World J Microbiol Biotechnol

DOI 10.1007/s11274-015-1903-5

ORIGINAL PAPER

Production and secretion of naphthoquinones is mediated

by the MFS transporter MFS1 in the entomopathogenic fungus
Ophiocordyceps sp. BCC1869
Pratoomporn Khaokhajorn1,2 • Sompid Samipak2 • Sutichai Nithithanasilp1 •

Morakot Tanticharoen1,3 • Alongkorn Amnuaykanjanasin1

Received: 17 November 2014 / Accepted: 14 July 2015

Ó Springer Science+Business Media Dordrecht 2015

Abstract Naphthoquinones are deep red polyketide pig-
ments produced by the ant-pathogenic fungus Ophiocordy-
ceps sp. BCC1869. In culture, biosynthesis of these
naphthoquinones remains at a low level during the first
20 days and reaches its maximum production level at
approximately 50 days. The MFS transporter gene MFS1
was previously identified in Ophiocordyceps sp. BCC1869
from a subtractive EST library between the fungus grown
under naphthoquinone-inductive and naphthoquinone-re-
pressive conditions. We cloned and sequenced this trans-
porter gene, which has an open reading frame of 1505 bp and
three introns (48, 52, and 58 bp). Phylogenetic analysis
showed this MFS transporter was tightly clustered with
fungal riboflavin transporters. Functional analysis of this
gene was performed by overexpression of MFS1 under the
control of a strong, constitutive promoter. We successfully
transformed the fungus with this overexpression plasmid
using PEG-protoplast transformation, which generated nine
transformants per lg of plasmid. RT-PCR indicated that the
MFS1 expression level in the overexpressing strains
increased 3- to 10-fold compared to the wild type. HPLC
analysis of crude extracts of mutants and wild type demon-
strated that four naphthoquinone derivatives, erythrostomi-
none, epierythrostominol, deoxyerythrostominone, and
deoxyerythrostominol, were the major naphthoquinones
produced and excreted in staggering quantities (20- to
2300-fold) in 7-day old liquid cultures by the mutant C7,
compared to the wild type. High resolution electrospray
ionization mass spectrometry verified mass spectra of these
purified metabolites. Three other naphthoquinone deriva-
tives, whose structures have not been identified, were also
detected in high amount in the mutant liquid cultures.
Keywords Naphthoquinone  MFS transporter 
Overexpression  Ophiocordyceps  Secretion 
Erythrostominone

Electronic supplementary material The online version of this

article (doi:10.1007/s11274-015-1903-5) contains supplementary
material, which is available to authorized users. Introduction

& Alongkorn Amnuaykanjanasin Ophiocordyceps sp. BCC1869 is an insect pathogenic fungus

alongkorn@biotec.or.th
that specifically infects ants. The species name has been
1
Bioresources Technology Unit, National Center for Genetic revised from O. unilateralis following molecular reclassifi-
Engineering and Biotechnology (BIOTEC), National Science cation of isolates in this genus (Kobmoo et al. 2012). This
and Technology Development Agency (NSTDA), 113 fungus produces six red naphthoquinone derivatives in cul-
Thailand Science Park, Thanon Phahonyothin, Tambon
Khlong Nueng, Amphoe Khlong Luang, Pathumthani 12120,
ture: erythrostominone, deoxyerythrostominone, 4-O-methyl
Thailand erythrostominone, epierythrostominol, deoxyerythrostomi-
2 nol, and 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-di-
Department of Genetics, Faculty of Science, Kasetsart
University, Bangkok 10900, Thailand enyl)-1,4-naphthoquinone (3,5,8-TMON) in culture
3 (Kittakoop et al. 1999). There have been attempts to enhance
School of Bioresources and Technology, King Mongkut’s
University of Technology Thonburi, Bangkok 10140, naphthoquinone production in this fungus by optimization of
Thailand culture conditions, including aeration, initial pH of medium,

