini wesayensi (https://microbiomics.io/ocean/).Sithole ukuthi okuningi kwe-phylogenetic kanye nobusha obusebenzayo kungatholakala kuphela ngokwakha kabusha ama-MAG kanye nama-SAG, ikakhulukazi emiphakathini yama-microbial engasetshenziswa kancane engaqondisa imizamo yesikhathi esizayo ye-bioprospecting.Nakuba sizogxila lapha ku-'Ca.I-Eudormicrobiaceae” njengozalo ikakhulukazi “obanethalente” le-biosynthetically, ama-BGC amaningi abikezelwa ku-microbiota engatholakali okungenzeka ihlanganise ama-enzymologies ayengachazwa ngaphambilini akhiqiza izinhlanganisela ezinezenzo ezibalulekile zemvelo kanye/noma ze-biotechnological.
Amasethi edatha e-Metagenomic avela ezifundweni ezinkulu ze-oceanographic kanye nochungechunge lwesikhathi onokujula okwanele kokulandelana afakiwe ukuze kwandiswe ukusabalala kwemiphakathi yasolwandle yasolwandle emifuleni yolwandle, izendlalelo ezijulile kanye nesikhathi.Lawa madathasethi (Ithebula Lokwengeza 1 kanye Nomfanekiso 1) ahlanganisa ama-metagenomics asuka kumasampula aqoqwe olwandle lwase-Tara (igciwane elinothisiwe, n=190; i-prokaryotic inothisiwe, n=180)12,22 kanye nohambo lwe-BioGEOTRACES (n=480).I-Hawaiian Oceanic Time Series (HOT, n = 68), Bermuda-Atlantic Time Series (BATS, n = 62)21 kanye ne-Malaspina Expedition (n = 58)23.Ukufundwa okulandelanayo kuzo zonke izingcezu ze-metagenomic kwahlungwa ngekhwalithi kusetshenziswa i-BBMap (v.38.71) ngokususa ama-adaptha alandelanayo ekufundweni, ukususa okufundiwe okufakwe kumephu ngokulandelana kokulawula ikhwalithi (PhiX genomes), nokusebenzisa i-trimq=14, maq=20 ilahla ikhwalithi yokufunda ephansi, maxns = 0 kanye nobude bendawo = 45. Ukuhlaziya okwalandela kwaqalwa noma kwahlanganiswa nokufundwa kwe-QC uma kucacisiwe (bbmerge.sh minoverlap=16).Ukufundwa kwe-QC kwenziwa ngendlela evamile (i-bbnorm.sh target = 40, minddepth = 0) ngaphambi kokwakha kusetshenziswa ama-metaSPAdes (v.3.11.1 noma v.3.12 uma kudingeka)53.Ama-scaffold contigs (okuzobizwa kamuva ngokuthi ama-scaffold) agcine ehlungwe ngobude (≥1 kb).
Amasampula e-metagenomic angu-1038 ahlukaniswa ngamaqembu, futhi eqenjini ngalinye lamasampula, ukufundwa kokulawulwa kwekhwalithi ye-metagenomic kwawo wonke amasampula kwafaniswa kubakaki besampula ngayinye ngokuhlukana, okuholele ekutheni inani elilandelayo leqembu elikubakaki ngababili lifundeke kanje: I-Tara Marine Virus - Enriched (190×190), Prokaryotes Enriched (180×180), BioGEOTRACES, HOT kanye BATS (610×610) kanye Malaspina (58×58).Ukwenza imephu kwenziwe kusetshenziswa i-Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 evumela ukufundwa ukuthi kuqhathaniswe nezingosi zesibili (kusetshenziswa ifulegi -a).Ukuqondanisa kwahlungwa ukuze okungenani kube izisekelo ezingu-45 ubude, zibe nobunikazi obungu-≥97%, kanye nokufundwa kwe-span ≥80%.Amafayela e-BAM angumphumela acutshungulwa kusetshenziswa iskripthi esithi jgi_summarize_bam_contig_depths se-MetaBAT2 (v.2.12.1)55 ukuze kuhlinzekwe ukumbozwa kwesampula kwangaphakathi naphakathi kweqembu ngalinye.Ekugcineni, abakaki baqoqwe ukuze kukhuliswe ukuzwela ngokusebenzisa i-MetaBAT2 ngayinye kuwo wonke amasampuli nge--minContig 2000 kanye -maxEdges 500. Sisebenzisa i-MetaBAT2 esikhundleni se-ensemble boxer ngoba iboniswe ekuhlolweni okuzimele ukuthi ingumshayisibhakela oyedwa osebenza kahle kakhulu.kanye nezikhathi ezi-10 kuye kwezingama-50 ngokushesha kunabanye abashayisibhakela abavame ukusetshenziswa57.Ukuze kuhlolwe umthelela wokuhlobana kobuningi, isampula elincanyana elikhethwe ngokungahleliwe le-metagenomics (i-10 kudathasethi yedatha ye-Tara Ocean amabili, i-10 ye-BioGEOTRACES, engu-5 yochungechunge lwesikhathi ngasinye, kanye ne-5 ye-Malaspina) iphinde isebenzise amasampula kuphela.Amasampula angaphakathi aqoqwa ukuze kutholwe ulwazi lokukhava.(Ulwazi Olwengeziwe).
