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
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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
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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
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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
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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
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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

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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
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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.
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Bilyk e al. Mic ob Cell Fac (2017) 16:5
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