ma ine d ugs
Communica ion
He e ologous Exp ession o he Nybomycin Gene
Clus e om he Ma ine S ain S ep omyces albus
subsp. chlo inus NRRL B-24108
Ma a Rod íguez Es é ez 1, Maksym My ono skyi 1, Nils Gumme lich 1,
Su d Nadmid 1and And iy Luzhe skyy 1,2,*
1Pha mazeu ische Bio echnologie, Uni e si ä des Saa landes, 66123 Saa b ücken, Ge many;
[email p o ec ed] (M.R.E.); m.my [email p o ec ed] (M.M.);
[email p o ec ed] (N.G.); [email p o ec ed] (S.N.)
2Helmhol z-Ins i u ü Pha mazeu ische Fo schung Saa land, 66123 Saa b ücken, Ge many
*Co espondence: [email p o ec ed]; Tel.: +49-0681-70223
Recei ed: 5 Oc obe 2018; Accep ed: 31 Oc obe 2018; Published: 4 No embe 2018


Abs ac :
S ep omyce es ep esen an impo an ese oi o ac i e seconda y me aboli es wi h
po en ial applica ions in he pha maceu ical indus y. The gene clus e s esponsible o hei
p oduc ion a e o en c yp ic unde labo a o y g ow h condi ions. Cha ac e iza ion o hese clus e s
is he e o e essen ial o he disco e y o new mic obial pha maceu ical d ugs. He e, we epo he
iden i ica ion o he p e iously uncha ac e ized nybomycin gene clus e om he ma ine ac inomyce e
S ep omyces albus subsp. chlo inus h ough i s he e ologous exp ession. Nybomycin has p e iously
been epo ed o ac agains quinolone- esis an S aphylococcus au eus s ains ha bo ing a mu a ed
gy A gene bu no agains hose wi h in ac gy A. The nybomycin- esis an mu an s gene a ed om
quinolone- esis an mu an s ha e been epo ed o be caused by a back-mu a ion in he gy A gene ha
es o es suscep ibili y o quinolones. On he basis o gene unc ion assignmen om bioin o ma ics
analysis, we sugges a model o nybomycin biosyn hesis.
Keywo ds:
s ep omyce es; seconda y me aboli es; nybomycin gene clus e ; he e ologous exp ession;
nybomycin biosyn hesis
1. In oduc ion
Ac inobac e ia ep esen a p ominen sou ce o na u al p oduc s wi h po en ial indus ial
applica ions. The genus S ep omyces is especially well known o p oduce a di e se spec um o
compounds wi h an ibac e ial, an i ungal, an i umo and e en insec icide and he bicide ac i i y [
1
–
3
].
The inc easing amoun o sequenced mic obial genomes has p o ided insigh in o he unp eceden ed
po en ial o ac inobac e ia o biosyn hesize na u al p oduc s [
4
,
5
]. Gene ally, dozens o a ious
seconda y me aboli e clus e s a e encoded in hei genomes. Howe e , hese clus e s a e o en
poo ly exp essed unde s anda d cul i a ion condi ions o e en emain silen , hus p e en ing
he isola ion and cha ac e iza ion o he encoded compounds. Such uncha ac e ized clus e s
wi h unknown biosyn he ic p oduc s a e usually ega ded as c yp ic. Di e en app oaches can
be used o cha ac e ize c yp ic clus e s, including changing cul i a ion pa ame e s (OSMAC
app oach), exp ession o pleio opic egula o y genes, in oduc ion o an ibio ic- esis an mu a ions,
and e ac o ing o he biosyn he ic pa hways [
6
–
9
]. Cu en ly, cha ac e iza ion o he c yp ic gene
clus e s encoding na u al p oduc s o en elies on exp ession o hei biosyn he ic pa hways in
he op imized su oga e s ains called he e ologous hos s o chassis s ains. The he e ologous
exp ession app oach has a numbe o ad an ages compa ed o o he clus e cha ac e iza ion me hods.
The simpli ied me abolic backg ound o he chassis s ains acili a es he iden i ica ion o na u al
Ma . D ugs 2018,16, 435; doi:10.3390/md16110435 www.mdpi.com/jou nal/ma ined ugs
Ma . D ugs 2018,16, 435 2 o 10
p oduc s; as DNA- ecombinee ing me hods in E. coli and DNA ans e me hods in o s ep omyce es
simpli y biosyn he ic s udies, and high p oduc ion yields enable p oduc supply o s uc u e
elucida ion and biological ac i i y s udies.
In his s udy, we epo he iden i ica ion and cha ac e iza ion o he p e iously uncha ac e ized
nybomycin gene clus e om he ma ine s ain S ep omyces albus subsp. chlo inus NRRL B-24108
h ough he e ologous exp ession in S ep omyces albus Del14. Nybomycin was i s isola ed in 1955;
howe e , he unique biological ac i i y o he an ibio ic was disco e ed only ecen ly [
10
]. Nybomycin
inhibi s g ow h o quinolone- esis an S aphylococcus au eus by a ge ing he mu a ed enzyme gy ase.
In e es ingly, he in ac gy ase encoded by he gy A gene wi hou he esis ance mu a ion is no
inhibi ed by he an ibio ic. The a e nybomycin- esis an mu an s de i ed om quinolone- esis an
S. au eus ha e all been epo ed o con ain he e e se mu a ion in he gy A gene, causing loss o
quinolone esis ance. Despi e his in e es ing mode o ac ion, he biosyn he ic gene clus e leading
o nybomycin p oduc ion emains unknown. Based on he clus e analysis, we also p opose he
biosyn he ic ou e leading o he p oduc ion o nybomycin.