123

and temperature (Unagul et al. 2005) or carbon and nitrogen
sources (Prathumpai et al. 2007). Production of naphtho-
quinones is widespread in fungi, although predominantly in
Fusarium species (Medentsev et al. 2005). The structures of
fungal naphthoquinones range from a simple one, 6-methyl-1,
4-naphthoquinone found in Marasmius graminum (Bendz
1948) to a more complex structure like dermocanarin found in
Dermocybe canaria (Gill and Gimenez 1995). Fungal naph-
thoquinones have a broad range of biological activities,
including antibacterial (Haraguchi et al. 1997), insecticidal
(Medentsev and Akimenko 1998), and anti-malarial activities
(Kittakoop et al. 1999). Interestingly, the chemical structure of
the naphthoquinones produced by Ophiocordyceps sp.
BCC1869 is similar to that of the commercial red pigments
shikonin and alkanin, which were derived from the root of
Alkanna tinctoria (Papageorgiou et al. 1979) and Lithosper-
mum erythrorhizo (Mizukami et al. 1978). Shikonin and
alkanin have been widely used in pharmaceuticals as wound-
healing agents and anti-microbial compounds (Papageorgiou
et al. 2008).
Transport systems are crucial in the uptake of essential
nutrients and ions as well as the export of waste compounds
and secondary metabolites. Two major classes of trans-
porters are the ATP-binding cassette (ABC) transporter
family and the major facilitator superfamily (MFS) (Ster-
giopoulos et al. 2002). The MFS transporters are a ubiquitous
group of proteins. They are composed of 12-14 hydrophobic
transmembrane domains (TMDs) (Paulsen et al. 1996; Pao
et al. 1998). MFS transporters are involved in transport of
sugars, drugs, polyols, vitamins, neurotransmitters, Krebs-
cycle metabolites, phosphorylated glycolytic intermediates,
amino acids, peptides, osmolites, iron-siderophores, nucle-
osides, anions, and cations (Stergiopoulos et al. 2002). In
filamentous fungi, MFS transporters are also involved in the
transport of secondary metabolites, including toxins; such is
the case for fumonisins and cercosporin produced by
Fusarium verticillioides and Cercospora nicotinaea
respectively (Proctor et al. 2003; López-Errasquı́n et al.
2006; Choquer et al. 2007; Amnuaykanjanasin and Daub
2009). Export of toxins can be regarded as a mechanism of
self- protection of the producing organism, which leads to
reduction of intracellular toxin accumulation (Gardiner et al.
2005). The export of toxins in pathogenic fungi might act as
virulence factors and, therefore, toxin transporters could be
useful targets to devise strategies that reduce disease and
toxin production (Del Sorbo et al. 2000; Stergiopoulos et al.
2002). In F. fujikuroi, the MFS transporter gene bik6 in the
biosynthetic cluster is involved in the biosynthesis of bika-
verin, a fungal naphthoquinone pigment (Wiemann et al.
2009). Overexpression of transporter genes could enhance
production levels of target compounds in producing
microbes. For instance, the MFS transporter CefT is located
near the polyketide synthase gene in the penicillin
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World J Microbiol Biotechnol
biosynthetic cluster. In one cefT-overexpressing transfor-
mant where three extra copies of this transporter gene were
integrated in the genome of the fungus Acremonium
chrysogenum, the level of penicillin N was increased by
100 % (Ullán et al. 2002). In the bacterium Pseudomonas sp.
M18, when genes for a three-component ABC export system,
pltHIJKN, were overexpressed, the production of antibiotic
pyoluteorin could be significantly increased (Huang et al.
2006). Strikingly, overexpression of a transporter gene is
naturally found in many microbial and cancer cells that are
resistant to therapeutic drugs. Overexpression of the ABC
transporters Cdr1p and Cdr2p led to clinical azole resistance
in Candida albicans (Tsao et al. 2009). Increased-tolerant
mutants of Staphylococcus aureus overexpressing mepA,
mdeA, norA and norC appeared after exposure of clinical
isolates to low concentrations of biocides and dyes, even in a
single exposure (Huet et al. 2008).
Previous work in our laboratory identified transporter
genes that could be involved in efflux of naphthoquinones
using suppressive subtractive hybridization (SSH). This
method could enrich the Ophiocordyceps sp. genes
specifically found in the naphthoquinone production stage,
but not (or less) in the non-production stage. Two detected
transporter genes encode the monocarboxylate transporter
MFS1 and the ABC transporter ABC1. Expression of
MFS1 was increased 29-fold in the production stage
compared with that in the non-production stage (Amnu-
aykanjanasin et al. 2011). In this study, MFS1 was over-
expressed to enhance production of naphthoquinones in the
insect pathogenic fungus, Ophiocordyceps sp. BCC1869.
Materials and methods
Fungal and bacterial cultures and conditions
Ophiocordyceps sp. BCC1869 was obtained from the BIO-
TEC Culture Collection (BIOTEC, Thailand) and maintained
on potato dextrose agar (PDA; Thermo Scientific, USA) at
25 °C. The fungus does not produce conidia on potato dex-
trose medium or other standard culture media. For liquid
cultures, seven hyphal plugs (5 mm diameters) from a 7-day
old PDA culture were inoculated into a 50 mL PD broth in
