Identification of the lomofungin biosynthesis gene cluster and associated flavin-dependent monooxygenase gene in Streptomyces lomondensis S015

Article abstract of DOI:10.1371/journal.pone.0136228

Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces.

Full text of DOI:10.1371/journal.pone.0136228

RESEARCH ARTICLE
Identification of the Lomofungin
Biosynthesis Gene Cluster and Associated
Flavin-Dependent Monooxygenase Gene in
Streptomyces lomondensis S015
Chunxiao Zhang, Chaolan Sheng, Wei Wang*, Hongbo Hu, Huasong Peng,
Xuehong Zhang
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao
Tong University, 800 Dongchuan Road, Shanghai, 200240, China
a11111
* weiwang100@sjtu.edu.cn
Abstract
OPEN ACCESS
Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic
lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting
of 23 open reading frames that includes the core phenazine biosynthesis gene cluster
lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also
identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the bio-
synthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phena-
zine, along with the absence of lomofungin. This result suggests that lomo10 is responsible
for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phena-
zine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for
further investigation of phenazine metabolite biosynthesis in Streptomyces.
Citation: Zhang C, Sheng C, Wang W, Hu H, Peng
H, Zhang X (2015) Identification of the Lomofungin
Biosynthesis Gene Cluster and Associated Flavin-
Dependent Monooxygenase Gene in Streptomyces
lomondensis S015. PLoS ONE 10(8): e0136228.
doi:10.1371/journal.pone.0136228
Editor: Marie-Joelle Virolle, University Paris South,
FRANCE
Received: April 19, 2015
Accepted: July 30, 2015
Published: August 25, 2015
Copyright: © 2015 Zhang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Natural phenazine compounds, a class of secondary metabolites containing a phenazine
nucleus, are mainly produced by Streptomyces and Pseudomonas species [1,2]. Phenazines have
many biological functions, and demonstrate antimicrobial, antifungal, anti-tumor, antimalar-
ial, and antiparasitic activities. The phenazine compounds produced by Pseudomonas species
usually have simple structures, such as phenazine-1-carboxyl acid (PCA) [3], 1-hydroxyphena-
zine [4], phenazine-1-carboxamide [5], and pyocyanin [6]. In addition to simple phenazines,
Streptomyces can biosynthesize phenazine derivatives with more complex structures, such as
diphenazines, terpenoidal phenazines, carbohydrate-containing phenazines, and saphenic acid
derived phenazines [1]. The biological activity of phenazine derivatives varies with the type
and number of functional groups attached to the phenazine nucleus [1,7]. For example, PCA
showed higher inhibitory activity than 1-hydroxyphenazine against plant disease pathogens
Data Availability Statement: All gene sequence files
are available from the NCBI database (accession
number KP721214.1).
Funding: This study was supported by grants from
the 863 Program of China (Grant No.
2012AA022107), the National Natural Science
Foundation of China (NSFC project No. 20706037),
and the 973 Project of China (Grant No.
2012CB721005).
Competing Interests: The authors have declared
that no competing interests exist.
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A Gene Function Research in Streptomyces lomondensis S015
such as Alternaria solani and Fusarium oxysporum [8]. Thus, investigation of side chain modi-
fication during the biosynthesis of phenazine derivatives is very important.
In Streptomyces and Pseudomonas, the biosynthetic process of phenazine products begins
with shikimic acid. In following steps, chorismic acid is synthesized through the shikimate
pathway then converted into intermediates containing a phenazine nucleus [9]. Two interme-
diates for all phenazine products, PCA and phenazine-1,6-dicarboxyl acid (PDC), are synthe-
sized via this pathway by the highly conserved phz gene cluster [9,10]. The phzABCDEFG gene
cluster was first sequenced in Pseudomonas fluorescens 2–79 [11], and has since been
sequenced in many other Pseudomonas species, including Pseudomonas aeruginosa strains
PAO1 [12] and M18 [13], and Pseudomonas chlororaphis strains PCL 1391 [14], 30–84 [15],
GP72 [16], and 2–79 [11]. The first sequenced phenazine biosynthesis gene cluster in Strepto-
myces was ephzBCDEGA, which contributes to the biosynthesis of endophenazines in Strepto-
myces cinnamonesis DSM1042 [17]. Until now, six other phz gene clusters have been described
in Streptomyces [18].