2. Resul s
Iden i ica ion o he Nybomycin Gene Clus e h ough I s He e ologous Exp ession
In he cou se o sys ema ic ac i a ion o c yp ic seconda y me aboli e clus e s om S ep omyces
albus subsp. chlo inus NRRL B-24108 [
3
], a clus e anno a ed by he an iSMASH genome mining
so wa e [
11
] as “ a y acid me abolism clus e ” was exp essed in he he e ologous hos s ains. Fo his
pu pose, a BAC 4N24 con aining he clus e was isola ed om he p e iously cons uc ed genomic
lib a y o S. albus subsp. chlo inus and ans e ed in o S ep omyces albus Del14 and S ep omyces li idans
TK24 [
12
,
13
]. The ob ained exconjugan s ains S ep omyces albus 4N24 and S ep omyces li idans 4N24
as well as he co esponding con ol s ains wi hou he BAC S. albus Del14 and S. li idans TK24
we e e men ed in he p oduc ion medium. LC-MS analysis o he exconjugan s ains con aining
he he e ologous clus e con i med i s success ul exp ession in S. albus 4N24, as indica ed by a new
peak ha was obse ed in he ex ac o he s ain (Figu e 1A,B and Figu e S1). Exp ession o he
clus e in S. li idans 4N24 did no lead o he p oduc ion o any new compounds compa ed wi h he
con ol s ain.
Ma . D ugs 2018, 16, x FOR PEER REVIEW 2 o 10
na u al p oduc s; as DNA- ecombinee ing me hods in E. coli and DNA ans e me hods in o
s ep omyce es simpli y biosyn he ic s udies, and high p oduc ion yields enable p oduc supply o
s uc u e elucida ion and biological ac i i y s udies.
In his s udy, we epo he iden i ica ion and cha ac e iza ion o he p e iously
uncha ac e ized nybomycin gene clus e om he ma ine s ain S ep omyces albus subsp. chlo inus
NRRL B-24108 h ough he e ologous exp ession in S ep omyces albus Del14. Nybomycin was i s
isola ed in 1955; howe e , he unique biological ac i i y o he an ibio ic was disco e ed only
ecen ly [10]. Nybomycin inhibi s g ow h o quinolone- esis an S aphylococcus au eus by a ge ing
he mu a ed enzyme gy ase. In e es ingly, he in ac gy ase encoded by he gy A gene wi hou he
esis ance mu a ion is no inhibi ed by he an ibio ic. The a e nybomycin- esis an mu an s de i ed
om quinolone- esis an S. au eus ha e all been epo ed o con ain he e e se mu a ion in he gy A
gene, causing loss o quinolone esis ance. Despi e his in e es ing mode o ac ion, he biosyn he ic
gene clus e leading o nybomycin p oduc ion emains unknown. Based on he clus e analysis, we
also p opose he biosyn he ic ou e leading o he p oduc ion o nybomycin.
2. Resul s
Iden i ica ion o he Nybomycin Gene Clus e h ough I s He e ologous Exp ession
In he cou se o sys ema ic ac i a ion o c yp ic seconda y me aboli e clus e s om S ep omyces
albus subsp. chlo inus NRRL B-24108 [3], a clus e anno a ed by he an iSMASH genome mining
so wa e [11] as “ a y acid me abolism clus e ” was exp essed in he he e ologous hos s ains. Fo
his pu pose, a BAC 4N24 con aining he clus e was isola ed om he p e iously cons uc ed
genomic lib a y o S. albus subsp. chlo inus and ans e ed in o S ep omyces albus Del14 and
S ep omyces li idans TK24 [12,13]. The ob ained exconjugan s ains S ep omyces albus 4N24 and
S ep omyces li idans 4N24 as well as he co esponding con ol s ains wi hou he BAC S. albus
Del14 and S. li idans TK24 we e e men ed in he p oduc ion medium. LC-MS analysis o he
exconjugan s ains con aining he he e ologous clus e con i med i s success ul exp ession in
S. albus 4N24, as indica ed by a new peak ha was obse ed in he ex ac o he s ain (Figu es 1A,B
and S1). Exp ession o he clus e in S. li idans 4N24 did no lead o he p oduc ion o any new
compounds compa ed wi h he con ol s ain.
Figu e 1. LC-MS ch oma og ams o c ude ex ac s om S. albus 4N24 and S. albus Del14. The new
peak ound in S. albus 4N24 c ude ex ac is indica ed wi h an as e isk (*). (A) Base peak
ch oma og ams; (B) ex ac ed ion ch oma og ams (299.10 ± 0.1 Da); (C) mass spec um associa ed o
R = 4.7 min om S. albus 4N24 LC-MS ch oma og am.
Figu e 1.
LC-MS ch oma og ams o c ude ex ac s om S. albus 4N24 and S. albus Del14. The new peak
ound in S. albus 4N24 c ude ex ac is indica ed wi h an as e isk (*). (
A
) Base peak ch oma og ams;
