Blockage of the early step of lankacidin biosynthesis caused a large production of pentamycin, citreodiol and epi-citreodiol in Streptomyces rochei

Article abstract of DOI:10.1038/ja.2014.160

In our effort to find the key intermediates of lankacidin biosynthesis in Streptomyces rochei, three UV-active compounds were isolated from mutant FS18, a gene disruptant of lkcA encoding a non-ribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrid enzyme. Their structures were elucidated on the basis of spectroscopic data of NMR and MS. Two compounds of a higher mobile spot on silica gel TLC (R_f =0.45 in CHCl₃ -MeOH=20:1) were determined to be an epimeric mixture of citreodiol and epi-citreodiol at the C-6 position in the ratio of 2:1. In contrast, the compound of a lower mobile spot (R_f =0 in CHCl₃ -MeOH=20:1) was identical to a 28-membered polyene macrolide pentamycin. The yields of citreodiols and pentamycin in FS18 were 5- and 250-fold higher compared with the parent strain. Introduction of a second mutation of srrX, coding a biosynthetic gene of the signaling molecules SRBs, into mutant FS18 did not affect the production of three metabolites. Thus, their production was not regulated by the SRB signaling molecules in contrast to lankacidin or lankamycin.

Full text of DOI:10.1038/ja.2014.160

The Journal of Antibiotics (2014), 1–6
2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14
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&
ORIGINAL ARTICLE
Blockage of the early step of lankacidin biosynthesis
caused a large production of pentamycin, citreodiol
and epi-citreodiol in Streptomyces rochei
Zhisheng Cao, Ryuhei Yoshida, Haruyasu Kinashi and Kenji Arakawa
In our effort to find the key intermediates of lankacidin biosynthesis in Streptomyces rochei, three UV-active compounds were
isolated from mutant FS18, a gene disruptant of lkcA encoding a non-ribosomal peptide synthetase (NRPS)-polyketide synthase
(
PKS) hybrid enzyme. Their structures were elucidated on the basis of spectroscopic data of NMR and MS. Two compounds of a
higher mobile spot on silica gel TLC (R = 0.45 in CHCl -MeOH = 20:1) were determined to be an epimeric mixture of citreodiol
f
3
and epi-citreodiol at the C-6 position in the ratio of 2:1. In contrast, the compound of a lower mobile spot (R = ~ 0 in CHCl -
f
3
MeOH = 20:1) was identical to a 28-membered polyene macrolide pentamycin. The yields of citreodiols and pentamycin in
FS18 were 5- and 250-fold higher compared with the parent strain. Introduction of a second mutation of srrX, coding a
biosynthetic gene of the signaling molecules SRBs, into mutant FS18 did not affect the production of three metabolites.
Thus, their production was not regulated by the SRB signaling molecules in contrast to lankacidin or lankamycin.
The Journal of Antibiotics advance online publication, 3 December 2014; doi:10.1038/ja.2014.160
INTRODUCTION
biosynthesis (lkcK-lkcO). Recently, involvement of an additional
The filamentous soil bacteria Streptomyces species produce a vast dehydratase activity in lankacidin biosynthesis was reported by
array of secondary metabolites including antibacterial, antifungal, Dickschat et al.11
anticancer, antiparasitic, herbicidic and immunosuppressant agents.
It is noteworthy that the lkc cluster contains only five ketosynthase
Recent development of the genome sequencing strategy revealed that domains, although eight rounds of condensation are required for the
Streptomyces strains usually contain over 20 secondary metabolite construction of the lankacidin skeleton, suggesting the modular-
biosynthetic gene clusters, many of which are cryptic and silent under iterative mixed polyketide biosynthesis of lankacidin. We carried out
laboratory culture condition.1 Hence, Streptomyces genome is well extensive mutations on the lkc-PKS genes to isolate the key inter-
considered to be an utmost important reservoir of natural products for mediates supporting this hypothesis.8 Although we have not yet
,10
new pharmaceutical applications.
