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Identification of the nonribosomal peptide
synthetase gene responsible for bassianolide
synthesis in wood-decaying fungus Xylaria sp.
BCC1067
Jiraporn Jirakkakul,
1
Juntira Punya,
2
Somchai Pongpattanakitshote,
2
Porntip Paungmoung,
1
Namol Vorapreeda,
1
Anuwat Tachaleat,
1
Cheeranun Klomnara,
1
Morakot Tanticharoen
2
and Supapon Cheevadhanarak
1,3
Correspondence
Supapon Cheevadhanarak
supaponche@gmail.com
1
School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi,
Bangkok 10140, Thailand
2
National Center for Genetic Engineering and Biotechnology, Thailand Science Park, 113
Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
3
Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi,
Bangkok 10150, Thailand
Received 11 October 2007
Revised 2 January 2008
Accepted 3 January 2008
Intensive study of gene diversity of bioactive compounds in a wood-rot fungus, Xylaria sp.
BCC1067, has made it possible to identify polyketides and nonribosomal peptides (NRPs)
unaccounted for by conventional chemical screening methods. Here we report the complete
nonribosomal peptide synthetase (NRPS) gene responsible for the biosynthesis of an NRP,
bassianolide, using a genetic approach. Isolation of the bassianolide biosynthetic gene, nrpsxy,
was achieved using degenerate primers specific to the adenylation domain of NRPS. The
complete ORF of nrpsxy is 10.6 kb in length. Based on comparisons with other known NRPSs,
the domain arrangement of NRPSXY is most likely to be C-A-T-C-A-M-T-T-C-R. The other ORF
found upstream of nrpsxy, designated efxy, is 1.8 kb in length and shows high similarity to
members of the major facilitator superfamily of transporters. Functional analysis of the nrpsxy gene
was conducted by gene disruption, and the missing metabolite in the mutant was identified.
Chemical analysis revealed the structure of the metabolite to be a cyclooctadepsipeptide,
bassianolide, which has been found in other fungi. A bioassay of bassianolide revealed a wide
range of biological activities other than insecticidal uses, which have been previously reported,
thus making bassianolide an interesting candidate for future structural modification. This study is
the first evidence for a gene involved in the biosynthesis of bassianolide.
INTRODUCTION
Bassianolide is an octacyclod epsipeptide consisting of four
molecules each of D-hydroxyisovaleric acid and L-N-
methylleucine, and has been reported to be an insecticide
obtained from Beauveria bassia na and Verticillium lecanii
(Suzuki et al., 1977). The structure of bassianolide is shown
in Fig. 1. Bassianolide belongs to a family of structurally
related cyclodepsipeptide compounds, which have emerged
as a broad family of compounds characterized by at least
one ester linkage. Great interest in this class of natural
products has stemmed from their diverse range of
biological activities, as has been shown for cryptophycins,
didemnins, dolastatins, PF1022, enniatin, destruxin,
Abbreviations: ESITOF, electrospray ionization–time of flight; ESYN,
enniatin synthetase; HTS, HC-toxin synthetase; KPR, ketopantoate
reductase; MFS, major facilitator superfamily; NRP, nonribosomal
peptide; NRPS, nonribosomal peptide synthetase.
The GenBank/EMBL/DDBJ accession numbers for the nrpsxy and efxy
sequences of Xylaria sp. BCC1067 are EF456733 and EF456734,
respectively.
A supplementary table showing
13
C NMR data for substance A in CDCl
3
,
and three supplementary figures showing
1
H NMR and
13
C NMR
spectra of the substance A molecule purified from Xylaria sp. BCC1067
in CDCl
3
solution, ESITOF MS data for substance A, and an HPLC
chromatogram of the acid hydrolysate of substance A, are available with
the online version of this paper.
Microbiology (2008), 154, 995–1006 DOI 10.1099/mic.0.2007/013995-0
2007/013995
G
2008 SGM Printed in Great Britain 995
beauvericin and valinomycin (Scherkenbeck et al., 2002;
Sarabia et al., 2004).