(
B
) ex ac ed ion ch oma og ams (299.10
±
0.1 Da); (
C
) mass spec um associa ed o
R
= 4.7 min om
S. albus 4N24 LC-MS ch oma og am.
Ma . D ugs 2018,16, 435 3 o 10
Analysis o he S. albus 4N24 ex ac by high- esolu ion MS analysis e ealed ha he iden i ied
peak co esponded o he compound wi h an [M + H]
+
o 299.102 m/zand he deduced molecula
o mula C
16
H
15
N
2
O
4
(Figu e 1C). A sea ch in a na u al p oduc da abase e ealed ha he iden i ied
compound migh co espond o he an ibio ic nybomycin (Figu e 2). To e i y his, a nybomycin
s anda d (San a C uz Bio echnology, Inc., Dallas, TX, USA) was used. LC-MS analysis o he pu e
nybomycin, he S. albus 4N24 ex ac , and he S. albus 4N24 ex ac spiked wi h pu e nybomycin
con i med ha he new compound co esponded o nybomycin; he e en ion ime and he mass
spec um o he new compound we e iden ical o hose o he pu e s anda d (Figu e S2). Addi ionally,
o alida e he iden i y o he de ec ed compound as nybomycin, he o me was pu i ied. Fo his
pu pose, S. albus 4N24 was inocula ed in o 10 L o DNPM medium, and he cul u e b o h o he s ain
was ex ac ed wi h e hyl ace a e. The compound was pu i ied om he ex ac using no mal-phase,
size-exclusion, and e e se-phase ch oma og aphy. The pu i ied compound, as well as he pu e
nybomycin s anda d, was used o NMR measu emen s. The eco ded
1
H-NMR spec a o he pu i ied
compound and o nybomycin we e iden ical (Table S1; Figu e S3), which unambiguously p o ed he
iden i y o he o me as nybomycin.
Ma . D ugs 2018, 16, x FOR PEER REVIEW 3 o 10
Analysis o he S. albus 4N24 ex ac by high- esolu ion MS analysis e ealed ha he iden i ied
peak co esponded o he compound wi h an [M + H]+ o 299.102 m/z and he deduced molecula
o mula C16H15N2O4 (Figu e 1C). A sea ch in a na u al p oduc da abase e ealed ha he iden i ied
compound migh co espond o he an ibio ic nybomycin (Figu e 2). To e i y his, a nybomycin
s anda d (San a C uz Bio echnology, Inc., Dallas, TX, USA) was used. LC-MS analysis o he pu e
nybomycin, he S. albus 4N24 ex ac , and he S. albus 4N24 ex ac spiked wi h pu e nybomycin
con i med ha he new compound co esponded o nybomycin; he e en ion ime and he mass
spec um o he new compound we e iden ical o hose o he pu e s anda d (Figu e S2).
Addi ionally, o alida e he iden i y o he de ec ed compound as nybomycin, he o me was
pu i ied. Fo his pu pose, S. albus 4N24 was inocula ed in o 10 L o DNPM medium, and he cul u e
b o h o he s ain was ex ac ed wi h e hyl ace a e. The compound was pu i ied om he ex ac
using no mal-phase, size-exclusion, and e e se-phase ch oma og aphy. The pu i ied compound, as
well as he pu e nybomycin s anda d, was used o NMR measu emen s. The eco ded 1H-NMR
spec a o he pu i ied compound and o nybomycin we e iden ical (Table S1; Figu e S3), which
unambiguously p o ed he iden i y o he o me as nybomycin.
Figu e 2. Chemical s uc u es o nybomycin, s ep onig in, and 4-aminoan h anilic acid. The
common co e s uc u e o nybomycin and s ep onig in a e highligh ed in ed.
BAC 4N24, which con ains he nybomycin biosyn he ic genes, comp ises a 36 kb genomic DNA
egion wi h 33 open eading ames (Figu e 3, Table 1). A sequence simila i y sea ch e ealed ha
nine open eading ames wi hin his egion sha ed homology a he p o ein le el wi h he genes
in ol ed in he biosyn hesis o s ep onig in, which is s uc u ally ela ed o nybomycin (Figu e 2).
Sequence analysis o he DNA agmen cloned in BAC 4N24 e ealed he pu a i e
s ep omycin- esis an gene, a hypo he ical gene, a gene encoding an ATP-binding p o ein, and ou
addi ional hypo he ical genes ollowed by he nybA gene encoding a pu a i e
3-ca boxy-cis,cis-mucona e cycloisome ase a i s 5’ end (Table 1), which implied ha he nybA gene
migh cons i u e he 5’ end o he nybomycin clus e . The 3’ end o he cloned egion comp ised he
genes encoding a pu a i e me hyl ans e ase, wo isopenicillin N syn hases, a anspo e , h ee
ansc ip ional egula o s, and a hypo he ical p o ein (nybS, nybT, nybU, nybV, nybW, nybX, nybZ,
and nybY, espec i ely). To cla i y whe he hese genes we e pa o he nybomycin biosyn he ic
clus e , wo BACs 4M14 and 6M11 ha pa ially o e lap wi h BAC 4N24 we e isola ed om he
genomic lib a y o S. albus subsp. chlo inus and exp essed in S. albus Del14. Bo h he isola ed 4M14
and 6M11 BACs comple ely co e ed he 5’ end o he agmen cloned in he o iginal BAC 4N24,
which led o nybomycin p oduc ion. Compa ed wi h BAC 4N24, BAC 4M14 lacked he egion
downs eam o he nybR gene, while BAC 6M11 lacked he egion downs eam o he nybL gene
(Figu e 3). The ob ained s ains S. albus 4M14 and S. albus 6M11 we e analyzed oge he wi h S. albus
Figu e 2.
Chemical s uc u es o nybomycin, s ep onig in, and 4-aminoan h anilic acid. The common
co e s uc u e o nybomycin and s ep onig in a e highligh ed in ed.
