mic oo ganisms
A icle
Dudomycins: New Seconda y Me aboli es P oduced
a e He e ologous Exp ession o an N ps Clus e
om S ep omyces albus ssp. Chlo inus N l B-24108
Cons anze Lasch 1, Ma c S ie ho 1, Ma a Rod íguez Es é ez 1, Maksym My ono skyi 1,
Jose Zapp 2and And iy Luzhe skyy 1,3,*
1Depa men o Pha maceu ical Bio echnology, Saa land Uni e si y, 66123 Saa b uecken, Ge many;
[email p o ec ed] (C.L.); [email p o ec ed] (M.S.);
[email p o ec ed] (M.R.E.); maksym.my [email p o ec ed] (M.M.)
2Depa men o Pha maceu ical Biology, Saa land Uni e si y, 66123 Saa b uecken, Ge many;
[email p o ec ed]
3AMEG Depa men , Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land,
66123 Saa b uecken, Ge many
*Co espondence: [email p o ec ed]; Tel.: +49-681-302-70200
Recei ed: 15 Oc obe 2020; Accep ed: 10 No embe 2020; Published: 16 No embe 2020


Abs ac :
Since he 1950s, na u al p oduc s o bac e ial o igin we e sys ema ically de eloped o
be used as d ugs wi h a wide ange o medical applica ions. The a ailable ea men op ions
o many diseases a e s ill no sa is ying, whe e o e, he disco e y o new s uc u es has no los
any o i s impo ance. Beyond he g ea a ie y o al eady isola ed and cha ac e ized me aboli es,
S ep omyce es s ill ha bo unin es iga ed gene clus e s whose p oduc s can be accessed using
he e ologous exp ession in hos o ganisms. This wo ks p esen s he disco e y o a se o s uc u ally
no el seconda y me aboli es, dudomycins A o D, h ough he exp ession o a c yp ic NRPS clus e
om S ep omyces albus ssp. Chlo inus NRRL B-24108 in he he e ologous hos s ain S ep omyces albus
Del14. A minimal se o genes, equi ed o he p oduc ion o dudomycins, was de ined h ough gene
inac i a ion expe imen s. This pape also p oposes a model o dudomycin biosyn hesis.
Keywo ds: S ep omyces; NRPS; he e ologous exp ession
1. In oduc ion
Biologically ac i e na u al p oduc s o mic obial o igin a e he esul o na u al design and
e olu iona y op imiza ion o a ge essen ial biological p ocesses, and a e, he e o e, a aluable sou ce
o po en ial d ug leads [
1
–
3
]. Since he 1950s, he bac e ial genus o S ep omyces la gely con ibu ed
o he pool o di e se s uc u al sca olds, some o which we e de eloped as success ul d ugs,
e.g., ancomycin (an ibac e ial) [
4
], a e mec in (an ipa asi ic) [
5
,
6
] and ac inomycin D (an icance ) [
7
,
8
],
e c. As a esul o ex ensi e sc eening, he disco e y o s uc u ally no el compounds wi h new
biological a ge s has become a challenging ask nowadays. Howe e , only a small pa o he
biosyn he ic gene clus e s encoded in mic obial genomes is eadily exp essed in he labo a o y,
while he es emain silen . I seems ha unde s anda d condi ions, only a con ined numbe o clus e s
leads o he p oduc ion o na u al p oduc s, which a e hen o en edisco e ed. Iden i ica ion o unique
biosyn he ic pa hways wi hin genome sequence da a and hei a ge ed exp ession in op imized chassis
s ains is ega ded as a mos p omising app oach o access new biologically ac i e sca olds [9,10].
Recen lyweha e epo ed he i s s udies on hegenomeminingo hes ainS ep omyces albus ssp.
Chlo inus NRRL B-24108. He e ologous exp ession o S. albus ssp. Chlo inus genes enabled iden i ying
he biosyn he ic gene clus e s o he an ibio ic nybomycin and he he bicide albucidin, as well as he
Mic oo ganisms 2020,8, 1800; doi:10.3390/mic oo ganisms8111800 www.mdpi.com/jou nal/mic oo ganisms
Mic oo ganisms 2020,8, 1800 2 o 11
isola ion and cha ac e iza ion o he no el compounds benzan h ic acid and ede icamycin C2 [
11
–
14
].
This s udy epo s on a biosyn he ic gene clus e o S ep omyces albus ssp. Chlo inus encoding an
uncha ac e ized non ibosomal pep ide syn he ase. The bioin o ma ic analysis did no e eal any
cha ac e ized homologs o he s udied clus e , implying ha i migh encode a no el na u al p oduc .
He e ologous exp ession o he NRPS clus e in ou op imized hos s S ep omyces albus Del14 [
15
] and
S ep omyces li idans Del8 [
16
] led o iden i ying a se o ou new compounds we named dudomycins.