Amagenome engeziwe (angaphandle) afakiwe ekuhlaziyweni okwalandela, okungama-MAG angama-830 akhethwe ngesandla avela kusethi yedatha ye-Tara Oceans26, ama-SAG angama-5287 avela kudathasethi ye-GORG20, kanye nedatha evela kusizindalwazi se-MAR (MarDB v. 4) kusukela kuma-REF ahlukanisiwe angu-1707 kanye 682 SAGs) 27. Kudathasethi ye-MarDB, ama-genome akhethwa ngokusekelwe kumethadatha etholakalayo uma uhlobo lwesampula lufana nenkulumo evamile elandelayo: '[S|s]ingle.?[C|c]ell|[C|c]ulture| [I|i] ngedwa'.
Ikhwalithi yesiqukathi ngasinye se-metagenomic kanye nama-genome angaphandle ahlolwe kusetshenziswa i-CheckM (v.1.0.13) kanye ne-Anvi'o's Lineage Workflow (v.5.5.0)58,59.Uma i-CheckM noma i-Anvi'o ibika ≥50% ukuphelela/ukuphelela kanye ≤10% nokungcola/ukuphelelwa umsebenzi, bese ulondoloza amaseli e-metagenomic nama-genome angaphandle ukuze ahlaziywe kamuva.Lawa maphuzu abe esehlanganiswa abe kusho ukuphelela (mcpl) kanye nokungcola okusho (mctn) ukuze kuhlukaniswe ikhwalithi yofuzo ngokwemibandela yomphakathi60 ngale ndlela elandelayo: ikhwalithi ephezulu: mcpl ≥ 90% kanye ne-mctn ≤ 5%;ikhwalithi enhle: mcpl ≥ 70%, mctn ≤ 10%, ikhwalithi ephakathi: mcpl ≥ 50% ne-mctn ≤ 10%, ikhwalithi efanelekile: mcpl ≤ 90% noma mctn ≥ 10%.Ama-genome ahlungiwe abe esehlotshaniswa nezikolo zekhwalithi (Q kanye ne-Q') ngendlela elandelayo: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (ukuhlukahluka kohlobo)/100 + 0.5 x log[N50] .(yenziwe ku-dRep61).
Ukuze kuvunyelwe ukuhlaziya okuqhathanisayo phakathi kwemithombo yedatha ehlukene nezinhlobo zofuzo (i-MAG, i-SAG ne-REF), ama-genome angu-34,799 aye asuswa ngokusekelwe ku-genome-wide average nucleotide identity (ANI) kusetshenziswa i-dRep (v.2.5.4).Iyaphinda)61 ​​eno-95% we-ANI thresholds28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) kanye nofuzo lomaka wekhophi eyodwa usebenzisa i-SpecI63 ehlinzeka ngokuhlangana kofuzo ezingeni lezinhlobo zezilwane.Kukhethwe i-genome emele iqoqo ngalinye le-dRep ngokuya ngesilinganiso sekhwalithi ephezulu (Q') echazwe ngenhla, esibhekwe njengemele uhlobo lohlobo.
Ukuze kuhlolwe isivinini semephu, i-BWA (v.0.7.17-r1188, -a) isetshenziselwe ukwenza imephu wonke amasethi angu-1038 okufundwa kwe-metagenomic nama-genomes angu-34,799 aqukethwe ku-OMD.Ukufundwa okulawulwa yikhwalithi kwenziwe imephu ngemodi enesiphetho esisodwa futhi ukuqondanisa okwaba umphumela kwahlungwa ukuze kugcinwe ukuqondanisa kuphela ≥45 bp ubude.kanye nobunikazi ≥95%.Isilinganiso sokubonisa sesampula ngayinye iphesenti lokufunda elisele ngemva kokuhlunga lihlukaniswe ngenani eliphelele lokufundwa kokulawulwa kwekhwalithi.Ngokusebenzisa indlela efanayo, i-metagenomes ngayinye ye-1038 yehliswa yaba yizigidi ezi-5 ezifakiwe (idatha enwetshiwe, i-Fig. 1c) futhi ifaniswe ne-GORG SAG ku-OMD nakuyo yonke i-GEM16.Inani lama-MAGs atholwe emanzini olwandle kukhathalogi ye-GEM16 lanqunywa imibuzo yamagama angukhiye wemithombo ye-metagenomic, ukukhetha amasampula amanzi olwandle (isb., ngokungafani nezinsalela zasolwandle).Ngokukhethekile, sikhetha okuthi “aquatic” njengokuthi “ecosystem_category”, “marine” as “ecosystem_type”, bese sihlunga “indawo yokuhlala” njengokuthi “deep ocean”, “marine”, “maritime oceanic”, “pelagic marine”, “marine water” , "Ulwandle", "Amanzi Olwandle", "Amanzi Olwandle Angaphezulu", "Amanzi Olwandle Angaphezulu".Lokhu kuholele ekutheni ama-MAG angu-5903 (ikhwalithi ephezulu engu-734) asatshalaliswe kuma-OTU angu-1823 (ukubukwa lapha).