succeeded to isolate such intermediates, we noticed that the lkcA
Streptomyces rochei 7434AN4 produces two structurally unrelated disruptant FS18 produced large amounts of UV-active compounds
polyketide antibiotics, lankacidin and lankamycin (Figure 1), and compared with the parent strain. We reported here the isolation and
2
carries three linear plasmids, pSLA2-L, -M and -S. Lankacidin and structural elucidation of citreodiol (5a), epi-citreodiol (5b) and
lankamycin inhibit peptide synthesis synergistically by binding to the pentamycin (6) from this mutant. In addition, we also describe that
3
,4
neighboring sites in the large ribosomal subunit, and their bio- the production of the three metabolites was not regulated by the SRB
syntheses are strictly controlled by the small diffusible signaling signaling molecules based on the result of an additional mutation
molecules termed SRBs (Streptomyces rochei butenolides) in of srrX.
5
S. rochei. In particular, lankacidin is a unique 17-membered
6
carbocyclic polyketide with various biological activities. Nucleotide RESULTS
sequencing of the largest plasmid pSLA2-L (210 614 bp) together with Metabolites from the lkcA disruptant FS18
extensive mutational analysis revealed that the lankacidin biosynthetic The lkcA mutant, termed FS18, exhibited complex metabolic changes
7–10
gene (lkc) cluster was located on pSLA2-L.
The lkc cluster contains in comparison with the parent and other lkc-PKS mutants. Strain FS18
a non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) failed to produce lankacidin and its derivatives due to the mutation in
hybrid gene (lkcA), three multidomain PKSs (lkcC, lkcF, lkcG), discrete the early biosynthetic step of lankacidin (Figure 2). As for the
dehydratase (lkcB) and acyltransferase (lkcD) genes, an amine oxidase lankamycin production, FS18 produced a similar level of lankamycin
gene (lkcE) and a gene cluster for pyrroloquinoline quinone (PQQ) (8.2 mg l− ) compared with the parent strain 51252 (8.5 mg l ).
1
− 1
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Hiroshima, Japan
Correspondence: Dr K Arakawa, Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama,
Higashi-Hiroshima, Hiroshima 739-8530, Japan.
E-mail: karakawa@hiroshima-u.ac.jp
Received 23 June 2014; revised 23 October 2014; accepted 6 November 2014

Metabolites in mutant of lankacidin biosynthesis
Z Cao et al
2
Figure 1 Structures of lankacidin C (1), lankacidinol (2), lankacidinol A (3), lankamycin (4), citreodiol (5a), epi-citreodiol (5b) and pentamycin (6) isolated
from Streptomyces rochei strains 51252 (parent) and FS18 (ΔlkcA). Ac, acetyl; Me, methyl.
Figure 2 Analysis of the metabolites produced by strain FS18. (a) HPLC chromatograms acquired using photodiode array detector. (I) strain 51252 (parent),
(
II) strain FS18 (ΔlkcA). The crude extract was applied on a COSMOSIL 5C18-MS-II column (4.6× 250 mm) and eluted with a mixture of acetonitrile-10 mM
−
1
sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml min . (b) The UV-visible spectra of compounds 1-3, 5a, 5b and 6. (c) Silica gel TLC
analysis of metabolites from 51252 (parent), FS18 (ΔlkcA) and KA78 (ΔlkcAΔsrrX) when irradiated with UV lamp at 254 nm (I) and stained with
anisaldehyde (II). TLC plate was developed with a mixture of CHCl3-MeOH (15:1, v/v). (d) Bioassay of crude extracts against Aspergillus niger. The plate was
incubated at 28 °C for 5 days.
The Journal of Antibiotics

Metabolites in mutant of lankacidin biosynthesis
Z Cao et al
3
Table 1 NMR data of citreodiol (5a) and epi-citreodiol (5b) in CDCl3
Citreodiol (5a)
epi-Citreodiol (5b)
Position δC (Mult.)
δH (Mult., J in Hz)
δC (Mult.)