Cyclodepsipeptides are not synthesized by ribosomes in the
same way as general peptides, but are instead pro duced by
a nonribosomal peptide synthetase (NRPS). NRPSs are
multimodular enzymes in which each module is respons -
ible for the addition of a single amino acid. These enzymes
produce nonribosomal peptides (NRPs) by a thiotemplate
mechanism. NRPSs can incorporate proteinogenic and
non-protein amino acids, as well as carboxy and hydroxy
acids. Further modifications, such as N-methylation,
epimerization, cyclization and heterocyclic ring formation,
can also be carried out by these enzymes (von Do
¨
hren et al.,
1997; Marahiel et al., 1997; Mootz et al., 2002).
NRPs occur in a wide range of organisms, including
bacteria, fungi, plants and marine organisms (Keller &
Schauwecker, 2003). These compounds often possess
desirable pharmaceutical characteristics, and have thus
been used for many applications, including use as
siderophores, antibiotics, immunosuppressants and anti-
cancer drugs (Konz & Marahiel, 1999; von Do
¨
hren &
Grafe, 1997).
So far, the majo rity of NRPSs and their products have
been characterized in bacteria, especially Bacillus and
Streptomyces species. Fewer fungal genes encoding NRPS
have been fully sequenced and characterized experiment-
ally. These genes include acvA from Aspergillus nidulans
and Penicillium chrysogenum, which controls the produc-
tion of the precurso r of
b-lactam antibiotics such as
penicillin (Brakhage, 1997); simA from Tolypocladium
inflatum, which controls the production of cyclosporin A
and is used as an immunosuppressive drug in organ
transplant surgery (Weber et al., 1994); and sidA, which
controls production of the siderophores N9,N9,N999-
triacetylfusarinine C (TAF) and ferricrocin, which are
virulence factors in Aspergillus fumigatus (Hissen et al.,
2005). To date, the genomic sequences of fungi appear to
have the potential for faster discovery of novel NRPS genes.
For example, 12 NPRS genes have be en found in the
Cochliobolus heterostrophus genome (Lee et al., 2005); 15
putative NRPS genes have been found in Fusarium
graminearum (Tobiasen et al., 2007); and 14, 22, 14, 20
and 18 NRPS genes have been found in the genome
sequences of A. fumigatus, Aspergillus terreus, A. nidulans,
Aspergillus flavus and Aspergillus oryzae, respectively
(Cramer et al., 2006a). The functions of some gen es have
been determined by gene disruption; for instance, NPS6
from C. heterostrophus is involved in virulence and
tolerance to oxidative stress (Lee et al., 2005; Oide et al.,
2006), and gliP from A. fumigatus is involved in gliotox in
production (Cramer et al. , 2006b). However, the functions
of most NRPS genes in fungi remain unknown, requiring
further inves tigation for greater understanding of NRPSs in
these organisms (Stack et al., 2007).
Xylaria sp. BCC1067 has been reported to be a rich source
of bioactive secondary metabolites (Isaka et al., 2000). One
of the major compounds, 19,20-epoxycytochalasin Q, has
revealed an interesting characteristic, as it contains
polyketide and an amino acid in its core structure (hybrid
polyketide–NRP). From our search for additional polyke-
tide synthases (PKSs) and NRPSs in this fungus using a
genetic approach via PCR, at least 10 PKS, one hybrid
PKS–NRPS (Amnuaykanjanasin et al., 2005) and seven
NRPS (Paungmoung et al., 2007) genes were found. These
results implied that Xylaria sp. BCC1067 should have the
genetic capacity to produce a number of natural products
and that the total amount of these compounds is likely to
be much greater than that which has already been reported
for this fungus.
In this study, we identified and analysed the complete
sequence of the bassianolide synthetase gene, nrpsxy, and
confirmed its role in bassianolide bio synthesis by inser-
tional mutagenesis, as shown by the absence of bassianolide
production following disruption of the nrpsxy
locus in
Xylaria sp. BCC1067. Moreover, an ORF upstream of
nrpsxy was also identified, revealing high similarity to
members of the major facilitator superfamily (MFS) of
transporters.
METHODS
Media and strains. Xylaria sp. BCC1067 (wild-type) and transfor-
mants of this strain were kept on malt extract agar (MEA) at 25 uC.
Seven-day-old culture agar discs (3 mm diameter) of either the wild-
type or the mutant strain were inoculated into 50 ml malt extract
broth (Oxoid) for 14 days, before harvesting for DNA isolation.
Escherichia coli LE392 was used as the host for lambda Fix II genomic
library construction. Subclones were propagated in E. coli strain
DH5
a (Woodcock et al., 1989).
