Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

ARTICLE Exploi ing gene ic di e si y and gene syn hesis o iden i y supe io ni ogenase Ni H p o ein a ian s o enginee N 2 -fixa ion in plan s Xi Jiang 1,2, Lucía Payá-To mo 1,2, Diana Co oian1, Inés Ga cía-Rubio 3, Rocío Cas ellanos-Rueda 1,4, Ál a o Ese e i 1,2, Gema López-To ejón1,2, S e an Bu én 1✉& Luis Manuel Rubio 1,2✉ Enginee ing ni ogen fixa ion in euka yo es equi es high exp ession o unc ional ni ogenase s uc u al p o eins, a goal ha has no ye been achie ed. He e we build a knowledge-based lib a y con aining 32 ni ogenase ni H sequences om p oka yo es o di e se ecological niches and me abolic ea u es and combine wi h apid sc eening in obacco o iden i y supe io Ni H a ian s o plan mi ochond ia exp ession. Th ee Ni H a ian s ou pe o m in obacco mi ochond ia and a e u he es ed in yeas . Hyd ogenobac e he mophilus (Aquifi- cae) Ni H is isola ed in la ge quan i ies om yeas mi ochond ia and ulfills Ni H p o ein equi emen s o e ficien N 2 fixa ion, including elec on ans e o subs a e educ ion, P- clus e ma u a ion, and FeMo-co biosyn hesis. H. he mophilus Ni H exp essed in obacco lea es shows lowe ni ogenase ac i i y han ha om yeas . Howe e , ans e o [Fe 4 S 4 ] clus e s om Ni U o Ni H in i o inc eases 10- old he ac i i y o he obacco-isola ed Ni H, e ealing ha plan mi ochond ia [Fe-S] clus e a ailabili y cons i u es a bo leneck o enginee plan ni ogenases. h ps://doi.o g/10.1038/s42003-020-01536-6 OPEN 1Cen o de Bio ecnología y Genómica de Plan as, Uni e sidad Poli écnica de Mad id, Ins i u o Nacional de In es igación y Tecnología Ag a ia y Alimen a ia, Pozuelo de Ala cón, 28223 Mad id, Spain. 2Depa amen o de Bio ecnología-Biología Vege al, Escuela Técnica Supe io de Ingenie ía Ag onómica, Alimen a ía y de Biosis emas, Uni e sidad Poli écnica de Mad id, 28040 Mad id, Spain. 3Cen o Uni e si a io de la De ensa, C a. de Huesca s/n, 50090 Za agoza, Spain. 4Depa men o Biosys ems Science and Enginee ing, ETH Zü ich, 4058 Basel, Swi ze land. ✉email: s e an.bu [email protected];lm. [email protected] COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio 1 1234567890():,; Ni ogen (N) e ilize s used o inc ease c op p oduc i i y in in ensi e ag icul u e p ac ices pollu e g oundwa e and elease g eenhouse gasses1. On he o he hand, sub- sis ence ag icul u e p ac ices including poo N e iliza ion p o- duce low and inconsis en yields causing malnu i ion and po e y2,3. The e is la ge in e es in enginee ing ce eal c op a ie ies capable o acqui ing hei own N4. One app oach o his ou come elies on unc ional exp ession o a ni ogenase enzyme by he ce eal plan 5. Ni ogenases a e p oka yo ic, O 2 -sensi i e, wo-componen me allop o eins ha con e ine N 2 in o bio- logically use ul NH 3 6–8. The mos e ficien and widesp ead a - ian , he molybdenum ni ogenase, is composed o an Fe p o ein (ni H-encoded) and a MoFe p o ein (encoded by ni D and ni K). The Fe p o ein (Ni H) dona es elec ons o he MoFe p o ein (Ni DK) ha in u n educes N 2 . Nascen Ni H and Ni DK polypep ides need o acqui e p ope qua e na y s uc u e and o ecei e me al clus e s, one [Fe 4 S 4 ] clus e pe Ni H homodime and wo pai s o P-clus e and FeMo-co pe Ni DK he e o- e ame , o unc ionali y. We ha e ecen ly e iewed he mechanisms and gene ic equi emen s o assemble hese co ac o s and o ma u e Ni H and Ni DK in o ac i e Mo ni ogenase9. The la ge numbe o ni ogen fixa ion (ni ) genes in ol ed, and he sensi i i y o mos o he p o ein p oduc s owa ds O 2 , makes ni ogenase enginee ing a daun ing ask wi h issues ha need o be sol ed s epwise. To da e, unc ional Ni H, Ni U, and Ni B ha e been pu ified om mi ochond ia o ae obically cul u ed Saccha omyces ce e i- siae cells10,11, while ac i e Ni U and Ni H we e isola ed om chlo oplas s o Nico iana ben hamiana a he end o he da k pe iod12. Also, he epo ed low s abili y o he Ni D p o ein13 has now been imp o ed in wo ecen s udies ha iden ified key esidues in he Ni D sequence as suscep ible o clea age upon mi ochond ia impo 14,15. No wi hs anding hese achie emen s, de ailed analysis o yeas mi ochond ia- a ge ed Azo obac e inelandii Ni H has been hampe ed by low p o ein solubili y esul ing in subop imal yields10. Accumula ion o mos ly insoluble Ni H was also epo ed when Klebsiella oxy oca Ni H was a ge ed o he obacco mi ochond ia16. The di ficul y o exp essing high le els o soluble and unc ional Ni H in yeas and obacco poses a majo p oblem o euka yo ic ni ogenase enginee ing as i is he mos abundan Ni p o ein du ing N 2 fixa ion17. The p oblem is exace ba ed because, in addi ion o se ing Ni DK wi h elec ons o subs a e educ ion, Ni H is equi ed o ma u e P-clus e s on o Ni DK and o he final s eps o FeMo-co biosyn hesis in complex wi h Ni EN9. Fo hese easons i is essen ial o iden i y a Ni H a ian ha is highly soluble and s able when exp essed a e y high le els in a plan cell, and ha can pe o m all h ee Ni H- dependen ac i i ies. One app oach o achie e his ou come would be p o ein enginee ing o well-s udied Ni H om model diazo- ophs (e.g., A. inelandii o K. oxy oca) aimed o in oduce sequences ha imp o e s abili y in he mi ochond ia18. P o ein enginee ing has been ex ensi ely employed o ob ain glyphosa e esis ance19, ano he impo an ai o c ops. Al e na i ely, mining o phylogene ically di e se ni H sou ces can be unde aken in o de o find na u al Ni H p o eins wi h supe io p ope ies, a s a egy ha was success ul o Ni B11 and o inc easing ca - o enoid le els in “Golden Rice”20. He e, 32 dis inc ni H genes we e sc eened o exp ession le el and solubili y in mi ochond ia o N. ben hamiana. The ni M, ni U, and ni S genes we e co-exp essed because hei p o ein p oduc s a e in ol ed in Ni H olding and in he biosyn hesis and deli e y o i s [Fe 4 S 4 ] clus e 9. The Hyd ogenobac e he mophilus Ni H was iden ified as as ly supe io o he A. inelandii Ni H in e ms o exp ession le els, solubili y, and unc ionali y bo h in obacco and yeas mi ochond ia. Mi ochond ia- a ge ed H. he - mophilus Ni H sa isfied all unc ional and spec oscopic equi emen s o a ni ogenase Fe p o ein when pu ified om yeas . The sc eening also pinpoin ed he plan mi ochond ia [Fe- S] clus e assembly as a bo leneck o u he enginee ing. Resul s Lib a y design and s a egy o exp ession o mi ochond ia- a ge ed Ni H in N. ben hamiana. A lib a y o 32 ni H sequences om phylogene ically di e se p oka yo es was designed con- side ing one o se e al o he ollowing c i e ia: (i)ni H genes ound in confi med diazo ophs; (ii)ni H genes om pho o ophs o plan -associa ed bac e ia; (iii)ni H genes om ae obic o gan- isms; (i ) g ow h empe a u e o he ni H hos ; ( )ni H genes om a chaeal ep esen a i es (Supplemen a y Da a 1). O ganized by phyla, he selec ion included genes om 1 Aquificae, 4 