δH (Mult., J in Hz)
1
2
3
4
5
6
7
8
2
6
168.8 (s)
127.6 (s)
137.4 (d)
—
—
168.9 (s)
127.4 (s)
137.6 (d)
—
—
7.19 (d, 11)
7.21 (d, 11)
124.8 (d) 6.68 (dd, 15 and 11) 125.1 (d) 6.66 (dd, 15 and 11)
145.1 (d)
75.7 (s)
73.1 (d)
16.9 (q)
12.8 (q)
22.0 (q)
51.9 (q)
6.10 (d, 15)
—
142.9 (d)
75.6 (s)
74.1 (d)
18.1 (q)
12.8 (q)
24.8 (q)
51.9 (q)
6.12 (d, 15)
—
3.70 (q, 6.4)
1.18 (d, 6.4)
1.98 (d, 1.0)
1.28 (s)
3.71 (q, 6.4)
1.18 (d, 6.4)
1.98 (d,1.5)
1.34 (s)
Figure 3 Scheme of a protection-deprotection strategy for purification of
compounds 5a and 5b. Treatment: (a) carbonyldiimidazole, toluene;
-Me
-Me
(b) separation by silica gel chromatography; (c) 0.5 M NaOH, THF-MeOH
1:1, v/v).
(
OMe
3.76 (s)
3.76 (s)
Spectra were recorded at 500 MHz for 1H and 125 MHz for 13C.
The residential solvent signal (δC =77) and internal standard tetramethylsilane (δH = 0) were
used as a reference.
Remarkably, this strain accumulated the large amounts of UV-active
compounds (5 and 6) (Figures 2a and c) and exhibited significant
antifungal activity against Aspergillus niger (Figure 2d).
The UV-active compound 5, a spot with strong UV-absorbance on
TLC (R = 0.45 in CHCl -MeOH = 20:1), was successively purified by
Sephadex LH20 with MeOH and silica gel chromatography with
pentamycin.15,16 This compound was successively purified by Sepha-
dex LH20 with MeOH and a preparative HPLC with acetonitrile-
f
3
1
0 mM sodium phosphate buffer (pH 8.2). Its molecular formula was
CHCl -MeOH = 50:1-30:1. This compound was detected at 5.9 min
3
determined by high-resolution positive ESI-MS to be C H O
3
5 58 12
on HPLC with a maximum absorbance at 267 nm, which differed
from that of lankacidin antibiotics (λmax = 230 nm) (Figures 2a and b).
High-resolution positive ESI-MS gave a distinct molecular ion peak at
+
(
observed m/z of 693.3810, calcd. for C H O Na [M+Na]
35 58 12
=
693.3821). In its 13C NMR, 35 carbons were classified into 3
methyl, 9 methylene, 21 methine and 2 quaternary carbons. Methine
carbons were further classified into 9 pentaene carbons and 12
deshielded carbons. All spectral data (Supplementary Table S1) agreed
2
2
37.1096 [M+Na]+ (calcd. for C H O Na, 237.1097). However,
2 carbon signals were observed in its C-NMR spectrum, which
11 18 4
1
3
suggests that this compound is a mixture of inseparable isomers in the
ratio of 2:1 (5a:5b). To separate 5a and 5b, a protection–deprotection
strategy was carried out using carbonyldiimidazole in toluene accord-
well with the reported data of pentamycin ( = fungichromin)
7,18
(
6, Figure 1).1
Pentamycin is a 28-membered pentaene macrolide
1
6
with a strong antifungal activity, suggesting that the observed
inhibitory zone against A. niger in Figure 2d was caused by the action
of 6. Average yield of 6 in FS18 was 5.0 mg l− , while the relative yield
1
2
ing to Shizuri et al. as shown in Figure 3. The first step quantitatively
yielded a mixture of carbonate derivatives 7a and 7b, which were then
1
separated by silica gel chromatography (R = 0.55 for 7a and 0.50 for
f
− 1
in strain 51252 was estimated to be 0.02 mg l from the calibration
7b in hexane-EtOAc = 2:1). Deprotection of the carbonate group by
curve of 6. Hence, mutation in the early step of lankacidin biosynthesis
showed 250-fold improvement in pentamycin production.
