Chromosomal position effect influences the heterologous expression of genes and biosynthetic gene clusters in Streptomyces albus J1074.

Bilyk e al. Mic ob Cell Fac (2017) 16:5 DOI 10.1186/s12934-016-0619-z RESEARCH Ch omosomal posi ion e ec in luences he he e ologous exp ession o genes andbiosyn he ic gene clus e s inS ep omyces albus J1074 Bohdan Bilyk1,2, Liliya Ho bal3 and And iy Luzhe skyy1,2,3* Abs ac Backg ound: E o s o cons uc he S ep omyces hos s ain wi h enhanced yields o he e ologous p oduc ha e ocussed mos ly on enginee ing o p ima y me abolism and/o he dele ion o endogenous biosyn he ic gene clus- e s. Howe e , o he ac o s, such as ch omosome compac iza ion, ha e been shown o ha e a signi ican in luence on gene exp ession le els in bac e ia and ungi. The exp ession o genes and biosyn he ic gene clus e s may a y signi ican ly depending on hei loca ion wi hin he ch omosome. Li le is known abou he posi ion e ec in ac ino- myce es, which a e impo an p oduce s o a ious indus ially ele an bioac i e molecules. Resul s: To demons a e an impac o he ch omosomal posi ion e ec on he he e ologous exp ession o genes and gene clus e s in S ep omyces albus J1074, a ansposon mu an lib a y wi h andomly dis ibu ed ansposon ha includes a β-glucu onidase epo e gene was gene a ed. Repo e gene exp ession le els ha e been shown o depend on he posi ion on he ch omosome. Using a combina ion o he ansposon sys em and a φC31-based ec- o , he a anciamycin biosyn he ic clus e was in oduced andomly in o he S. albus genome. The p oduc ion le els o a anciamycin a ied up o eigh old depending on he loca ion o he gene clus e wi hin he ch omosome o S. albus J1074. One o he isola ed mu an s ains wi h an a i icially in oduced a achmen si e p oduced app oxima ely 50% mo e a anciamycin han s ains wi h endogenous a Bs. Conclusions: In his s udy, we demons a e ha exp ession o he epo e gene and a anciamycin biosyn he ic clus e in S ep omyces albus J1074 a ies up o eigh old depending on i s posi ion on he ch omosome. The in eg a- ion o he he e ologous clus e in o di e en loca ions on he ch omosome may signi ican ly in luence he i e o he p oduced subs ance. This knowledge can be used o he mo e e icien enginee ing o Ac inobac e ia ia he eloca ion o he biosyn he ic gene clus e s and inse ion o addi ional copies o he e ologous cons uc s in a sui able ch omosomal posi ion. Keywo ds: S ep omyces albus J1074, He e ologous exp ession, Ch omosomal posi ion e ec , φC31-a achmen si e © The Au ho (s) 2017. This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/ publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Backg ound Du ing he pas ew decades, s ep omyce es ha e been ex ensi ely employed o he he e ologous exp ession o bioac i e na u al p oduc s. He e ologous exp es- sion con ibu es subs an ially o he disco e y and cha ac e isa ion o seconda y me aboli es. The inc ease in he numbe o sequenced mic obial genomes dem- ons a es ha mos o he biosyn he ic clus e s emain silen unde s anda d labo a o y condi ions. The exp es- sion o silen gene clus e s in a well-cha ac e ised he e - ologous hos is a alida ed app oach o he disco e y o new na u al p oduc s [1]. Howe e , insu icien yields o he e ologously p oduced molecules o en p e en hei u he pu i ica ion and cha ac e isa ion. Cu en ly, Open Access Mic obial Cell Fac o ies *Co espondence: [email p o ec ed]land.de 1 Pha mBioTec GmbH, Science Pa k 1, 66123 Saa b ücken, Ge many Full lis o au ho in o ma ion is a ailable a he end o he a icle Page 2 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 se e al S ep omyces s ains ha e been u ilised as hos s o he p oduc ion o na u al p oduc s. The mos widely used a e S ep omyces coelicolo , S ep omyces a e - mi illis and S ep omyces albus J1074 [2]. Howe e , he p oduc ion le el o he he e ologously p oduced com- pounds o en needs o be u he op imised. Indus i- ally ele an i es o some na u al p oduc s may each he mul i-g am pe li e scale (e.g.,≥10g/L e y h omy- cin;≥30g/L e acycline;≥80g/L salinomycin; pe sonal communica ion wi h P o . H oje Pe ko ic, Ljubljana Uni e si y), whe eas he yields