Fi micu es, 1 Ac inobac e ia, 15 P o eobac e ia, 6 Cyanobac e ia, 1 Chlo obi, 1 Chlo oflexi, and 3 Eu ya chaeo a (Fig. 1a). The wo kflow o his s udy is desc ibed in Fig. 1b. The gene sequences encoding he 32 Ni H a ian s we e cloned in o plan ec o s o Ag obac e ium ume aciens infil a ion-media ed Ni H exp ession in N. ben hamiana lea es (Supplemen a y Table 1, see Me hods sec ion o de ails). The ni H sequences we e codon- op imized o S. ce e isiae because codon-usage is simila o obacco21 and he wo kflow included downs eam exp ession o obacco-selec ed Ni H a ian s in yeas o biochemical cha ac e - iza ion. The genes we e unde con ol o he s ong and cons i u i e E35S p omo e . Amino- e minal COX4-TS ex ensions we e added o Ni H p o eins. COX4 is he 29 amino acid ansi pep ide o he S. ce e isiae mi ochond ia p o ein cy och ome c oxidase subuni IV (MLSLRQSIRFFKPATRTLCSSRYLLQQKP), whe eas TS deno es he 28 amino acid Twin-S ep-Tag pep ide (WSHPQFEKGGGSGGGSGGSAWSHPQFEK)22. COX4 a ge ed Ni H p o eins o he mi ochond ia ma ix and TS was used o enable a ian -independen immunoblo de ec ion o Ni H and o acili a e i s pu ifica ion. Impo an ly, he TS- ag has been shown o no significan ly a ec Ni H unc ionali y12. COX4-TS-Ni H a ian s a e he ea e deno ed as NbNi HXx whe e Nb s ands o he hos N. ben hamiana,Xx deno es a ian s collec i ely, and o he supe sc ip s indica e he species om which Ni H sequence was ob ained. Vec o s wi h NbNi HXx cons uc s addi ionally con ained a ansc ip ional uni o exp ession o he g een fluo escen p o ein (GFP) ha was used as indica o o success ul lea infil a ion (Supplemen a y Table 1). An auxilia y ec o was cons uc ed o co-exp ess A. inelandii ni M,ni U, and ni S and a ge hei p o ein p oduc s o mi ochond ia ia N- e minal SU9 ex ensions. Simila o COX4, he mi ochond ial p esequence o subuni 9 o he Neu ospo a c assa F 0 -ATPase23 (SU9) has been shown o deli e Ni p o eins o N. ben hamiana mi ochond ia24. Ni U and Ni S assemble [Fe- S] clus e s des ined o Ni p o eins in A. inelandii25. While no essen ial o exp ession o unc ional Ni HA in S. ce e isiae mi ochond ia10 hey we e equi ed o gene a e high amoun s o ac i e Ni B in yeas 11. As we aimed o iden i y Ni H a ian s accumula ing a highe le els han Ni HA , Ni UA and Ni SA we e included in his s udy. In A. inelandii and o he well- s udied diazo ophs Ni M is in ol ed in Ni H olding o dime iza ion p io [Fe 4 S 4 ] clus e acquisi ion9,26. Despi e ni M no being p esen in o ganisms o some selec ed ni H a ian s (Supplemen a y Da a 1), his gene was always included in infil a ion expe imen s o consis ency. Iden ifica ion o Ni H p o eins sui able o exp ession in N. ben hamiana.N. ben hamiana lea es we e co-infil a ed wi h a 1:1:1 mix u e o h ee dis inc A. ume aciens cul u es o exp ession o , espec i ely, one NbNi HXx a ian plus GFP, he auxilia y p o eins NbNi MA ,NbNi UA , and NbNi SA , and he ARTICLE COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 2COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio RNA silencing supp esso p19 o enhance he ni ansgene exp ession (Fig. 1b)27. P o ein ex ac s we e p epa ed om he N. ben hamiana lea es h ee days a e infil a ion and analyzed o accumula ion o soluble NbNi HXx using an ibodies ecognizing he TS- ag. Only wo Ni H a ian s we e consis en ly de ec ed among expe imen s (Fig. 1c, Supplemen a y Fig. 1a), namely hose o igina ing om Me hanocaldococcus in e nus (NbNi HMi) and Hyd ogenobac e he mophilus (NbNi HH ). A hi d Ni H a ian om Me hano he mobac e ma bu gensis (NbNi HMm) was de ec ed a low le els a one occasion. In con as , analysis o o al ex ac s p epa ed om he infil a ed obacco lea es showed ha , al hough accumula ion le els o he NbNi HXx p o eins a ied significan ly, 25 o he 32 a ian s could be de ec ed (Supplemen a y Fig. 1b). Only NbNi H exp ession o a ian s om B ady hizobium japonicum,Rhizobium leguminosa um b . i olii,He baspi illum se opedicae,Gloeo hece sp. KO68DGA, Rhodopseudomonas palus is,Me hano he mobac e he mau o- ophicus, and F ankia sp. (s ain FaC1) could no be demon- s a ed. Sequence alignmen s and 3D-modeling o Ni HMi, Ni HH , and Ni HMm a e shown in Supplemen a y Fig. 2. The 3D-models did no e eal any specific ea u e ha would explain hei supe io accumula ion as soluble p o ein in obacco mi o- chond ia, bu all h ee p o eins o igina e om he mophilic o ganisms (Supplemen a y Da a 1) which could possibly explain hei s abili y and solubili y. Ac i i y o Ni H a ian s isola ed om mi ochond ia o ae obically cul u ed S. ce e isiae.N. ben hamiana sc eening- iden ified a ian s and Ni HA we e exp essed in S. ce e isiae and pu ified by S ep- ag a fini y ch oma og aphy (STAC) o e alua e unc ionali y when a ge ed o mi ochond ia. Fo his, genes encoding COX4-TS-Ni H cons uc s we e ans e ed o exp es- sion ec o s oge he wi h su9-ni MA ,su9-ni UA , and su9-ni SA unde he con ol o galac ose-inducible GAL1 o GAL10 p o- mo e s (Supplemen a y Table 2, Supplemen a y Fig. 3a). These COX4-TS-Ni H a ian s exp essed in ae obic S. ce e isiae cul- u es a e he ea e deno ed ScNi HMm,ScNi HMi,ScNi HH , and ScNi HA (ScNi HXx collec i ely). While ScNi HMm,ScNi HMi, and ScNi HH we e pu ified o nea homogenei y (Fig. 2a), SDS-PAGE analysis o ScNi HA showed addi ional slowe mig a ing co-elu ing p o eins. Mass spec ome y confi med ha hese we e con aminan s (Fig. 2a). ScNi HA solubili y was low and much p o ein was los o he pelle ac ion when p epa ing he soluble cell- ee ex ac (CFE) explaining i s poo pu ifica ion yield (abou 11 mg pe kg o S. ce e isiae cells) (Supplemen a y Fig. 3b–e, Supplemen a y Table 3). The yield o ScNi HMm was also ela i ely low, in line wi h he in e io esul in he N. ben hamiana sc eening. In con as , he yields o ScNi HMi and ScNi HH we e ca. 20 imes highe . I on (Fe) quan ifica ion o pu ified samples was a iable bu indica ed ha ScNi HH was isola ed la gely as holo-p o ein con aining one [Fe 4 S 4 ] clus e pe dime (Supplemen a y Table 3). Consis en ly, immunoblo analysis showed ha ScNi HH , ScNi MA ,ScNi UA , and ScNi SA had been e ficien ly a ge ed o he mi ochond ia (Supplemen a y Fig. 4). Ac i i ies o pu ified ScNi HXx a ian s we e de e mined in i o using he ace ylene educ ion assay (ARA) and compa ed Euka ya Eu ya chaeo a (3) C ena chaeo a Nanoa chaeo a Thauma chaeo a Ko a chaeo a P o eobac e ia (15) (8) (2) (2) (3) Ac inobac e ia (1) Fi micu es (4) Bac e oides Aqui icae (1) Chlo o lexi (1) Chlo obi (1) Cyanobac e ia (6) LUCA Bac e ia (29) The mo ogae To al (32) ab 48 - 35 - Ponceau 28 - α-GFP 48 - 35 - α-Ni S 35 - α-Ni U 35 - α-Ni M 35 - kDa C l S ep-HRP (NbNi HXx) : Ni H : Ni U/Ni S/Ni M c+++++ -A Mm Mi H Ni H lib a y design (N. ben hamiana) s nai a H iN 3s nai a H iN 23 Pu i ica ion Scale up (I) (III) Lea in il a ion (A. ume