250 mL Erlenmeyer flask. A liquid culture was shaken on a
rotary shaker at 150 rpm, 25 °C for 7 days and a solid culture
was incubated at the same temperature for 14 days. The
mycelia were used for genomic DNA extraction. For total
RNA isolation, the fungus was grown on a 145 mm diameter
petri dish filled with a 50 mL solid medium. Mycelia for
protoplast preparation were grown in the Grace’s insect
medium (Life Technologies, USA). This culture medium was
used for generation of blastospores of insect pathogenic fungi
(Wongsa et al. 2005). The fungal mycelia were dislodged in
World J Microbiol Biotechnol
sterile water from a 7-day old PDA culture and used as an
inoculum. Five hundred lL of this mycelial suspension was
inoculated into 2.5 mL of the Grace’s insect medium and the
culture was incubated at 25 °C for 24 h. The young mycelia
were then collected by centrifugation at 9450g, rinsed twice
with sterile water, and homogenized with Silamat S6 (Ivoclar
Vivadent, Principality of Liechtenstein). The homogenized
mycelium was resuspended in 1 mL of the Grace’s insect
medium, then mixed with 2 mL of the same culture medium,
and incubated under the same conditions.
Recombinant plasmid for fungal transformation was
constructed based on the recipient vector, pTxA (previ-
ously constructed by Amnuaykanjanasin and Daub (2009))
containing the hygromycin B phosphotransferase gene
(hph) for selection in fungi. Escherichia coli strain DH5-a
was used to maintain plasmid vector. Standard molecular
methods (Sambrook and Russel 2001) were used for
purification of plasmid DNA, restriction enzyme analysis,
and cloning.
Cloning of the MFS transporter gene MFS1
in Ophiocordyceps sp. BCC1869
Genomic DNA was isolated from mycelium using the
method previously described (Reader and Broda 1985).
Chromosome walking of the transporter gene MFS1 was
performed using inverse PCR (Keim et al. 2004) with two
primers, MFS-Inv-1 (50 -GGACCATATCGATCGTAGAC
TCTG-30 ) and MFS-Inv-2 (50 -CATCGTTGCAGCTG
GATCTAGC-30 ) derived from the originally identified
MFS1 sequence (Amnuaykanjanasin et al. 2011). PCR
reaction was performed using High Fidelity Taq DNA
Polymerase (Thermo Scientific, USA). The thermal cycling
program for inverse PCR consisted of: 5 min of 95 °C; 5
cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for
1 min; and 30 cycles of 94 °C for 30 s, 55 °C for 1 min
and 72 °C for 1 min, followed with the final extension at
72 °C for 10 min. The full-length open reading frame of
MFS1 was submitted for sequencing at Macrogen (South
Korea). DNA and protein sequence analyses were per-
formed using BLAST software (the USA’s National
Center for Biotechnology Information), ExPASy pro-
teomic tools (Swiss Institute of Bioinformatics, Geneva,
Switzerland), and TMHMM server V.2.0-CBS program
(Center for Biological Sequence Analysis, Denmark).
Nucleotide and deduced amino acid sequences of MFS1
are in the GenBank database with the accession number
KJ466963.
Construction of MFS1-overexpression plasmid
For construction of MFS1-overexpression plasmid, the
pTxA vector containing the Pyrenopora tritici-repentis
ToxA promoter (Lorang et al. 2001) was used as a recipient
vector for cloning of the MFS1 coding sequence under a
strong and constitutive promoter (Amnuaykanjanasin and
Daub 2009). The MFS1 fragment was amplified from the
Ophiocordyceps sp. BCC1869 genomic DNA using the
forward primer MFS-F-Sma (50 -CCCGGGATTGACTCT
TCAAGATGTCAG-30 ) and the reverse primer MFS-R-
Hind (50 -GCAAGCTTG- CACCAATTTACAGTGTTC
ACC-30 ). Both primers have SmaI and HindIII restriction
sites respectively, facilitating the cloning of the transporter
gene fragment into the recipient vector. The MFS1 frag-
ment was digested with SmaI and HindIII, then ligated into
SmaI and HindIII-restricted pTxA vector. The MFS1-
overexpression plasmid was then called pTxA-MFS1
(Fig. 1a).
PEG-mediated protoplast transformation
of Ophiocordyceps sp. BCC1869
Protoplast preparation and Ophiocordyceps sp. transfor-
mation were modified from the method previously descri-
bed (Tilburn et al. 1983). The young Ophiocordyceps sp.
A
hph
6.57 kb
B
kb
2.5
2.0 ToxA-MFS1
0.7 hph
0.5
M WT C1 C4 C5 C7 C8 C32 C54 P
Fig. 1 a Schematic representation of MFS1-overexpressing plasmid
pTxA-MFS1. The 1.8-kb Ophiocordyceps sp BCC1869 MFS1
fragment was inserted in SmaI and HindIII-restricted pTxA vector,
which has the Pyrenopora tritici-repentis ToxA promoter (Lorang
et al. 2001) and a selection marker, hygromycin resistance cassette
(hph). b Determination of integration of the overexpression vector in
genomes of wild type (WT) and seven hygromycin-resistant isolates
(C1, C4, C5, C7, C8, C32, and C54). Two primer pairs, ‘hygB-F and
hygB-R’ and ‘ToxA-F and MFS-R-Hind’ were used to amplify the
hph cassette and the ToxA-MFS1 overexpressing cassette respec-
tively. M, 1 kb plus DNA ladder; P, plasmid pTxA-MFS1
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BCC1869 mycelia were prepared as described above and
then rinsed with 0.7 M NaCl. For generation of protoplast,
young mycelia were digested with 40 mg mL-1 of Glu-
canex (Novozyme, Denmark) dissolved in 0.7 M NaCl.
The mycelium-wall lysing enzyme mixture was shaken at