treatment with 0.5 M NaOH in THF-MeOH (1:1, v/v) afforded
diastereomerically pure 5a and 5b. From their spectral data, com-
pounds 5a and 5b were identical to citreodiol (5a; Figure 1) and epi-
citreodiol (5b), both of which were originally isolated from Penicillium
Synthesis of pentamycin, citreodiol and epi-citreodiol was not
regulated by the SRB signaling molecules
1
2
citreoviride B. As no complete set of NMR assignments has been
In many Streptomyces species, antibiotic production is strictly
controlled by the small diffusible signaling molecules such as A-factor
1
13
reported, the H and C spectral data were summarized in Table 1.
2
5
The optical rotation values of 5a and 5b were [α] = − 4.8 (c = 0.21,
CHCl ) and [α] = +6.1 (c = 0.16, CHCl ), respectively. All spectral
19
D
for streptomycin production in Streptomyces griseus and virginia
2
6
20
3
D
3
butanolides for virginiamycin production in Streptomyces virginiae.
data of 5a and 5b were identical to the reported values for synthetic
Two signaling molecules, SRB1 and SRB2, were isolated from S. rochei
and their structures of SRB1 and SRB2 were determined to be
(1′R)2-(1′-hydroxyl-6′-oxo-8′-methylnonyl)-3-methyl-4-hydroxybut-2-
(
6S,7S)(+)-citreodiol and (6R,7S)(-)-epi-citreodiol, except for their
2
2
opposite rotation values; [α] = +4.3 (c = 1.0, CHCl ) for (+)-citreo-
diol and [α]D = − 7.1 (c = 2.3, CHCl ) for (− )-epi-citreodiol.
D
3
2
1
12–14
3
en-1,4-olide and (1′R)2-(1′-hydroxyl-6′-oxo-8′-methyldecyl)-3-methyl-
Thus, compounds 5a and 5b were elucidated to be (6R,7R,2E,4E)-
,7-dihydroxy-2,6-dimethylocta-2,4-dienoic acid methyl ester and
6S,7R,2E,4E)-6,7-dihydroxy-2,6-dimethylocta-2,4-dienoic acid methyl
5
4
-hydroxybut-2-en-1,4-olide, respectively. SRB1 and SRB2 induced
6
(
5
lankacidin and lankamycin production at 40 nM concentration. It is
noteworthy that the biosynthetic gene clusters of lankacidin and
ester, respectively. This is the first discovery of 5a and 5b from lankamycin as well as their regulatory genes including srrX, which
Streptomyces species. Average yield of 5 in FS18 was calculated to be encodes a synthetic enzyme for SRBs, are located on pSLA2-L in
−
1
1
.2 mg l from the HPLC peak intensity, which was fivefold larger S. rochei.
compared with the parent strain 51252.