Ama-genome e-Prokaryotic achazwe ngokwe-taxonomic kusetshenziswa i-GTDB-Tk (v.1.0.2)64 enemingcele ezenzakalelayo eqondise i-GTDB r89 inguqulo 13. I-Anvi'o yasetshenziselwa ukukhomba ama-eukaryotic genome ngokusekelwe ekuqaguleni kwesizinda nokukhumbula ≥50% kanye nokungafuneki ≤ 10%.Isichasiselo se-taxonomic sohlobo oluthile sichazwa njengenye yezakhi zofuzo ezimele.Ngaphandle kwama-eukaryotes (148 MAG), igenomu ngayinye yaqale yachazelwa ngokusebenza kusetshenziswa i-prokka (v.1.14.5)65, iqamba izakhi zofuzo eziphelele, echaza imingcele ye-“archaea” noma “bacteria” njengoba kudingeka, nayo ebikwe kwabangewona amakhodi wofuzo.kanye nezifunda ze-CRISPR, phakathi kwezinye izici ze-genomic.Chasisa izakhi zofuzo ezibikezelwe ngokukhomba izakhi zofuzo zomaka wekhophi eyodwa (uscMG) usebenzisa i-fetchMG (v.1.2)66, yabela amaqembu e-ortholog kanye nombuzo usebenzisa i-emapper (v.2.0.1)67 esekelwe ku-eggNOG (v.5.0)68.Isizindalwazi se-KEGG (eshicilelwe ngoFebruwari 10, 2020) 69. Isinyathelo sokugcina senziwa ngokufanisa amaprotheni kusizindalwazi se-KEGG kusetshenziswa i-DIAMOND (v.0.9.30)70 enombuzo nesihloko esihlanganisa ≥70%.Imiphumela yaphinde yahlungwa ngokuvumelana ne-NCBI Prokaryotic Genome Annotation Pipeline71 ngokusekelwe ku-bitrate ≥ 50% yomkhawulo we-bitrate olindelwe (isixhumanisi ngokwaso).Ukulandelana kwezakhi zofuzo kuphinde kwasetshenziswa njengokufakwayo ukuze kukhonjwe ama-BGC kugenome kusetshenziswa i-antiSMASH (v.5.1.0)72 namapharamitha azenzakalelayo nokuqhuma kweqoqo okuhlukile.Wonke ama-genome nezichasiselo zihlanganiswe zaba yi-OMD kanye nemethadatha yomongo etholakala kuwebhu (https://microbiomics.io/ocean/).
Ngokufana nezindlela ezichazwe ngaphambilini12,22 sasebenzisa i-CD-HIT (v.4.8.1) ukuze sihlanganise > ama-protein-coding ayizigidi ezingama-56.6 asuka kumagciwane nama-archaeal genomes asuka ku-OMD aye ku-95% wobunikazi kanye nezakhi zofuzo ezimfishane (90%) > 17.7 million amaqoqo ofuzo.Ukulandelana okude kakhulu kwakhethwa njengofuzo olumele iqoqo ngalinye lofuzo.Ama-metagenome angu-1038 abe esefaniswa namalungu eqoqo angu->17.7 wezigidi ze-BWA (-a) futhi amafayela angumphumela we-BAM ahlungwa ukuze kugcinwe ukuqondanisa kuphela ngo-≥95% wobunikazi bamaphesenti kanye ≥45 nokuqondanisa kwesisekelo.Ubuningi bezakhi zofuzo ezijwayeleke ngobude bubalwe ngokubala kokuqala okufakwayo kusukela ekuqondaniseni okuyingqayizivele okungcono kakhulu bese, kokufakwayo okunemephu engaqondakali, kwenezela izibalo zamaqhezu ezakhini zofuzo eziqondiwe ezihambisanayo ngokulingana nenani lazo lokufaka okuhlukile.