aciens) Ni H pu i ica ions (S. ce e isiae)(II) Ni H + GFP (pN2XJ81-pN2XJ112) Ni U/Ni S/Ni M (pN2XJ165) p19 (GB1203) M. ma bu gensis M. in e nus H. he mophilus Solubili y sc eening (N. ben hamiana) H. he mophilus Ni H Ni H cha ac e iza ion ( H. he mophilus ) In i o complemen a ion (A DJ77) (V) 3 Ni H a ian s Ni H + Ni U/Ni S/Ni M H. he mophilus (XJ1Y) M. ma bu gensis (XJ2Y) M. in e nus (XJ3Y) Ace ylene educ ion analysis (yeas pu i ied Ni H) H. he mophilus Ni H unc ional N i H inac i e Ni H + Ni U/Ni S/Ni M + GFP + p19 (pAE382) Ac i i y analysis ( obacco pu i ied H. he mophilus Ni H) Fe e iliza ion Ex ended da k pe iod (N. ben hamiana) (VIII) N i H ac i e Ni H + Ni U/Ni S/Ni M + GFP + p19 (pAE382) H. he mophilus Ni H pu i ica ion (N. ben hamiana) (VII) (IV) [Fe 4 S 4 ] analysis (yeas Ni H) i) P-clus e ma u a ion ii) FeMo-co syn hesis iii) N 2 educ ion (yeas Ni H) (VI) Fig. 1 Ni H lib a y design and sc eening. a Phylogene ic ee indica ing he dis ibu ion and numbe o he es ed Ni H a ian s among bac e ial phyla (blue) and a chaeal lineage (magen a). bExpe imen al wo kflow o his s udy. cImmunoblo s o N. ben hamiana soluble p o ein ex ac s de eloped wi h an ibodies agains TS, GFP, Ni U, Ni S, and Ni M (s ep I o panel b). GFP de ec ion was used as con ol o no malize e ec i eness o infil a ion expe imen s. Ponceau s aining panel is shown as loading and memb ane ans e con ol. Do ed line indica es di e en exposu es o he same memb ane. Cell- ee ex ac s o A. inelandii DJ ( o Ni U and Ni S) o E. coli Rose a (DE3) o e exp essing Ni MA ( o Ni M, as Ni M exp ession le els in A. inelandii is low) we e used as size con ols. Unc opped immunoblo s a e shown in Supplemen a y Fig. 8. COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 ARTICLE COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio 3 o ha o Ni H pu ified om A. inelandii (deno ed Ni HA ). In all cases Ni DK pu ified om A. inelandii (deno ed Ni DKA ) was used as MoFe p o ein componen . ScNi HA ac i i y was 85% o Ni HA (Fig. 2b), suppo ing p e ious obse a ions ha STAC is sui able o pu ifica ion o me al-clus e con aining Ni p o eins exp essed in yeas 11,28.ScNi HH specific ac i i y was abou hal o ScNi HA , while ScNi HMm and ScNi HMi showed e y low ac i i ies (Fig. 2b). The assay did no de e mine whe he lowe ac i i ies we e due o Ni H a ian de ec s, o o incompa ibili y wi h ScNi USA in i o ( esul ing in apo-Ni H p o ein wi h low [Fe 4 S 4 ] clus e occupancy) o Ni DKA in i o ( esul ing in poo elec on dona ion). Recons i u ion o ScNi HMm [Fe 4 S 4 ] clus e s in i o by ei he mixing wi h Fe, L-cys eine, DTT, and EcNi SA (di ec econs i u ion) o by incuba ing wi h [Fe 4 S 4 ] clus e - loaded EcNi UA (Ni U-media ed econs i u ion) did no ac i a e he p o ein (Supplemen a y Fig. 5), indica ing ha his Ni H a ian is no compa ible wi h Ni DKA . In con as , ScNi HMi was ac i a ed o some ex en by Ni UA , and u he by di ec econs i u ion, indica ing ha he A. inelandii Ni US machine y is no op imal o Ni HMi (Supplemen a y Fig. 5). Howe e , ac i i ies we e e y low compa ed o he as-isola ed ScNi HH p o ein (Fig. 2b). This could be explained by Ni HH ha bo ing mo e o he conse ed amino acid esidues known o be impo an o he in e ac ion wi h Ni DKA (Supplemen a y Fig. 2). Impo an ly, soluble accumula ion o ScNi HH in mi ochon- d ia was 20- old highe han ScNi HA (Supplemen a y Table 3), which ansla es in o a leas 10- old highe in i o ac i i y and ulfills Ni H quan i y equi emen s o ni ogenase enginee ing. Thus, ScNi HH was u he cha ac e ized. ScNi HH exhibi s Ni H-cha ac e is ic spec oscopic signals and is unc ional in i o. Pu ified ScNi HH p o ein p esen ed ul a iole – isible (UV– is) abso p ion spec a ypical o O 2 - sensi i e [Fe-S] clus e -con aining p o eins (Fig. 3a). Amino- e minal sequencing e ealed ha amino acid esidues EQKP emained a e COX4 p ocessing (Fig. 3b), whe e con e sion o glu amine (Q) o glu amic acid (E) could be due o deamina ion pe o med by he mi ochond ial ma ix N- e minal amidase NTA129. Elec on pa amagne ic esonance (EPR) confi med ha ScNi HH p o ein con ained an [Fe 4 S 4 ] clus e wi h simila signal in ensi y and g- alues as Ni HA (Fig. 3c), sugges i e o success ul ma u a ion in o unc ional Fe p o ein. The Ni H a ian chosen o enginee N 2 -fixing plan s mus pe o m P-clus e ma u a ion and FeMo-co biosyn hesis in addi ion o se e as elec on dono o subs a e educ ion. We he e o e es ed whe he H. he mophilus Ni H could e e he Ni −pheno ype o A. inelandii DJ77 (Δni H s ain)30. Fo his, s-ni HH was in oduced by ans o ma ion in o DJ77 and he esul ing s ain UW481 was es ed o diazo ophic g ow h and in i o ace ylene educ ion ac i i y. UW481 showed diazo ophic g ow h bo h in solid and liquid media (Supplemen a y Fig. 6a, b), and immunoblo analysis demons a ed sus ained A Ni HH exp ession and ace ylene educing ac i i y indica i e o ac i e ni ogenase (Supplemen a y Fig. 6c, d). These da a s ongly indica e ha Ni HH can eplace he unc ions o na i e A. inelandii Ni H o some ex en , which equi es p oduc i e in e ac ions wi h a leas apo-Ni DKA , Ni DKA , and Ni ENA p o eins. ScNi HH is ac i e in subs a e educ ion, P-clus e o ma ion and FeMo-co syn hesis. Each indi idual Ni H-dependen ac i i y was hen analyzed in i o using pu e ScNi HH p epa a ions (Fig. 3d). P-clus e ma u a ion was de e mined by supplemen ing CFE o A. inelandii DJ77 (Δni H) wi h ScNi HH . The DJ77 ex ac is de oid o FeMo-co and con ains inac i e apo-Ni DKA wi h imma u e P-clus e s. The P-clus e ma u a ion assay using DJ77 CFE elies on posi i e ou comes o h ee dis inc ac i i ies pe o med in wo sequen ial eac ions (Fig. 3d). In he fi s eac ion (S ep I +II) pu e Ni H and FeMo-co a e added o DJ77 CFE esul ing in Ni H-dependen educ i e coupling o he wo [Fe 4 S 4 ] P-clus e p ecu so s o o m ma u e P-clus e s (S ep I), ollowed by FeMo-co inse ion in o P-clus e con aining apo- Ni DKA o gene a e ac i e Ni DKA (S ep II) (Fig. 3d). Te a- hiomolybda e is hen added o p e en u he FeMo-co inse - ion, sepa a ing he ma u a ion (S ep I +II) and ac i i y (S ep III) eac ions. Ac i a ion o DJ77 apo-Ni DKA by ScNi HH demons a ed i s P-clus e ma u a ion ac i i y (Fig. 3e). In i o FeMo-co syn hesis (Fig. 3d, S ep II)9was de e mined by combining pu ified p epa a ions o ScNi HH , apo-Ni DKA con aining P-clus e s bu de oid o FeMo-co31, apo-Ni ENA con aining pe manen [Fe 4 S 4 ] clus e s bu lacking FeMo-co p ecu so 32, Mo, homoci a e, and ei he he FeMo-co p ecu so ba kDa 130 - 100 - 75 - 63 - 48 - 35 - 28 - - - - - - - - - - - - - - - - - - - - - - 1 2 3 4 5 Coomassie ScNi HA ScNi HMi ScNi HMm ScNi HH A Mm Mi H -Ni H 0 500 1000 1500 2000 2500 E hylene p oduc ion (nmol C 2 H 4 ·min -1 ·mg -1 o Ni DK A ) ScNi H A Mm Mi H -Ni H 0 10 20 30 O e O e Fig. 2 Ac i i y o selec