PCA is the core structure for all phenazine biosynthesis products in Pseudomonas [19],
whereas both PCA [20] and PDC [21] can form the core structure in Streptomyces. All phena-
zine derivatives are further biosynthesized from PCA or PDC by modification of the side
chains. The phenazine-modifying genes in Pseudomonas, including the phzM methyltransfer-
ase and phzS salicylate hydroxylase genes from P. aeruginosa [22], and the phzH asparagine
synthetase gene from P. chlororaphis PCL1391 [14], have been extensively studied. Because of
the complicated structure of phenazine derivatives in Streptomyces, only a few genes for phena-
zine-modifying proteins, such as the prenyltransferase genes, have been examined in Strepto-
myces [9].
Monooxygenases play an important role in oxidation reactions in a number of secondary
metabolite biosynthesis pathways in Streptomyces [23–25]. A FMN-dependent monooxygenase
is involved in dihydrokalafungin oxidation catalysis, the last step in the biosynthesis of the nat-
ural antibiotic actinorhodin in S. coelicolor [26]. A P450 monooxygenase NysL is responsible
for C-10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in S.
noursei [27]. Monooxygenases are also involved in the biosynthesis of phenazine compounds
[12,15,16]. A FAD-dependent monooxygenase PhzS from P. aeruginosa PAO1 catalyzes
hydroxylative decarboxylation of PCA to yield 1-OH-Phz [12]. phzO, a gene encodes a flavin-
dependent aromatic monooxygenase that hydroxylates PCA to produce 2-hydroxyphenazine-
1-carboxylic acid (2-OH-PCA), has been found in both P. aureofaciens 30–84 [15] and P. chlor-
oraphis GP72 [16]. Until now, no monooxygenase for the hydroxylation of phenazine com-
pounds in Streptomyces has been reported.
Lomofungin is an olive-yellow phenazine antibiotic that was first discovered in Streptomyces
lomondensis sp. n. [28]. This antibiotic has broad-spectrum antibacterial activity against both
Gram-positive and Gram-negative bacteria, as well as pathogenic fungi [29–32]. However,
despite these advantageous properties, the application of lomofungin has been limited by the
low production titer during strain cultivation. S. lomondensis S015, which can biosynthesize
lomofungin, was isolated from rhizosphere soil in Shanghai, China, by our group. We have
since worked to improve lomofungin production in this strain, both by optimization of fer-
mentation conditions and by overexpression of regulatory genes [33,34].
In this study, the lomofungin biosynthesis genes were examined after the whole genome
sequenc of S. lomodensis S105 by comparison with available known sequences in P. chlorora-
phis GP72 (GenBank: HM594285.1). In addition to the phenazine biosynthesis core gene clus-
ter, a putative flavin-dependent monooxygenase (lomo10), responsible for the hydroxylation of
lomofungin, was also identified, and was further characterized by in-frame partial deletion.
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A Gene Function Research in Streptomyces lomondensis S015
Materials and Methods
2.1 Bacterial strains, plasmids, and growth conditions
The bacterial strains and plasmids used in this study are described in Table 1 [35,36,37]. The
primers used for polymerase chain reaction (PCR) assays are described in Table 2 [36].
S. lomondensis S015 (China Center for Type Culture Collection No: M2013140) and its
mutants were cultivated at 28°C according to Wang et al. [33]. Seed cultures and fermentations
were performed using mannitol soybean (MS) medium (2% mannitol, 2% soybean powder, 2%
agar, pH 7.2) and yeast malt (YM) medium (0.4% yeast extract, 1% malt extract, 0.4% glucose,
pH 7.2), respectively.
All Escherichia coli strains were grown in Luria-Bertani (LB) medium (1% tryptone, 0.5%
yeast extract, 1% NaCl, pH 7.2) at 37°C with appropriate antibiotics, as described by Kieser
et al. [38].
2.2 DNA isolation, manipulation, and sequencing
Genomic DNA was isolated using the method described by Hopwood et al. [38], and DNA was
further manipulated according to Maniatis et al. [39,40]. PCR amplicons were isolated from
agarose gel using a DNA Gel Extraction Kit (TranGen Biotech, Beijing, China). PCRs were per-
formed in a 25 μl volume using PrimerSTAR HS DNA polymerase (Takara Bio, Dalian, China)
with genomic DNA as template. PCR products were purified using an EasyPure PCR Purifica-
tion Kit (TranGen Biotech). Primers were synthesized by Invitrogen, Shanghai, China. DNA
was sequenced by Huada, Shenzhen, China.