o na u al p oduc s in he e ologous hos s emain in he millig am ange [2]. One o he ac o s signi ican ly in luencing he exp es- sion o genes and biosyn he ic gene clus e s is hei loca- ion on he ch omosome, he so-called ‘ch omosomal posi ion e ec ’. This e m desc ibes di e ences in he exp ession o a gene depending on i s ch omosomal loca ion. S udies o gene exp ession in Saccha omyces ce e isiae demons a ed ha he exp ession o he e - ologous genes was ep essed when in eg a ed a silen loci, indica ing ha ep ession occu ed due o a posi- ional e ec [3]. In ano he s udy [4], i was shown ha an Aspe gillus nidulans mu an , de ec i e in he sys em esponsible o his one me hyla ion, ac i a es silen sec- onda y me aboli e clus e s. The posi ion e ec can in luence no only he exp es- sion le el o a na i e gene a e spon aneous ansloca- ions, bu also ansgene exp ession a e inse ion in o di e en egions o a genome, leading o changes in he - e ologous p oduc ion. I was demons a ed ha a ia- ions in β-galac osidase ac i i y can each 300- old in esponse o ansloca ions o he β-galac osidase gene in he Esche ichia coli ch omosome [5]. Howe e , Schmid and Ro h demons a ed [6] only a h ee old a ia ion in he exp ession le el o he his ope on clus e andomly dis ibu ed wi hin he Salmonella yphimu ium ch omo- some. Addi ionally, Lac obacillus lac is mu an s showed a h ee old di e ence in he le els o gus(a) exp ession [7]. The main ac o s causing such a iabili y in gene exp ession a e: (i) he le el o DNA compac iza ion [8]; (ii) a ia ions in he p omo e s eng h; and (iii) he dis- ance o he o igin o eplica ion [7]. The i s wo ac o s a e mo e c i ical o euka yo ic o ganisms, which ha e high le els o DNA compac iza ion and g ea e a ia- ion in p omo e s eng hs, which may in luence a down- s eam he e ologous gene. By con as , he dis ance o he eplica ion o igin is he majo ac o o a iabili y in gene exp ession in p oka yo ic cells, because hey con- ain a single o igin o eplica ion pe genopho e. Thus, a gene placed close o he o igin o eplica ion is epli- ca ed be o e a gene loca ed nea he e minus and he e- o e has an ope a i e inc ease in gene dosage [9]. Despi e he impo ance o s ep omyce es as p oduc- e s o na u al p oduc s, he posi ion e ec has no ye been in es iga ed in hem. The ep esen a i es o his genus ha e a complex li e cycle. These bac e ia colonise he en i onmen by g owing b anching, mul igenomic hyphae while simul aneously o ming unigenomic spo es o achie e dispe sion. Recen s udies iden i ied a no el p o ein speci ic o Ac inobac e ia ha is esponsible o DNA compac iza ion, ch omosome seg ega ion and an ibio ic p oduc ion [10]. In o he s udies [11], a his- one-like p o ein was cha ac e ised ha can s uc u ally couple changes in DNA con o ma ion and ansc ip ion in esponse o s ess. Such complexi y in nucleoid o gani- sa ion leads o a ia ions in exp ession o di e en DNA egions and causes a ch omosomal posi ion e ec . How- e e , o da e, a lack o e icien ools o he explo a ion o s ep omyce es genomics has limi ed any a emp s o e alua e he impac o his ac o on he exp ession o he e ologous genes and clus e s. He e, we ex end p e ious s udies on he ch omosomal posi ion e ec o he mycelial mic oo ganism S ep omy- ces albus J1074, which is a ypical ep esen a i e o he S ep omyces genus and is well known o i s ou s and- ing po en ial as a hos o he he e ologous p oduc ion o bioac i e small molecules [12]. Du ing he pas decade, S. albus J1074 has been used o he he e ologous exp es- sion o a ious an ibio ic biosyn he ic clus e s, e.g. hio- co aline, cyclooc a in and s e imycin. Nume ous gene ic ools ha e ecen ly been de eloped o he s ain, making i s genome enginee ing a he s aigh o wa d [13–15]. To e alua e he impac o he ch omosomal posi ion e ec on he e ologous exp ession in he genome o S. albus J1074, a ansposon mu an lib a y wi h andomly dis ibu ed gus(a), he β-glucu onidase epo e gene, was gene a ed. The exp ession o he epo e gene in ecombinan s ains was analysed, and a ious ac o s in luencing he exp ession o he gusA gene a e dis- cussed. In addi ion, using a combina ion o a ansposon