ed Ni H a ian s pu ified om mi ochond ia o ae obically cul u ed S. ce e isiae enginee ed s ains. a STAC-pu ified ScNi HA (s ain XJ4Y), ScNi HMm (s ain XJ2Y), ScNi HMi (s ain XJ3Y), and ScNi HH (s ain XJ1Y) p o eins. The low solubili y o ScNi HA p omo ed binding o con amina ing p o eins (as column was no sa u a ed wi h ScNi HA ) ha we e iden ified by pep ide mass finge p in ing as: 1) Acc1p (N1P4Q3), 2 and 3) py u a e ca boxylase (N1P377), and 4) HSP70 (D2J4C2). No iden ifica ion was possible o band numbe 5. Unc opped gels a e shown in Supplemen a y Fig. 9. bARA o STAC-pu ified ScNi HXx a ian s. Ac i i y using Ni HA and Ni DKA (posi i e con ol) was 2406 ± 53 uni s (nmol e hylene o med pe min and mg o Ni DKA ). Da a ep esen mean alues (n=2 echnical eplica es). ARTICLE COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 4COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio Ni B-co bound o he ca ie p o ein Ni XA 33 o Ni B p o ein supplemen ed wi h Fe and S34. As o he P-clus e ma u a ion assay, e a hiomolybda e was added be o e he ARA (Fig. 3d, S ep III). Figu e 3 shows ha ScNi HH suppo ed FeMo-co syn hesis in i o. Impo an ly, ScNi HH and ScNi BM (Me ha- no he mobac e he mau o ophicus Ni B isola ed om S. ce e i- siae)11 ac ed oge he in he Ni B-dependen in i o FeMo-co syn hesis assay in which Ni B-co was concomi an ly syn hesized by ScNi BM a he han added in pu ified o m. This esul p o ed compa ibili y o wo essen ial p o eins o N 2 fixa ion, ScNi HH and ScNi BM , when p oduced in yeas mi ochond ia. I also showed in e species compa ibili y wi h Ni DKA and Ni ENA , al oge he cons i u ing he conse ed biochemical co e o ni ogenase. ScNi HH ac i i y in subs a e educ ion was demons a ed by he ARA and by educ ion o N 2 in o NH 3 . ARA i a ion was ca ied ou wi h a fixed quan i y o Ni DKA and inc easing amoun s o ScNi HH . Maximum Ni DKA ac i i y was achie ed a mola ScNi HH o Ni DKA a ios la ge han 40 (Fig. 3g), simila o eac ions wi h he na u al coun e pa Ni HA 35. This esul sugges s ha he maximum ac i i y ha can be achie ed combining ScNi HH wi h Ni DKA is 1000 uni s (i.e., hal o he ac i i y wi h Ni HA ). In addi ion, ScNi HH suppo ed N 2 educ ion in o NH 3 by Ni DKA . Impo an ly, he a io o NH 3 o e hylene p oduced by Ni DKA was simila independen ly o using Ni HA o ScNi HH (Fig. 3h). As-isola ed NbNi HH was inac i e bu could be ac i a ed by [Fe 4 S 4 ] clus e econs i u ion.NbNi HH was pu ified om A. ume aciens-infil a ed lea es o N. ben hamiana. Plan s we e g own unde long-day condi ions (16 h ligh /8 h da k) and lea es we e p ocessed a he end o he da k pe iod. Genes encoding K K D D K K D D FeMo-co K K D D P-clus e [Fe 4 S 4 ] Open FeMo-co si e HH H H MoFe p o ein Fe p o ein ac S ep I P-clus e ma u a ion S ep II FeMo-co biosyn hesis N 2 C 2 H 2 HH S ep III Ni ogenase ac i i y d 300 400 500 600 700 800 0 0.2 0.4 0.6 0.8 1.0 Wa eleng h (nm) No malized abso bance As-isola ed Ai -exposed YLL EQKPSAWSH TS- ag COX4 300 400390380370360350340330320310 B (mT) EPR in ensi y (a.u.) ScNi HH Ni HA g = 2.04 g = 1.94 g = 1.84 g = 2.05 g = 1.94 g = 1.86 NH 3 C 2 H 4 hg e E hylene p oduc ion (nmol C2H4 · min-1 · assay-1) S ep I+II: S ep III: + + Ni HA ScNi HH - -+ -+ + - - + - 0 5 10 15 20 b* 0 100 200 300 E hylene p oduc ion (nmol C2H4 · min-1 · mg-1 o Ni DKA ) Ni B-co + Ni HA Ni B-co + ScNi HH Ni BA + Ni HA ScNi BM + ScNi HH ScNi BM ScNi HH 5 10 20 40 60 80 100 120 140 160 0 250 500 750 1000 1250 Ra io ScNi HH : Ni DKA E hylene p oduc ion (nmol C2H4·min -1·mg-1o Ni DKA ) Ni H A ScNi H H 0 300 600 900 1200 1500 1800 2100 0 150 300 450 600 E hylene p oduc ion (nmol C 2 H 4 ·m in -1 ·mg -1 o Ni DK A ) Ammonia p oduc ion (nmol NH 3 ·mi n -1 ·m g -1 o Ni DK A ) Fig. 3 Cha ac e iza ion o ScNi HH .aUV– is abso p ion spec a o as-isola ed and ai -exposed ScNi HH .bP ocessing si e (black a ow) o COX4 mi ochond ia a ge ing signal (blue) as de e mined by N- e minal sequencing o ScNi HH . The con e sion o Q o E (ma ked by *) could be due o deamina ion p ocesses. cEPR signal o as-isola ed ScNi HH (190 μM) compa ed o Ni HA (71.2 μM). Expe imen al da a (black lines) and simula ions (blue lines) o each p o ein a e shown oge he wi h g alues. dSchema ic ep esen a ion o Ni H-dependen ac i i ies es ed in e–h. Figu e adap ed om Bu én e al.9. Copy igh 2020 ACS unde CC BY 4.0 h p://c ea i ecommons.o g/licenses/by/4.0.eIn i o P-clus e ma u a ion o apo-Ni DKA p esen in CFE o A. inelandii DJ77 (Δni H) a e addi ion o ScNi HH o Ni HA as indica ed (±). Te a hiomolybda e was added ( ollowing s ep I +II) o inhibi u he apo-Ni DKA ac i a ion du ing he ARA (s ep III). Da a ep esen mean alues (n=2 echnical eplica es). Ni B-co and Ni B-dependen in i o FeMo-co syn hesis using ScNi HH o Ni HA . Te a hiomolybda e was added ( ollowing s ep II) o inhibi u he apo-Ni DKA ac i a ion du ing he ARA (s ep III). A 20:1 mola a io o ScNi HH o Ni DKA was used in he ARA (s ep III). Da a ep esen mean alues (n=2 echnical eplica es (Ni B-co +Ni HA ), n=3 echnical eplica es (Ni B-co +ScNi HH ), n=4 echnical eplica es (Ni BA +Ni HA ), n=5 echnical eplica es (ScNi BM +ScNi HH ), n=3 echnical eplica es (ScNi BM ), n=4 echnical eplica es (ScNi HH )). Blue and ed do s co espond o independen expe imen s. gTi a ion o Ni DKA ac i i y wi h ScNi HH . Posi i e con ol eac ions pe o med wi h Ni HA and Ni DKA a 40:1 mola a io ga e 1692 ± 4 uni s (nmol e hylene o med pe min and mg o Ni DKA ). Reac ions lacking Ni H (nega i e con ol) ga e 2.5 ± 0.8 uni s. Da a ep esen mean alues (n=2 echnical eplica es). hARA ( ed do s, le y-axis) and N 2 - educ ion assay (blue squa es, igh y-axis) using ScNi HH and Ni DKA (s ep III). Ni HA was used as con ol. Da a ep esen mean alues (n=4 echnical eplica es). COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 ARTICLE COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio 5 NbNi HH ,NbNi MA ,NbNi UA , and NbNi SA ( oge he wi h p19 and GFP) we e piled up in a single plan -exp ession ec o o co-exp ession (Me hods sec ion and Supplemen a y Table 1). Pu ified NbNi HH did no exhibi b own colo o [Fe-S] clus e s and was inac i e in he ARA when combined wi h Ni DKA (Fig. 4a). The e o e, we econs i u ed NbNi HH [Fe 4 S 4 ] clus e in i o ei he by mixing wi h Fe, L-cys eine, DTT, and EcNi SA (di ec econs i u ion), o by incuba ing wi h [Fe 4 S 4 ] clus e - loaded EcNi UA (Ni U-media ed econs i u ion). Bo h me hods ac i a ed he NbNi HH as de e mined by he ARA (Fig. 4a), demons a ing ha he p o ein was co ec ly olded bu lacked i s [Fe 4 S 4 ] clus e . This esul sugges ed ha inse ion and/o s abi- li y o NbNi HH [Fe 4 S 4 ] clus e was poo in mi ochond ia o lea es. cb kDa QQKPS AWSHPQ FEK TS- agCOX4 130 - 100 - 75 - 63 - 48 - 35 - 28 - 180 - * Coomassie d S ep -HRP (NbNi HH ) kDa 8 h 16 h Ligh