The compounds we e pu i ied, and hei s uc u es we e elucida ed in
1
H and
13
C nuclea magne ic
esonance (NMR) expe imen s. The iden i ied compounds a e s uc u ally ela ed and consis o a co e
lysine and h ee b anched-chain hyd oxy a y acid esidues. A hypo hesis on dudomycin biosyn hesis
is p oposed based on he esul s o gene dele ion expe imen s.
2. Ma e ials and Me hods
2.1. Gene al Expe imen al P ocedu es
The s ains, bac e ial a i icial ch omosomes (BACs), and plasmids used in his wo k a e lis ed in
Table S3. Esche ichia coli s ains we e cul u ed in lysogeny b o h (LB) medium [
17
]. S ep omyces s ains
we e g own on soy lou manni ol (MS) aga [
18
] and in liquid yp ic soy b o h (TSB; Sigma-Ald ich,
S . Louis, MO, USA). 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 as aqueous solu ion) and de ined medium DM (manni ol 5 g/L, amino acid
0.5 g/L, K2PO4 0.5 g/L, MgSO4
×
7 H2O 0.2 g/L, FeSO4
×
7 H2O 0.01 g/L) we e used o me aboli e
exp ession. When DM was used o p oduc ion, he cells o he p ecul u e we e washed h ee imes
using amino acid- ee de ined medium p io o inocula ion. Amino acids l- al, l-ile, l-lys, and d-lys
we e supplied o a de ined medium as needed. The an ibio ics kanamycin, ap amycin, hyg omycin,
ampicillin, and nalidixic acid we e added when equi ed.
2.2. Isola ion and Manipula ion o DNA
DNA manipula ion, ans o ma ion in o E. coli, as well as in e gene ic conjuga ion be ween E.
coli and S ep omyces, we e pe o med acco ding o s anda d p o ocols [
17
–
19
]. BAC DNA om a
cons uc ed genomic lib a y o S ep omyces albus ssp. Chlo inus NRRL B-24108 was isola ed wi h
he BACMAX
™
DNA pu i ica ion ki (Lucigen, Middle on, WI, USA). Dele ion o se e al genes was
pe o med on he BAC 4E8 i sel using a wo-s ep app oach. S ep one ocused on dele ing all genes
1 o 21, leading o BAC 4E8_del1, s ep wo add essed u he dele ion o genes 25 and 26 on BAC
4E8_del1, esul ing in BAC 4E8_del2. The genes we e eplaced by esis ance ma ke s ampicillin and
hyg omycin h ough homologous ecombina ion using he Red/ET sys em [
20
]. PCR was pe o med o
ampli ica iono he espec i egenecasse es omplasmidspUC19andpXCMhyg o max. PCRp ime s
20190429_1_ w, 20190429_1_ e , 20190429_2_ w, and 20190429_2_ e we e cons uc ed wi h o e hang
egions o si e-speci ic in oduc ion o he casse es le o igh om he dudomycin clus e and
simul aneous emo al o he genes 1 o 21 o 25 and 26. Res ic ion mapping and sequencing we e
used o con ol he success o he ecombina ion. Res ic ion enzymes om The moFishe Scien i ic
(Wal ham, MA, USA) o New England BioLabs NEB (Ipswich, MA, USA) we e used acco ding o he
manu ac u e ’s ins uc ion.
2.3. Me aboli e Ex ac ion
Fo me aboli e ex ac ion, S ep omyces s ains we e g own in 15 mL o TSB in a 100 mL ba led
lask o 1 o 2 days, and 1 mL o seed cul u e was used o inocula e 100 mL o p oduc ion medium in a
500 mL ba led lask. Cul u es we e g own o 7 days a 28
◦
C and 180 pm in an In o s mul i on shake
(In o s AG, Basel, Swi ze land). Me aboli es we e ex ac ed om he cul u e supe na an wi h an equal
amoun o ei he n-bu anol o e hyl ace a e, e apo a ed a 40 ◦C and kep a s o age condi ion 4 ◦C.
Mic oo ganisms 2020,8, 1800 3 o 11
2.4. Mass Spec ome y (MS) Me aboli e Analysis
D ied ex ac s we e dissol ed in me hanol p io o he mass spec ome y (MS) analysis.
MS expe imen s we e ca ied ou on a Dionex Ul ima e 3000 UPLC sys em (The moFishe Scien i ic,
Wal ham, MA, USA) coupled o PDA de ec o (s a iona y phase 30 o 100 mm ACQUITY UPLC BEH
C18 1.7
µ
m column (Wa e s Co po a ion, Mil o d, MA, USA), mobile phase: Linea g adien o [A]
ddH
2
O+0.1% o mic acid/[B] ace oni ile +0.1% o mic acid, 5% o 95% a low a e o 0.6 mL/min).
Fu he mass de ec ion was pe o med coupling ei he an amaZon speed (B uke , Bille ica, MA, USA)
o LTQ O bi ap XL mass spec ome e (The moFishe Scien i ic, Wal ham, MA, USA) using posi i e
ioniza ion mode and mass ange de ec ion o m/z 200 o 2000. Da a analysis was pe o med using
so wa e Compass Da a Analysis . 4.1 (B uke ) and Xcalibu . 3.0 (The moFishe Scien i ic).