80 rpm, 28 °C for 2 h. Protoplasts were then washed and
resuspended with STC solution (1 M sorbitol, 10 mM Tris-
HCl pH 7.0, and 10 mM CaCl2). Transformation of
Ophiocordyceps sp. BCC1869 was performed as follows;
250 lL of protoplasts (107 protoplasts mL-1) were mixed
with 5 lg of the plasmid pTxA-MFS1 and 50 lL of a
polyethylene glycol (PEG) solution (60 % PEG-4000,
10 mM Tris-HCl pH 7.0, and 10 mM CaCl2). The mixture
was incubated on ice for 20 min. Then, 200, 400, or
700 lL of the PEG solution were added into each reaction
tube separately. These transformation reactions were
incubated at room temperature for 5 min and immediately
mixed with a selective medium (PDA containing 1 M
sucrose, 50 lg mL-1 hygromycin B, and 0.7 % agar,).
Transformants were then maintained on PDA containing
50 lg mL-1 hygromycin B for any further experiments.
After two rounds of hygromycin-resistance selection,
hygromycin-resistant transformants were determined for
the integration of the overexpressing vector in the fungal
genome. Two primer pairs, ToxA-F (50 -ATAAGAATGCG
GCGGCTGGAATCCATGGAGGAGT-30 ) vs MFS-R-
Hind (described above) and hygB-F (50 -TCAGCGAG
AGCCTGACCTATTGC-30 ) vs hygB-R (50 -CGAGTACT
TCTACACAGCCATCG-30 ), were used in PCR amplifi-
cation of the ToxA-MFS1 cassette and the hph cassette in
these transformants.
Complementary DNA synthesis, cDNA sequencing,
and gene expression analysis
Total RNA was extracted from mycelia shaken in PDB for
7 days. RNA extraction method was performed as previ-
ously described (Porebski et al. 1997). Contaminating
genomic DNA was removed by treatment with DNase I
(Thermo Scientific, USA). Reverse transcription-PCR was
performed to determine expression of MFS1 in transfor-
mants compared to Ophiocordyceps wild type. Two lg of
total RNA were used in reverse transcription. Reverse
transcription was performed as follows; 1 9 reverse tran-
scription buffer, 1.5 mM MgCl2, 2 mM of each dNTP,
2.5 lM random hexamers, 1 unit of RiboLock RNase
inhibitor, and 2.5 units of RevertAid Reverse Transcriptase
(Thermo scientific, USA). The mixture was incubated at
42 °C for 90 min to generate first-strand cDNAs which
were used as template for amplification of a MFS1 gene
fragment using the primers RT-MFS1-F (50 -GATGCA
CCTCCAGTCAATCCTTGG-30 ) and RT-MFS1-R (50 -
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World J Microbiol Biotechnol
GGACCATATCGATGGTAGACTCTG-30 ). This targeted
MFS1 genomic region has one intron. In a separate tube,
the genomic DNA of Ophiocordyceps sp. BCC1869 was
used as a template. PCR products from genomic DNA and
cDNA templates were sequenced to verify the presence of
introns. As a control, total RNA was used as template in the
PCR without reverse transcription.
Quantitative RT-PCR was conducted to determine the
expression levels of MFS1 in the overexpressing mutants
and wild type grown in PDB. The quantitative RT-PCR
experiments were performed using Maxima SYBRÒ
Green/Fluorescein qPCR Master Mix (Thermo Scientific,
USA). Reverse transcription was performed as described
above. The primer pair RT-MFS1-F and RT-MFS1-R (as
described above) was used to determine MFS1 expression
level. Thermal cycling parameters were set as follows:
95 °C for 1 min; followed by 35 cycles of 95 °C for 30 s,
55 °C for 30 s, and 72 °C for 30 s; and fluorescence
acquisition at 77 °C for 1 s. The 18S rRNA primer pair
18S-F (50 -TCCGTAGGTGAACCTGCGG-30 ) and 18S-R
(50 -TCCTCCGCTTATTGATATGC-30 ) were used to
amplify 18S rRNA gene, and used as an internal control to
normalize the MFS1 expression level. Fold change relative
to wild type was calculated according to the 2-DDCT
methods (Livak and Schmittgen 2001). Quantitative RT-
PCR experiments were repeated at least twice.
Naphthoquinone extraction and HPLC analysis
Naphthoquinone quantity was determined using high per-
formance liquid chromatography (HPLC) analysis of crude
extracts from liquid (PDB) cultures (Kittakoop et al. 1999).
For liquid culture, a 7-day old PDB culture was centrifuged
at 9450g to separate the mycelial fraction from the super-
natant. These two fractions were dried with Savant-Super
Modulyo (Thermo Electron, USA) and extracted with
dichloromethane: methanol (1:1). All crude extracts con-
taining naphthoquinones were dried and initially resus-
pended in 1 mL methanol, adjusted to 5 lg mL-1.
Ten microliters of the extract were subjected to HPLC (a
WATERS e 2695 separation module with a WATERS
2998 photodiode array detector). A reverse phase C18
column (4.6 9 250 mm, 5 lm, VERTICALÒ, VertiSepTM)
was employed. The analysis condition was set to a flow rate
of 0.5 mL min-1 at wavelength of 500 nm, with a water:
acetonitrile step gradient used in the separation method and
consisting of 20 % acetonitrite for 20 min, gradient to
45 % acetonitrite for 15 min, gradient to 100 % for
15 min, and gradient to 20 % for 10 min. Comparison of
HPLC peak area was used to determine production of
naphthoquinones in wild type and mutants. The experiment
was repeated three times.
World J Microbiol Biotechnol
Mass spectrometry analysis
Crude extracts of the secreted fractions of wild type and the
overexpressing-mutant C7 were analyzed for the mass data.
Each of the major peaks was collected using HPLC. ESI-