sys em and he φC31-based ec o , he a anciamycin biosyn he ic clus e was in oduced andomly in o he S. albus genome. Analysis o a anciamycin p oduce s also demons a ed an eigh old a ia ion in he p oduc- ion le el o he an ibio ic depending on i s ch omosomal loca ion. One o he isola ed mu an s ains wi h an a i- icially in oduced a achmen si e p oduced 53% mo e a anciamycin han s ains wi h endogenous a Bs. Resul s anddiscussion Impac o  he ch omosomal posi ion e ec ongene exp ession To examine an impac o he ch omosomal posi ion e ec on he exp ession o he e ologous genes in S. albus J1074, a ansposon ha bou ing he epo e gene Page 3 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 was cons uc ed (Fig.1). The ansposon con ained he gus(a) epo e gene, encoding β-glucu onidase unde he con ol o he e mEp1 p omo e [16]. The ac i i y o β-glucu onidase can be spec opho ome ically meas- u ed. The gus(a) gene is lanked by wo d-phage e mina- o s [17], which p e en ansc ip ion in bo h di ec ions wi h an e iciency o up o 85% (Addi ional ile1: Figu e S1). The ap amycin esis ance gene was cloned ups eam and in he opposi e di ec ion o gus(a) o exclude any impac on exp ession o he epo e gene. Then he ansposon was cloned in o he plasmid con aining syn- he ic gene o Hima 1- ansposase and empe a u e sensi i e pSG5 eplicon. The ec o was in oduced in o genome S. albus J1074 and lib a y o ansposon mu an s was gene a ed as desc ibed p e iously [14]. Eigh een mu an s om his lib a y wi h he andomly dis ibu ed epo e gene we e isola ed and analysed. The inse ions we e mapped on he ch omosome using escue cloning, as desc ibed elsewhe e [14] (Table1; Fig.2). All iden i ied inse ions we e localised in a co e egion o he ch omo- some. A measu emen o he GusA ac i i ies was made a e 48h o g ow h (Table1; Fig.4). The a e age alue o ac i i y was 8.6U/mg, and eigh o he analysed mu an s (44%) had ac i i ies ha lay wi hin his ange (mu an s # 1, 2, 4, 5, 14, 16, 17 and 18). Fi e mu an s (28%) had ac i - i y alues abo e a e age, and ano he i e mu an s (28%), loca ed mos ly in he cen e o he ch omosome, and had alues below a e age. The a ia ion in he epo e gene exp ession was six- old ( he lowes alue obse ed was 2.7U/mg, and he highes was 17.3U/mg), which was compa able o esul s desc ibed o o he p oka yo es, whe e wo- o h ee old a ia ions in ac i i y ha e been obse ed [6, 7, 18]. Owing o he isola ion o gus(a) by he wo e mina- o s, he ac i i y o he local p omo e s should no ha e had a signi ican impac on i s exp ession. To con i m his, he GusA ac i i ies o he mu an s we e compa ed wi h S. albus J1074 RNA-seq da a a e 48h o cul i a- ion in liquid medium. The RPKM ( eads pe kilobase pe million eads) alues o he genes, which a e loca ed in he genome o S. albus J1074 ups eam and in he same o ien a ion as gus(a), we e analysed. The esul s dem- ons a ed ha , in he ob ained ansposon mu an s, local p omo e s had a mino e ec on he le el o gus(a) Fig. 1 The map o he plasmid con aining gus(a) in he ansposon, pALG. Plasmids con ains ollowing ea u es: hima 1(a)—syn he ic ansposase gene, unde con ol o p1— ipAp, hios ep on inducible p omo e ; gus(a)— epo e gene o glucu onidase; p2—e mEp1, p omo e 1 o e y h omycin esis ance gene; d e mina o o d- phage, pSG5 ep ac inomyce es empe a u e-sensi i e eplicon; aac(3) IV ap amycin esis ance ma ke , hph hyg omycin esis ance ma ke , ITR in e ed e minal epea s; R6Ko i o igin o escue cloning Table 1 Loci o pALG- ansposon inse ions inS. albus J1074 iden i ied by escue plasmid sequencing M Locus (XNR) Gene unc ion GUS ac i i y (U/mg) S . de . (U/mg) Exp ession le el (RPKM) 1 1302 U ease subuni α 1 8.2 0.6 1.9 2 1853 Bi- unc ional ans e ase/deace ylase 7.7 0.4 2.2 3 1880 Phospha ase 15.4 2.1 42.5 4 2035 Cons. hyp. p o ./biosyn hesis docking sca old p o . 9.1 1.3 5.7 5 2089 In eg al memb ane p o ein 8.1 0.1 24.7 6 2358 Reg. p o ./Me R- amily ansc ip ional eg. 11.8 1.1 22.7 7 2599 P edic ed p o ein/adenyla e cyclase 17.3 1.8 3.5 8 2683 Be a-lac amase 3.4 1.7 31.7 9 2883 Te R- amily ansc ip ional egula o 6.2 1.2 6.0 10 2981 In eg al memb ane p o ein 3.5 0.5 102.6 11 3133 Pep idase C14 caspase ca aly ic subuni 3.3 0.1 97.1 12 3300 Conse ed hypo he ical p o ein 2.7 0.9 2.3 13 3826 Conse ed hypo he ical p o ein/DNA-binding p o . 