2.3 Genome and protein sequence analysis
The lomofungin biosynthesis gene cluster was identified from the whole genome sequencing
results of S. lomondensis S015 and analyzed using the antiSmash program (http://antismash.
secondarymetabolites.org, accessed on June 25th, 2013) [41]. The identified sequence was then
aligned with the phenazine biosynthesis gene cluster from P. chlororaphis GP72 [3,18] for con-
firmation. The sequences of the surrounding genes were subjected to similarity comparisons
and functional predictions using the BLAST program of the NCBI GenBank database (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). The website was accessed on April 10th, 2013.
2.4 Construction of the lomo10 deletion mutant strain DCC601
The lomo10 gene was disrupted using the pKC1139-lomo10 inactivation plasmid. Two flanking
regions or “arms” (1,996 and 1,784 bp), containing the upstream and downstream regions of
lomo10, were amplified by PCR (30 cycles, 98°C for 10 s, 55°C for 15 s, and 68°C for 2 min)
from S. lomondensis S015 genomic DNA using the primers lomo10 left arm For/Rev and
lomo10 right arm For/Rev, respectively. The resulting products were individually ligated into
the TA cloning vector pMD19-T to yield pCC601 and pCC602, respectively. Both insertions
were verified by DNA sequencing. The downstream fragment from pCC602 was then excised
using HindIII and BglII and ligated into the corresponding restriction sites in pCC601 to gener-
ate pCC603. The complete 3780 bp fragment from pCC603 was then excised and ligated to the
corresponding HindIII and XbaI sites of pKC1139, generating inactivation plasmid pKC1139-
lomo10.
pKC1139-lomo10 was first introduced into E. coli ET12567(pUZ8002) via heat shock trans-
formation to generate the donor strain, then introduced into S. lomondensis S015 by conjuga-
tion [42]. Following incubation of the transconjugants at 28°C for 18 h, 1 ml of sterile water
containing nalidixic acid and apramycin, both at a final concentration of 50 μg/ml, was spread
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A Gene Function Research in Streptomyces lomondensis S015
Table 1. Bacterial strains and plasmids used in this study.
Strains
Characteristics
Reference/
source
Escherichia coli
DH5α
Host for general cloning
Donor strain for intergeneric conjugation KmR, CmR
TranGen Biotech
MacNeil et al. [35]
ET12567
(pUZ8002)
Streptomyces lomondensis
S015
Wild-type, lomofungin-producing strain
Our laboratory
This study
DCC601
DCC602
Plasmids
pKC1139
pIB139
Δlomo10 mutant
lomo10-complemented strain DCC601 (containing pIB139-lomo10), Aprr
This study
E. coli-Streptomyces shuttle vector temperature-sensitive, Aprr
Bierman et al. [36]
E. coli-Streptomyces erythraea integrative shuttle vector containing a strong constitutive PermE* promoter,
Wilkinson et al.
[37]
Aprr
pMD19-T
General cloning plasmid, Amr
Takara Bio
This study
This study
This study
This study
pCC601
Left arm of lomo10 subcloned into pMD19-T, Amr
Right arm of lomo10 subcloned into pMD19-T, Amr
Left and right arms ligated into pMD19-T, Amr
pCC602
pCC603
pKC1139-lomo10
Plasmid for in-frame partial deletion of lomo10, left and right arms ligated into pKC1139 at the HindIII/BglII/
XbaI sites, Aprr
pCC604
lomo10 ligated into pMD19-T, Amr
This study
This study
pIB139-lomo10
Vector for lomo10 complementation, Aprr
doi:10.1371/journal.pone.0136228.t001
onto the surface of the MS plate. Transconjugants were incubated for a further 2–4 days at
28°C, and resulting colonies were streaked onto solid MS medium containing 50 μg/ml apra-
mycin at 37°C to yield single-crossover homologous recombination mutants. To inactivate
lomo10, single crossover mutants were cultured at 37°C with shaking at 220 rpm for 3 days in a
250 ml flask containing 50 ml YM liquid medium. These mutants were cultured for five succes-
sive generations without apramycin to generate a double cross-over mutant that was sensitive
to apramycin. In-frame deletion of lomo10 in the resulting positive mutant was confirmed by
PCR (30 cycles, 98°C for 10 s, 55°C for 15 s, and 68°C for 2 min) with primers lomo10 left arm-
For and lomo10 right arm-Rev.
Table 2. Primers used in this study.