Construction of a lambda genomic DNA library. A genomic DNA
from Xylaria sp. BCC1067 was prepared according to the method of
Raeder & Broda (1985). The genomic library of Xylaria sp. BCC1067
Fig. 1. Structure of bassianolide. The structure was obtained
from PubChem Compound (http://www.ncbi.nlm.nih.gov/sites/
entrez?db=pccompound).
J. Jirakkakul and others
996 Microbiology 154
was constructed following the protocol for Gigapack III XL Packaging
Extract provided by the manufacturer (Stratagene).
Identification, cloning and sequencing of an NRPS gene.
Degenerate oligonucleotide primers targeting highly conserved motifs
of known NRPSs (Turgay & Marahiel, 1994) were used for
amplification and identification by PCR of putative NRPS gene
fragments in Xylaria sp. BCC1067. The sequences of the oligonucleo-
tides used were as follows: forward primer A3, 59-TA(C/T) AC(T/C/
G) TC(A/T/C) GGI (A/T)CI AA(G/A) GC-39; and reverse primer A5,
59-(C/T)TC (T/C/G)GT IGG (T/C/G)CC (A/G)TA (T/G)GC-39. The
NRPS PCR product (EN
11
) labelled with
[
32
P
]
dATP was used as a
probe for screening the genomic library of Xylaria sp. BCC1067 by
plaque hybridization.
Construction of a bassianolide-deficient Xylaria sp. BCC1067
mutant. Disruption of the nrpsxy gene was achieved by inserting a
2.05 kb phleomycin-resistance cassette (ble), which was derived from
the vector pOBT (Cheevadhanarak et al., 1991), into the first
adenylation domain-coding region. The disruption construct was
created by cloning a HincII fragment of the nrpsxy gene from the
DNA insert of lambda
lXyENRPS I into vector pUC18. The resulting
plasmid pEN3.8 was inserted by ble cassette into an SmaI site
corresponding to the adenylation domain of the cloned fragment; as a
result, a new plasmid, pDEN3.8, was obtained. The disruption
plasmid pDEN3.8 was then cut with EcoRI to obtain a linear 4.554 kb
DNA fragment. This fragment contained 1.048 and 1.451 kb segments
of the nrpsxy gene flanking the ble cassette (Fig. 3a) and was used to
transform protoplasts of Xylaria sp. BCC1067 according to the
method of Tilburn et al. (1983).
Substrate specificity of the adenylation domain prediction. The
substrate specificity-conferring amino acid in the adenylation
domains was determined according to the method of Stachelhaus et
al. (1999)
HPLC analysis of metabolite profile. HPLC was conducted using a
reverse-phase column (Inertsil ODS-3, GL Sciences) and diode array
detector (996 Photodiode Array Detector, Waters). An isolated
culture of either the Xylaria sp. BCC1067 wild-type or the mutant
NTT4 was grown in 250 ml malt extract broth. The cultures were
incubated at 25 uC for 14 days. Subsequently, the harvested mycelia
were extracted with 50 ml methanol for 2 days. After discarding the
mycelium, the lipid portion of the extract was removed by extraction
with 30 ml hexane, and the methanol layer was concentrated under
reduced pressure to obtain a brown semisolid. This mycelial extract
was dissolved in methanol to a final concentration of 100 mg ml
21
.
Separation of metabolites from 20
ml of extract was performed on an
Inertsil ODS-3 reverse-phase column. Analysis was done at a flow rate
of 1 ml min
21
at 210 nm with a water–acetonitrile step gradient as
follows: 0 min/5 % acetonitrile, 10 min/5 % acetonitrile, 15 min/50 %
acetonitrile, 25 min/50 % acetonitrile, 30 min/80 % acetonitrile,
40 min/80 % acetonitrile, 45 min/100 % acetonitrile, and 60 min/
100 % acetonitrile, following the method of Weckwerth et al. (2000).
The water used in the analysis contained 0.05 % (v/v) trifluoroacetic
acid (TFA).