TOF mass spectra were determined by using a micrOTOF
mass spectrometer (Bruker, Germany).
Results
MFS1 is a monocarboxylate transporter similar
to riboflavin transporters
Sequence analysis of the MFS transporter MFS1 in
Ophiocordyceps sp. BCC1869 identified a 2022 bp geno-
mic sequence. The gene has an open reading frame of
1505 bp long. MFS1 contains three introns (48, 52 and
55 bp) that were identified by comparison between its
genomic and cDNA sequences. These introns have the
characteristic 50 (GT) and 30 (AG) intron splicing sites of
eukaryotic genes (Jacobs and Stahl 1995). In our compar-
ative genome analysis, this MFS transporter gene was
searched against the sequenced genome of a closely related
ant pathogen, Ophiocordyceps polyrachis-furcata (Wicha-
dakul et al. personal communication). The result showed
that this transporter is not in a polyketide biosynthetic
cluster or a cluster for biosynthesis of a secondary
metabolite.
This transporter gene encodes a putative 449-amino acid
protein that contains 12 transmembrane domains predicted
by TMHMM program. It has an isoelectric point of 7.52
and a molecular weight of 49.08 kDa. MFS1 showed
similarity to various MFS transporters, mostly of unknown
function. The homolog of the highest similarity was Nec-
tria haematococca hypothetical protein NECHA-
DRAFT_72176 (XP_003039753.1) (64 % identity in
amino acid sequence). The sequence was also similar to
several monocarboxylate transporters (MCTs) and ribo-
flavin transporters from various fungi including insect
pathogenic fungi. The MCT transporters are involved in the
proton-linked transport of monocarboxylates (e.g., L-lac-
tate, pyruvate), the ketone bodies (Halestrap 2012), aro-
matic amino acid (Kim et al. 2001), and riboflavin (Reihl
and Stolz 2005) across the plasma membrane. In addition, a
MCT transporter gene GzMCT is located next to the
polyketide synthase gene in a zearalenone biosynthetic
cluster in Gibberella zeae (Kim et al. 2005).
We constructed a phylogeny of MFS1 and other fungal
MFS transporters, including monocarboxylate-, riboflavin
transporters and other fungal transporters of known com-
pounds. The accession numbers of all the sequences used in
this phylogenetic analysis are provided in the supplementary
table (online resource 1). In agreement with the similarity
search, the Ophiocordyceps MFS1 was tightly clustered
with a group of fungal MFS transporters, in which Saccha-
romyces cerevisiae Mch5p is basal to this clade (100 %
bootstrap; Fig. 2). ScMch5p is involved in the uptake of
riboflavin in the yeast cells (Reihl and Stolz 2005). How-
ever, the mammalian riboflavin transporters were separated
in another branch sistered to this fungal riboflavin trans-
porter group, suggesting that they may have different evo-
lutionary origins. In contrast, the MFS transporters for other
fungal compounds were placed separately from the ribo-
flavin transporters with 99 % bootstrap support. These
included the cercosporin transporter CFP in Cercospora
kikuchii and the MFS transporter Bik6 for transport of
another type of naphthoquinone, bikaverin, in Fusarium
fujikuroi (Fig. 2).
The fastidious fungus Ophiocordyceps sp. BCC1869
was successfully transformed with optimizations
Several attempts have been made for optimization of pro-
toplast preparation and transformation of this fastidious
fungus. To our knowledge, there is no report of transfor-
mation of Ophiocordyceps species. Recently, transforma-
tion of a closely-related species Cordyceps militaris was
performed (Zheng et al. 2011; Rachamawati et al. 2013).
Ophiocordyceps sp. BCC1869 grows very slowly; reaching
20–25 mm of diameter on PDA in 14 days, similar to other
species in the genus Ophiocordyceps. First, we have
determined the inhibitory concentration of hygromycin B
in this fungus. The result showed that the fungal growth
was inhibited at 50–250 lg mL-1 (data not shown). Only
hyphae from the Grace’s insect medium culture, not those
from PDB cuture, resulted in a high number of protoplasts
after the same digestion with wall-lysing enzymes
(Fig. 3a). PEG solution was added twice during transfor-
mation. First, 50 lL of PEG were added with the DNA-
protoplast mixture. Then, an extra volume of 200, 400, or
700 lL of the PEG solution was added in the final incu-
bation at room temperature, which made the final con-
centration of PEG at 30, 39, or 45 %, respectively. Seven
to nine transformants per lg of DNA were obtained in the
treatment with 39 and 45 % PEG (Fig. 3b). We then col-
lected 80 transformants after two rounds of selection on the
selective medium containing hygromycin.
MFS1-overexpressing mutants had a marked
increase in MFS1 expression levels compared to wild
type
Ten hygromycin B-resistant isolates were determined for
integration of the overexpressing vector in their genomes.
The PCR amplification results demonstrated that both the
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0.05
99 EdMCP
AgMCH5
PoMCH5
100 PbMCH5
67
AdMCH5
CmMCP
OpMFS1 ET
84
AoMCP
AcMCP
100 BbMCP
100 MoMCH5
100 BbMFS
100
CmMFS1
100 100 MaMCP
100 CmMFS2
100 BbMCT
MrMCT
ScMch5p RB
100 HmRFVT3 Riboflavin transporters
66
99 MmRFVT2 in mammals
FgTri102 TC
CkCFP CC Transporters for other
FfBik6
BK fungal compounds