9.9 1.1 27.5 14 4204/5 IGR (in e genic egion) b w. wo p edic ed p o eins 8.6 1.1 25.4 15 4675/6 IGR b w. cons. hyp. p o ein and phospholipase 14.7 1.5 19.6 16 5073/4 IGR b w. ansmemb . anspo e and in eg al memb . p o . 8.8 1.3 3.5 17 5168 Glyce ol kinase 1/2 7.8 1.0 27.9 18 5663 Succina e dehyd ogenase la op o ein subuni 8.0 1.3 29.9 Page 4 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 exp ession, e.g., mu an 10, which exhibi ed one o he lowes le els o GusA ac i i y, was lanked ups eam by he egion wi h a high RPKM alue (Fig.3). Concu en ly, mu an s wi h a ela i ely low RPKM o lanking genes (e.g. mu an s 7 and 15) we e cha ac e ised by a high GusA ac i i y le el (Fig.3). Ch omosomal posi ion e ec does no co ela e wi hdis ance oo iC P e ious s udies on he posi ion e ec in E. coli and Sal- monella demons a ed ha a ia ions in he exp ession le el o epo e genes a e caused mainly by gene dosage [6, 19–21]. Unlike mos bac e ia ha possess a ci cula ch omosome, s ep omyce es ca y a linea ch omosome wi h a cen ally loca ed o iC. Thus, i he gene dosage plays a signi ican ole in he exp ession le el o he e - ologous genes, he GusA ac i i y in mu an s wi h inse - ions localised close o he ch omosome cen e should be highe han in hose wi h inse ions localised close o he ch omosome ends. Howe e , plo ing he GusA ac i i ies wi h posi ions o he epo e gene showed ha mu an s wi h in eg a ion nea he o iC demons a ed GusA ac i i y below a e age (mu an s 8, 9, 10, 11 and 12), whe eas s ains wi h an inse ion loca ed some dis ance om he o iC, close o cen es o he ch omosomal shoulde s, demons a ed abo e-a e age esul s (mu an s 3, 6 and 15). Reloca ion o  he si e o gene clus e in eg a ion The ob ained esul s demons a ed ha he ansloca ion o he he e ologous cons uc may signi ican ly in luence i s exp ession. This esul should also be alid o mo e complex gene ic s uc u es, such as an an ibio ic bio- syn he ic gene clus e . I is di icul o deli e an ibio ic biosyn he ic gene clus e s in o a s ep omyce es ch omo- some ia a ansposon, due o hei la ge size. Mos o he ec o s ca ying biosyn he ic gene clus e s a e in eg a ed in o he ch omosome ia he phage φC31 in eg a ion sys- em. Thus, we ansloca ed he a B a achmen si e o φC31 in o a ious loca ions o he S. albus J1074 ch o- mosome. A ansposon wi h an a achmen si e o he φC31-phage was cons uc ed and cloned in o a non- ep- lica i e ec o con aining hima 1(a), he syn he ic ans- posase gene. The ob ained plasmid pAHT (Fig.4) did no equi e any addi ional induc ion s eps o cu ing o he plasmid backbone, because he exconjugan s ob ained al eady con ained a copy o he ansposon inse ed in he genome. This ec o was han in oduced in o S. albus SAM3 (Δa B·ΔpseB4), lacking bo h endogenous a B si es [22], by in e gene ic conjuga ion om E. coli. The a anciamycin gene clus e was in eg a ed in o a i- icially dis ibu ed a Bs. This 35.9-kb la ge clus e was isola ed om S ep omyces echina us [23] and was p e i- ously exp essed in di e en s ep omyce es [24]. The cos- mid wi h he a anciamycin clus e was in oduced in o wen y- ou ansposon mu an s. In his way, a lib a y o s ains ca ying he a anciamycin biosyn he ic clus e a di e en ch omosomal posi ions was ob ained. As a con- ol, wo s ains con aining na u ally loca ed a Bs (Δa B and ΔpseB4 [22]) we e used. Al oge he , he mu an s demons a ed an eigh old a ia ion in a anciamycin p oduc ion. Such a a ia ion in he exp ession le el o a he e ologous clus e co ela ed wi h esul s ob ained o gus(a)-gene exp ession desc ibed he ein. The a e age p oduc ion o a anciamycin o he mu an s ains was 40.53U/mg. Howe e , in his case, we obse ed s onge a ia ion in he esul s: only one o he 24 mu an s p o- duced in he ange o 40.53±10% (mu an 24). Thi een mu an s (54%) demons a ed below-a e age esul s, and 10 mu an s (42%) e inced abo e-a e age esul s (Fig.5). The p oduc ion o a anciamycin om ou a i icially in eg a ed a Bs was mo e han 20% abo e he a e age p oduc