Ama-genomes asuka ku-OMD enwetshiwe (anama-MAG engeziwe avela ku-“Ca. Eudormicrobiaceae”, bheka ngezansi) engezwe kusizindalwazi sethuluzi lokuhlaziya i-moTUs74 metagenomic (v.2.5.1) ukuze kwakhiwe idatha egciniwe yereferensi ye-mOTU enwetshiwe.Ayisithupha kuphela ama-genome ekhophi eyodwa (23,528 genomes) asinda kuma-uscMG ayishumi.Ukwandiswa kwesizindalwazi kuholele kumaqoqo engeziwe angu-4,494 ezingeni lezinhlobo zezilwane.Ama-metagenome angu-1038 ahlaziywa kusetshenziswa amapharamitha we-moTU azenzakalelayo (v.2).Isamba sama-genomes angama-989 aqukethwe kumaqoqo e-moTU angama-644 (95% REF, 5% SAG kanye nama-99.9% okungeye-MarDB) awatholwanga yiphrofayela ye-moTU.Lokhu kukhombisa imithombo ehlukahlukene eyengeziwe yokuhlukaniswa kwasolwandle kwama-genome e-MarDB (iningi lama-genomes angatholakali ahlotshaniswa nezinto eziphilayo ezihlukaniswe nentlenga, izidalwa zasolwandle, njll.).Ukuze siqhubeke sigxile endaweni evulekile yolwandle kulolu cwaningo, asiwafaki ekuhlaziyeni umfula ngaphandle uma etholwe noma afakwa kusizindalwazi esinwetshiwe se-moTU esidalwe kulolu cwaningo.
Wonke ama-BGC asuka ku-MAG, SAG kanye ne-REF ku-OMD (bona ngenhla) ahlanganiswe nama-BGC akhonjwe kuzo zonke izikafula ze-metagenomic (antiSMASH v.5.0, amapharamitha azenzakalelayo) futhi abonakala kusetshenziswa i-BiG-SLICE (v.1.1) (isizinda se-PFAM)75.Ngokusekelwe kulezi zici, sibale wonke amabanga e-cosine phakathi kwama-BGC futhi sawaqoqa (izixhumanisi eziqondile) zaba i-GCF ne-GCC sisebenzisa ama-threshold webanga angu-0.2 no-0.8 ngokulandelanayo.Le mikhawulo iwukushintshwa kwama-threshold asetshenziswa ngaphambilini kusetshenziswa i-Euclidean distance75 kanye nebanga le-cosine, elidambisa elinye iphutha kuqhinga lokuqala lokuhlanganisa le-BiG-SLICE (Ulwazi Olwengeziwe).
Ama-BGC abe esehlungwa ukuze kugcinwe kuphela u-≥5 kb obethelwe kuzikafula ukuze kwehliswe ubungozi bokuhlukana njengoba kuchazwe ngaphambilini16 kanye nokukhipha ama-MarDB REF nama-SAG angatholakali kuma-metagenomes angu-1038 (bona ngenhla).Lokhu kuholele ekutheni inani eliphelele lama-BGC angu-39,055 afakwe ikhodi yi-OMD genome, nokunye okungu-14,106 okukhonjwe kuzingcezu ze-metagenomic (okungukuthi azihlanganisiwe kuma-MAG).Lawa ma-BGC “e-metagenomic” asetshenziselwe ukulinganisa ingxenye yamandla e-marine microbiome biosynthesis engafakwanga kusizindalwazi (Ulwazi Olwengeziwe).I-BGC ngayinye yayisebenza ngokusebenza ngokuvumelana nezinhlobo zomkhiqizo oqagelayo ochazwa izigaba zomkhiqizo ezimelene ne-SMASH noma ezimaholoholo ezichazwe ku-BiG-SCAPE76.Ukuvimbela ukuchema kwesampula ekubalweni kwenani (i-taxonomic kanye nokusebenza kwe-GCC/GCF, ibanga le-GCF ne-GCC kumininingwane egciniwe, kanye nobuningi be-metagenomic ye-GCF), ngokugcina kuphela i-BGC ende kunazo zonke nge-GCF ngayinye yohlobo ngalunye, ama-BGC angama-39,055 aphinde aphindaphindwa, okuholele ekutheni kube nesamba esiyi-17,689 BGC.
Ubusha be-GCC ne-GCF buhlolwe ngokusekelwe ebangeni eliphakathi kwesizindalwazi esibaliwe (isizindalwazi se-RefSeq ku-BiG-FAM)29 kanye nokuhlolwa okuqinisekisiwe (MIBIG 2.0)30 BGC.Ku-BGC ngayinye yabamele abangu-17,689, sikhethe ibanga elincane kakhulu le-cosine kusizindalwazi esifanele.Lawa mabanga amancane abe eselinganiswa (okushoyo) ngokwe-GCF noma i-GCC, njengoba kufanele.I-GCF ibhekwa njengentsha uma ibanga lesizindalwazi likhulu kuno-0.2, elihambisana nokuhlukaniswa okuhle phakathi kwe-GCF (isilinganiso) nesithenjwa.Ku-GCC, sikhetha u-0.4, okuwumkhawulo ophindwe kabili ochazwe yi-GCF, ukukhiya ubudlelwano besikhathi eside nezixhumanisi.