BAC 4N24, which con ains he nybomycin biosyn he ic genes, comp ises a 36 kb genomic DNA
egion wi h 33 open eading ames (Figu e 3, Table 1). A sequence simila i y sea ch e ealed ha nine
open eading ames wi hin his egion sha ed homology a he p o ein le el wi h he genes in ol ed
in he biosyn hesis o s ep onig in, which is s uc u ally ela ed o nybomycin (Figu e 2). Sequence
analysis o he DNA agmen cloned in BAC 4N24 e ealed he pu a i e s ep omycin- esis an gene,
a hypo he ical gene, a gene encoding an ATP-binding p o ein, and ou addi ional hypo he ical genes
ollowed by he nybA gene encoding a pu a i e 3-ca boxy-cis,cis-mucona e cycloisome ase a i s 5’
end (Table 1), which implied ha he nybA gene migh cons i u e he 5’ end o he nybomycin clus e .
The 3’ end o he cloned egion comp ised he genes encoding a pu a i e me hyl ans e ase, wo
isopenicillin N syn hases, a anspo e , h ee ansc ip ional egula o s, and a hypo he ical p o ein
(nybS,nybT,nybU,nybV,nybW,nybX,nybZ, and nybY, espec i ely). To cla i y whe he hese genes
we e pa o he nybomycin biosyn he ic clus e , wo BACs 4M14 and 6M11 ha pa ially o e lap wi h
BAC 4N24 we e isola ed om he genomic lib a y o S. albus subsp. chlo inus and exp essed in S. albus
Del14. Bo h he isola ed 4M14 and 6M11 BACs comple ely co e ed he 5’ end o he agmen cloned
in he o iginal BAC 4N24, which led o nybomycin p oduc ion. Compa ed wi h BAC 4N24, BAC 4M14
lacked he egion downs eam o he nybR gene, while BAC 6M11 lacked he egion downs eam
o he nybL gene (Figu e 3). The ob ained s ains S. albus 4M14 and S. albus 6M11 we e analyzed
Ma . D ugs 2018,16, 435 4 o 10
oge he wi h S. albus 4N24 o nybomycin p oduc ion. Du ing he LC-MS analysis, no nybomycin was
de ec ed in he ex ac s o ei he he S. albus 4M14 o S. albus 6M11 s ains (Figu e S4). Nybomycin was
eadily de ec able in he ex ac o S. albus 4N24. These esul s gi e e idence ha he 3’- e minal egion
o 4N24, which con ains he genes downs eam o nybR (nybS o nybZ), is essen ial o nybomycin
p oduc ion. Taken oge he , ou esul s sugges ha he genes om nybA o nybZ migh cons i u e he
nybomycin gene clus e , which is u he suppo ed by he ac ha he genes om nybA o nybF and
nybN o nybP sha e high le els o homology wi h genes in he biosyn he ic clus e o s ep onig in
(Table 1), a compound ha is s uc u ally simila o nybomycin (Figu e 2).
Ma . D ugs 2018, 16, x FOR PEER REVIEW 4 o 10
4N24 o nybomycin p oduc ion. Du ing he LC-MS analysis, no nybomycin was de ec ed in he
ex ac s o ei he he S. albus 4M14 o S. albus 6M11 s ains (Figu e S4). Nybomycin was eadily
de ec able in he ex ac o S. albus 4N24. These esul s gi e e idence ha he 3’- e minal egion o
4N24, which con ains he genes downs eam o nybR (nybS o nybZ), is essen ial o nybomycin
p oduc ion. Taken oge he , ou esul s sugges ha he genes om nybA onybZ migh cons i u e
he nybomycin gene clus e , which is u he suppo ed by he ac ha he genes om nybA onybF
and nybN onybP sha e high le els o homology wi h genes in he biosyn he ic clus e o
s ep onig in (Table 1), a compound ha is s uc u ally simila o nybomycin (Figu e 2).
Figu e 3. Nybomycin biosyn he ic gene clus e . Gene ic o ganiza ion is shown. Below, maps o h ee
di e en BAC clones a e displayed: BAC 4N24 con ains he ull sequence o he nybomycin gene
clus e . BAC 4M14 lacks he genes om nybS onybZ; and BAC 6M11 lacks he genes om nybM
onybZ.
Table 1. P oposed unc ions o genes in nybomycin biosyn he ic gene clus e and homology wi h
s ep onig in biosyn he ic genes.
Gene Size
(aa) P oposed Func ion GenBank
homologue 1
Iden i y/
Simila i y (%)
S ep onig in
Gene Clus e
Homologue 2
Iden i y/
Simila i y (%)
o -07 268 S ep omycin
3’’-adenylyl ans e ase WP_037865927.1 71/77 - -
o -06 159 Hypo he ical p o ein WP_027736486.1 86/93 - -
o
-
05
163 ATP-binding p o ein WP_052413949.1 76/83 - -
o -04 332 Hypo he ical p o ein WP_055499466.1 74/82 - -
o -03 341 Hypo he ical p o ein WP_030379123.1 71/80 - -
o
-
02
494 Hypo he ical p o ein WP_078869279.1 70/81 - -
o -01 242 Hypo he ical p o ein - - - -
nybA 475 3-ca boxy-cis,cis-mucona e
cycloisome ase WP_066029238.1 66/85 s nL (AFW04563.1) 71/76
nybB 669 FAD-binding p o ein WP_066029239.1 77/84 s nK1
(AFW04562.1) 66/75
nybC 325 NADPH:quinone educ ase WP_066029240.1 81/87 s nH1
(AFW04558.1) 63/71
nybD 638 An h anila e syn hase WP_079145437.1 81/87 s nM1
(AFW04564.1) 65/75
nybE 227 Isocho isma ase WP_066029243.1 84/89
s nM2
(AFW04565.1) 66/76
nybF 402 DAHP syn hase WP_066029245.1 81/87 s nM3
(AFW04567.1) 62/69
nybG 279 Hypo he ical p o ein - - - -
nybH 257 Vicinal oxygen chela e p o ein WP_066029246.1 64/72 - -
nybI 222 NAD(P)H:dehyd ogenase WP_066029248.1 90/95 - -
nybJ
135 Hypo he ical p o ein WP_066029250.1 76/86 - -
nybK 266 N-ace yl ans e ase WP_066029251.1 80/88 - -
nybL 333 Amidohyd olase WP_066029254.1 84/90 - -
nybM
354 Ace oace yl-CoA syn hase WP_066029256.1 82/87 - -
nybN 182 A oma ase/cyclase WP_066029258.1 78/85 s nI (AFW04559.1) 54/67
nybO 550 Long-chain acyl-CoA syn he ase WP_066029260.1 85/90 s nJ (AFW04560.1) 68/81
nybP 476 Salicyla e hyd oxylase WP_079145438.0 74/79
s nH2
(AFW04561.1) 62/72
nybQ 376 Hypo he ical p o ein WP_030685222.1 57/69 - -
nybR 238 NAD-dependen epime ase WP_066029265.1 82/89 - -
nybS 253 SAM-dependen
me hyl ans e ase WP_079145439.1 89/94 - -
nybT 333 Isopenicillin N syn hase amily
oxygenase WP_078974705.1 80/89 - -
nybU 342 Isopenicillin N syn hase amily
oxygenase WP_078974705.1 72/83 - -
Figu e 3.