ion om endogenous a achmen si es (Fig. 5, mu an s 1, 2, 4 and 21). The yield o a anciamycin in one o hose mu an s (mu an 2) was 56% highe han in he con ol s ains. This esul demons a ed ha he anslo- ca ion o he clus e may esul in signi ican a ia ion in an ibio ic p oduc ion. These da a sugges ha he cen al egions o he ch omosomal “shoulde s” a e mo e p e e able loci o he exp ession o he e ologous cons uc s, because GusA ac i i y in mu an s wi h hese in eg a ions was highe han a e age. Gi en ha s ep omyce es a e well- es ablished cell ac o ies o he biosyn hesis o na u al p oduc s, i is impo an o s udy he in luence o ch o- mosomal localisa ion on he exp ession o biosyn he ic gene clus e s. Indeed, he eigh old a ia ion in an ibio ic p oduc ion was obse ed in he lib a y o mu an s ca ying co e- sponding gene clus e s in di e en loca ions o he ch o- mosome. Fou o he 24 isola ed mu an s demons a ed mo e han a 20% inc ease in an ibio ic p oduc ion, and Fig. 2 Dis ibu ion o inse ion loci o pALG- ansposons in S. albus J1074. Blue hombs ep esen posi ions o he mapped ansposon inse ions; ed ci cle ep esen s posi ion o o iC o S. albus-ch omosome Page 5 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 one o hese mu an s showed a 56.5% inc ease in he yield o a anciamycin (82.4U/mg) compa ed wi h he a e age p oduc ion o he con ol s ains (52.65 U/mg). Thus, he p oposed app oach can be used in combi- na ion wi h o he me hods ha a e employed o he op imisa ion o he he e ologous p oduc ion o na u- al p oduc s. This app oach can be used no only o ansloca ion o he a achmen si e, bu also o in o- duce addi ional in eg a ion si es in o he genome, hus esul ing in an inc ease in he numbe o clus e s pe genome [25]. We belie e ha he gene ic ools desc ibed he ein will ind wide applica ions in he cons uc ion and manipula ion o ac inobac e ial he e ologous hos s. Conclusions Ou s udy demons a es he e ec o he ch omosomal posi ion e ec on a he e ologously exp essed epo e gene and on mo e complex gene ic cons uc s, such as an ibio ic biosyn he ic gene clus e s in S. albus J1074. Based on his phenomenon, we de eloped an app oach ha can be used o he cons uc ion o hos s ains wi h imp o ed he e ologous p oduc ion ea u es. Me hods Bac e ial s ains andcul i a ion condi ions The cul i a ion o he S. albus s ains was conduc ed on a soy-manni ol aga o in a liquid TSB medium a 29°C. The cul i a ion o E. coli was conduc ed in an LB medium a 37°C. The in e gene ic conjuga ion o he plasmids was pe o med as p e iously desc ibed [26]. The lib a y Fig. 3 The compa ison o GusA-ac i i y le els wi h exp ession le el o adjacen genes (co ela ion coe icien is equal—0.34). Blue columns co - espond o alues o GusA-ac i i y. S ains we e g own o 48 h a 28 °C; ligh g ey columns co espond o RPKM a e 48 h o cul i a ion a 28 °C. Mu an s a e placed acco ding o loca ion o hei ansposons on he ch omosome Fig. 4 The map o he plasmid con aining phage a achmen si e in he ansposon, pAHT. Plasmids con ains ollowing ea u es: hima 1(a)—syn he ic ansposase gene, unde con ol o p1— ipAp, hios ep on inducible p omo e , aac(3)IV ap amycin esis ance ma ke , hph hyg omycin esis ance ma ke , ITR in e ed e minal epea s, R6Ko i o igin o escue cloning, a B a achmen si e o φC31- phage Page 6 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 o ansposon mu an s was ob ained as desc ibed else- whe e [14]. Recombinan DNA echniques Lambda- ed-media ed ecombina ion was conduc ed as desc ibed in he s anda d p o ocol [27]. The isola ion o genomic DNA om S. albus s ains was pe o med acco ding o a p e iously desc ibed me hod [26]. PCR using Phusion polyme ase (The mo Scien i ic), sepa a- ion o DNA by aga ose gel elec opho esis and Sou h- e n blo hyb idisa ion we e pe o med as p e iously desc ibed [28]. DNA agmen s and PCR p oduc s we e pu i ied using PROMEGA™ ki s. Oligonucleo ides we e syn hesised by Eu o ins MWG Ope on. DNA sequenc- ing was pe o med by GATC-Bio ec. Isola ion o  escue plasmids Ch omosomal DNA was isola ed om he desi ed s ains and diges ed wi h SacII o pALG ansconjugan s, espec i ely. The agmen s we e hen sel -liga