Ubuningi be-metagenomic ye-BGC buhlawumbiselwe njengenala emaphakathi yezakhi zayo zofuzo ze-biosynthetic (njengoba kunqunywa i-anti-SMASH) etholakala kumaphrofayela zeleveli yofuzo.Ubuningi be-metagenomic ye-GCF ngayinye noma i-GCC yabe isibalwa njengesamba sama-BGC amele (kuyi-17,689).Lawa mamephu amaningi abe esejwayela ukwakheka kwamaselula kusetshenziswa isampula ye-moTU count ngayinye, ephinde yaba nemizamo yokulandelanisa (idatha enwetshiwe, i-Fig. 1d).Ukuvama kwe-GCF noma i-GCC kubalwe njengephesenti lamasampuli anensada > 0.
Ibanga le-Euclidean phakathi kwamasampuli libalwe kusukela kuphrofayela ye-GCF evamile.Lawa mabanga ehlisiwe ngosayizi kusetshenziswa i-UMAP77 futhi ukushumeka okwaba umphumela kwasetshenziselwa ukuhlanganisa okungagadwanga okususelwe ekuminyaneni kusetshenziswa i-HDBSCAN78.Inani eliphelele lamaphoyinti amaphoyinti eqoqo (futhi yingakho nenani lamaqoqo) elisetshenziswa i-HDBSCAN linqunywa ngokukhulisa amathuba akhulayo obulungu beqoqo.Amaqoqo ahlonziwe (kanye nesampula elinganiselwe engahleliwe yalawa maqoqo ukuze aphendule ngokuchema ekuhlaziyweni kwe-permutational multivariate of variance (PERMANOVA)) ahlolelwa ukubaluleka kwamabanga e-Euclidean angehlisiwe kusetshenziswa i-PERMANOVA.Isilinganiso sikasayizi we-genome wamasampuli sibalwe ngokusekelwe kubuningi obuhlobene be-moTU kanye nosayizi olinganiselwe we-genome wamalungu e-genome.Ikakhulukazi, isilinganiso sosayizi wofuzo we-moTU ngayinye salinganiselwa njengesilinganiso sobukhulu bofuzo lwamalungu alo alungiswa ukuze aphelele (ngemuva kokuhlunga) (isibonelo, i-genome ephelele engu-75% enobude obuyi-3 Mb inosayizi olungisiwe ongu-4. Mb).kuma-genome amaphakathi anobuqotho ≥70%.Usayizi omaphakathi we-genome wesampula ngayinye wabe esebalwa njengesamba sosayizi wegenome we-moTU esindwa ngobuningi obuhlobene.
Isethi ehlungiwe yama-BGC afakwe ikhodi ye-genome ku-OMD iboniswa ezihlahleni ze-GTDB zebhaktheriya nezakudala (ezinhlakeni ezingu-≥5 kb, ngaphandle kwe-REF ne-SAG MarDB engatholakali kuma-metagenomes angu-1038, bheka ngenhla) kanye nezigaba zawo zomkhiqizo ezibikezelwe ngokusekelwe ku-phylogenetic. isikhundla se-genome (bheka ngenhla).Siqale sanciphisa idatha ngezinhlobo zezinto eziphilayo, sisebenzisa i-genome enama-BGC amaningi kulolo hlobo njengabameleli.Ukuze kubonwe ngeso lengqondo, abameleli baphinde bahlukaniswa ngamaqembu ezihlahla, futhi futhi, ku-clade ngayinye eneseli, i-genome equkethe inombolo enkulu yama-BGC yakhethwa njengommeleli.Izinhlobo ezithuthukisiwe ze-BGC (okungenani i-genome eyodwa ene>15 BGCs) zabuye zahlaziywa ngokubala i-Shannon Diversity Index yezinhlobo zemikhiqizo efakwe ikhodi kulawo ma-BGC.Uma zonke izinhlobo zomkhiqizo ezibikezelwe zifana, ama-chemical hybrids kanye namanye ama-BGC ayinkimbinkimbi (njengoba kubikezelwe i-anti-SMAH) athathwa njengawohlobo lomkhiqizo ofanayo, kungakhathaliseki ukuhleleka kwawo kuqoqo (isb. i-protein-bacteriocin ne-bacteriocin-proteoprotein fusion umzimba).ihybrid).