To analyze whether the synthesis of compounds 5a, 5b and 6 is
Compound 6 with strong UV absorbance was detected as a blue- regulated by the SRB signaling molecules, a second mutation of srrX
gray spot on TLC when baked after anisaldehyde-H SO spraying was introduced into strain FS18. The BamHI site near the 5ʹ-end of
2
4
(
R s = ~ 0 in CHCl -MeOH = 20:1 and 0.44 in CHCl -EtOAc-MeOH- srrX was filled-in by Klenow enzyme (Figure 4a), which caused a frame
f
3
3
H O = 22:22:44:11), and appeared as a distinct peak at 28.8 min on shift in srrX. Plasmid pKAR3069 containing the mutated srrX gene on
2
HPLC (Figures 2a and c). Its UV spectrum (λmax at 322, 338, 356 nm) the Streptomyces-E. coli shuttle vector pRES1821 was transformed into
(Figure 2b) was characteristic of methylpentaenes such as filipin and strain FS18 to give a lkcA-srrX double mutant KA78. The frame-shift
The Journal of Antibiotics

Metabolites in mutant of lankacidin biosynthesis
Z Cao et al
4
Figure 4 Construction of the lkcA-srrX double mutant KA78 and analysis of its metabolites. (a) Construction of the lkcA-srrX double mutant KA78. Ec,
EcoRI; Ba, BamHI, Hi, HindIII; tsr, thiostrepton resistant gene. (b) Southern blot analysis of total DNA. Lane 1, λ/HindIII; lane 2, strain FS18/BamHI; lane
3
, strain KA78/BamHI. (c) HPLC chromatograms of metabolites from strains 51252, FS18, KA78 and KY85 (the srrX single mutant) monitored at 267 nm
(
I) and at 338 nm (II). The crude extract was applied on a COSMOSIL 5C18-MS-II column (4.6× 250 mm) and eluted with a mixture of acetonitrile-10 mM
−
1
sodium phosphate buffer (pH 8.2) (3:7, v/v) at a flow rate of 1.0 ml min
.
mutation on srrX was confirmed by Southern hybridization analysis. As DISCUSSION
shown in Figure 4b, the 5.6-kb BamHI and 1.9-kb BamHI-EcoRI In this study, we analyzed complex metabolic changes and the
fragments in strain FS18 were replaced by the 7.5-kb BamHI-EcoRI structures of citreodiol (5a), epi-citreodiol (5b) and pentamycin (6)
fragment in KA78. Metabolites produced by strain KA78 were analyzed overproduced by the lkcA disruptant FS18 of S. rochei. The LkcA
by TLC, HPLC and bioassay against A. niger (Figures 2 and 4). This protein plays an important role in both recognition of glycine as a
strain produced 5a, 5b and the antifungal agent 6 at a similar level to starter unit and its condensation with a malonyl CoA extender unit in
8
the single lkcA mutant FS18 (Figure 4c), while failed to accumulate the early step of lankacidin biosynthesis. Altering the metabolic flux
lankacidins 1-3 or lankamycin 4 (Figure 2c). The single srrX mutant of fatty acid precursors sometimes causes an enhanced production of
could not produce compounds 5a, 5b and 6 as well as 1-4. This result polyketides in the producing microorganisms.22 In the case of the
showed that production of three compounds (5a, 5b, 6) was not polyether antibiotic nanchangmycin in Streptomyces nanchangensis,
regulated by the SRB signaling molecules.
its titer was improved threefold by gene inactivation of the
The Journal of Antibiotics

Metabolites in mutant of lankacidin biosynthesis
Z Cao et al
5
uncharacterized PKS clusters.23 For heterologous expression of sec- the latter does the methylenomycin biosynthetic genes coded on the
2
4
ondary metabolite gene clusters, Komatsu et al. constructed versatile linear plasmid SCP1. Hence, it is plausible that production of the three
model host Streptomyces avermitilis SUKA17, whose chromosomal metabolites is controlled by an unidentified signaling molecule/
region including principal biosynthetic genes for avermectin and
oligomycin was extensively deleted. Consequently, expression of the
introduced oxytetracycline genes in the SUKA17 transformant was
increased to yield a fivefold titer compared with the wild-type
transformant. However, the overproduction of pentamycin and
citreodiols in S. rochei seems different from these cases, because it
occurred only in the lkcA mutant. None of the other genes in the lkc
cluster, including three ketoreductase domains (lkcC-KR, lkcF-KR1
and lkcF-KR2), a C-methyltransferase gene (lkcC-MT), discrete
acyltransferase (lkcD) and dehydratase (lkcB) genes, caused an over-
production when they were disrupted. These results suggest that the
improved production of 5a, 5b and 6 in FS18 is not related to the
cease of lankacidin production, though they are synthesized from a
common precursor, malonyl CoA. The molecular mechanism of
complex metabolic changes in FS18 remains to be clarified.
receptor system coded on the S. rochei chromosome. Together with
the synergistic action of lankacidin and lankamycin to the neighboring
sites on ribosome, the location of their biosynthetic clusters and
regulatory genes on the linear plasmid pSLA2-L show ingenious
strategies of Streptomyces in genome evolution and environmental
adaptation.