2.5. Pu i ica ion
Theex ac om he10Lcul u ewasdissol edinme hanol. A i s pu i ica ions epwasca iedou
using Size Exclusion Ch oma og aphy (SEC; s a iona y phase: Sephadex-LH20; mobile phase: isoc a ic
elu ion using 100% me hanol). F ac ions con aining dudomycins we e pooled, d ied, edissol ed in
me hanol and unde gone a second pu i ica ion s ep: Re e sed Phase (RP) HPLC (Agilen In ini y
1200 se ies HPLC sys em; s a iona y phase: Syne gi
TM
4
µ
m Fusion-RP 80 Å 250
×
10 (Phenomenex,
To ance, CA, USA); mobile phase: Linea g adien o [A] H
2
O+0.1% o mic acid/[B] ace oni ile +
0.1% o mic acid, 30% o 95% [B] in 14.5 min a low a e o 4 mL/min, column o en empe a u e 45
◦
C,
de ec ion UV 210 nm ollowed by ac ion con ol on HPLC-MS). F ac ions we e pooled o ob ain he
ou pu e dudomycin isola es A o D.
2.6. Nuclea Magne ic Resonance (NMR) Spec oscopy
The
1
H-NMR spec um in CDCl
3
(Deu e o, Kas ellaun, Ge many) was eco ded on a B uke
A ance 500 spec ome e (B uke , BioSpin GmbH, Rheins e en, Ge many) equipped wi h a 5 mm
BBO p obe a 298 K. The chemical shi s we e epo ed in pa s pe million (ppm) ela i e o TMS.
The spec a we e eco ded wi h he s anda d
1
H pulse p og am using 64 scans. All o he NMR spec a
we e acqui ed on a B uke Ascend 700 MHz NMR spec ome e a 298 K equipped wi h a 5 mm TXI
c yop obe. As a sol en , deu e a ed CD
3
OD was used. HSQC, HMBC, 1H-1H COSY spec a we e
eco ded using he s anda d pulse p og ams om he TOPSPIN . 3.6 so wa e. Selec i e 1D TOCSY
expe imen s we e pe o med using mixing imes o 120 ms.
2.7. Genome Mining and Bioin o ma ic Analysis
The genome o S. albus ssp. Chlo inus was sc eened o seconda y me aboli e biosyn he ic gene
clus e s using he an iSMASH online ool (h ps://an ismash.seconda yme aboli es.o g/#!/s a ) [
21
].
Analysis o gene ic da a was pe o med using Geneious so wa e, . 11.0.3 [
22
]. The genomic sequence
o S ep omyces albus ssp. Chlo inus was deposi ed in GenBank unde accession numbe VJOK00000000.
Fo de eplica ion, he Dic iona y o Na u al P oduc s (DNP) 28.1 was used as a da abase o known
na u al p oduc s.
3. Resul s and Discussion
3.1. Iden i ica ion and Exp ession o he NRPS Gene Clus e
Genome mining o S ep omyces albus ssp. Chlo inus NRRL B-24108 using An iSMASH so wa e
e ealed se e al c yp ic biosyn he ic gene clus e s (BGCs) wi hin i s ch omosome [
22
]. A BGC encoding
a pu a i e non ibosomal pep ide syn he ase (NRPS) caugh ou a en ion as he so wa e did no de ec
any homology o al eady cha ac e ized BGCs [
23
]. A BAC 4E8 co e ing he en i e NRPS clus e was
iden i ied in he p e iously cons uc ed genomic lib a y o S. albus ssp. Chlo inus (GenBank accession
numbe VJOK00000000). The BAC 4E8 was ans e ed in o he op imized he e ologous hos s ains
Mic oo ganisms 2020,8, 1800 4 o 11
S ep omyces albus Del14 and S ep omyces li idans Del8, leading o he espec i e exconjugan s ains
S ep omyces albus 4E8 and S ep omyces li idans 4E8. The ob ained exconjugan s ains we e cul i a ed
in he p oduc ion medium DNPM, and he me aboli es we e ex ac ed om he cul u e supe na an
wi h e hyl ace a e o N-bu anol. High- esolu ion HPLC-MS analysis e ealed he p esence o ou new
peaks in he e hyl ace a e and N-bu anol ex ac s o bo h S. albus and S. li idans ha bo ing he BAC 4E8
(Figu e 1; Figu es S1 and S2). The iden i ied peaks could no be obse ed in he ex ac s o he con ol
s ains wi hou he BAC. Analysis o he mass spec a o he iden i ied peaks e ealed he molecula
ions [M +H
+
] wi h he masses 573.411, 587.426, 601.442, and 615.457 Da. A sea ch in he DNP da abase
o na u al p oduc s o he iden i ied high- esolu ion masses did no gene a e any ma ches, implying
ha he iden i ied compounds migh be new. The mass di e ences o 14 Da be ween he indi idual
compounds imply ha hey migh di e by he p esence o addi ional CH
2
g oups. The iden i ied
compounds we e named dudomycins A, B, C, and D.