Primers
Sequence (50–30)
Enzyme sites
lomo10 left arm-For
lomo10 left arm-Rev
lomo10 right arm-For
lomo10 right arm-Rev
lomo10-For
AAATTTGAATTCTGGATGATCGCGACGATTTC
AAAAAAAAAAAAATTTAGATCTCATCTCCTGCAGACCCCGAGT
AAATTTAGATCTGAGTTCGTCAAGGTCAGCTCCC
AAATTTTCTAGACAGTCGGGGAAGCACTTGAG
ACGCATATGGTGGTGCTCGGGGCCAGCATCG
ACGTCTAGATTCCGATTTCTCAGCGCTGTC
TTGCGCCCGATGCTAGTCG
HindIII
BglII
BglII
XbaI
NdeI
XbaI
lomo10-Rev
pIB-F [36]
pIB-R [36]
GCACGACAGGTTTCCCGACTG
doi:10.1371/journal.pone.0136228.t002
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A Gene Function Research in Streptomyces lomondensis S015
2.5 Construction of the lomo10 self-complementation strain DCC602
The 1,326 bp lomo10 region was amplified (30 cycles, 98°C for 10 s, 55°C for 15 s, and 68°C for
1 min) from S. lomondensis S015 genomic DNA using the primers lomo10-For and lomo10-
Rev. The PCR product was gel-purified and ligated into pMD19-T to form plasmid pCC604,
which was confirmed by DNA sequencing. The lomo10 fragment was then excised from
pCC604 using restriction enzymes NdeI/XbaI, and ligated into the corresponding sites of the
integrative vector pIB139 [37] to yield the self-complementary plasmid pIB139-lomo10.
pIB139-lomo10 was then introduced into the S. lomondensis S015 lomo10 deletion mutant
strain, DCC601, by conjugation from E. coli ET12567(pUZ8002). A positive exconjugant was
obtained by apramycin resistance screening and confirmed by PCR amplification and DNA
sequencing using the primer pair pIB-F/pIB-R [43] and the thermal cycler parameters men-
tioned above.
2.6 High performance liquid chromatography (HPLC) analysis
Phenazine products in wild-type S015, lomo10-inactivated mutant DCC601, and lomo10-com-
plemented strain DC602 were analyzed by HPLC as described by Wang et al. [33]. For sam-
pling, up to 5 ml of culture broth were centrifuged at 10,800 × g for 8 min. The supernatant
was adjusted to pH 2.0 using an aqueous HCl solution (6 M), and mixed with 5 ml of pure
butanone. The resulting mixture was centrifuged at 10,800 × g for 5 min and the upper layer
was collected. The water layer (lower layer) was extracted a second time using 5 ml of pure
butanone, and the combined extracts were dried using a rotary vacuum dryer (Christ RVC
2–18, Osterode, Germany) at 33°C. The resulting residue was dissolved in 5 ml of HPLC grade
solvent, a 1:1 (v/v) mixture of 0.1% formic acid and acetonitrile, and filtered through a 0.22 μm
polyvinylidene difluoride syringe filter (Millipore, Shanghai, China). A 20:l sample of the
resulting filtrate was analyzed by HPLC using an Agilent 1260 HPLC system (Agilent, Beijing,
China) equipped with a DAD detector and an Agilent Eclipse Plus C18 column (250 × 4.6 mm;
5 μm), which was used at 30°C. The mobile phase consisted of solvents A (0.1% formic acid)
and B (acetonitrile), which were used with the following gradient profile: 0–4 min, 80–60% A;
4–20 min, 60% A; and 20–30 min, 60–80% A. The HPLC system was operated at a constant
flow rate of 1 ml/min.
2.7 Purification and structural analysis of the new phenazine product
Mutant DCC601 was used for large-scale fermentation (10 l) in yeast extract-malt extract
broth at 28°C and 180 rpm for 96 h [33]. The liquid culture was centrifuged at 7,104 × g for 30
min, and then adjusted to pH 2.0 using an aqueous HCl solution (6 M). The supernatant was
extracted four times with 2.5 l butanone. All four supernatants were combined and then con-
centrated using a vacuum evaporator at 33°C to remove the organic phase. For further purifica-
tion, the raw extract was dissolved in 0.1% formic acid/acetonitrile (1:1, v/v) and purified by
preparative HPLC using an Agilent 1200 series apparatus with a C18 column (ZOBRAX-C18
column, 5 μm, 10.0 × 250 mm, Elite, Dalian, China). The mobile phase consisted of 0.1% for-
mic acid/acetonitrile (60:40, v/v) with a flow rate of 1 ml/min (detection by absorbance at 270
nm). The peak containing the pure compound was collected and dried under vacuum and
freezing.