MATERIALS AND METHODS
Strains and culture conditions
S. rochei wild-type strain 7434AN4 and strain 51252 that carries only pSLA2-L
were described previously.2,7 Strain FS18, a disruptant of the NRPS/PKS hybrid
gene lkcA, was constructed previously (Supplementary Figure S1).8 In strain
FS18, the 1.6-kb kanamycin cassette was introduced into the BamHI site
(
nt 33 166 of pSLA2-L) on the lkcA gene (nt 31 013–35 881 complement of
pSLA2-L). YEME liquid medium (0.3% yeast extract, 0.5% polypeptone, 0.3%
Citreodiol (5a) and epi-citreodiol (5b) were originally isolated from
ascomycetous fungi Penicillium citreoviride B with a concomitant
production of citreoviridin, a potent inhibitor of ATP synthesis and
malt extract, 1% D-glucose and 34% sucrose) was used for preparation of
4
0
4
1
S
.
r
o
c
h
e
i
p
r
o
t
o
p
l
a
s
t
s
.
P
r
o
t
o
p
l
a
s
t
s
w
e
r
e
r
e
g
e
n
e
r
a
t
e
d
o
n
R
1
M
a
g
a
r
m
e
d
i
u
m
.
YM medium (0.4% yeast extract, 1.0% malt extract and 0.4% D-glucose, pH
1
2
ATP hydrolysis. Some of the secondary metabolic pathways are
7.3) was used for antibiotic production. Tryptic soy broth (TSB) medium was
common in bacteria and fungi; for example, cephalosporins (a used for preparation of Aspergillus niger spore suspension and bioassay.
member of β-lactam antibiotics) are produced by both actinomycetes Escherichia coli XL1-Blue (Agilent Technologies, Santa Clara, CA, USA) was
(
Streptomyces clavuligerus) and fungi (Acremonium chrysogenum).25 used for construction of targeting plasmids. E. coli strains were grown in Luria
Biosynthetic genes for citreodiols have not been identified in Bertani (LB) medium supplemented with ampicillin (100 μg ml− 1) for plasmid
P. citreoviride B or S. rochei. Comparative study will provide intriguing maintenance when necessary.
insights how secondary metabolite clusters have evolved among
prokaryotic Streptomyces and eukaryotic fungi. Compounds 5a and Detection and isolation of metabolites
b were produced even by the pSLA2-L-deficient strain UV1-2,2 Detailed procedures were described in Supplementary Information.
5
harboring pSLA2-M and -S (data not shown). Sequence analysis
revealed that pSLA2-M and -S have no structural genes for polyketide Construction of the lkcA-srrX double mutant KA78
synthesis (accession numbers; AB597522 and AB905437, respec- A 1.8-kb PCR fragment containing the srrX gene was amplified using cosmid
tively). Thus, the biosynthetic genes for 5a and 5b may be located C7 (nt 143 101–184 445 of pSLA2-L)7 as a template and a pair of primers,
2
6
on the S. rochei chromosome.
srrX-f1 (5′-GACGAATTCGTGAACGCGCCGGCCACCAG-3′, where italics
Pentamycin (6) was originally isolated from Streptomyces cellulosae indicate an EcoRI site) and srrX-r1 (5′-CTGAAGCTTGTAGCCGCG
2
7
CTCGTTGCGCC-3′, where italics indicate a HindIII site). This fragment was
digested with EcoRI and HindIII and cloned into pUC19 to give pKAR3067.
The BamHI restriction site near the 5′-end of srrX was eliminated by filling-in
and self-ligating reactions to give pKAR3068, the vector of which was replaced
by E. coli-Streptomyces shuttle vector pRES1821 to give a targeting plasmid,
pKAR3069.
This plasmid was transformed into protoplasts of S. rochei FS18.