Figu e 1.
HPLC-MS analysis o dudomycin p oduc ion by S. albus Del14 s ain ha bo ing he BAC
(bac e ial a i icial ch omosomes) 4E8. The s ain S. albus Del14 wi hou he BAC was used as a con ol.
The ex ac ed base peak ch oma og ams 573-574 Da, 587-588 Da, 601-602 Da, 615-616 Da a e shown.
3.2. Pu i ica ion and S uc u e Elucida ion
To ge insigh s in o he s uc u es o he p oduced compounds, he p oduce s ain S. albus 4E8
was cul i a ed in 10 L o he p oduc ion medium DNPM, and he me aboli es we e ex ac ed om he
cul u e supe na an wi h e hyl ace a e. The s ain S. albus 4E8 was p e e ed o S. li idans 4E8, due o
i s highe p oduc ion le el. The dudomycins A o D co esponding o he iden i ied molecula ions
[M +H
+
] wi h he masses 573.411, 587.426, 601.442, and 615.457 Da we e success ully pu i ied om
he ex ac : 1.2 mg o dudomycin A, 1.1 mg o dudomycin B, 0.7 mg o dudomycin C and 0.5 mg o
dudomycin D we e ob ained. The s uc u e elucida ion o he isola ed dudomycins was pe o med
using 1D and 2D NMR wi h a special ocus on 1D TOCSY expe imen s.
Due o i s small molecula mass, dudomycin A was he i s compound used o he s uc u e
elucida ion. The molecula o mula was calcula ed as C
30
H
56
O
8
N
2
based on he calcula ed
high- esolu ion mass o 572.403 Da, indica ing ou deg ees o unsa u a ion. Analysis o
1
H,
13
C NMR,
and edi ed HSQC led o 6 me hyls, 13 me hylenes, 7 me hines, and 4 qua e na y ca bons. Two o he
emaining i e p o ons belonged o NH-g oups, as he 1H NMR measu emen in CDCl3 e ealed he
p esence o wo signals a
δ
H 6.81 and
δ
H 7.12 ppm (Figu e S3), which disappea ed in p o ic sol en s.
Due o b oad signals and he compound’s poo solubili y in CDCl
3
, all o he spec a we e eco ded in
CD3OD.
In e p e a ion o HHCOSY e ealed ou disc e e spin sys ems in he molecule. The i s sequence
s a ing om he me hine signal a
δ
H 4.26 ollowed by ou me hylenes a
δ
H 1.83, 1.38, 1.51, and 3.17
was assigned o lysine, which was suppo ed by HMBC da a. The emaining h ee spin sys ems
we e mos ly close o each o he , leading o many o e laps o me hylene and me hyl esonances
in he a ea o
δ
H 0.8–1.7 (Figu e S4). Howe e , well-sepa a ed me hine p o on signals a
δ
H 5.20,
Mic oo ganisms 2020,8, 1800 5 o 11
3.95, 3.92 enabled selec i e 1D TOCSY expe imen s using hese esonances as i adia ion poin s.
Thei ca e ul in e p e a ion enabled he ull assignmen o all spin sys ems leading o h ee di e en
3-hyd oxy-6-me hyl hep anoic acid moie ies (HMH1 o HMH3).
Long- ange HMBC co ela ions o Lys-H-2 (
δ
H 4.26,
δ
C 56.2) o HMH1-C-1
0
(
δ
C 171.9) and
Lys-H-6 (
δ
H 3.17,
δ
C 40.4) o C-1
0
(
δ
C 174.3) p o ed connec ions o HMH1 and HMH2 ia amide bonds.
The ca bonyl g oup o lysine (
δ
C 178.9) did no show any ex e nal connec ions, hence i was assigned
as a ee ca boxyl g oup. The emaining long- ange HMBC co ela ions be ween H-3
0
(
δ
H 5.20,
δ
C 73.3)
and C-1”’ (
δ
C 173.2) indica ed an es e bond be ween HMH1 and HMH3 leading o he inal s uc u e
o dudomycin A (Figu e 2; Figu es S3–S10; Table S1).
Figu e 2.
Obse ed HMBC (a ows) and selec i e 1D TOCSY (bold lines) key co ela ions o
dudomycin A.
The esul s o NMR analysis indica e ha he dudomycins B (C
31
H
58
O
8
N
2
), C (C
32
H
60
O
8
N
2
),
and D (C
33
H
62
O
8
N
2
) a e s uc u ally ela ed o dudomycin A. F om hese compounds, only dudomycin
D (Table S2) was a pu e subs ance, while dudomycins B and C we e he mix u es o se e al isome s.