Isolation and characterization of pure substance A. The mycelial
extract was fractionated by Sephadex LH20 column chromatography
(using methanol as an eluent) in order to obtain the fraction
containing substance A. Subsequently, substance A was purified by
step-gradient HPLC, as described above. Structural documentation
was performed by NMR and MS analysis. Proton NMR (
1
H NMR)
and carbon-13 NMR (
13
C NMR) spectra were measured in CDCl
3
on
a Bruker DRX400 spectrometer, and electrospray ionization–time of
flight (ESITOF) mass spectra were obtained on a Micromass LCT
mass spectrometer (Isaka et al., 2005).
Determination of D and L configuration of amino acids in
substance A by acid hydrolysis.
Substance A (0.5 mg) was
hydrolysed with 6 M HCL (0.4 ml) at 110–120 uC for 15 h. After
concentration to dryness, the residue was dissolved in 100
ml
methanol. Twenty microlitres of hydrolysed substance A was
subjected to HPLC using a ligand-exchange-type chiral column
(SUMICHIRAL OA-5000, 5
mm bead size, 4.66150 mm internal
diameter6length; Sumika Chemical Analysis Service) with 5 %
CH
3
OH in 2 mM CuSO
4
as the system eluent (flow rate 1 ml min
21
,
UV wavelength 235 nm).
D and L configurations of a-hydroxyisova-
leric acid and N-methylleucine were used as reference standards.
Biological assay. Biological activities of substance A against human
epidermoid carcinoma (KB cells), human breast cancer (BC-1 cells),
human small cell lung cancer (NCI-H187 cells), Mycobacterium
tuberculosis H37Ra, Plasmodium falciparum K1, and African Green
Monkey kidney fibroblasts (Vero cells) were performed by the
Bioassay Laboratory at the National Center for Genetic Engineering
and Biotechnology (BIOTEC), Thailand. Antibacterial activity was
assessed using the disc diffusion assay (McGaw et al., 2000). The
bacterial strains tested included Staphylococcus aureus ATCC 29213,
Bacillus subtilis ATCC 6633, E. coli ATCC 25922 and Pseudomonas
aeruginosa ATCC 27853.
RESULTS
Identification of the NRPS gene
To identify the NRPS gene(s) in Xylaria sp. BCC1067,
degenerate PCR primers specific to conserved sequence
motifs of NRPS genes were used in the amplification of
genomic DNA from Xylaria sp. BCC1067. One product
(~400 bp), EN
11
, showed the highly conserved core motifs
A3–A5 of typical NRPS adenylation domains. Probing the
genomic library of Xylaria sp. BCC1067 with EN
11
allowed
us to identify two overlapping phages,
lXyENRPS I and
lXyEN9.1, which covered 7.24 kb corresponding to a
partial NRPS gene lacking its 39-terminal region. To
identify the remaining part of this gene, an additional step
of chromosomal walking was performed. A SalI fragment
of the 39-terminal end of
lXyEN9.1, namely EN
643
,was
used as a probe to screen the genomic library (Fig. 2a). One
positive phage,
lEN643T113, was isolated, and its DNA
insert was subjected to sequencing. The results of sequence
analysis revealed a complete ORF (10 641 bp), designated
nrpsxy, which was interposed by a 61 bp intron located
near the 39 end. In addition, we also found a smaller
complete ORF (1806 bp), designated efxy, 5.7 kb upstream
of nrpsxy, which was transcribed in the opposite direction.