Thiostrepton-resistant transformants were subjected to sequential cultivation
in YM liquid medium supplemented with thiostrepton (10 μg ml− 1) to give the
plasmid-integrated (single crossover) strain, which was then cultured in the
absence of thiostrepton to give the double crossover strain. Mutation was
confirmed by Southern hybridization using a DIG DNA Labeling and Detection
Kit (Roche Diagnostics, Germany) according to the manufacturer’s protocol.
ATCC12625 (as fungichromin) and Streptomyces penticus (as penta-
1
6
mycin), and its absolute configuration was determined by spectral
comparison of the degradation products and partial synthesis.2
A trace amount of pentamycin was also isolated from a lankacidin-
producing strain of Streptomyces rochei var. volubilis. Filipin III,
a 14-deoxy congener of pentamycin, is the most studied pentaene
and biosynthetic aspects. Its biosyn-
thetic gene cluster (pte) in Streptomyces avermitilis spans over 80 kb
and contains 13 genes. Homologous genes with the pte cluster were
not found on pSLA2-L, -M and -S, indicating the chromosomal
location of the pentamycin gene (pem) cluster. Indeed, the pem cluster
was identified in the contig sequences of strain 7434AN4, which
contains 15 genes with an additional P450 monooxygenase gene pemI
and a ferredoxin gene pemJ (data not shown). Further studies on the
pem cluster are in progress in our laboratory.
Mutation of the srrX gene in strain FS18 revealed that the
production of citreodiol, epi-citreodiol and pentamycin is not regu-
lated by the SRB signaling molecules. In S. rochei, the signaling
molecules/receptor system, SRBs/SrrA, strictly controls lankacidin and
8,29
3
0
antibiotic from biological3
1,32
33
3
4
Antifungal activity
Aspergillus niger was used as test microorganism. A spore suspension of A. niger
was obtained by washing a well-grown Petri dish culture (90ø × 8 mm) with a
sterile aqueous solution containing 1% Tween 20 (0.5 ml). The resulting
suspension was diluted with sterile water (10 ml) and filtered through a cotton
plug. The bioassay plate was composed of two layers; the bottom layer
contained TSB medium with 1.5% agar, while the top agar contained TSB-
agar (0.8%) supplemented with 1% spore suspension of A. niger. Crude extracts
3
5–37
lankamycin production.
All of the genes responsible for biosynth-
esis and regulation of the two antibiotics are coded on the linear
plasmid pSLA2-L. Streptomyces coelicolor A3(2) contains two signaling from 100-ml broth (40 μl) were dropped onto paper disks (diameter; 0.8 cm),
3
8
molecule/receptor systems, SCB1/ScbR and methylenomycin furan put on the bioassay plates and incubated at 28 °C for 5 days. The relative yield
3
9
(
MMF)/MmfR. The former regulates the cryptic modular PKS gene of 6 in strain 51252 was estimated by the calibration curve of inhibition zones
cluster (kas; SCO6273-SCO6288) coded on the chromosome, while obtained by purified 6.
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Metabolites in mutant of lankacidin biosynthesis
Z Cao et al
6
ACKNOWLEDGEMENTS
20 Kawachi, R. et al. Identification of an AfsA homologue (BarX) from Streptomyces
virginiae as a pleiotropic regulator controlling autoregulator biosynthesis, virginiamycin
biosynthesis and virginiamycin M1 resistance. Mol. Microbiol. 36, 302–313 (2000).
We thank Professor John C. Vederas (University of Alberta) for a kind gift
of natural pentamycin. We thank Mrs. Tomoko Amimoto (Natural Science
Center for Basic Research and Development, Hiroshima University) for the
measurement of high-resolution mass spectra. This work was supported by a
Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (MEXT), and a
Charitable Trust Araki Medical and Biochemical Research Memorial Fund.
2
2
2
2
1 Ishikawa, J., Niino, Y. & Hotta, K. Construction of pRES18 and pRES19, Streptomyces-
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja)
The Journal of Antibiotics