Simila o dudomycin A, he s uc u e o dudomycin D con ains a lysine co e, wi h h ee hyd oxy a y
acids a ached. Howe e , in con as o dudomycin, A h ee esidues o 3-hyd oxy-6-me hyl oc anoic
acid (HMO1 o HMO3) can be ound in he s uc u e o dudomycin D ins ead o h ee esidues o
3-hyd oxy-6-me hyl hep anoic acid (HMH1 o HMH3). The alkyl chain o he 3-hyd oxy-6-me hyl
oc anoic acid is ex ended by one addi ional CH
2
g oup compa ed o 3-hyd oxy-6-me hyl hep anoic
acid wha explains he o e all mass di e ence o 42 Da be ween dudomycins A and D.
NMR spec a o dudomycins B and C e ealed ha bo h samples consis o h ee isome s each.
Simila o dudomycins A and D, dudomycins B and C also con ain a lysine co e in hei s uc u es.
In con as o dudomycins A and D, which con ain ei he HMH o HMO esidues bound o he co e,
dudomycins B and C con ain a mix u e o HMH and HMO esidues in hei s uc u es. The esul s o
1
H NMR and a mul i ude o 1D TOCSY measu emen s indica e ha dudomycin B con ains wo HMH
and one HMO esidues, while dudomycin C con ains one HMH and wo HMO esidues. These esul s
a e in acco dance wi h he obse ed mass di e ence o 14 Da be ween Dudomycin A and B and o
28 Da be ween dudomycins A and C. Th ee isome ic o ms a e possible o bo h dudomycin B and C
(Figu e 3, Figu es S11–S24).
Figu e 3.
The s uc u es o isola ed dudomycins. In he case o dudomycin A, all h ee R g oups
co espond o he CH
3
g oup. Dudomycin B is a mix u e o 3 cons i u ional isome s wi h wo R g oups
co esponding o CH
3
and one R g oup— o C
2
H
5
. Dudomycin C is a mix u e o 3 cons i u ional
isome s wi h one R g oup co esponding o CH
3
and wo R g oups— o C
2
H
5
. In dudomycin D,
all h ee R g oups co espond o C2H5.
Expe imen s o de e mine he absolu e con igu a ion o dudomycins ha e no been pe o med,
due o low amoun s o he isola ed compounds. The esul s o a eeding expe imen imply ha l-lysine

Mic oo ganisms 2020,8, 1800 6 o 11
is used as a p ecu so o dudomycin biosyn hesis (Figu e S25). A highe dudomycin p oduc ion le el
was obse ed upon he cul i a ion o S. albus 4E8 in a de ined medium wi h l-lysine as a ni ogen
sou ce han in he medium wi h d-lysine.
The occu ence o HMH and HMO in na u al p oduc s is e y a e, and o he bes o ou
knowledge, he s uc u es o dudomycin A o D ha e no been epo ed be o e.
3.3. De e mina ion o he Minimal Dudomycin Gene Clus e
The 30 kb DNA agmen cloned in he BAC 4E8 con ains 26 genes (Figu e 4; Table 1). Gene 22
encodes a pu a i e NRPS comp ising condensa ion (C), adenyla ion (A), pep idyl ca ie p o ein (PCP),
and hioes e ase (TE) domains. The A domain was p edic ed o ha e a weak p e e ence o ecogni ion
o he amino acid o ni hine. Due o he s uc u al simila i y o o ni hine and lysine gene 22 encoding,
an NRPS enzyme was ega ded o be in ol ed in dudomycin biosyn hesis. A sequence analysis o he
egions ups eam and downs eam o gene 22 was pe o med o iden i y he genes possibly in ol ed
in he p oduc ion o dudomycins. This analysis did no e eal any gene whose p oduc could be
enzyma ically in ol ed in dudomycin biosyn hesis. Sequence homology analysis e ealed ha he
homologs o genes 22, 23, and 24 a e clus e ed oge he in he genomes o se en di e en s ains
implying ha hose genes migh be in ol ed in he same pa hway. Genes 23 and 24 encode a pu a i e
anspo p o ein and a pu a i e ansc ip ional egula o , espec i ely. To con i m ha he genes
ups eam, gene 22 a e no in ol ed in dudomycin biosyn hesis, he genes 1 o 21 we e subs i u ed in
he BAC 4E8 wi h an ampicillin casse e using Red/ET. The cons uc ed BAC 4E8_del1 was ans e ed
in o S. albus Del14 s ain yielding S. albus 4E8_del1. HPLC-MS analysis o he me aboli e p oduc ion by
he ob ained s ains did no de ec any di e ences in dudomycin p oduc ion compa ed o he S. albus
4E8 s ain. This clea ly indica ed ha he genes 1 o 21 encoded in he BAC 4E8 a e no in ol ed in he
biosyn hesis o dudomycins.
To ind ou i genes 25 and 26 a e in ol ed in dudomycin p oduc ion, hey we e eplaced in he
BAC 4E8_del1 wi h a hyg omycin esis ance casse e using Red/ET. The cons uc ed BAC 4E8_del2
con ains genes 22, 23, and 24 only. The BAC was ans e ed in o S. albus Del14, and he ob ained s ain
S. albus 4E8_del2 was checked o dudomycin p oduc ion. HPLC-MS analysis e ealed ha dele ing
genes 25 and 26 did no a ec he p oduc ion o dudomycins (Figu e S26), indica ing ha hese genes
do no ake pa in he biosyn hesis o he compounds.