I-DNA esele (ilinganiselwa ku-6 ng) kusukela kusampula ye-Malaspina MP1648, ehambisana nesampula yezinto eziphilayo SAMN05421555 futhi ifaniswe ne-Illumina SRR3962772 ukufundwa kwemetagenomic isethelwe ukufundwa okufushane, icutshungulwe ngokuya ngephrothokholi yokulandelanisa ye-PacBio enokufaka okuphansi kakhulu ukuze kusetshenziswe isampula ye-DNA ye-GSMBio ikhithi (100-980-000) kanye nekhithi yokulungiselela isifanekiso ye-SMRTbell Express 2.0 (100-938-900).Kafushane, i-DNA esele yasikwa, yalungiswa futhi yahlanzwa (i-ProNex ubuhlalu) kusetshenziswa i-Covaris (g-TUBE, 52104).I-Purified DNA ibe isingaphansi kokulungiswa komtapo wezincwadi, ukukhuliswa, ukucwengwa (ubuhlalu be-ProNex) nokukhetha usayizi (>6 kb, Blue Pippin) ngaphambi kwesinyathelo sokugcina sokuhlanza (i-ProNex ubuhlalu) nokulandelana kuplathifomu ye-Sequel II.
Ukwakhiwa kabusha ezimbili zokuqala ca.Ku-MAG Eremiobacterota, sihlonze ama-ANI angeziwe ayisithupha >99% (lawa afakwe kuMfanekiso 3), ahlungwa ekuqaleni ngokusekelwe kumaphuzu okungcola (kamuva ahlonzwa njengokuphindaphinda kwezakhi zofuzo, bheka ngezansi).Sithole nethreyi elibhalwe ukuthi “Ca”.I-Eremiobacterota” evela ezifundweni ezehlukene23 futhi yawasebenzisa kanye nama-MAG ayisishiyagalombili ocwaningo lwethu njengereferensi yokufundwa kwe-metagenomic kusuka kumasampuli angama-633 eukaryotic athuthukisiwe (>0.8 µm) kusetshenziswa i-BWA (v.0.7.17) Ref -r1188, – ifulegi) ukuze kuthathwe isampula ehlisiwe. ukwenza imephu (izigidi ezi-5 zifundiwe).Ngokusekelwe kumamephu aqondene nokucebisa (ahlungwe ngobunikazi bokuqondanisa obungu-95% kanye nokufakwa okungu-80% okufundiwe), ama-metagenome angu-10 (ukufakwa okulindelekile ≥5×) akhethelwe ukuhlanganiswa kanye nama-metagenome angu-49 engeziwe (ukufakwa okulindelekile ≥1×) ukuze kuhlanganiswe okuqukethwe.Kusetshenziswa amapharamitha afanayo nangenhla, lawa masampuli aboshwa futhi kwengezwa ama-'Ca angu-10.I-MAG Eremiobacterota ibuyiselwe.Lawa ma-MAG ayi-16 (ngaphandle kwalawo amabili asevele egciniwe) aletha inani eliphelele lama-genomes ku-OMD enwetshiwe liye ku-34,815.Ama-MAG anikezwa amazinga e-taxonomic ngokususelwe ekufaneni kwawo kwe-genomic kanye nesikhundla ku-GTDB.Ama-MAG ayi-18 ahoxiswa kusetshenziswa i-dRep abe yizinhlobo ezi-5 (intraspecific ANI>99%) kanye ne-3 genera (intrageneric ANI 85% kuya ku-94%) emndenini ofanayo79.Abamele izinhlobo zezilwane bakhethwa ngesandla ngokusekelwe kubuqotho, ukungcola, kanye ne-N50.I-nomenclature ephakanyisiwe inikezwa Ulwazi Olwengeziwe.
Hlola ubuqotho nokungcoliswa kwe-'Ca.I-MAG Eremiobacterota, sihlole ubukhona be-uscMG, kanye nohlu- kanye namasethi ofuzo omaka wekhophi eyodwa asetshenziswa yi-CheckM kanye ne-Anvi'o.Ukuhlonzwa kwezimpinda ezingu-2 kuma-uscMG angu-40 kwaqinisekiswa ukwakhiwa kabusha kwe-phylogenetic (bona ngezansi) ukuze kukhishwe noma yikuphi ukungcola okungase kube khona (lokhu kuhambisana no-5% ngokusekelwe kulezi zinhlobo zomaka ezingu-40).Ucwaningo olwengeziwe lwabamele i-MAGAs 'Ca.Izinga eliphansi lokungcola kulawa ma-genome akhiwe kabusha laqinisekiswa ezinhlotsheni ze-Eremiobacterota kusetshenziswa isixhumi esibonakalayo se-Anvi'o esisekelwe ebuningini nasekulandeleni ukwakheka kokuxhumana (Ulwazi Olwengeziwe)59.