OH O
O CH3 O OH O OCH3
OH O OH H H
H3C N OH H3C O
NH
O O CH3
O O CH3
H3C N N O H3CO O OCH3 O CH3 O
H3CO O H3C O H
CH2 OH O CH3
OH O OH H
H OH OCH3
H OH OH O
H OH
CH2
OH
Erythrostominone (ET) Riboflavin (RB) Bikaverin (BK) Cercosporin (CC) Trichothecene (TC)

Fig. 2 Neighbor-joining (NJ) phylogeny of Ophiocordyceps MFS conducted with 1000 replicates. Bootstrap values of 60 % or more are
transporter MFS1 and some fungal monocarboxylate transporters. provided at nodes. The polyketides, substrates of the transporters, and
Human and mouse riboflavin transporters are included as an outgroup their structures are shown at the clades
for the riboflavin transporter group. Bootstrap analysis using NJ was

ToxA-MFS1 and hph fragments were found in five isolates:
C1, C4, C5, C7, and C8 (Fig. 1b). Neither of the fragments
was detected from the isolates C32 and C54, which
appeared after 14 days post transformation. These two
isolates showed similar characteristics to the wild type in
growth, colony color, and naphthoquinone production at a
macroscopic level (Fig. 4b). Two mutants, C7 and C8,
were selected to investigate the MFS1 gene expression
level. The MFS1 gene expression analysis was performed
using RNA extracted from the fungus grown in PDB cul-
ture. The quantitative RT- PCR result indicated that the
two mutants C7 and C8 had increased MFS1 expression
levels by 10.2- and 3.2-fold respectively compared with
wild type grown in PDB culture (Fig. 4a).
Overexpression of MFS1 led to enhanced production
of naphthoquinones
The MFS1-overexpressing strains C7 and C8 produced and
secreted a noticeably increased amount of red naphtho-
quinones on 14-day old potato dextrose agar (Fig. 4b). Deep
red guttation droplets of naphthoquinones were found on
tops of the mutants’ colonies, particularly the mutant C7.
Furthermore, the hyphae of mutants C7 and C8 appeared
mostly white, compared to the purple mycelia of wild type
and the wild type-like strain C32 (Fig. 4b). Quantitative
measurement of naphthoquinones was conducted using liq-
uid cultures of wild type and the mutants. HPLC analysis of
the extracts indicated that four naphthoquinone derivatives,
erythrostominone, epierythrostominol, deoxyerythrostomi-
nol, and deoxyerythrostominone, were the major compounds
produced by the wild type and the overexpressing mutants
(Fig. 5). In addition, three unknown naphthoquinone
derivatives having similar UV–VIS absorption spectra to
those of six previously identified naphthoquinones (NQ1–6)
(Kittakoop et al. 1999) were found in the overexpressing
strains C7 and C8 and, to a lesser level, in wild type (Fig. 5).
Relative quantification of these naphthoquinones was
determined by comparison of the peak area of each naph-
thoquinone in the HPLC chromatogram. Total naphtho-
quinone production was calculated by summation of all the
peak areas of these seven naphthoquinones including the

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Grace’s insect medium
A B
100 μm
Fig. 3 PEG-protoplast transformation of Ophiocordyceps sp.BCC
1869. a The fungus was incubated in two different media, PDB and
Grace’s insect medium, for protoplast generation. Only the fungal
cells grown in Grace’s insect medium gave rise to a huge number of
protoplasts (indicated by arrows), whereas those from PDB generated
newly identified naphthoquinones, referred to in this study
as, NQ7, 8, and 9.
The MFS1-overexpressing mutants produced and
secreted a staggering amount of each naphthoquinone in
liquid culture compared with wild type (Figs. 6, 7). It is
noted that the wild type’s production of erythrostominone,
epierythrostominol, deoxyerythrostominol, and deoxyery-
throstominone, remained at low production levels during
the first 20 days (Kittakoop et al. 1999; Unagul et al. 2005),
consistent with the four low-leveled naphthoquinones
detected in this study. By contrast, within 7 days in PDB,
the MFS1-overexpressing mutants produced a huge amount
of these naphthoquinones. The mutant C7 produced the
highest amount of each of seven naphthoquinones found in
this study, compared to those produced by the wild type.
Although the mutant C8 produced a lower amount of these
naphthoquinones compared to C7, the levels were still
considerably higher than those of the wild type. For ery-
throstominone (NQ1), we detected none of this naphtho-
quinone in wild type’s 7-day old PDB culture. By contrast,
erythrostominone was the most abundant compound in the
crude extract of mutant C7 and the major one in those of
C8 (Fig. 6), which led to an increase of erythrostominone
relative quantity by 2,308- and 276-fold in C7 and C8,
respectively. The levels of deoxyerythrostominol (NQ3)
and deoxyerythrostominone (NQ4) were fairly similar in
No plasmid control 30% PEG
39% PEG 45% PEG
only a few protoplasts and a significant number of hyphae remained
after cell wall lysis (data not shown). b The fungal colonies first
appeared on day 5 of incubation. The 9-day old transformation
cultures are shown. The mixing of protoplasts and DNA in 39 and
49 % PEG solution generated the highest number of transformants
each mutant; 24- and 33-fold increase in C7 and 5- and
14-fold increase in C8 compared with those of the wild
type. Quantities of the four naphthoquinones in the wild
type-like mutant C32 were similar to those of the wild type
(Fig. 6).
For all the naphthoquinones, the compounds were
mostly found in the secreted fraction (supernatant of liquid
culture) (Fig. 6). The amount of total naphthoquinones in
the MFS1-overexpressing mutants C7 and C8 was 69.5 and
12.0 times of that of the wild type respectively.
High resolution ESI–MS analysis of erythrostominone,
epierythrostominol, deoxyerythrostominone and deoxy-
erythrostominol, initially purified by HPLC, verified the
integrity of these naphthoquinones with m/z of 371.0737,
373.0894, 355.0788, and 357.0945 [M?Na]?, respectively.
Discussion
In this study, overproduction of the valuable naphtho-
quinones was induced by overexpression of a MFS trans-
porter gene MFS1 in the entomopathogenic fungus
Ophiocordyceps sp. BCC1869. This transporter gene was
originally identified using a cDNA subtraction method
between the fungus growing in naphthoquinone-producing
condition and that in naphthoquinone-repressive condition
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Fig. 4 MFS1 expression level 14

and naphthoquinone production A
in the MFS1-overexpressing
*
12 **
mutants C7 and C8 and wild