Da k Ponceau LighLighLighLighLigh Da kDa kDa kDa k NbNi HH kDa 180 - α-GFP QQKPSA WSHPQ FEKGG TS- agCOX4 130 - 100 - 75 - 63 - 48 - 35 - 28 - Coomassie e 12345 a As-isola ed Di ec EcNi UA - Ni H NbNi HH pu .: NbNi UA kDa 48 - 35 - 48 - 35 - 48 - 35 - S ep-HRP (NbNi UA ) α-Ni S Ponceau Fe e iliza ion +- E hylene p oduc ion (nmol C2H4 · min-1 · mg-1 o Ni DKA ) Recons . h g 28 - 48 - 28 - 35 - 35 - 63 - 100 - 75 - 48 - 35 - 63 - 75 - E hylene p oduc ion (nmol C2H4 · min-1 · mg-1 o Ni DKA ) 0 10 20 30 Da k 0 100 200 300 400 500 ARTICLE COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 6COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio Fe e iliza ion o he soil inc eases soluble Ni U in mi o- chond ia o N. ben hamiana. One explana ion o he low [Fe 4 S 4 ] clus e con en o NbNi HH could be insu ficien Fe a ailabili y in he soil. We obse ed ha accumula ion o NbNi- UA , bu no o NbNi SA , inc eased when he wa e used o i iga e he A. ume aciens-infil a ed plan s was supplemen ed wi h Fe (Fig. 4b). Sul u was no supplemen ed in soil as he infil a ion solu ion con ained Mg 2 SO 4 . Al hough Fe e iliza ion ipled he yield o STAC-isola ed NbNi UA , he a e age Fe con en o 2 Fe a oms pe p o ein was no a ec ed (Supple- men a y Fig. 7a, b, Supplemen a y Table 4). This could be due o he loss o ansien Ni U [Fe-S] clus e s du ing pu ifica ion, and i is no a su p ising ou come as isola ion o EcNi UA con aining only he pe manen [Fe 2 S 2 ] clus e s has been p e iously obse ed36. Immunoblo s de ec ed wo di e en ly mig a ing NbNi UA species in pu ifica ions om obacco lea es (Fig. 4c). Amino- e minal sequencing showed ha bo h species we e clea ed ei he one o se en amino acids in o he TS- ag (Fig. 4d). As bo h NbNi UA species showed he same N- e mini p oces- sing, we concluded ha he as e mig a ing polypep ide was unca ed a he C- e minus. Ex ended da k pe iod combined wi h Fe e iliza ion p oduced ac i e NbNi HH in mi ochond ia o N. ben hamiana lea es. The soil o N. ben hamiana plan s exp essing NbNi HH was e ilized wi h Fe o inc ease Fe a ailabili y. In addi ion, he da k pe iod p eceding lea ha es was ex ended om 8 h o 16 h hypo hesizing ha longe da kness would lowe in acellula O 2 and s abilize NbNi HH [Fe 4 S 4 ] clus e . Da k pe iod ex ension did no inc ease NbNi HH accumula ion (Fig. 4e) bu allowed o isola ion o ac i e p o ein as shown below. Abou 6 mg o NbNi HH was consis en ly isola ed pe kg o N. ben hamiana lea es (Fig. 4 , Supplemen a y Fig. 7c, Supplemen a y Table 5). Amino- e minal sequencing showed ha mi ochond ia- a ge ed NbNi HH accumula ed as wo species (simila o NbNi UA ), one in which wo amino acid esidues om he TS- ag we e emo ed wi h he COX4 signal and ano he ha was p ocessed fi e amino acid esidues u he in o he TS- ag (Fig. 4g). Func ionali y o NbNi HH isola ed om lea es o Fe e ilized obacco plan s ollowing 16 h o da kness was de e mined using ARA. NbNi HH p epa a ions consis en ly showed ac i i ies bu hese we e low compa ed o hose o [Fe 4 S 4 ] clus e - econs i u ed NbNi HH (Fig. 4h). This esul sugges ed ha NbNi HH accumula ed as wo species in obacco mi ochond ia, whe e inac i e p o ein likely lacking [Fe 4 S 4 ] clus e was mo e abundan han unc ional and [Fe 4 S 4 ] clus e -con aining NbNi HH . Con- sis en ly, he Fe con en o pu ified NbNi HH p epa a ions we e below de ec ion limi (Supplemen a y Table 5). Al oge he he esul s indica e ha while soluble NbNi HH accumula es in good quan i y in mi ochond ia o N. ben hamiana lea es, enginee ing o addi ional p o ein componen s o biosyn he ic pa hways will be equi ed o imp o e [Fe 4 S 4 ] clus e acquisi ion o s abili y. Discussion The fi s s udy epo ing p oduc ion o ac i e Ni H in yeas p o ed ha mi ochond ia is a sui able o ganelle o hos ing O 2 - sensi i e Ni p o eins unde ae obic g ow h condi ions10. Despi e being a alid p oo -o -concep , u he de elopmen s wi h A. inelandii Ni H we e limi ed by low yields as only a small po ion was soluble in he mi ochond ial ma ix. Simila solubili y issues we e la e epo ed o K. oxy oca Ni H a ge ed o N. ben- hamiana mi ochond ia16 and a e confi med in his s udy using immunoblo sc eening and STAC. Iden i ying he bes possible Ni H p o ein o euka yo ic (plan ) exp ession was he e o e o u e mos impo ance. Ni H is he mos abundan Ni p o ein equi ed o N 2 fixa ion in A. inelandii17. Besides being he Fe p o ein componen o Mo ni ogenase, Ni H is essen ial o he assembly o bo h Ni DK co ac o s, namely he P-clus e and he FeMo-co9. Ni H p o eins o ni ogenase enginee ing in plan s should: (i) be s able and soluble a high le els in he mi ochond ial ma ix, and (ii) be compa ible wi h he Ni DK componen om a well- s udied model-diazo oph i hei own Ni DK componen s a e no a ailable in pu ified o m. Compa ibili y is impo an when e alua ing unc ion o candida e Ni H a ian s. In ou case i mean ha any selec ed Ni HXx should be compa ible wi h Ni - MA (i Ni HXx is no Ni M-independen ), Ni USA o ma u a- ion and [Fe 4 S 4 ] clus e syn hesis/inse ion, and Ni DKA o ni ogenase ac i i y measu emen s. We no e ha his equi e- men in oduces a selec ion bias and ha he sc eening could ha e o e looked Ni H a ian s ha we e supe io o ha o H. he mophilus i combined wi h di e en Ni DK. The Ni H a ian s es ed in his s udy we e selec ed om a cu a ed da ase o hund eds o Ni H sequences by a o ing ae obic o plan -associa ed o igins, o o e come he inhe en O 2 - sensi i i y o Ni H, and unc ionali y a mode a e empe a u es. We also hypo hesized ha Ni H a ian s om a chaea could unc ion be e in a euka yo ic en i onmen as his domain o li e is belie ed o be mo e closely ela ed o he Euka yo a37, and because ou p e ious wo k exp essing a chaeal Ni B a ian s in yeas had shown hem o be supe io o hose o bac e ial o igin11. We expec ed ha mos Ni H a ian s would be pa ly soluble in obacco mi ochond ia when exp essed oge he wi h he accesso y p o eins Ni UA , Ni SA , and Ni MA . Howe e , only Ni H om M. in e nus and H. he mophilus we e consis en ly de ec ed in soluble obacco ex ac s, in addi ion o M. ma bu - gensis ha was occasionally de ec ed a lowe le els. Two o hese Ni H p o eins o igina ed om a chaea and he hi d om a bac e ium. One possibili y could be ha he Ni MA p o ein was no exp essed a su ficien le els in he obacco mi ochond ia and ha only hese h ee Ni H a ian s did no equi e Ni M o ma u a ion. Howe e , low le els o Ni M exp ession appea o be enough o Ni H ma u a ion in K. oxy oca38,39. A mo e plausible explana ion can be ound in he he mophilic na u e o M. in e nus,H. he mophilus, and M. ma bu gensis. I has ecen ly Fig. 4 Cha ac e iza ion o he NbNi HH p o ein. a Ac i a ion o as-isola ed NbNi HH p o ein wi h [Fe 4 S 4 ] clus e s ei he by di ec chemical syn hesis o by EcNi UA -media ed econs i u ion. Ac