The esul s o gene dele ion expe imen s demons a e ha genes 22, 23, and 24 (designa ed as
dudA,dudB, and dudC), encoding he pu a i e NRPS, anspo p o ein, and ansc ip ional egula o
(Table 1, Figu e 4), su ice o he p oduc ion o dudomycins. Since he p oduc s o dudB and dudC
do no ha e an enzyma ic unc ion, we suppose ha only he p oduc o dudA is esponsible o he
biosyn hesis o dudomycins. The p oduc s o dudB and dudC a e likely in ol ed in he anspo o
he biosyn he ic p oduc s and in he egula ion o dudomycin p oduc ion, espec i ely. Since he
inac i a ion o he genes dudB and dudC was no pe o med, he possibili y ha he genes a e no
essen ial o dudomycin p oduc ion canno be comple ely excluded.
Figu e 4.
The ch omosomal agmen o S ep omyces albus ssp. Chlo inus NRRL B-24108 con aining he
dudomycin gene clus e . The genes pu a i ely in ol ed in dudomycin biosyn hesis a e highligh ed in
da k g ey. The ch omosomal agmen s cloned in BACs 4E8, 4E8_del1 and 4E8_del2 a e shown wi h
black ba s.
Mic oo ganisms 2020,8, 1800 7 o 11
Table 1.
P oposed unc ions o he genes in he DNA agmen con aining he dudomycin gene clus e .
Gene # Locus Tag Pu a i e Func ion
1 SACHL_42600 glycosyl ans e ase
2 SACHL_42610 hypo he ical p o ein
3 SACHL_42620 phospha ase
4 SACHL_42630 N,N’-diace yllegionaminic acid syn hase
5 SACHL_42640 hypo he ical p o ein
6 SACHL_42650 hyd olase
7 SACHL_42660 memb ane lipop o ein p ecu so
8 SACHL_42670 galac ose/me hyl galac oside impo ATP-binding
p o ein
9 SACHL_42680 ibose anspo sys em pe mease p o ein
10 SACHL_42690 b anched-chain amino acid anspo sys em/pe mease
componen
11 SACHL_42700 cy idine deaminase
12 SACHL_42710 py imidine-nucleoside phospho ylase
13 SACHL_42720 hypo he ical p o ein
14 SACHL_42730 hypo he ical p o ein
15 SACHL_42740 hypo he ical p o ein
16 SACHL_42750 hypo he ical p o ein
17 SACHL_42760 hypo he ical p o ein
18 SACHL_42770 hypo he ical p o ein
19 SACHL_42780 ubiquinone biosyn hesis O-me hyl ans e ase
20 SACHL_42790 zinc me allo-pep idase
21 SACHL_42800 hypo he ical p o ein
22 [dudA] SACHL_42810 dimodula non ibosomal pep ide syn hase
23 [dudB] SACHL_42820 inne memb ane anspo p o ein
24 [dudC] SACHL_42830 ansc ip ional egula o
25 SACHL_42840 deme hyl ebeccamycin-d-glucose O-me hyl ans e ase
26 SACHL_42850 hypo he ical p o ein
3.4. Biosyn hesis o Dudomycins
S uc u ally dudomycins consis o a lysine co e and h ee hyd oxy a y acid esidues, wo o
which a e a ached di ec ly o he lysine moie y h ough amide bonds, while he hi d esidue is
es e i ied wi h one o he lysine bound hyd oxy a y acids (Figu e 3). Two ypes o hyd oxy a y acids
a e used o he biosyn hesis o dudomycins: The sho e HMH and he longe HMO. Th ee HMH
esidues a e ound in he smalles compound—dudomycin A, while h ee HMO esidues a e used o
o m he la ges compound—dudomycin D. Dudomycins B and C con ain bo h ypes o hyd oxy a y
acids in hei s uc u es. The esul s o gene inac i a ion s udies and sequence analysis demons a ed
ha only he p oduc o he dudA gene is esponsible o biosyn hesis o dudomycins. The gene encodes
an NRPS comp ising ou domains only: C, A, PCP, and TE. We p opose ha he A domain ac i a es he
l-lysine and loads i on he PCP module. The C domain hen a aches wo hyd oxy a y acid moie ies
o he wo a ailable amino g oups o PCP-bound lysine. The p oduc is eleased h ough he hyd oly ic
ac i i y o he TE domain. We p opose ha he hi d hyd oxy a y acid is spon aneously es e i ied
wi h HMH1 o HMO1, espec i ely (Figu es 2and 5). I emains unclea i he a achmen o he hi d
hyd oxy a y acid occu s be o e o a e he elease o he NRPS p oduc . No peaks co esponding
o he dudomycin de i a i es wi hou HMH3 o HMO3 esidue could be iden i ied by HPLC-MS
analysis in he ex ac s o S ep omyces s ains ha bo ing he dudomycin biosyn he ic clus e .