Rao of MFS1 expression level

type (WT). a Quantitative RT-
PCR demonstrated that the 10

relave to wild type

mutants had increased gene
expression compared to wild
8
type grown in 7-day old PDB
cultures. This experiment was
repeated at least twice. Data are 6
the means from these
experiments and shown as fold-
4
change relative to wild
type ± standard error.
*P \ 0.05 and **P \ 0.01 2
using t test. b Naphthoquinone
production of Ophiocordyceps
0
sp. wild type (BCC1869) and
WT C7 C8
the MFS1-overexpressing
strains on 14-day old potato B
dextrose agar. The wild type
and the wild type-like strain
C32’s colonies had purple
hyphae (white arrows) and
secreted numerous red droplets
on tops of colonies. By contrast,
the MFS1-overexpressing
mutants C7 and C8’s colonies
had mostly white hyphae (black
arrows). These mutants also
produced and secreted deep red
drops on the tops of colonies.
(Color figure online)

WT C7

C32 C8

(Amnuaykanjanasin et al. 2011). Our original assumption
was that when the transporter gene is overexpressed, the
secretion (S) fraction to the total (T) amount (secretion and
mycelial fractions) of the overexpressing mutants should
be higher than that of the wild type. Nonetheless, the ratios
were quite similar between those of the overexpressing
strains and that of the wild type and both were in the range
of 82–100 % (Table 1). These data suggested the following
points. First, most of the produced naphthoquinones are
secreted outside the cells. Second, the staggering quantities
of naphthoquinones in the mutant C7 could be attributed to
the continuous functioning of this transporter (driven by
the constitutive promoter), leading to continuous produc-
tion of these naphthoquinones in this fungus. Having the
transporter that can readily pump out the compounds once
they are synthesized, preventing naphthoquinones to be
accumulated to a detrimental level, allows the fungal cells
to stably produce these metabolites. For biosynthesis of
daunorubicin (DNR) and its hydroxy derivative doxoru-
bicin (DXR) in Streptomyces peucetius, overexpression of

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MFS1-overexpressing mutant C7
A erythrostominone (NQ1) B
1.00 Peak of 25.950 min (NQ1)
deoxyerythrostominone (NQ2) 0.40 230.9
0.20 277.0 315.1 509.0
0.80
0.00
Peak of 27.472 min (NQ4)
0.60 0.40 230.9
deoxyerythrostominol
AU

(NQ5) 0.20 277.0 316.3 510.2

0.00
0.40
NQ8
Peak of 34.782 min (‘NQ7’)
NQ9 233.2
0.04
epierythrostominol
0.20 317.4 506.6
(NQ4) 0.02
NQ7
0.00
0.00 Peak of 35.152 min (NQ5)
1.00
0.00 25.00 30.00 35.00 40.00 45.00 233.2

wild type 318.6 505.4

0.00
1.00 Peak of 35.826 min (‘NQ8’)
232.0
0.02 275.8 315.1 509.0
0.80
0.00

Peak of 36.597 min (‘NQ9’)

0.60 233.2 297.2 484.4
0.00
AU

-0.02
0.40
Peak of 36.947 min (NQ2)
0.40 233.2
0.20 0.20 317.4 505.4

0.00
250 300 350 400 450 500 550 600
0.00
0.00 25.00 30.00 35.00 40.00 45.00
Retenon me (minutes)

Fig. 5 HPLC profile and UV–VIS absorption spectra of naphtho-
quinone derivatives. a HPLC chromatogram clearly showed that the
mutant C7’s crude extract had four major naphthoquinones [ery-
throstominone (NQ1), epierythrostominol (NQ4), deoxyerythrostomi-
nol (NQ5), deoxyerythrostominone (NQ2)], as previously reported
(Kittakoop et al. 1999) and three newly identified naphthoquinone
derivatives (NQ7, NQ8, and NQ9). By contrast, the wild type extract
contained only tiny amount of these compounds at the same
incubation time (7 days). b All the nine naphthoquinone derivatives
had similar UV–VIS absorption spectra

the transporter genes (drrA and drrB) led to a 2.2-fold
increase in the amount of DNR (Malla et al. 2009) and a
twofold increase in the amount of DXR (Song et al. 2011).
On the other hand, drrA–drrB null mutant had a tenfold
decrease in the DNR production (Srinivasan et al. 2010).
The authors also found that the regulatory gene dnrI and
the polyketide synthase gene dpsA were markedly down-
regulated to 1/8th and 1/16th respectively in the mutant
compared to the wild type, suggesting the feedback regu-
lation of DNR biosynthesis.
Our phylogeny suggested that MFS1 could belong to
the riboflavin transporter clade. The Ophiocordyceps
MFS1 could probably have a similar function with the
proteins in this group, which includes Saccharomyces
cerevisiae monocarboxylate transporter Mch5. In the
yeast S. cerevisiae, Mch5 plays an important role as a
facilitator to uptake riboflavin from the environment into
the cytoplasm (Reihl and Stolz 2005). MFS1 from
Ophiocordyceps sp. probably has a substrate of similar
molecular structure to that of Mch5. It is interesting to
note that the riboflavin structure is similar to those of
Ophiocordyceps naphthoquinones, in which both of them
contain three aromatic rings (Kittakoop et al. 1999).
Transporters that can transport several compounds with
similar structures were also detected in other fungi as well
as other organisms. The transporter UapC from Asper-
gillus brasiliensis can bind and transport uric acid and
xanthine with high affinity (Krypotou et al. 2015). Simi-
larly, the human hOAT1 transporter could strongly bind
uric acid, xanthine, and their related compounds as well
as benzbromaron, colchicine, omeprazole, caffeine, and
theobromine, all of which have the purine-based structure