i i y using Ni HA (posi i e con ol) was 1773 ± 10 uni s (nmol e hylene o med pe min and mg o Ni DKA ). Da a ep esen mean alues (n=2 echnical eplica es). bImmunoblo s showing he e ec o Fe e iliza ion on NbNi UA and NbNi SA o al p o ein exp ession. Ni S an ibody was p obed on he same memb ane a e incuba ion wi h S ep ac in-HRP. cNbNi UA p o ein pu ified om obacco plan s. The lowe band (ma ked by *) indica es a as e mig a ing NbNi UA polypep ide. dP ocessing si es (black a ows) o he COX4 mi ochond ia a ge ing signal (blue) as de e mined by N- e minal sequencing o he ull-leng h and he as e mig a ing NbNi UA .eImmunoblo s showing accumula ion o NbNi HH a he end o 8 h o 16 h nigh (da k pe iod). NbNi HH p o ein pu ified om obacco lea es. gP ocessing si es (black a ows) o COX4 signal (blue) as de e mined by N- e minal sequencing o NbNi HH .hARA o fi e independen NbNi HH STAC-pu ifica ions (1–5). Measu ed ac i i ies using Ni HA (posi i e con ols) and wi hou Ni H (nega i e con ols) we e, espec i ely, 2406 ± 53 and 4.6 ± 1.3 (pu ifica ion 1), 1773 ± 10 and 3.2 ± 2.2 (pu ifica ions 2 and 3), and 1692 ± 3.9 and 2.5 ± 0.8 (pu ifica ions 4 and 5). All ac i i ies a e in nmol e hylene o med pe min and mg o Ni DKA . Da a ep esen mean alues (n=2 echnical eplica es). Unc opped immunoblo s and gels a e shown in Supplemen a y Fig. 10. COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 ARTICLE COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio 7 been epo ed ha he empe a u e inside espi ing mi ochond ia o cul u ed human cells is a ound 50 °C40, e en when he ex e nal medium is main ained a 38 °C. Whe he he same d as ic e ec on empe a u e holds ue o mi ochond ia o a lea cell is no known o us, bu i could explain in pa he ou come o ou Ni H sc eening. None o he wo highes exp essed Ni H p o eins o igina ed om p o en diazo ophs. We a e no awa e o any s udy in es iga ing diazo ophy in he a chaeon M. in e nus. Howe e , Ni BMi cu ed he Ni −pheno ype o an A. inelandii ni B mu an s ain41 and, as Ni B has no o he known unc ion han biosyn hesis o ni ogenase ac i e-si e co ac o s, i is likely ha M. in e nus is in ac a diazo oph. On he o he hand, N 2 - fixa ion has been es ed bu no obse ed in H. he mophilus TK- 642. In e es ingly only six Ni H a ian s in ou lib a y o igina ed om o ganisms ha ing genes wi h high simila i y o A. inelandii ni M. Pe haps o he p olyl isome ases could subs i u e o Ni M in hese o ganisms. Whe he Ni M (and he Ni US machine y) is equi ed o ma u a ion o he h ee selec ed Ni H p o eins (especially Ni HH ) in mi ochond ia will be in es iga ed in u u e wo k. Mi ochond ia-exp essed ScNi HH was he only a ian ha suppo ed ele an ni ogenase ac i i y when combined wi h Ni DKA . I s ac i i y co esponded o oughly hal o ha using ScNi HA e en i he ScNi HH o Ni DKA mola a io was inc eased well abo e 40 no mally used o ARA. Eme ich and Bu is showed ha Ni H p o eins can unc ion wi h Ni DK om o he o ganisms35, bu his s udy only combined p o eins om bac e ia. An op imal g ow h empe a u e o 72 °C has been epo ed o H. he mophilus TK-643, which could explain lowe ScNi HH ac i i y in subs a e educ ion assays. Howe e , ou p edic ion om his s udy and p e ious wo k on Ni B is ha subop imal wo king empe a u e o Ni p o eins om he mo- philes is a p ice wo h paying when enginee ing ni ogenase in euka yo es, as solubili y and s abili y o hese a ian s is so much imp o ed. One obse a ion o his s udy was ha he specific ac i i y o as-isola ed NbNi HH p o ein was lowe han ScNi HH . We hink his was caused by poo [Fe 4 S 4 ] clus e a ailabili y –and hence ine ficien inco po a ion –o poo Ni H [Fe 4 S 4 ] clus e s abili y wi hin he lea cell mi ochond ia. In his con ex , i is no known how Fe e iliza ion inc eased accumula ion o soluble NbNi UA . Mo e a ailable Fe could inc ease mi ochond ia [Fe-S] clus e s biosyn hesis and [Fe 2 S 2 ] clus e occupancy in NbNi UA which, in u n, would p o ide s abili y o he p o ein. A compa ibili y issue be ween NbNi HH and NbNi UA and NbNi SA is unlikely since EcNi UA could e ec i ely ac i a e NbNi HH in i o. NbNi HH mis olding in mi ochond ia is also unlikely as i was e ficien ly ac i a ed by econs i u ion o i s [Fe 4 S 4 ] clus e . I is howe e likely ha p o ec ion by espi a o y O 2 -consump ion in lea is lowe han in yeas . NbNi HH exposu e o O 2 du ing lea p o- cessing is also a possibili y making his a pu ely echnical p o- blem. While lea es we e kep in liquid ni ogen and lysis and pu ifica ion we e pe o med inside an anae obic glo e box, i is di ficul o comple ely ule ou ha some O 2 apped wi hin he lea was eleased du ing issue dis up ion. In conclusion, his s udy shows ha gene ic di e si y can be exploi ed o iden i y, om a e y la ge pool o sequences, he mos adequa e Ni p o ein componen s o enginee a euka yo ic ni ogenase. Modula cloning echniques, gene syn hesis wi h codon op imiza ion, and o he syn he ic biology ools pe mi building mul i-p o ein pa hways wi h componen s o e y di e se o igin. In his case he Ni H p o ein om H. he mophilus was iden ified as soluble in mi ochond ia o bo h S. ce e isiae and N. ben hamiana accumula ing a much highe le els han he A. inelandii homologue. This example is ele an no only because he iden ified a ian pe o med all h ee Ni H-essen ial eac ions, namely P-clus e ma u a ion, FeMo-co biosyn hesis, and Ni D- KA educ ion, bu also because Ni HH o med unc ional in e species in e ac ions wi h Ni B, Ni EN, and Ni DK p o eins, al oge he ep esen ing he ou p o eins cons i u ing he co e o diazo ophy. Me hods Design, assembly, and cloning o he ni H lib a y. A cu a ed da ase o diazo- ophs41 was used o collec ni H candida es and design he lib a y. Genes encoding ni H a ian s we e codon op imized o exp ession in S. ce e isiae and he sequence encoding pE35S::cox4- wins ep was codon op imized o exp ession in obacco (Supplemen a y Da a 1). All gene ic pa s we e op imized using he GeneOp imize ool (The moFishe ) and syn hesized by The moFishe ia he Enginee ing Ni ogen Symbiosis o A ica (ENSA) p ojec . The ni H genes we e syn hesized and cloned in o pMA cloning ec o wi h BamHI and Bs EII es ic ion si es flanking each gene. The pE35S::cox4- wins ep sequence was flanked by HindIII and BglII es ic ion si es. pGFPGUSplus (plasmid #64401, Addgene) and he pMA ec o con aining pE35S::cox4- wins ep we e diges ed wi h HindIII and BglII and used o gene a e he pa en al ec o pN2SB41, con aining a pE35S::cox4- wins ep-gus- NOS ansc ip ional uni in which gus was flanked by BamHI and Bs EII es ic ion si es. The pa en al ec o pN2SB41 and all pMA ec o s con aining ni H a ian s we e diges ed wi h BamHI and Bs EII and used o gene a e ec o s pN2XJ81-pN2XJ112 (Supplemen a y Table 1). pGFPGUSplus was used o gene a e ec o pN2XJ165 con aining