Ukuhlaziywa kwe-phylogenomic, sikhethe ama-MAG amele amahlanu "Ca".I-Eudormicrobiaceae”, zonke izinhlobo “Ca.I-genome ye-Eremiobacterota kanye namalungu enye i-phyla (okuhlanganisa i-UBP13, i-Armatimonadota, i-Patescibacteria, i-Dormibacterota, i-Chloroflexota, i-Cyanobacteria, i-Actinobacteria ne-Planctomycetota) iyatholakala ku-GTDB (r89)13.Wonke lawa ma genome aye achazelwa njengoba kuchazwe ngaphambilini ekukhishweni kofuzo lwekhophi eyodwa kanye nesichasiselo se-BGC.Ama-genome e-GTDB alondolozwe ngokuvumelana nemibandela engenhla yobuqotho nokungcoliswa.Ukuhlaziywa kwe-Phylogenetic kwenziwa kusetshenziswa ukuhamba komsebenzi kwe-Anvi'o Phylogenetics59.Isihlahla sakhiwe kusetshenziswa i-IQTREE (v.2.0.3) (izinketho ezizenzakalelayo kanye -bb 1000)80 ekuqondaneni kwe-39 tandem ribosomal proteins ekhonjwe ngu-Anvi'o (MUSCLE, v.3.8.1551)81.Izikhundla zakhe zehliswa.ukuvala okungenani u-50% we-genome82 kanye ne-Planctomycecota yasetshenziswa njengeqembu elisekelwe phezu kwe-GTDB yezihlahla ze-topology.Isihlahla esisodwa sama-uscMG angama-40 sakhiwe kusetshenziswa amathuluzi afanayo namapharamitha.
Sisebenzise i-Traitar (v.1.1.2) namapharamitha azenzakalelayo (i-phenotype, evela kuma-nucleotides)83 ukuze sibikezele izici ezivamile zama-microbial.Sihlole indlela yokuphila engase ibe khona yokubamba inkunzi ngokusekelwe kunkomba yezilwane ezidlayo eyakhiwe ngaphambilini84 encike kokuqukethwe isakhi seprotein-coding ku-genome.Ngokukhethekile, sisebenzisa i-DIAMOND ukuze siqhathanise amaprotheni ku-genome ngokumelene nesizindalwazi se-OrthoMCL (v.4)85 sisebenzisa izinketho -okuzwela kakhulu -id 25 -query-cover 70 -ikhava yesihloko 70 -top 20 FUTHI sibale izakhi zofuzo ezihambisana izakhi zofuzo zomaka ezizingelayo nezingezona ezidla ezinye.Inkomba ingumehluko phakathi kwenani lezimpawu ezidlayo nezingezona ezokudla.Njengokulawula okwengeziwe, siphinde sahlaziya i-"Ca" genome.Isici se-Entotheonella TSY118 sisekelwe ekuhlotshaneni kwayo ne-Ca.I-Eudoremicrobium (usayizi omkhulu we-genome namandla e-biosynthetic).Okulandelayo, sihlole izixhumanisi ezingaba khona phakathi kofuzo lomaka wezilwane ezizingelayo nezingezona izilwane ezizingelayo kanye namandla e-biosynthetic we-Ca.I-Eudormicrobiaceae” futhi yathola ukuthi alukho ufuzo olungaphezu kolulodwa (kusuka kunoma yiluphi uhlobo lofuzo lomaka, okungukuthi i-predator/non-predator gene) olugqagqana ne-BGC, okusikisela ukuthi i-BGC ayihlanganisi amasiginali wokuzingela.Isichasiselo se-genomic esengeziwe se-replicons ehlutshiwe senziwe kusetshenziswa i-TXSSCAN (v.1.0.2) ukuze kuhlolwe ngokukhethekile isistimu yokufihla, i-pili, ne-flagella86.
Abamele ama-'Ca abahlanu baklanywe ngokwenza imephu yama-metaranscriptoms angama-623 asuka kungxenyana ye-prokaryotic kanye ne-eukaryotic enothisayo yolwandle lwase-Tara22,40,87 (kusetshenziswa i-BWA, v.0.7.17-r1188, -a ifulege).I-Eudormicrobiaceae genome.Amafayela e-BAM acutshungulwe nge-FeatureCounts (v.2.0.1)88 ngemva kokufunda okungu-80% kanye nokuhlunga kobunikazi obungu-95% (okunezici zezinkethoIzibalo -primary -O -fraction -t CDS,tRNA -F GTF -g ID -p ) Ibala inombolo yokufaka isakhi sofuzo ngasinye.Amamephu akhiqiziwe enziwa abejwayelekile ukuze abe nobude bezakhi zofuzo kanye nobuningi bezakhi zofuzo zomaka i-moTU (isibalo sokufakwa esimaphakathi esijwayelekile sobude sezakhi zofuzo ezinenani lokufakwayo >0) futhi ilogi yaguqulwa yaba ngu-22.74 ukuze kutholwe isisho esihlobene ngeseli ngayinye ileveli yofuzo, echaza futhi ukuhlukahluka kusuka kusampula kuya kusampula ngesikhathi sokulandelana.Izilinganiso ezinjalo zivumela ukuhlaziywa okuqhathanisayo, ukunciphisa izinkinga zokuqamba lapho kusetshenziswa idatha yobuningi obuhlobene.Amasampuli kuphela ane>5 kwezingu-10 zofuzo zomaka we-moTU aye acatshangelwa ukuze ahlaziywe okwengeziwe ukuze kutholakale ingxenye enkulu eyanele ye-genome.