ed and ans o med in o E. coli T ans o max cells. The ans o - man s we e selec ed based on hei esis ance o ap amy- cin. The ob ained plasmids we e isola ed and sequenced using he P1-pALG-ch p ime . Plasmid cons uc ion The plasmids and p ime s used in his s udy a e lis ed below (see Addi ional ile1). The DNA manipula ions and cloning p ocedu es we e pe o med acco ding o s anda d p o ocols [29], and he plasmid cons uc s we e con i med by DNA sequencing. Cons uc ion o pALG The gus(a) gene was ampli ied using he pSETgus(a) plas- mid as a empla e, he o wa d p ime , F -XI-ep1-gus(a), ca ying he XbaI si e and e mEp1, a Saccha opolys- po a e y h aea e mEp1 gene p omo e and he e e se p ime , Rs-MI- d-gus(a), ca ying he d-phage e mi- na o and he MunI si e. The PCR agmen was liga ed in o pNheIaac [14] linea ised by XbaI and MunI leading o p11-8. The aac(3)IV gene was ampli ied using pIJ773 as a empla e, he o wa d p ime , F -ERI- d-aac, ca - ying he d e mina o and he e e se p ime , Rs-ERI- aac. Bo h p ime s ca y he EcoRI si e. The agmen was cloned in o he EcoRI si e o p11-8 eplacing an exis ing aac(3)IV, esul ing in p11-8aac d. The P uII agmen o p11-8aac d, con aining he ansposon, was liga ed o pALHim linea ised wi h EcoRV, o yield pALG (Fig.1). Cons uc ion o pAHT The hph gene was ampli ied using pAL1 as a empla e, wi h F -MI-a B-hph as he o wa d p ime , ca ying a B and he MunI es ic ion si e, and Rs-XI-hph as he e e se p ime ca ying he XbaI si e. The ampli- ied agmen was cloned in o he MunI and XbaI si es o pTn5Oks esul ing in pTn5Oksa Bhph(II). To con- s uc a backbone o he ansposon ec o , he hyg o- mycin esis ance gene in p31Him was eplaced by S. albus-mu an s 0 20 40 60 80 10 0 1/g Fig. 5 Rela i e concen a ion o a anciamycin in cul u e media o he e ologous a anciamycin p oduce s. da B—S. albus SAM1(Δa B)::p412C06; dpseB4—S. albus SAM2(ΔpseB4)::-p412C06; 1-24—S. albus SAM3 (Δa B·ΔpseB4)::pAHT::p412C06-mu an s. Do ed black line co esponds o a e age p oduc ion o a anciamycin by ansposon mu an s ains; do ed ed line co esponds o a e age p oduc ion o a anciamycin by con ol s ains wi h one in eg a ion si e. S ains we e g own o 120 h a 28 °C. Rela i e concen a ion o a anciamycin was calcula ed as peak a ea pe g am o d y mass Page 7 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 he ap amycin esis ance gene using λ- ed-media ed ecombina ion [30]. The p ime s F -hph/aac(3)IV and Rs-hph/aac(3)IV we e used o ampli y he agmen o ecombina ion. The ob ained plasmid, pAHS, was lin- ea ized by EcoRV and liga ed wi h he ansposon om he pTn5Oksa Bhph(II) plasmid, gi ing pAHT (Fig.4). Measu emen o glucu onidase ac i i y A e a s a iona y phase o g ow h was eached, 10mL o he main cul u e was ans e ed in o 15mL-conical cen i uge ubes. Mycelium was pelle ed by cen i uga- ion, he supe na an was disca ded and he samples we e d ied o 24h a 65°C. A e 24h, he conical cen i uge ubes wi h d y mycelium we e weighed. The spec opho- ome ic measu emen o glucu onidase ac i i y was ca - ied ou as p e iously desc ibed [29]. S ain cul i a ion andHPLC analysis The S. albus s ains con aining he a anciamycin biosyn- he ic clus e we e g own in 20mL o a TSB medium o 48h a 30°C. Then, 200μL o p ecul u e we e ans e ed in o 15mL o a NL5+1% yeas ex ac medium and cul- i a ed o an addi ional 120h a 30°C. A e cul i a ion, 5mL o he mycelia we e ha es ed by cen i uga ion o 30min a 2000 c . The supe na an was ans e ed in o a new Falcon ube and mixed wi h 5mL o e hyl ace a e and o a ed on a o a o o 30min. Then, he samples we e cen i uged a 4000 pm o 10min, and he uppe phase o he samples was ans e ed o glass ials and d ied unde N2. The d y pelle was dissol ed in ace oni- ile and cen i uged a 20,000 c o 10min o emo e he cell deb is. Then, 80μL o he ex ac s we e ans- e ed in o HPLC ials and analysed by HPLC. The HPLC–ESI–MS-UV–Vis analysis was pe o med on a Dionex Ul ima e 3000 HPLC sys em (The mo Fishe Scien i ic) ha was connec ed o an ESI–MS Amazon mass spec ome e (B uke ). The HPLC sys em was equipped wi h a 100×2.1mm, 1.7-µm BEH C18 col- umn (Wa e s); a 50×2.1mm, 1.7-µm BEH C18 column (Wa e s); o