ansc ip ional uni s o mi ochond ia- a ge ed accesso y Ni p o eins (A. inelandii Ni U, Ni S, and Ni M). The su9-ni UA (AAAAGGATCCAATGGCCTCCACTCGTGTCCT CG, AAAAAAGGTCACCTTAGACTTCCATTTGGGCGTGTGCG) and su9- ni SA (AAACTAGTATGGCCTCCACTCGTGTCCTCG, AAAAGAGCTCTTAAC CATAGACAGGAGCAAAGGCTTTACC) genes we e amplified by PCR om he yeas ec o pN2GLT410. Amplifica ion eac ions added flanking BamHI and Bs EII ( o su9-ni U)o SpeI and SacI ( o su9-ni SA ) si es. The DNA agmen con aining he su9-ni MA sequence was c ea ed by o e lapping PCR using p ime s in oducing sequences homologous o hose flanking he XhoI si e o pGFPGUSplus (ATTATG GAGAAACTCGAGTTAACCATGTGCTAAGTTTTCC, TACAAATCTATCTCT CTCGAGATGGCCTCCACTCGTG, CTTTCTGAGGCCATGGAAGAGTAGGCG CGCTTCTGG, CGCGCCTACTCTTCCATGGCCTCAGAAAGATTAGCTGA TG). pGFPGUSplus was fi s diges ed wi h BglII and Bs EII o inse su9-ni UA , hen wi h XbaI and SacI o inse su9-ni SA , and finally diges ed wi h XhoI o inse su9-ni MA by homologous ecombina ion44. All DNA diges ions we e pe o med using enzymes om New England Biolabs. Liga ed p oduc s (T4 ligase, P omega) we e in oduced in o E. coli DH5α chemically compe en cells and selec ed on LB (Lysogenic b o h) supplemen ed wi h app op ia e an ibio ics. Plasmid ex ac ion was pe o med using Qiap ep Spin Minip ep ki (QIAGEN) and co ec cloning was confi med by Sange sequencing (Mac ogen). G ow h o S. ce e isiae, mi ochond ia isola ions, and ScNi H pu ifica ions.S. ce e isiae o galac ose-induced exp ession o ScNi HMm,ScNi HMi,ScNi HH , and ScNi HA oge he wi h SU9-Ni UA , SU9-Ni SA , and SU9-Ni MA (XJ1Y-XJ4Y, Supplemen a y Table 3) we e cul u ed in 4-l e men e s unde ae obic condi ions (0.625 l o ai pe minu e and l o cul u e, 250 pm s i ing) and used o mi o- chond ia isola ions o Ni H pu ifica ions as p e iously desc ibed11. P epa a ion o CFE and STAC pu ifica ions we e pe o med a O 2 -le els below 1 ppm in anae- obic chambe s (Coy sys ems o MB aun). Typically, cells we e esuspended in lysis bu e (100 mM T is-HCl (pH 8.6), 200 mM NaCl, 10% glyce ol, 2 mM sodium di hioni e (DTH), 1 mM PMSF, 1 μg/ml leupep in, 5 μg/ml DNAse I) a a a io o 1:2 (w/ ). To al ex ac s (TE) we e p epa ed by lysis o he cell suspensions unde anae obic a mosphe e using an EmulsiFlex-C5 homogenize (A es in Inc.) ope a ing a 20,000 psi. The TE was ans e ed o cen i uge ubes equipped wi h sealing closu es (Beckman Coul e ) and cen i uged a 50,000 g o 1 h a 4 °C (A an i J-26 XP). The supe na an was fil e ed using fil e ing cups wi h a po e size o 0.2 μm, ende ing cell- ee ex ac (CFE) o soluble p o eins ha was loaded a 2.5 ml/min in o a 5 ml S ep-Tac in XP column (IBA Li eSciences) a ached o an ÄKTA FPLC (GE Hea hca e). The column was washed using 75 ml washing bu e (100 mM T is-HCl pH 8.0, 200 mM NaCl, 10% glyce ol, 2 mM DTH). S ep-Tac in XP column-bound p o eins we e elu ed wi h 15 ml washing bu e supplemen ed wi h 50 mM bio in (IBA Li eSciences). The elu ion ac ion was concen a ed, and bio in emo ed, by passing he p o ein h ough PD-10 desal ing columns (GE Heal hca e). Desal ed elua e was u he concen a ed using cen i ugal fil e s (Amicon, Millipo e) wi h 30 kDa cu o . Finally, he concen a ed p o ein was snap- ozen in c yo ials (Nalgene) and s o ed in liquid N 2 . Soil Fe e iliza ion, p epa a ion o anae obic N. ben hamiana lea cell- ee ex ac s, and pu ifica ion o NbNi HH and NbNi UA .N. ben hamiana plan s we e g own unde long day condi ions (16 h ligh /8 h da k) wi h suppo ing ligh om 17:00 o 00:00 o 4 weeks. Fo Fe e iliza ion expe imen s, plan s we e i iga ed (2l pe week) wi h ap wa e supplemen ed wi h 1 g/l Seques ene G100 ARTICLE COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 8COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio (Syngen a). Lea es ha es ed a e ex ended da k pe iod (16 h) we e kep in da kness om 17:00 (p e ious day) un il sample collec ion (09:00 ollowing mo ning). Pu ifica ions o NbNi HH and NbNi UA we e pe o med a O 2 -le els below 1 ppm inside anae obic chambe s (Coy sys ema o MB aun). Typically, 200 g o lea ma e ial was ha es ed and ozen in liquid N 2 . Lea ma e ial was ans e ed in o an anae obic chambe in ozen condi ion and dis up ed in equal amoun (w/ ) o lysis bu e (100 mM T is-HCl pH 8.6, 200 mM NaCl, 10% glyce ol, 2 mM DTH, 1 mM PMSF, 1 μg/ml leupep in, 5 μg/ml DNAseI) using a blende (Os e Classic 4655) ope a ing a maximum powe and main ained a 4 °C using a ci cula ing wa e ba h. TE was fil e ed h ough cheese clo h o emo e la ge deb is. P epa a ion CFE by cen i uga ion, S ep-Tac in a fini y ch oma og aphy, p o ein elu ion, concen a ion, and s o age was iden ical as o yeas -exp essed ScNi H p o eins. The pu ifica ion p ocedu e o NbNi UA only di e ed in ha no DTH was p esen in he bu e s. P o ein me hods, an ibodies, UV– is abso p ion spec um, and elec on pa amagne ic esonance. P o ein concen a ions we e measu ed using he BCA p o ein assay (PIERCE) in combina ion wi h iodoace amide o elimina e he in e e ing e ec o DTH45. Colo ime ic Fe de e mina ion was pe o med as epo ed46, and he N- e minal amino acid sequences we e de e mined by Edman deg ada ion (P o eome Fac o y AG). An ibodies used in his s udy and hei dilu ions o immunoblo ing we e as ollows: polyclonal an ibodies de ec ing Ni UA (used a 1:2,000 in 5% BSA), Ni SA (used a 1:1,000 in 5% BSA), Ni HA (used a 1:5,000 in 5% BSA), Ni MA (used a 1:2,000 in 5% BSA) we e aised agains pu ified p epa a ions o he co esponding A. inelandii p o eins (gene a ed in house). S ep- ag II (“S ep-MAB”, 2-1507-001, IBA Li esciences, 1:2,000 in 5% BSA), S ep-Tac in conju aged o HRP (“S ep- HRP”, 2-1502-001, IBA Li esciences, 1:50,000 in TBS- T), GFP (sc-9996, San a C uz Bio echnology, 1:2,000 in 5% BSA), HSP60 (LK-2, ab59458, Abcam, 1:1,000 in 5% BSA), and Tubulin (3H3087, sc-69971, San a C uz Bio echnology, 1:500 in 5% BSA) specific an ibodies a e comme cially a ailable. The UV– is abso p ion spec a we e eco ded a e emo al o he DTH om he p o ein samples using PD-10 desal ing columns (GE Heal hca e) equilib a ed wi h he co esponding p o ein bu e wih ou DTH. DTH- ee p o ein samples we e hen dilu ed in he same bu e s and ans e ed o a Q6 spec oscopy cu e es wi h sealing closu es. Abso p ion (280 nm o 800 nm) was eco ded using a UV- 2600 spec opho ome e (Shimadzu). EPR measu emen s we e pe o med in a B uke E500 spec ome e equipped wi h a esona o ope a ing in he TE 102 mode a 9.47 GHz. Tempe a u e was se and s abilized o 10 K by an Ox o d empe a u e con olle egula ing a gas-flow c yos a e ige a ed wi h helium. Fo measu emen s, a mic owa e powe o 2.5 mW and a magne ic field modula ion ampli ude o 1 mT was used. Expe imen al condi ions we e ca e ully moni o ed o a oid o e -modula ion o sa u a ion e ec s. Simula ions o he EPR spec a we e pe o med using