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A 2750 *** Secreon

2,308
Relave quanty of each naphthoquinone (x 108) 2500 Mycelium

2250
in 50 mL shaken culture in 7 days

2000

1750

1500
***
33
1250
**
1000 24
750 ***
14
500 ***
** 276 *
197 5
250 ** NS NS NS NS
*** 47 1 2 1 1
*** *** *** NS NS *
0 *

ERN ERL DEL DEN ERN ERL DEL DEN ERN ERL DEL DEN ERN ERL DEL DEN

WT C7 C8 C32

B
Fungal strain WT C7 C8 C32

NQ7 NQ8 NQ9 NQ7 NQ8 NQ9 NQ7 NQ8 NQ9 NQ7 NQ8 NQ9
*** * *** *
Mycelium 0.4 + 0.3 0.0 + 0.0 0.1 + 0.0 12.4 + 0.8 1.0 + 0.0 4.7 + 0.1 0.7 + 0.4 0.5 + 0.1 0.2 + 0.0 0.1 + 0.1 0.1 + 0.1 0.0 + 0.0

*** *** *** ** ***

Secretion 3.4 + 1.6 0.2 + 0.3 2.2 + 1.2 283.1 + 32.5 561.1 + 50.0 96.1 + 0.4 26.0 + 2.7 43.6 + 6.1 23.0 + 10.4 6.0 + 1.5 0.6 + 0.1 3.6 + 0.8

Total relative 3.8 0.2 2.3 295.5 562.1 100.8 26.7 44.1 23.2 6.1 0.7 3.6
quantity
Fold change to 76.9 3156.7 44.5 7.0 247.7 10.2 1.6 3.5 1.6
WT

Fig. 6 Naphthoquinone production in MFS1-overexpression mutants total amount of each naphthoquinone relative to that of wild type.
(C7 and C8), wild type (WT)-like transformant C32, and wild type b The relative quantity of each of the three newly identified
grown in PDB for 7 days. a The quantity of each of the four naphthoquinones (NQ7, NQ8, and NQ9) is shown. This experiment
naphthoquinones [erythrostominone (ERN), epierythrostominol was repeated three times. Data are the means from these three
(ERL), deoxyerythrostominol (DEL), deoxyerythrostominone experiments and shown as peak area (9 108) ± standard error.
(DEN)] was determined by the peak area of the chromatogram, *P \ 0.05, **P \ 0.01, ***P \ 0.001, and NS, not significant using
which was then adjusted to the total amount per 50 mL liquid culture. t test
The number indicated on top of each tubular show a fold change of

Table 1 The ratio between the relative amount (the HPLC peak area) for each naphthoquinone in MFS1-overexpressing mutants (C7 and
from the secretion (S) fraction and the total amount (T; the sum of C8), a wild type-like transformant (C32), and wild type
peak areas from both secretion and mycelial fractions) in percentage
Fungal Erythrostominone Epierythrostominol NQ7 Deoxy- NQ8 NQ9 Deoxy-
strain erythrostominol erythrostominone

WT 100.0a 100.0 ± 0.0 87.7 ± 6.6 91.7 ± 4.9 94.0a 95.7 ± 0.3 86.3 ± 8.8
C7 99.9 ± 0.0 99.8 ± 0.0 95.8 ± 0.2 94.4 ± 0.2 99.8 ± 0.0 95.3 ± 0.1 90.7 ± 0.2
C8 99.1 ± 0.7 99.3 ± 0.2 82.5 ± 19.0 91.0 ± 4.8 98.9 ± 0.1 93.6 ± 7.0 89.6 ± 2.4
C32 100.0a 100.0 ± 0.0 98.0 ± 2.0 98.2 ± 1.6 100.0 ± 0.0 98.8 ± 1.4 95.9 ± 4.8

The experiment was repeated three times. Data are the means from these three experiments and shown as S:T ratio ± standard error. There were
no significant differences among the strains
a
We obtained only a single value due to the extremely low or zero amount of these compounds in the samples. Only once were these
naphthoquinones detected in three independent experiments

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(a pyrimidine ring fused to an imidazole ring) (Sugawara
et al. 2005).
The MFS1 overexpression in the overexpressing
mutants was correlated with the naphthoquinone quantities
in liquid culture. This indicated that overexpression of this
transporter gene markedly enhanced naphthoquinone pro-
duction in this fungus. Our result also revealed that the
MFS1 expression was correlated with production of these
red naphthoquinone pigments under the aerated condition,
further supporting that MFS1 encodes a MFS transporter
required for naphthoquinone production in Ophiocordyceps
sp.
In summary, we have demonstrated that genetic
manipulation of a transporter gene can improve a fungal
strain’s polyketide production. Efflux of target compounds
is likely to be one bottleneck in the production process. A
continuous flux of compounds is released out of the cells to
prevent the compound level from reaching toxicity levels,
so that the production can be sustained. Using this strategy
‘efflux-mediated overproduction’, we can harness more
from natural products of filamentous fungi.
Acknowledgments We are indebted to Chakapong Intaraudom and
Drs. Juntira Panya and Taridaporn Bunyapaiboonsri for great help and
advice in chemical analysis. We thank Wiwat Somyong for help in
compound extraction. We are also grateful to Dr. Chanikul Chutrakul
and Sarocha Panchanawaporn for advice in fungal transformation.
This work was supported by the National Center for Genetic Engi-
neering and Biotechnology’s Platform Program.
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