Nybomycin biosyn he ic gene clus e . Gene ic o ganiza ion is shown. Below, maps o h ee
di e en BAC clones a e displayed: BAC 4N24 con ains he ull sequence o he nybomycin gene clus e .
BAC 4M14 lacks he genes om nybS o nybZ; and BAC 6M11 lacks he genes om nybM o nybZ.
Table 1.
P oposed unc ions o genes in nybomycin biosyn he ic gene clus e and homology wi h
s ep onig in biosyn he ic genes.
Gene Size (aa) P oposed Func ion GenBank
homologue 1
Iden i y/
Simila i y (%)
S ep onig in Gene
Clus e Homologue 2
Iden i y/
Simila i y (%)
o -07 268 S ep omycin 3”-adenylyl ans e ase WP_037865927.1 71/77 - -
o -06 159 Hypo he ical p o ein WP_027736486.1 86/93 - -
o -05 163 ATP-binding p o ein WP_052413949.1 76/83 - -
o -04 332 Hypo he ical p o ein WP_055499466.1 74/82 - -
o -03 341 Hypo he ical p o ein WP_030379123.1 71/80 - -
o -02 494 Hypo he ical p o ein WP_078869279.1 70/81 - -
o -01 242 Hypo he ical p o ein - - - -
nybA 475 3-ca boxy-cis,cis-mucona e cycloisome ase WP_066029238.1 66/85 s nL (AFW04563.1) 71/76
nybB 669 FAD-binding p o ein WP_066029239.1 77/84 s nK1 (AFW04562.1) 66/75
nybC 325 NADPH:quinone educ ase WP_066029240.1 81/87 s nH1 (AFW04558.1) 63/71
nybD 638 An h anila e syn hase WP_079145437.1 81/87 s nM1 (AFW04564.1) 65/75
nybE 227 Isocho isma ase WP_066029243.1 84/89 s nM2 (AFW04565.1) 66/76
nybF 402 DAHP syn hase WP_066029245.1 81/87 s nM3 (AFW04567.1) 62/69
nybG 279 Hypo he ical p o ein - - - -
nybH 257 Vicinal oxygen chela e p o ein WP_066029246.1 64/72 - -
nybI 222 NAD(P)H:dehyd ogenase WP_066029248.1 90/95 - -
nybJ 135 Hypo he ical p o ein WP_066029250.1 76/86 - -
nybK 266 N-ace yl ans e ase WP_066029251.1 80/88 - -
nybL 333 Amidohyd olase WP_066029254.1 84/90 - -
nybM 354 Ace oace yl-CoA syn hase WP_066029256.1 82/87 - -
nybN 182 A oma ase/cyclase WP_066029258.1 78/85 s nI (AFW04559.1) 54/67
nybO 550 Long-chain acyl-CoA syn he ase WP_066029260.1 85/90 s nJ (AFW04560.1) 68/81
nybP 476 Salicyla e hyd oxylase WP_079145438.0 74/79 s nH2 (AFW04561.1) 62/72
nybQ 376 Hypo he ical p o ein WP_030685222.1 57/69 - -
nybR 238 NAD-dependen epime ase WP_066029265.1 82/89 - -
nybS 253 SAM-dependen me hyl ans e ase WP_079145439.1 89/94 - -
nybT 333 Isopenicillin N syn hase amily oxygenase WP_078974705.1 80/89 - -
nybU 342 Isopenicillin N syn hase amily oxygenase WP_078974705.1 72/83 - -
nybV 495 MFS anspo e WP_079145411.1 81/87 - -
nybW 243 T ansc ip ional egula o WP_079145410.1 82/93 - -
nybX 197 T ansc ip ional egula o WP_025356654.1 89/93 - -
nybY 87 Hypo he ical p o ein WP_086560781.1 68/81 - -
nybZ 219 T ansc ip ional egula o WP_057613815.1 79/86 - -
1NCBI accession numbe s a e gi en. 2NCBI accession numbe s a e shown in pa en heses.
3. Discussion
The an ibio ic nybomycin was disco e ed in 1955 in he cul u e b o h o s ep omyce es A 717 [
14
].