he Ma lab oolbox Easyspin47. In i o Ni H ac i i y. Ni H ac i i y was de e mined as desc ibed by Shah e al. wi h sligh modifica ions48. Reac ions we e p epa ed inside anae obic chambe s. Pu ified Ni H p o eins we e analyzed by ARA a e addi ion o Ni DKA and ATP- egene a ing mix u e (1.23 mM ATP, 18 mM phosphoc ea ine, 2.2 mM MgCl 2 , 3 mM DTH and 46 μg/ml o c ea ine phosphokinase, 22 mM T is-HCl pH 7.5) in a final olume o 600 μl inside 9 ml se um ials unde A a mosphe e con aining 500 μl o ace ylene (1 a m). The a io o Ni H o Ni DK in he assays was 40:1 unless o he wise indica ed. The ARA we e pe o med a 30 °C in a shaking wa e ba h o 15 min. Reac ions we e s opped by adding 100 μl o 8 M NaOH. Posi i e con ol eac ions o ace ylene educ ion we e ca ied ou wi h Ni HA . E hylene o med was measu ed in 50 μl gas phase samples using a Po apak N 80/100 column in a gas ch oma og aph (Shimadzu). Reduc ion o N 2 o NH 3 was de e mined in eac ion mix u es p epa ed as o he ARA bu con aining 100 mM 3-(N-mo pholino)p opanesul onic acid (MOPS), pH 7.8, as bu e . Mix u es we e p epa ed in olumes o 750 μl, om which 100 μl was emo ed a assay s a o se e as backg ound ( o ) o NH 3 measu emen s. A e exchanging ial a mosphe e o N 2 , mix u es we e incuba ed a 30 °C o 30 min, and eac ions we e s opped by addi ion o 100 μl 5 M EDTA. Twen y-fi e μl o he blank ( o ) and he eac ion ( 30 ) we e added in duplica es o 200 μlo- ph haldialdehyde eagen solu ion (The moFishe Scien ific) in 96-well mic opla e o fluo escence-based assays (Nunc). Fluo escence (Ex 390 nm, Em 472 nm) was measu ed using a Va ioskan LUX pla e eade (The moFishe Scien ific). NH 3 p oduc ion was de e mined om he inc ease in fluo escence ( 30 - o ) agains s anda ds p epa ed wi h NH 4 Cl and eco ded in he same pla e. In i o P-clus e ma u a ion. P-clus e ma u a ion assays we e pe o med inside anae obic chambe s. The in i o assay combined isola ed Ni H o be es ed (50 μg) wi h A. inelandii DJ77 (Δni H) CFE (4.34 mg o al p o ein) and an excess o pu e FeMo-co (0.85 μM) in 500 μl ATP- egene a ing mix u e as desc ibed abo e. Reac ions we e incuba ed a 30 °C o 30 min. Fo y μl o 1 mM (NH 4 ) 2 MoS 4 ( e a hiomolybda e) we e hen added and mix u es we e incuba ed o 10 min a oom empe a u e o p e en u he FeMo-co inco po a ion in o Ni DKA du ing he ARA. Apo-Ni DKA ac i a ion a e P-clus e ma u a ion and FeMo-co inse ion was analyzed by ARA a e addi ion o an excess o he same Ni H species (100 μg) and ATP- egene a ing mix u e in a final eac ion olume o 1 ml. ARA was ca ied ou in 9 ml se um ials con aining A and 500 μl o ace ylene (1 a m) in he headspace o 15 min a 30 °C. Posi i e con ol eac ions o in i o P-clus e ma u a ion and ARA con ained pu ified Ni HA . E hylene o med was measu ed in 50 μl gas phase samples using a Po apak N 80/100 column in a gas ch oma og aph (Shimadzu). In i o FeMo-co syn hesis and apo-Ni DKA econs i u ion. Ni B-co-dependen FeMo-co syn hesis assays we e pe o med inside anae obic chambe s as desc ibed by Cu a i e al., wi h sligh modifica ions34. One hund ed μl eac ions con ained 3.0 μM Ni H, GST-Ni X-Ni B-co (20.4 μM Fe), 1.5 μM apo-Ni ENA , 0.6 μM apo- Ni DKA , 17.5 μMNa 2 MoO 4 , 175 μMR-homoci a e, 1 mg/ml BSA, and ATP- egene a ing mix u e (1.23 mM ATP, 18 mM phosphoc ea ine disodium sal , 2.2 mM MgCl 2 , 3 mM DTH, 46 μg/ml c ea ine phosphokinase, final concen a ions in 22 mM T is-HCl (pH 7.5) bu e a 30 °C o 60 min. Ni B-dependen FeMo-co syn hesis assays we e pe o med as he abo e desc ibed Ni B-co-dependen assay eplacing GST-Ni X-Ni B-co by 10.0 μM Ni B monome , 125 μM FeSO 4 , 125 μMNa 2 S, and 125 μM SAM. Following in i o syn hesis o FeMo-co, 17.5 μM (NH 4 ) 2 MoS 4 was added o p e en u he FeMo-co inco po a ion in o apo-Ni DKA , and incuba ed o 10 min a 25 °C. Ac i a ion o apo-Ni DKA was analyzed by addi ion o 500 μl ATP- egene a ing mix u e and ScNi HH (2.0 μMfinal concen a ion) in 9 ml ials con aining A and 500 μl ace ylene. The ARA we e pe o med a 30 °C o 20 min. Posi i e con ol eac ions o ARA con ained Ni DKA and Ni HA . E hylene o med was measu ed in 50 μl gas phase samples using a Po apak N 80/100 column in a gas ch oma og aph (Shimadzu). In i o [Fe-S] clus e econs i u ion and Ni H ac i i y. In i o [Fe-S] clus e econs i u ions o Ni H and Ni UA pu ified om E. coli (EcNi UA )10 we e pe - o med in anae obic chambe s as desc ibed by Zheng and Dean49 wi h sligh modifica ions. Ni H o Ni U (20 μM) was added o 22 mM T is-HCl (pH 7.5) bu e supplemen ed wi h 8 mM 1,4-di hio h ei ol (DTT) in a final olume o 100 μl and incuba ed a 37 °C o 30 min. Then, eac ions we e supplemen ed wi h 1 mM L-cys eine, 1 mM DTT, 400 μM (NH 4 ) 2 Fe(SO 4 ) 2 , and 225 nM Ni SA pu ified om E. coli (EcNi SA )10, and incuba ed a 37 °C o e nigh . Finally, he p o eins we e dilu ed 1000- old in 22 mM T is-HCl (pH 7.5) bu e , and hen concen a ed using cen i ugal fil e s (Amicon, Millipo e) wi h 30 kDa cu o o emo e excess eagen s. Fo “di ec econs i u ion”ac i i y assays, he ac i i y o [Fe 4 S 4 ] clus e econs i u ed Ni H p o ein was de e mined using ARA. Fo “Ni U-media ed econs i u ion”, as-isola ed Ni H p o ein was mixed wi h [Fe-S] clus e econs i u ed EcNi UA , and hen immedia ely used o ARA. S a is ics and ep oducibili y. Dis inc samples we e used o in i o ac i i y measu emen s and sample sizes a e indica ed by n, whe e each dis inc sample was measu ed a leas wo imes. Mean o measu ed ac i i ies a e shown. The da a p esen ed in he figu e g aphs a e lis ed in Supplemen a y Da a 2. Da a a ailabili y The au ho s decla e ha he da a suppo ing he findings o his s udy a e a ailable wi hin he a icle, i s supplemen a y in o ma ion and da a, and upon eques . Recei ed: 15 June 2020; Accep ed: 25 No embe 2020; Re e ences 1. E isman, J. W. e al. Ni ogen: Too much o a i al esou ce. (Science B ie . WWF Ne he lands, Zeis , The Ne he lands, 2015). 2. Bo laug, N. E. Feeding a wo ld o 10 billion people: he mi acle ahead. In Vi o Cell De . Biol. 38, 221–228 (2002). 3. Sibha u, K. T. & Qaim, M. Ru al ood secu i y, subsis ence ag icul u e, and seasonali y. PLoS One 12, e0186406 (2017). 4. Bloch, S. E., Ryu, M. H., Ozaydin, B. & B oglie, R. Ha nessing a mosphe ic ni ogen o ce eal c op p oduc ion. Cu . Opin. Bio echnol. 62, 181–188 (2020). 5. Bu én, S. & Rubio, L. M. S a e o he a in euka yo ic ni ogenase enginee ing. FEMS Mic obiol. Le . 365, nx274– nx274 (2018). 6. Bulen, W. A. & LeCom e, J. R. The ni ogenase sys em om Azo obac e : wo- enzyme equi emen o N2 educ ion, ATP-dependen H2 e olu ion, and ATP hyd olysis. P oc. Na l Acad. Sci. USA 56, 979–986 (1966). COMMUNICATIONS BIOLOGY | h ps://doi.o g/10.1038/s42003-020-01536-6 ARTICLE COMMUNICATIONS BIOLOGY | (2021)4:4 | h ps://doi.o g/10.1038/s42003-020-01536-6 | www.na u e.com/commsbio 9