Iphrofayili ye-transcriptome ejwayelekile ye-'Ca.I-E. taraoceanii ibe ngaphansi kokuncishiswa kobukhulu kusetshenziswa i-UMAP futhi ukumelela okuwumphumela kwasetshenziselwa ukuhlanganisa okungagadiwe kusetshenziswa i-HDBSCAN (bona ngenhla) ukuze kunqunywe isimo senkulumo.I-PERMANOVA ihlola ukubaluleka komehluko phakathi kwamaqoqo ahlonziwe endaweni yokuqala (engancishisiwe) yebanga.Umehluko phakathi kwalezi zimo uhlolwe ku-genome yonkana (bheka ngenhla) kanye nezindlela ezingu-201 ze-KEGG zihlonzwe emaqenjini asebenzayo angu-6, okungukuthi: i-BGC, i-secretion system kanye nezakhi zofuzo ze-flagellar ezivela ku-TXSSCAN, ama-enzyme e-degradation (ama-protease nama-peptidase), kanye nezilwane ezidlayo nezingezona- izakhi zofuzo.izimpawu zenkomba ezidla inyama.Kusampula ngayinye, sibale isisho se-median esijwayelekile sekilasi ngalinye (qaphela ukuthi isisho se-BGC ngokwaso sibalwa njengokuvezwa kwe-median kwezakhi zofuzo ze-biosynthetic zaleyo BGC) futhi kuhlolelwe ukubaluleka kuzo zonke izifundazwe (uhlolo lwe-Kruskal-Wallis lulungiselwe i-FDR).
Izakhi zofuzo zokwenziwa zathengwa kwaGenScript kanye neziqalo ze-PCR zathengwa kwaMicrosynth.I-Phusion polymerase evela ku-Thermo Fisher Scientific yasetshenziselwa ukukhulisa i-DNA.I-NucleoSpin plasmids, ijeli ye-NucleoSpin kanye nekhithi yokuhlanza ye-PCR evela e-Macherey-Nagel yasetshenziselwa ukuhlanzwa kwe-DNA.Ama-enzyme okuvinjelwa kanye ne-T4 DNA ligase athengwe kwa-New England Biolabs.Amakhemikhali ngaphandle kwe-isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) kanye ne-1,4-dithiothreitol (DTT, AppliChem) athengwa ku-Sigma-Aldrich futhi asetshenziswa ngaphandle kokuhlanzwa okwengeziwe.Ama-antibiotics chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), kanye ne-carbenicillin (Cbn) athengwe kwa-AppliChem.Izingxenye zemidiya ze-Bacto Tryptone kanye ne-Bacto Yeast Extract zathengwa kwa-BD Biosciences.I-Trypsin yokulandelanisa ithengwe kwa-Promega.
Ukulandelana kofuzo kukhishwe ku-anti-SMASH ebikezelwe nge-BGC 75.1.E. malaspinii (Ulwazi olwengeziwe).
Izakhi zofuzo embA (locus, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (locus, MALA_SAMN05422137_METAG-framework_127-gene_4), kanye ne-embAM (okuhlanganisa izifunda ze-intergene) zakhiwe ngokulandelana kwe-p5 kanye ne-(A) njenge-pc. eyenzelwe ukuvezwa ku-E nini.Isakhi sofuzo se-embA safakwa ngaphansi kwesayithi lokuqala lokuhlanganisa okuningi (MCS1) ye-pACYCDuet-1(CmR) kanye ne-pCDFDuet-1(SmR) enezindawo zokuqhekeka ze-BamHI kanye ne-HindIII.Izakhi zofuzo ze-embM kanye ne-embMopt (i-codon-optimized) zafakwa ngaphansi kwe-MCS1 pCDFDuet-1(SmR) ne-BamHI ne-HindIII futhi zafakwa endaweni yesibili yokuhlanganisa okuningi kwe-pCDFDuet-1(SmR) kanye ne-pRSFDuet-1(KanR) (MCS2) nge NdeI/ChoI.Ikhasethi le-embAM lahlanganiswa laba pCDFDuet1(SmR) kanye nezindawo zokuhlukanisa i-BamHI kanye ne-HindIII.I-orf3/embI gene (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) yakhiwa ngokugqagqana isandiso se-PCR kusetshenziswa ama-primers EmbI_OE_F_NdeI kanye ne-EmbI_OE_R_XhoI, igaywe nge-NdeI/Xhoet-XhoI-Duba1M1CDM1I-ligay ama-enzyme (Okungeziwe ithebula).6).Ukuvinjelwa kokugayeka kokudla kanye nokuxhunywa kwe-enzyme kwenziwa ngokuya ngephrothokholi yomkhiqizi (i-New England Biolabs).