The s uc u al ea u es o nybomycin as a used py idoquinolone ing sys em and an angula ly used
oxazoline ing a e o pa icula biosyn he ic in e es as hey ha e no been epo ed o o he na u al
p oduc s [
15
]. Despi e he unique s uc u e o nybomycin, i s biosyn he ic clus e and biosyn he ic ou e
Ma . D ugs 2018,16, 435 5 o 10
ha e emained elusi e. Only he esul s o eeding s udies imply ha ace a e, me hionine, and some
non-iden i ied shikima e- ype in e media es se e as biosyn he ic p ecu so s o nybomycin [
15
,
16
].
In his a icle, we ha e desc ibed he iden i ica ion o he nybomycin biosyn he ic gene clus e om he
ma ine s ep omyce e S. albus subsp. chlo inus NRRL B-24108 h ough i s exp ession in he clus e - ee
he e ologous hos S. albus Del14.
Sequence analysis o he DNA agmen cloned in BAC 4N24 has e ealed ha a numbe o
genes wi hin his agmen a e highly homologous o he genes in ol ed in biosyn hesis o he
an ibio ic s ep onig in (Table 1) [
17
]. Di ec compa ison o nybomycin and s ep onig in has shown
dis inc s uc u al simila i y, wi h bo h s uc u es con aining a diamino-subs i u ed, six-membe ed
ing (Figu e 2). The s uc u al simila i y and he pa ial homology o he gene clus e s sugges ha
nybomycin and s ep onig in biosyn he ic ou es can ha e some simila biosyn he ic in e media es
and enzyma ic eac ions.
Based on he sequence analysis and he esul s o BAC exp ession, we p opose ha he genes
nybA o nybZ cons i u e he nybomycin gene clus e . The nybA gene, encoding a pu a i e 3-ca boxy-cis,
cis-mucona e cycloisome ase, is homologous o he s ep onig in biosyn he ic gene s nL. The nybZ gene
encodes a pu a i e ansc ip ional egula o ha migh also pa icipa e in nybomycin biosyn hesis.
Simila o he s ep onig in pa hway, he 3-deoxy-D-a abinohep ulosona e 7-phospha e (DAHP)
syn hase encoded by nybF is likely o ca alyze he i s eac ion in he nybomycin biosyn he ic
ou e. DAHP syn hase is esponsible o he i s eac ion o he shikima e pa hway—biosyn hesis o
3-deoxy-D-a abino-hep -2-ulosona e 7-phospha e (DAHP) om phosphoenolpy u a e and D-e y h ose
4-phospha e (Figu e 4) which is suppo ed by he esul s o eeding expe imen s ha ha e
demons a ed ha he ca bons o he cen al ing o nybomycin a e de i ed om a shikima e- ype
in e media e [
15
]. Ca alyzing he i s eac ion, DAHP syn hase egula es he amoun o ca bon
en e ing he shikima e pa hway and he e o e can be esponsible o i s up egula ion o p o ide
su icien amoun s o biosyn he ic p ecu so s o nybomycin. The genes encoding enzymes esponsible
o he con e sion o DAHP in o cho isma e a e absen in he nybomycin clus e and in he
s ep onig in pa hway [
17
]. The e o e, he hos ’s p ima y me abolism enzymes mos likely o e ake
hese biosyn he ic s eps. We p opose ha he second eac ion ca alyzed by he enzymes encoded
in he biosyn he ic clus e is he con e sion o cho isma e in o 4-aminoan h anilic acid (Figu e 4),
which is a key in e media e in bo h nybomycin and s ep onig in biosyn hesis (Figu e 2). Isola ion o
4-aminoan h anilic acid om he cul u e b o h o he s ep onig in p oduce S ep omyces locculus
suppo s his sugges ion [
18
]. Fu he mo e, 4-aminoan h anilic acid has also been shown o inco po a e
in o s ep onig in [
18
]. We p opose ha he p oduc s o he nybC,nybD,nybE, and nybL genes
migh be esponsible o he con e sion o cho isma e in o 4-aminoan h anilic acid. The nybL
gene, encoding pu a i e amidohyd olase [
19
], has no homologue in he s ep onig in biosyn he ic
pa hway. We sugges ha he p o ein p oduc o nybL may p o ide enough supply o ammonia o
he amina ion o cho isma e by an h anila e syn hase [
20
], encoded by nybD, du ing he biosyn hesis
o 4-aminoan h anilic acid. In he s ep onig in biosyn hesis, he unc ion o he nybL gene may be
o e aken by he one o he hos ’s p ima y me abolism enzymes.
A e o ma ion o 4-aminoan h anilic acid, i is hen hyd oxyla ed in he hi d posi ion and
deca boxyla ed, gene a ing 2,6-diaminophenol (Figu e 4). The nybP gene encoding pu a i e salicyla e
hyd oxylase migh be esponsible o he hyd oxyla ion eac ion. Then, wo ace oace a e uni s a e
a ached o he amino g oups o 2,6-diaminophenol h ough he ac ion o he N-ace yl ans e ase
encoded by he nybK gene (Figu e 4). The pu a i e ace oace yl-CoA syn hase encoded by he nybM gene
ca alyses he o ma ion o ace oace yl-CoA om ace yl-CoA and malonyl-CoA. The NybM enzyme is
likely esponsible o he p oduc ion o su icien amoun s o ace oace a e, which is used as a p ecu so
in nybomycin biosyn hesis. The p ecu so ole o ace oace a e in nybomycin biosyn hesis is suppo ed
by he esul s o he eeding expe imen s, which ha e unequi ocally de ined ace a e as he sou ce o he
ex e io ca bons o he py idone ings [
16
]. We specula e ha a e a achmen o he ace oace a e uni s,
he pu a i e cyclase encoded by he nybN gene ca alyses he closu e o he py idone ings, leading o

Ma . D ugs 2018,16, 435 6 o 10
o ma ion o in e media e
1
(Figu e 4). The me hyla ion o ni ogen wi hin he py idone ings is likely
o be ca alyzed by he SAM-dependen me hyl ans e ase encoded by nybS. We hypo hesize ha he
nex eac ion in nybomycin biosyn hesis is a closu e o he oxazoline ing (Figu e 4). This s ep migh
be ca alyzed by he p oduc o nybT o nybU, which code o isopenicillin N syn hases (IPNS). IPNS is
esponsible o he biosyn hesis o isopenicillin N h ough a bicycliza ion eac ion— i s , he o ma ion
o a C-N bond gene a es a
β
-lac am ing; hen, he closu e o a i e-membe ed hiazolidine ing is
accomplished by he o ma ion o a C-S bond [
21
]. Sul u and oxygen ha e simila p ope ies [
22
].