a 100×2mm, 2.5-µm Luna C18 column (Phenomenex), depending on he me hod. Fo unknown ex ac s, an 18-min g adien was pe o med, mainly on he long column, whe eas a 9-min g adien was used o he p epu i ied o known ex ac s. All g adien me h- ods began wi h 5% B and inc eased o 95% B o e 9 o 18min. Sol en A consis ed o H2O+0.1% o mic acid, whe eas sol en B con ained ACN+0.1% o mic acid. The low a e o he BEH C18 columns was 0.6 mL/ min, whe eas he low a e o he Luna C18 column was 0.4mL/min; UV–Vis de ec ion was pe o med om 210 o 600nm. Mos o he ime, he ESI–MS was used in al e na ing mode (posi i e and nega i e). Only a ull MS scan o an addi ional scan wi h MS2 da a was eco ded, depending on he me hod. Bo h HPLC and MS sys ems we e combined using he Hys a p og am (B uke ). As such, he s anda d LC–MS expe imen s could be selec ed as supe me hods. These expe imen s included he selec ed column (Luna=Luna C18; 100=BEH C18 100mm; 50=BEH C18 50mm), g adien (9, 18min) and MS mode (MS only, ms2 posneg). High- esolu ion ESI–MS was measu ed using a Maxis Q-To 4G (B uke ) o an O bi ap LTQ (The mo Fishe Scien i ic) mass spec ome e connec ed o he same HPLC sys em used o s anda d LC–MS. Abb e ia ions RPKM: eads pe kilobase o ansc ip pe million eads; Cons. hyp. p o .: conse a i e hypo he ical p o ein; Reg. p o .: egula o y p o ein; IGR: in e - genic egion; Memb . p o .: memb ane p o ein; ME: mosaic end; ITR: in e ed e minal epea . Au ho s’ con ibu ions BB pa icipa ed in he design o expe imen s, pe o med he expe imen s and analysed he da a. LH pe o med addi ional e i ica ion o he esul s and analysed he da a. AL designed he expe imen s and pe o med p ojec supe ision. BB and AL w o e he pape . All au ho s ead and app o ed he inal manusc ip . Au ho de ails 1 Pha mBioTec GmbH, Science Pa k 1, 66123 Saa b ücken, Ge many. 2 Helm- hol z-Ins i u e o Pha maceu ical Resea ch Saa land, Campus, Building C2.3, 66123 Saa b ücken, Ge many. 3 Depa men o Pha maceu ical Bio echnology, Saa land Uni e si y, 66123 Saa b ücken, Ge many. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. A ailabili y o da a and ma e ials The cons uc ed mu an s ains and plasmids a e a ailable a he Depa men o Pha maceu ical Bio echnology o he Saa land Uni e si y, Saa b ücken, Ge many. Funding This wo k was suppo ed h ough unding om DZIF g an and by he Eu o- pean Commission unde he 7 h F amewo k P og am h ough he “Collabo- a i e P ojec ” ac ion “STREPSYNTH” G an No. 613877 and h ough he ERC s a ing g an EXPLOGEN No. 281623 o AL. Recei ed: 9 June 2016 Accep ed: 20 Decembe 2016 Re e ences 1. Ongley SE, Bian X, Neilan BA, Mülle R. Recen ad ances in he he e olo- gous exp ession o mic obial na u al p oduc biosyn he ic pa hways. Na P od Rep. 2013;30:1121–38. 2. Bal z RH. Gene ic manipula ion o seconda y me aboli e biosyn hesis o imp o ed p oduc ion in S ep omyces and o he ac inomyce es. J Ind Mic obiol Bio echnol. 2016;43:343–70. Addi ional ile Addi ional ile1: Figu e S1. Glucu onidase ac i i y in cell lysa es o ecombinan S. albus s ains. Table S1. Plasmids used in his wo k. Table S2. P ime s used in his wo k. Page 8 o 8 Bilyk e al. Mic ob Cell Fac (2017) 16:5 • We accep p e-submission inqui ies • Ou selec o ool helps you o ind he mos ele an jou nal • We p o ide ound he clock cus ome suppo • Con enien online submission • Tho ough pee e iew • Inclusion in PubMed and all majo indexing se ices • Maximum isibili y o you esea ch Submi you manusc ip a www.biomedcen al.com/submi Submi you nex manusc ip o BioMed Cen al and we will help you a e e y s ep: 3. Schnell R, Rine J. A posi ion e ec on he exp ession o a RNA gene media ed by he SIR genes in Saccha omyces ce e isiae. Mol Cell Biol. 1986;6:494–501. 4. Bok JW, Chiang Y-M, Szewczyk E, Reyes-Dominguez Y, Da idson AD, Sanchez JF, Lo H-C, Wa anabe K, S auss J, Oakley BR, e al. Ch oma- in-le el egula ion o biosyn he ic gene clus e s. Na Chem Biol. 2009;5:462–4. 5. B yan JA, Sella s LE, Busby SJW, Lee DJ. Ch omosome posi ion e ec s on gene exp ession in Esche ichia coli K-12. Nucleic Acids Res. 2014;. doi:10.1093/na /gku828. 