The dried intermediate metabolite was then used for further liquid chromatography-high
resolution mass spectrometery (LC-HRMS) and nuclear magnetic resonance (NMR) analyses.
The LC-HRMS was performed on a Waters ACQUITY UPLC system (Waters Corporation,
Milford, MA) with an ACQUITY BEH C18 column (100 mm × 2.1 mm, 1.7 μm; Waters). The
column was eluted with gradient solvent of 20:80 solutions B:A (0–15 min), 40:60 solutions B:
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A Gene Function Research in Streptomyces lomondensis S015
A (15.01–25 min), and 20:80 solutions B:A (25.01–35 min) at a flow rate of 0.40 ml/min, where
solution A is 0.1% (v/v) formic acid and solution B is acetonitrile. The system was monitored
by measuring UV absorbance at 270 nm. The mass spectrometer was run in positive ion detec-
tion mode set to scan between 50 and 1,000 m/z. The NMR assay was performed with a Bruker
NMR spectrometer (Avance III 600 MHz; Bruker, Karlsruhe, Germany).
Results
Analysis of lomofungin biosynthesis genes based on whole genome
sequence of S. lomondensis S015
Sequencing and assembly of the S. lomondensis S015 genome resulted in a draft genome size of
9,448,526 bp and a GC content of 71.7%. The antiSmash program and alignment of the whole
genome sequence with the phenazine biosynthesis gene cluster of P. chlororaphis GP72 [3,18]
identified a putative gene cluster for the biosynthesis of lomofungin. This contiguous DNA
region contains 23 open reading frames (Fig 1, Table 3). Six of the genes, designated
lphzGFEDCB, showed high similarity (53.4–72.2%) at the amino acid level to the phenazine
biosynthesis genes phzBCDEFG of P. chlororaphis GP72 [3,18]. Except for the absence of a
phzA ortholog, and the order of the genes, the high similarity of these six genes to the corre-
sponding genes in P. chlororaphis GP72 suggested a close evolutionary relationship with this
species.
Sequences from Streptomyces lomondensis S015 have been deposited in the NCBI GenBank
database under accession number KP721214.
A set of eight genes, designated lomo1–8, were most likely to code for enzymes relating to
the shikimate pathway [2]. lomo9 showed 95% amino acid similarity to the acyl-CoA dehydro-
genase gene from Streptomyces sp. NRRL S-646, which oxidizes branched-chain acyl-CoA fatty
acid derivatives and macrolide antibiotics in Streptomyces coelicolor and Streptomyces avermiti-
lis [44]. lomo10 showed 95% amino acid similarity to monooxygenases genes from Streptomy-
ces acidiscabies and Streptomyces noursei, which are responsible for hydroxylation during the
biosynthesis of thaxtomin A [9] and the polyene macrolide antibiotic nystatin, respectively
[27,45]. lomo11 codes for a protein with moderate similarity (39%) to a putative methyltrans-
ferase that has been reported to catalyze the conversion of macrocin to tylosin [46]. Both
lomo12 and lomo17 showed high similarity to the transcriptional regulatory genes in Strepto-
myces sp. NRRL S-646 and they both belong to the AsnC family.
Inactivation and self-complementation of lomo10
As there are three hydroxyl groups included in the structure of lomofungin, the function of the
putative hydroxylation gene lomo10 was investigated by generating an in-frame partial deletion
mutant, DCC601, via double-crossover homologous recombination (Fig 2A). Strain DCC601
was obtained by deletion of the 1032 bp in the coding region of lomo10. No promoter was
found in this region by using online software (http://www.fruitfly.org/seq_tools/promoter.
html) analysis. Thus, the partial deletion of lomo10 might not affect the expression of other
genes, such as lomo11. The genotype of the DCC601 lomo10 deletion mutant, as well as that of
the lomo10 self-complementation strain DCC602, was confirmed by PCR analysis as shown in
Fig 2B and 2C, respectively and DNA sequencing. The phenotypes of wild-type strain S015,
DCC601, and DCC602 were examined following culture on solid MS medium (Fig 3). Wild-
type strain S015 produced the olive-yellow lomofungin (Fig 3A), whereas the lomo10 deletion
mutant (DCC601) produced light purple colored colonies (Fig 3B). Complementation with
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A Gene Function Research in Streptomyces lomondensis S015
Fig 1. Putative lomofungin biosynthesis gene cluster in Streptomyces lomondensis S015.
doi:10.1371/journal.pone.0136228.g001
lomo10 restored lomofungin production (Fig 3C). There was no obvious difference in the phe-
notype of mycelium between WT, mutant strain DCC601 and complementary strain DCC602.