By sequence analysis, we deduced that the nrpsxy gene
encoded a single ORF of two complete peptide synthetase
modules and an additional domain homologous to
ketopantoate reductase (KPR) at the C terminus of the
predicted protein (Fig. 2b). The predicted protein encoded
by the nrpsxy gene contained 3546 amino acids w ith a
calculated molecular mass of about 390 kDa. Almost the
entire length of the amino acid sequence of NRPSXY
(amino acid position 1–3135) displayed strong homology
NRPS gene in Xylaria for bassianolide synthesis
http://mic.sgmjournals.org 997
to the enniatin synthetase (ESYN) encoded by the esyn gene
of Fusarium equiseti (Q00869; 58 % identity and 74 %
similarity) and to the cyclosporin synthetase encoded by
the simA gene of T. inflatum (CAA82227; 48 % identity and
64 % similarity). The C-terminal domain of about 412
amino acids (position 3136–3546) did not show any
homology to known NRPSs in the databases, but displayed
significant similarity to several uncharacterized proteins
from various microbes, including KPRs such as AbpA
_Cof
Magnaporthe grisea 70-15 (XP_363700; 45 % identity and
66 % simil arity), AbpA of Pichia guilliermondii ATCC 6260
(XP
_001483630; 29 % identity and 50 % similarity), AbpA
of Ps. aeruginosa C3719 (ZP
_00965349; 30 % identity and
46 % similarity) and AbpA of E. coli F11 (ZP_00722740;
25 % identity and 42 % similarity). Detailed analysis of
NRPSXY showed that it had a domain architecture (C-A-
T-C-A-M-T-T-C-R) with corresponding regions of con-
densation (C) in amino acid residues 48–432, 1104–1442
and 2719–3082; adenylation (A) in amino acid residues
505–982 and 1582–2482; N-methylation (M) in amino acid
residues 2083–2399, where this domain was inserted into
the adenylation domain within the second module
(between motifs A8 and A9); and thiolation (T) in amino
acid residues 1017–1082, 2510–2571 and 2608–2670, as
well as a KPR (R) domain in amino acid residues 3174–
3507, which contained a putative NAD/FAD binding motif
(SRIHILGVGNLGKFV) (Fig. 2b ). These conserved resi-
dues are indicated in bold type in Table 1.
To predict the substrate specificity-co nferring amino acid
in the NRPSXY adenylation domains, two alternative
methods were used: (1)
BLAST analysis of the amino acid
sequence of each A domain against protein databases; and
(2) signature sequence analysis according to the method of
Stachelhaus et al. (1999). When using
BLAST analysis via the
NCBI website (http://www.ncbi.nlm.nih.gov/blast/
Blast.cgi), the first A domain of NRPSXY showed
homology to the
D-2-hydroxyisovaleric acid-activating
domain from ESYN of F. equiseti (68 % identity and
81 % simil arity), and the
L-alanine-activating domain from
HC-toxin synthetase (HTS) of Cochliobolus carbonum
(38 % identity and 58 % similarity). The second A domain
showed homology to the leucine-activating domain (63 %
identity and 77 % similarity) and to the valine-activating
domain (59 % identity and 77 % similarity) from cyclo-
sporin synthetase of T. inflatum. It also showed homology
to the valine-activating domain from ESYN of F. equis eti
(54 % identity and 71 % similarity) and fro m cereulide
synthetase B (CesB) of Bacillus cereus (38 % identity and
56 % similarity). However, the results from the second
method revealed that the signature sequence of the first A
domain showed significant matches to the signature
Fig. 2. Schematic map of the genomic region containing the nrpsxy gene and modular structure of NRPSXY. (a) Restriction
map and gene arrangement of efxy (GenBank accession no. EF456734) and nrpsxy (GenBank accession no. EF456733) in
Xylaria sp. BCC1067. Restriction site abbreviations: D, DraI; E, EcoRI; H, HincII; S, SmaI; Sa, SalI; X, XbaI. The orientations of
the genes are indicated by arrowheads, and the positions of introns are indicated by white bars. The nrpsxy gene is interrupted
by a putative intron of 61 bp. The lambda clone arrangements isolated in this work and the probes used for chromosomal
walking are marked (EN
11
,EN
643
). (b) Modular organization of NRPSXY as revealed by cDART (conserved Domain Architecture
Retrieval Tool; http://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi?cmd=rps) from NCBI. The domains within the
modules are indicated: A, adenylation domain; C, condensation domain; T, thiolation domain; M, N-methylation domain; R, KPR
domain; A
D-Hiv
,2-D-hydroxyisovaleric acid-activating domain; A
L-Leu
, N-methylleucine-activating domain. Both A
D-Hiv
and A
L-Leu
are annotated from the structure identification of substance A encoded by the nrpsxy gene.