6. Schmid MB, Ro h JR. Gene loca ion a ec s exp ession le el in Salmonella yphimu ium. J Bac e iol. 1987;169:2872–5. 7. Thompson A, Gasson MJ. Loca ion e ec s o a epo e gene on exp es- sion le els and on na i e p o ein syn hesis in Lac ococcus lac is and Saccha omyces ce e isiae. Appl En i on Mic obiol. 2001;67:3434–9. 8. Go schling DE, Apa icio OM, Billing on BL, Zakian VA. Posi ion e ec a S. ce e isiae elome es: e e sible ep ession o Pol II ansc ip ion. Cell. 1990;63:751–62. 9. Pa i GD, Higgins CF. Ch omosomal domains o supe coiling in Salmo- nella yphimu ium. Mol Mic obiol. 1993;10:685–96. 10. Swie cz JP, Nanji T, Gloyd M, Gua ne A, Ellio MA. A no el nucleoid- associa ed p o ein speci ic o he ac inobac e ia. Nucleic Acids Res. 2013;41:4171–84. 11. Ald idge M, Facey P, F ancis L, Bayliss S, Del Sol R, Dyson P. A no el bi unc- ional his one p o ein in S ep omyces: a candida e o s uc u al coupling be ween DNA con o ma ion and ansc ip ion du ing de elopmen and s ess? Nucleic Acids Res. 2013;41:4813–24. 12. Bal z R. S ep omyces and Saccha opolyspo a hos s o he e ologous exp ession o seconda y me aboli e gene clus e s. J Ind Mic obiol Bio- echnol. 2010;37:759–72. 13. Pe zke L, Luzhe skyy A. In i o Tn5-based ansposon mu agenesis o S ep omyce es. Appl Mic obiol Bio echnol. 2009;83:979–86. 14. Bilyk B, Webe S, My ono skyi M, Bilyk O, Pe zke L, Luzhe skyy A. In i o andom mu agenesis o s ep omyce es using ma ine -based ansposon Hima 1. Appl Mic obiol Bio echnol. 2013;97:351–9. 15. Siegl T, Luzhe skyy A. Ac inomyce es genome enginee ing app oaches. An onie Van Leeuwenhoek. 2012;102:503–16. 16. Bibb MJ, Whi e J, Wa d JM, Janssen GR. The mRNA o he 23S RNA me hylase encoded by he e mE gene o Saccha opolyspo a e y h aea is ansla ed in he absence o a con en ional ibosome-binding si e. Mol Mic obiol. 1994;14:533–45. 17. O suka J, Kunisawa T. Cha ac e is ic base sequence pa e ns o p omo e and e mina o si es in phi X174 and d phage DNAs. J Theo Biol. 1982;97:415–36. 18. Sousa C, de Lo enzo V, Cebolla A. Modula ion o gene exp ession h ough ch omosomal posi ioning in Esche ichia coli. Mic obiology. 1997;143:2071–8. 19. Beckwi h JR, Signe ER, Eps ein W. T ansposi ion o he lac egion o E. coli. Cold Sp ing Ha bo Symp Quan Biol. 1966;31:393–401. 20. Block DH, Hussein R, Liang LW, Lim HN. Regula o y consequences o gene ansloca ion in bac e ia. Nucleic Acids Res. 2012;40:8979–92. 21. Coope S, Helms e e CE. Ch omosome eplica ion and he di ision cycle o Esche ichia coli. J Mol Biol. 1968;31:519–40. 22. Bilyk B, Luzhe skyy A. Unusual si e-speci ic DNA in eg a ion in o he highly ac i e pseudo-a B o he S ep omyces albus J1074 genome. Appl Mic obiol Bio echnol. 2014;98:5095–104. 23. Kelle -Schie lein W, Mülle A. The suga componen o a anciamycin: 2-0-me hyl-l- hamnose. Expe ien ia. 1970;26:929–30. 24. Luzhe skyy A, Maye A, Ho mann J, Pelze S, Holzenkämpe M, Schmi B, Wohle SE, Ven e A, Bech hold A. Cloning and he e ologous exp ession o he a anciamycin biosyn he ic gene clus e e ealed a new lexible glycosyl ans e ase. ChemBioChem. 2007;8:599–602. 25. Mandelscheid N, Bilyk B, Busche T, Kalinowski J, Paulula T, Bech hold A, Pe zke L, Luzhe skyy A. An in luence o he copy numbe o biosyn he ic gene clus e s on he p oduc ion le el o an ibio ics in a he e ologous hos . J Bio echnol. 2016;20(232):110–7. doi:10.1016/j.jbio ec.2016.05.038. 26. Kiese T, Bibb MJ, Bu ne MJ, Cha e KF, Hopwood DA. P ac ical S ep o- myces gene ics. No wich: John Innes Founda ion; 2000. 27. Da senko KA, Wanne BL. One-s ep inac i a ion o ch omosomal genes in Esche ichia coli K-12 using PCR p oduc s. P oc Na l Acad Sci USA. 2000;97:6640–5. 28. Samb ook J, Russel DW. Molecula cloning: a labo a o y manual. 3 d ed. Cold Sp ing Ha bo : Cold Sp ing Ha bo Labo a o y P ess; 2001. 29. My ono skyi M, Welle E, Fedo enko V, Luzhe skyy A. β-Glucu onidase as a sensi i e and e sa ile epo e in ac inomyce es. Appl En i on Mic obiol. 2011;77:5370–83. 30. Gus B, Challis GL, Fowle K, Kiese T, Cha e KF. PCR- a ge ed S ep omyces gene eplacemen iden i ies a p o ein domain needed o biosyn he- sis o he sesqui e pene soil odo geosmin. P oc Na l Acad Sci USA. 2003;100:1541–6.