Mic oo ganisms 2020,8, 1800 8 o 11
Figu e 5. P oposed biosyn hesis exempla ily o dudomycin A.
A achmen o wo hyd oxy a y acid esidues o a lysine, which is ca alyzed by he single-module
NRPS encoded by dudA is no ypical. A leas ano he wo cases a e knownwhen a single NRPS module
pe o ms wo ounds o condensa ion. Du ing he biosyn hesis o myxochelins a single-module NRPS
encoded by he mxcG gene c ea es he amide linkage be ween wo 2,3-dihyd oxybenzoic acid esidues
and he wo amino g oups o lysine [
24
]. Du ing he biosyn hesis o ib iobac in he single-module
NRPS encoded by he ibF gene also ca alyzes wo condensa ion e en s. Howe e , in he case o
ib iobac in, wo condensa ion domains a e p esen wi hin VibF each o which is likely esponsible o
one condensa ion eac ion [25].
Th ee genes, dudA o dudC, a e equi ed o he biosyn hesis o dudomycins in S. albus Del14
and S. li idans Del8. The dudA gene encodes he abo e men ioned NRPS, and genes, dudB and
dudC, encode he pu a i e memb ane anspo e and ansc ip ional egula o . No genes in ol ed in
p ecu so supply could be iden i ied in he DNA egions lanking genes, dudA o dudC, implying ha
all p ecu so s equi ed o he dudomycin biosyn hesis, including he hyd oxy a y acids HMH and
HMO a e p o ided by he hos me abolism.
S ep omyces a e known o hei ex ao dina y p e alence o b anched-chain a y acids, which a e
syn hesized by ype II a y acid syn hases [
26
]. B anched-chain amino acids o en se e as p ecu so s
o biosyn hesis o iso and an eiso ca boxylic acids. Th ough oxida ion o he amine g oup, he amino
acids a e con e ed in o
α
-ke o acids, which a e hen used as s a e uni s in bac e ial a y acid
biosyn hesis [
27
]. Valine and isoleucine wi h high p obabili y se e as main biosyn he ic p ecu so s o
HMH and HMO, espec i ely. These amino acids a e con e ed o 3-me hyl-2-oxobu anoic acid and
3-me hyl-2-oxopen anoic acid h ough he ac ion o aline dehyd ogenase o homologous enzymes.
The o med 2-ke o ca boxylic acids a e deca boxyla ed and used as s a e uni s by a ype II a y acid
syn hase which con e s hem o HMH and HMO a e deca boxyla i e condensa ion o wo malonyl
uni s (Figu e 6). The p ecu so ole o l- aline and l-isoleucine in biosyn hesis o HMH and HMO is
indi ec ly con i med by eeding s udies [
28
]. Du ing cul i a ion in a de ined medium wi h l- aline as
ni ogen sou ce, he s ain S. albus 4E8_del2 p oduced mos ly he smalle dudomycins (dudomycins A
and B), which con ain mainly HMH, which is de i ed om l- aline. Wi h l-isoleucine, as a single
ni ogen sou ce, he s ain p oduced mos ly he bigge dudomycins C and D. These compounds
con ain mainly HMO, which is de i ed om l-isoleucine (Figu e S25). Fu he mo e, hea ily impai ed
p oduc ion o dudomycins was obse ed when BAC 4E8 was exp essed in an S. albus delVDH s ain [
29
]
wi h inac i a ed aline dehyd ogenase gene dh (Figu e S27). This esul sugges s ha he aline
dehyd ogenese is in ol ed in he supply o HMH and HMO by oxida ion o he b anched-chain
amino acids l- aline and l-isoleucine. Low dudomycin p oduc ion by S. albus delVDH 4E8_del2 also
indica es ha o he homologs o he aline dehyd ogenase wi h b oad subs a e speci ici y a e encoded
in he genome o S. albus, in acco dance wi h p e iously published da a [30].
Mic oo ganisms 2020,8, 1800 9 o 11
Figu e 6. P oposed biosyn hesis o HMH and HMO.
4. Conclusions
In his pape , we epo he iden i ica ion and success ul he e ologous exp ession o a new
NRPS clus e leading o he p oduc ion o a g oup o new compounds called dudomycins A o
D. The single-module NRPS which is esponsible o dudomycin p oduc ion ac i a es l-lysine and
ca alyzes i s condensa ion wi h wo hyd oxy a y acid CoA p ecu so s. Such examples whe e a single
condensa ion domain ca alyzes wo condensa ion s eps a e e y a e. The b anched hyd oxy a y
acids used o he dudomycin biosyn hesis a e p o ided by he hos ’s me abolism. The amino acids
l- aline and l-isoleucine se e as main p ecu so s o hei biosyn hesis.