The e o e, in a simila way o hiazolidine ing closu e, IPNS could be able o ca alyze he o ma ion o
a C-O bond be ween he ca bon a om o he N-me hyl g oup and he oxygen a om o he OH g oup
p esen in in e media e
2
, gene a ing an oxazoline ing (Figu e 4). Finally, a hyd oxyla ion eac ion
akes place a C-8’, possibly ca alyzed by he oxido educ ase NybB, gi ing ise o nybomycin inal
s uc u e. The compound is hen sec e ed o he ex acellula space, mos likely by he memb ane
anspo e encoded by nybV. Exp ession o seconda y me aboli e biosyn hesis genes is commonly
egula ed by ac i a o s and ep esso s coded by genes ha a e loca ed wi hin he same clus e .
We hypo hesize ha he p oduc s o nybW,nybX, and nybZ, which encode pu a i e ansc ip ional
egula o s, migh con ol he exp ession o he genes in ol ed in nybomycin biosyn hesis.
Ma . D ugs 2018, 16, x FOR PEER REVIEW 7 o 10
Figu e 4. P oposed biosyn he ic pa hway o nybomycin.
4. Ma e ials and Me hods
4.1. Gene al Expe imen al P ocedu es
All s ains and BACs used in his wo k a e lis ed in Table S2. Esche ichia coli s ains we e
cul u ed in LB medium [24]. S ep omyces s ains we e g own on soya lou manni ol aga (MS aga )
[25] o spo ula ion and conjuga ion and in liquid yp ic soy b o h (TSB; Sigma-Ald ich, S . Louis,
MO, USA). Fo seconda y me aboli e exp ession, liquid DNPM medium (40 g/L dex in, 7.5 g/L
soy one, 5 g/L baking yeas , and 21 g/L MOPS, pH 6.8) was used. The an ibio ics kanamycin,
ap amycin and nalidixic acid we e supplemen ed when equi ed.
4.2. Isola ion and Manipula ion o DNA
BAC ex ac ion om a S ep omyces albus subsp. chlo inus cons uc ed genomic lib a y (In ac
Genomics, USA), DNA manipula ion, E. coli ans o ma ion, and E. coli/S ep omyces in e gene ic
conjuga ion we e pe o med acco ding o s anda d p o ocols [24–26]. Plasmid DNA was pu i ied
wi h he BACMAX™ DNA pu i ica ion ki (Lucigen, Middle on, WI, USA). Res ic ion
Figu e 4. P oposed biosyn he ic pa hway o nybomycin.
Ma . D ugs 2018,16, 435 7 o 10
In his pape we epo he iden i ica ion o he gene clus e encoding p oduc ion o he s uc u ally
unique an ibio ic nybomycin. Biological ac i i y o nybomycin is also o pa icula in e es as i inhibi s
g ow h o quinolone- esis an S aphylococcus au eus, do man Mycobac e ium ube culosis, and o he
G am-posi i e and G am-nega i e bac e ia [
10
,
14
,
23
]. In e es ingly, nybomycin a ge s solely he
mu an , quinolone- esis an DNA gy ase wi h a Se 84Leu subs i u ion, while i is inac i e agains he
wild- ype, quinolone-sensi i e o m o he enzyme [
10
]. The mu a ion desc ibed hus a o cause
nybomycin esis ance is a e e se Leu84Se mu a ion wi hin he gy A gene ha es o es quinolone
sensi i i y. The iden i ica ion o he nybomycin clus e p esen ed in his pape enables biosyn he ic
s udies o nybomycin p oduc ion, gene a ion o new nybomycin de i a i es, and op imiza ion o
i s p oduc ion as well as a nybomycin supply o u he biological s udies. Toge he , hese wo ks
migh help igh he de elopmen o quinolone esis ance and e i e quinolones as an e ec i e class
o an ibio ics.
4. Ma e ials and Me hods
4.1. Gene al Expe imen al P ocedu es
All s ains and BACs used in his wo k a e lis ed in Table S2. Esche ichia coli s ains we e cul u ed
in LB medium [
24
]. S ep omyces s ains we e g own on soya lou manni ol aga (MS aga ) [
25
] o
spo ula ion and conjuga ion and in liquid yp ic soy b o h (TSB; Sigma-Ald ich, S . Louis, MO, USA).
Fo seconda y me aboli e exp ession, liquid DNPM medium (40 g/L dex in, 7.5 g/L soy one, 5 g/L
baking yeas , and 21 g/L MOPS, pH 6.8) was used. The an ibio ics kanamycin, ap amycin and nalidixic
acid we e supplemen ed when equi ed.