The HPLC profiles of the fermentation products of wild-type S015, DCC601, and DCC602
are illustrated in Fig 4. The peak corresponding to lomofungin appeared at a retention time of
17.3 min in the wild-type strain, but was eliminated in the knockout mutant, and a new peak
with a retention time of 14.6 min appeared. Comparison of the HPLC profiles of the fermenta-
tion products of extracts from complementation strain DCC602 showed that lomofungin pro-
duction had been restored in this strain. The new compound produced by the mutant strain,
Table 3. Genes in the putative lomofungin biosynthesis cluster.
ORFs
aa
Proposed function
Identity (%)
Ortholog identified by BLAST search
lomo1
lomo2
lomo3
lomo4
lomo5
lomo6
lomo7
lomo8
lphzG
201
378
272
306
203
398
201
162
213
278
598
111
368
160
354
442
273
342
484
314
340
435
337
Shikimate kinase
88%
97%
78%
90%
71%
90%
78%
85%
53%
66%
63%
65%
60%
72%
95%
48%
39%
94%
91%
89%
72%
86%
86%
Streptomyces fulvoviolaceus
Streptomyces fulvoviolaceus
Streptomyces sp. NRRL B-5680
Streptomyces sp. NRRL S-646
Streptomyces albaduncus
Chorismate synthase
3-phosphoshikimate- 1-carboxyvinyltransferase
5,10-methylenetetrahydrofolate reductase
Hexokinase
S-adenosylmethionine synthetase
Quinone oxidoreductase
Flavin reductase
Streptomyces sp. NRRL S-646
Streptomyces anulatus
Streptomyces sp. NRRL S-646
Pseudomonas chlororaphis GP72
Pseudomonas chlororaphis GP72
Pseudomonas chlororaphis GP72
Pseudomonas chlororaphis GP72
Pseudomonas chlororaphis GP72
Pseudomonas chlororaphis GP72
Streptomyces sp. NRRL S-646
Streptomyces viridosporus
phzG
lphzF
phzF
lphzE
phzE
lphzD
phzD
lphzC
phzC
lphzB
phzB
lomo9
lomo10
lomo11
lomo12
lomo13
lomo14
lomo15
lomo16
lomo17
Acyl-CoA dehydrogenase
Flavin-dependent monooxygenase
Methyltransferase
Streptomyces auratus
Transcriptional regulator
Transporter
Streptomyces sp. NRRL S-646
Streptomyces sp. NRRL S-646
Streptomyces sp. NRRL S-646
Streptomyces antibioticus
Alcohol dehydrogenase
EsmB1, phenazine antibiotic biosynthesis protein
Aldehyde dehydrogenase
Transcriptional regulator
Streptomyces sp. NRRL S-646
Streptomyces sp. NRRL S-646
doi:10.1371/journal.pone.0136228.t003
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A Gene Function Research in Streptomyces lomondensis S015
Fig 2. Inactivation and self-complementation of lomo10 in Streptomyces lomondensis S015. (A) Schematic of the in-frame partial deletion of 1032 bp in
lomo10 to generate the Δlomo10 mutant DCC601. Primers 1, 2, 3, and 4 were used to amplify the left and right homology arms. GTG and TGA were the start
and termination codons for lomo10, respectively. The expected PCR product from the wild-type (WT) strain was 5,106 bp, and that from DCC601 was 4,074
bp using the primers lomo10 left arm-For and lomo10 right arm-Rev. (B) PCR analysis of WT strain and DCC601. (C) PCR analysis of DCC601 and lomo10
complementation strain DCC602. The amplicon generated from the DCC602 genomic DNA gave the expected 1,726 bp fragment, but no band was amplified
from the DCC601 strain.
doi:10.1371/journal.pone.0136228.g002
compound A, was purified, and its structure was characterized by LC-HRMS and NMR
analyses.
The methods and results of whole-cell biotransformation of compound A by Lomo10 were
shown in S1 File and S1 Fig, respectively. After 2 h reaction, lomofungin was synthesized in the
reaction system of E.coli DH5μ/pMD-18T-lomo10 mixed with compound A. The results fur-
ther verified that Lomo10 could hydroxylates compound A to produce lomofungin.