Supplemen a y Ma e ials:
The ollowing a e a ailable online a h p://www.mdpi.com/2076-2607/8/11/1800/
s1. Figu e S1: P oduc ion o dudomycins by di e en hos s using di e en o ganic sol en s o ex ac ion,
Figu e S2: Mass spec a o dudomycins (
A
–
D
), Figu e S3:
1
HNMR (500 MHz, CDCl
3
) spec um o dudomycin
A, Figu e S4:
1
HNMR (500 MHz, CD
3
OD) spec um o dudomycin A, Figu e S5: 13C NMR (176 MHz, CD
3
OD)
spec um o dudomycin A, Figu e S6: DEPT-135 (176 MHz, CD
3
OD) spec um o dudomycin A, Figu e S7, HSQC
(700 MHz, CD
3
OD) spec um o dudomycin A, Figu e S8: COSY (700 MHz, CD
3
OD) spec um o dudomycin
A, Figu e S9: HMBC (700 MHz, CD
3
OD) spec um o dudomycin A, Figu e S10: (
A
)
1
HNMR spec um o
dudomycin A showing he i adia ion poin s B (Lys, H-2), C (HMH3, H-3
000
), D (HMH2, H-3
00
) and E (HMH1,
H 3
0
). (
B
–
E
) Selec i e 1D TOCSY (700 MHz, CD
3
OD) spec a o he co esponding i adia ion poin s, Figu e S11:
1
HNMR (700 MHz, CD
3
OD) spec um o dudomycin B, Figu e S12; DEPT-135 (176 MHz, CD
3
OD) spec um o
dudomycin B showing a 2/1 a io o he me hyl signals o HMH and HMO, Figu e S13: HSQC (700 MHz, CD
3
OD)
spec um o dudomycin B, Figu e S14: COSY (700 MHz, CD
3
OD) spec um o dudomycin B, Figu e S15: HMBC
(700 MHz, CD
3
OD) spec um o dudomycin B, Figu e S16: (
A
)
1
HNMR spec um o dudomycin B showing he
i adia ion poin s C (H-3
00
/H-3
000
) and C (H-3
0
). (
B
–
D
) Selec i e 1D TOCSY (700 MHz, CD
3
OD) spec a o he
co esponding i adia ion poin s, Figu e S17: H NMR (700 MHz, CD
3
OD) spec um o dudomycin C, Figu e S18:
DEPT-135 (176 MHz, CD
3
OD) spec um o dudomycin C showing an app oxima e 1/2 a io o he me hyl signals
o HMH and HMO, Figu e S19: HMBC (700 MHz, CD
3
OD) spec um o dudomycin C, Figu e S20: (
A
)
1
HNMR
spec um o dudomycin C showing he i adia ion poin s B (H-3
00
/H-3
000
) and C (H-3
0
). (
B
–
D
) Selec i e 1D
TOCSY (700 MHz, CD
3
OD) spec a o he co esponding i adia ion poin s, Figu e S21:
1
HNMR (700 MHz,
CD
3
OD) spec um o dudomycin D, Figu e S22: DEPT-135 (176 MHz, CD
3
OD) spec um o dudomycin D,
Figu e S23: HSQC (700 MHz, CD
3
OD) spec um o dudomycin D, Figu e S24: (
A
)
1
HNMR spec um o dudomycin
D showing he i adia ion poin s B (HMO2/3, H-3
00
/H-3
000
), C (HMO1, H-3
0
) and D (Lys, H-2). (
B
–
D
) Selec i e 1D
TOCSY (700 MHz, CD
3
OD) spec a o he co esponding i adia ion poin s; Figu e S25: P oduc ion o dudomycin
de i a i es in de ined medium (DM) wi h single sou ces o ni ogen, Figu e S26: P oduc ion o dudomycins
a e gene dele ion expe imen s, Figu e S27: Dec eased p oduc ion o dudomycin de i a i es by S. albus delVDH
4E8, Table S1: NMR da a o dudomycin A in CD
3
OD, Table S2: NMR da a o dudomycin D in CD
3
OD, Table S3:
S ains, BACs, plasmids and p ime s used in his wo k, Tables S4: P oposed unc ions o he genes in he DNA
agmen con aining he dudomycin gene clus e .
Au ho Con ibu ions:
Dudomycin peaks we e i s iden i ied by M.R.E. Excep om NMR analysis, he
expe imen s we e designed and e alua ed by C.L., M.M. and A.L. and he p ac ical wo k pe o med by C.L. NMR
expe imen s we e se up, ca ied ou and e alua ed by M.S. and he da a e iewed by J.Z. The manusc ip was
d a ed by C.L., M.S. and M.M. All au ho s ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This esea ch has ecei ed unding om BMBF unde G an “EXPLOMARE“ 031B0868A.
Acknowledgmen s:
We hank Helmhol z Ins i u e o Pha maceu ical Resea ch Saa land, Saa b uecken, Ge many
(HIPS) o suppo o NMR measu emen s. The s ain S. albus ssp. chlo inus NRRL B-24108 was p o ided by BASF
SE Ludwigsha en, Ge many.
Con lic s o In e es : The au ho s decla e no con lic o in e es .