4.2. Isola ion and Manipula ion o DNA
BAC ex ac ion om a S ep omyces albus subsp. chlo inus cons uc ed genomic lib a y
(In ac Genomics, USA), DNA manipula ion, E. coli ans o ma ion, and E. coli/S ep omyces
in e gene ic conjuga ion we e pe o med acco ding o s anda d p o ocols [
24
–
26
]. Plasmid DNA
was pu i ied wi h he BACMAX
™
DNA pu i ica ion ki (Lucigen, Middle on, WI, USA). Res ic ion
endonucleases we e used acco ding o manu ac u e ’s ecommenda ions (New England Biolabs,
Ipswich, MA, USA).
4.3. Me aboli e Ex ac ion and Analysis
S ep omyces s ains we e g own in 10 mL o TSB o 1 day, and 1 mL o each cul u e was used o
inocula e 50 mL o p oduc ion medium. Cul u es we e g own o 7 days a 28
◦
C. Me aboli es we e
ex ac ed wi h e hyl ace a e om he supe na an , e apo a ed, and dissol ed in me hanol. One
µ
L o
each sample was sepa a ed using a Dionex Ul ima e 3000 UPLC (The mo Fishe Scien i ic, Wal ham,
MA, USA), a 10-cm ACQUITY UPLC
®
BEH C18 column, 1.7
µ
m (Wa e s, Mil o d, MA, USA) and a
linea g adien o 0.1% o mic acid solu ion in ace oni ile agains 0.1% o mic acid solu ion in wa e
om 5% o 95% in 18 min a a low a e o 0.6 mL/min. Samples we e analyzed using an amaZon speed
mass spec ome e o maXis high- esolu ion LC-QTOF sys em (B uke , USA). Da a we e collec ed
and analyzed wi h he B uke Compass Da a Analysis so wa e, e sion 4.1 (B uke , Bille ica, MA,
USA). Monoiso opic mass was sea ched in he na u al p oduc da abase DNP (Dic iona y o Na u al
P oduc s [27]).
4.4. Nybomycin Isola ion and 1H-NMR Spec oscopy
S ep omyces albus 4N24 was g own in 30 mL o TSB o 1 day, and 1 mL o he p ecul u e was
used o inocula e 100 lasks con aining 100 mL o DNPM medium. Cul u es we e incuba ed a 28
◦
C
o 7 days. Me aboli es om he supe na an we e ex ac ed as desc ibed abo e. The c ude ex ac
was ac iona ed by no mal phase ch oma og aphy on a p epacked silica ca idge (Bio age, Uppsala,
Sweden) using hexane, dichlo ome hane, e hyl ace a e, and me hanol as he mobile phase. F ac ions
Ma . D ugs 2018,16, 435 8 o 10
con aining nybomycin we e de ec ed by LC-MS analysis. They we e pooled oge he , e apo a ed,
and dissol ed in me hanol. The sample was u he sepa a ed by size-exclusion ch oma og aphy on an
LH 20 Sephadex column (Sigma-Ald ich, USA) using me hanol as he sol en . Finally, he sample was
sepa a ed by semip epa a i e HPLC (Dionex Ul iMa e 3000, The mo Fishe Scien i ic, USA) using a
C18 column (Syne gi 10
µ
m, 250
×
10 mm; Phenomenex, Ascha enbu g, Ge many) and a 0.1% o mic
acid solu ion in ace oni ile as he mobile phase o ob ain nybomycin (0.1 mg). Indi idual peaks we e
collec ed and analyzed by LC-MS as desc ibed abo e. The
1
H-NMR spec a we e eco ded on a B uke
A ance 500 spec ome e (B uke , BioSpin GmbH, Rheins e en, Ge many) a 300 K equipped wi h
a 5 mm BBO p obe using deu e a ed i luo oace ic acid (Deu e o, Kas ellaun, Ge many) as sol en
con aining e ame hylsilane (TMS) as a e e ence. The chemical shi s we e epo ed in pa s pe
million (ppm) ela i e o TMS. All spec a we e eco ded wi h he s anda d
1
H pulse p og am using
128 scans.
4.5. Genome Mining and Bioin o ma ics Analysis
The S. albus subsp. chlo inus genome was sc eened o seconda y me aboli e biosyn he ic gene
clus e s using he an iSMASH [
11
] online ool (h ps://an ismash.seconda yme aboli es.o g/#!/s a )
and he so wa e Geneious 11.0.3 [
28
]. The DNA sequence o he nybomycin gene clus e was deposi ed
in o GenBank unde accession numbe MH924838.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/1660-3397/16/11/435/
s1, Table S1:
1
H-NMR da a o isola ed and s anda d nybomycin; Table S2: Bac e ial s ains and BACs used
in his wo k; Figu e S1: UV ch oma og ams o c ude ex ac s om S. albus 4N24 and S. albus Del14; Figu e S2:
Compa ison o LC-MS ch oma og ams o S ep omyces albus 4N24 c ude ex ac and a nybomycin s anda d;
Figu e S3:
1
H-NMR spec a o a nybomycin s anda d and nybomycin isola ed om S. albus 4N24; Figu e S4:
Ex ac ed ion ch oma og ams o S. albus 4M14, S. albus 6M11, and S. albus 4N24 c ude ex ac s.
Au ho Con ibu ions:
M.R.E., M.M., and A.L. designed expe imen s; M.R.E. pe o med expe imen s; N.G.
pe o med and e alua ed he NMR analysis; S.N. con ibu ed o compound iden i ica ion; M.R.E., M.M., and A.L.
analyzed he da a and w o e he manusc ip ; and all au ho s e iewed he manusc ip .
Funding: This esea ch was unded by he ERC s a ing g an EXPLOGEN o AL, g an numbe 281623.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
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