Purification and structural analysis of compound A
To confirm the structure of the new compound, compound A was purified from 10 l of fermen-
tation broth. In total, 80 mg of compound A was obtained following purification. The exact
Fig 3. Photographs of the different strains grown on MS medium. (A) Wild-type strain. (B) lomo10
inactivated mutant DCC601. (C) lomo10 complementation strain DCC602.
doi:10.1371/journal.pone.0136228.g003
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A Gene Function Research in Streptomyces lomondensis S015
Fig 4. HPLC profiles of fermentation products from different strains. (A) Wild-type strain. (B) lomo10
inactivated mutant DCC601. (C) lomo10 complementation strain DCC602. lomofungin (Rt = 17.3 min),
compound A (Rt = 14.6 min).
doi:10.1371/journal.pone.0136228.g004
mass of compound A obtained from LC-HRMS was 299.07 for [C15H10N2O5]+ (calculated
mass, 298.25; Fig 5). This calculated mass was 16 Da smaller than the known mass of lomofun-
gin, suggesting that compound A contained one less hydroxyl than lomofungin (C15H10N2O6,
MW 314 Da) [47]. Structural analysis was performed by 1H NMR, 13C NMR, distortionless
enhancement by polarization transfer-90, 1H-1H correlation spectroscopy, heteronuclear mul-
tiple bond correlation, and heteronuclear multiple quantum coherence analyses, and the pro-
ton and carbon chemical shifts of compound A are shown in Table 4 and in S2 Fig,
respectively. The 1H-NMR spectra contained four proton signals (δH, 7.39–8.41 ppm) that are
typical of double bonds (ring hydrogen), along with corresponding 13C-NMR spectra of carbon
atoms (δC, 110.1–134.5 ppm), suggesting that compound A has a phenazine ring in the core of
its structure. Compared with the reported NMR data for lomofungin [47], the main differences
were the absence of a hydroxyl hydrogen at δH 11.22, and the presence of hydrogen ring at δH
8.27. Therefore, compound A was predicted to be 1-carbomethoxy-6-formy-4,9-dihydroxy
phenazine, a new chemical compound missing the C-7 hydroxyl of lomofungin (Fig 6).
Discussion
Phenazine biosynthesis gene clusters are normally composed of seven genes arranged in order
(e.g. phzABCDEFG), and have been located downstream of shikimate pathway-related genes in
Pseudomonas species including P. fluorescens 2–79, P. aureofaciens 30–84, and P. chlororaphis
PCL1391 [11,14,15]. Two nearly identical core phz gene clusters, called phzA1-G1 and
phzA2-G2, with different promoters and flanking regions, have also been found in Pseudomo-
nas sp. M18 [48] and P. aeruginosa PAO1 [12].
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A Gene Function Research in Streptomyces lomondensis S015
Fig 5. Spectrum of compound A following LC-HRMS analysis. The caculated mass of compound 1 was
determined to be 298.25 (m/z 299.07).
doi:10.1371/journal.pone.0136228.g005
Compared with Pseudomonas, the number and order of genes within the phenazine biosyn-
thetic gene cluster in Streptomyces are more varied. Streptomyces anulatus 9663 also has a
seven-gene cluster for the biosynthesis of endophenazine A and endophenazine B, but unlike
the corresponding region in Pseudomonas species, phzA is located at the end of the cluster:
ppzBCDEFGA [9,49]. A six-gene cluster for prenylated phenazine biosynthesis in Streptomyces
cinnamonensis DSM 1042 is ordered ephzBCDEGA, and completely lacks the gene encoding
the PhzF protein [17]. Another gene cluster consisting of just epzAGFC was identified in S. cin-
namonensis DSM 1042, and is also involved in prenylated phenazine biosynthesis [50]. The
whole genome sequencing performed in the current study showed that the phenazine biosyn-
thetic gene cluster in S. lomondensis S015 is structured lphzGFEDCB, and while still located
downstream of the shikimate pathway-related genes, the gene order is completely reversed
compared with the cluster in Pseudomonas, and lacks phzA. In addition, we found another two
phenazine biosynthesis-related genes, phzC2 and phzE2, in another scaffold of the whole
genome of S. lomondensis S015. Further study is needed to confirm whether they are involved
in the biosynthesis of lomofungin.
Until now, monooxygenases for the hydroxylation of phenazine compounds have been
identified only in Pseudomonas. Most of these monooxygenases use PCA as their substrate
[12,15,16]. In the current study, alignment of the lomo10 amino acid sequence showed that
lomo10 encodes a putative flavin-dependent monooxygenase. Inactivation of lomo10 in S.
lomondensis S015 resulted in the production of a novel phenazine product containing a dele-
tion of the hydroxyl at the C-7 position of lomofungin. The four monooxygenases that cata-
lyzed the hydroxylation of phenazines were compared and analyzed using DNASTAR
Lasergene.v7.1 software and the results were shown in S3 Fig The monooxygenases in P. aureo-
faciens 30–84 and P. chlororaphis GP72 showed very high similarity and they both catalyze the
hydroxylation of PCA at its C-2 position. The monooxygenase in S. lomondensis S015 showed
low similarity to others might due to the differences in substrate structure and hydroxylation
position because it hydroxylates compound A at its C-7 position.
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A Gene Function Research in Streptomyces lomondensis S015
Table 4. Chemical shift summarized from 1H (DMSO-d6) and 13C (MeOD-d6) analyses recorded by 600 MHz NMR spectrometry.
1H NMR
Chemical shift δ (ppm)
13C NMR
Chemical shift δ (ppm)
-COOCH3
C-2
3.95, s
8.41, d
7.30, d
8.28, d
7.39, d
11.40,s
C-1
C-2
120.9
134.4
111.2
156.5
141.3
135.2
123.0
134.5
110.1
160.2
133.4
139.8
190.7
52.1
C-3
C-3
C-7
C-4
C-8
C-4a
C-5a
C-6
-CHO
C-7
C-8
C-9
C-9a
C-10a
-CHO
-COOCH3
-COOCH3
166.2
doi:10.1371/journal.pone.0136228.t004
There are three hydroxyl groups in lomofungin, located at the C-4, C-7, and C-9 positions.
Only the C-7 hydroxyl was deleted in the lomo10 mutant strain DCC601, suggesting that there
might be other hydroxylation genes in S. lomondensis S015. Two P450 monooxygenases,
named lomo56 and lomo57, were located downstream of the lomofungin biosynthesis gene
cluster in S. lomondensis S015. These two genes may be involved in the transfer of the C-4 and
C-7 hydroxyl groups of lomofungin.
Based on the alignment of the S. lomondensis S015 genes and the other results obtained in
this study, a putative lomofungin biosynthesis pathway is shown in Fig 7. Buckland et al. [51]
confirmed that lomofungin is biosynthesized from PDC. Thus, here we propose that the
lphzGFDECB gene cluster can biosynthesize PDC. The new compound obtained in this study,
1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine (compound A), might be synthesized
from PDC in several steps catalyzed by Lomo9 and Lomo11 et al., and then converted into
lomofungin by lomo10. Further validation of this pathway is required.
Fig 6. Chemical structure of compound A.
doi:10.1371/journal.pone.0136228.g006
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Fig 7. Proposed lomofungin biosynthetic pathway in Streptomyces lomondensis S015.
doi:10.1371/journal.pone.0136228.g007
Supporting Information
S1 Fig. HPLC profiles of whole-cell biotransformation of compound A by E.coli DH5μ/
pMD-18T-lomo10. (A) Lomofungin standard. (B) Biotransformation system with Lomo10.
(C) Control without Lomo10.
(TIF)
S2 Fig. NMR spectra of Compound A. (A) 1H NMR spectrum in DMSO-d6. (B) 13C NMR
spectrum in MeOD. (C) COSY spectrum in DMSO-d6. (D) HMBC spectrum in DMSO-d6.
(TIF)
S3 Fig. Amino acid blast of monooxygenases among different stains.
(TIF)
S1 File. Method of biotransformation of compound A.
(PDF)
Acknowledgments
The authors express their appreciation to the Instrumental Analysis Center of Shanghai Jiao
Tong University, particularly Dr. Lei Feng, for help with LC-HRMS analysis. We also thank
Prof. Shuangjun Lin for help analyzing the NMR results.
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A Gene Function Research in Streptomyces lomondensis S015
Author Contributions
Conceived and designed the experiments: CZ WW XZ. Performed the experiments: CZ CS
WW. Analyzed the data: CZ CS WW. Contributed reagents/materials/analysis tools: CZ CS
WW XZ HP. Wrote the paper: CZ CS WW XZ HH.
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