J. Jirakkakul and others
998 Microbiology 154
sequence for D-2-hydroxyisovaleric acid of ESYN (90 %)
and to
L-alanine of HTS (20 %). The signature sequence for
the second domain of NRPSXY revealed the highest
similarity to the N-methylleucine-activating domain found
in the second module of cyclosporin synthetase (CssA)
(90 %), the N-methylvaline-activating domain found in the
Fig. 3. Disruption of the gene encoding NRPSXY in Xylaria sp. BCC1067. (a) Schematic diagram of the insertion mutation of the
nrpsxy gene. The disruption fragment, the genotype of the wild-type and the genomic rearrangement after a successful gene
insertion by double-crossover integration in the mutant NTT4 are shown. The arrows marked with T6, T7, T10, T3, OliC and BleF
represent the primers used for verifying the presence of the insertion in the mutant via PCR amplification. (b) PCR analysis of the
mutant NTT4. Amplification of genomic DNA from the mutant NTT4 with the T6+T7 primer pair generated a PCR product of
~2.7 kb (lane 2) compared to the undisrupted size from wild-type genomic DNA (lane 1). OliC+T3 and T10+BleF primer pairs
showed PCR products of the predicted sizes ~1.9 (lane 3) and ~3.5 kb (lane 4), which were absent when amplified from wild-
type genomic DNA. The DNA ladder marker (lane M) shows the size in kilobases. (c) Southern blot analysis of the mutant NTT4.
DraI-digested genomic DNA of the wild-type (lane 1) and the mutant NTT4 (lane 2) was hybridized to a 2.5 kb probe located at the
59 end of the gene. A homologous crossover into the DraI fragment yielded a band of ~5.2 kb and absence of the 3.1 kb wild-type
band, as seen in mutant NTT4, indicating ble cassette insertion into the nrpsxy gene. Lane M, DNA ladder marker.
NRPS gene in Xylaria for bassianolide synthesis
http://mic.sgmjournals.org 999
fourth module of CssA of T. inflatum (50 %), and the
leucine-activating domai n of bacitracin synthetase (BacA)
(60 %) (Table 2). However, chemical analysis of the nrpsxy
gene product has now confirmed that the first A domain of
NRPSXY is responsible for the activation of
D-2-hydroxy-
isovaleric acid, while the second A do main activates N-
methylleucine.
Regarding the efxy gene, its deduced amino acid sequence
was composed of 602 amino acids, and was interrupted by
four introns of 63, 62, 58 and 64 bp in length. This protein
sequence showed similarity to many membrane transport
proteins of the MFS, including those from Bcmfs1 of
Botrytis cinerea (76 % similarity) (Hayashi et al., 2002),
AflT of Aspergillus clavatus (68 % similarity), TOXA of
Cochliobolus carbonum (56 % similarity) (Pitkin et al.,
1996) and the MFS multidrug transporter of A. fumigatus
AF293 (66 % similarity). Secondary structure analysis
by TMHMM (http://www.cbs.dtu.dk/services/TMHMM)
revealed that EFXY contained 14 membrane-spanning
domains.
Inactivation of the nrpsxy gene
To determine the function of the gene, insertional
mutagenesis of the nrpsxy gene was performed. To
interrupt the nrpsxy gene in the chromosomal locus of
Xylaria sp. BCC1067, we constructed a linear DNA
disruption fragment in which a phleomycin-resistance
cassette, ble, was inserted by blunt-end ligation in the SmaI
site of nrpsxy flanked by 1.048 kb EcoRI/SmaI and 1.451 kb
SmaI/EcoRI fragments of the nrpsxy ge ne (Fig. 3a).
Transformation of Xylaria sp. BCC1067 with the linear
DNA fragment and selection on phleomycin led to the
Table 1. Comparison of the conserved motifs of the catalytic NRPSXY domains with conserved core motifs previously defined by
Konz & Marahiel (1999)
Conserved residues are indicated in bold type. ND, Not detected.
Domain Consensus sequence
(Konz & Marahiel, 1999)
NRPSXY module 1
(this work)
NRPSXY module 2 (this work)
Adenylation
A1 L(TS)YxEL WTYNEL LTYAEL
A2 (core 1) LKAGxAYL(VL)P(LI)D LKTGRAFTLIDP LKASLAYLPFDV
A3 (core 2) LAYxxYTSG(ST)TGxPKG LAYVLFTSGSTGEPKG LAYVIFTSGSTGRPKG
A4 FDxS FDAC FDAA
A5 NxYGPTE NGYGQSENAYGPTE
A6 (core 3) GELxLxGxG(VL)ARGYL GELVIESPGIARGYI GELVVTGDGLARGYT
A7 (core 4) Y(RK)TGDL YRTGDL YRTGDR
A8 (core 5) GRxDxQVKIRGxRIELGEIE GRRDSQVKIRGQRVTSEVEGRMDQQVKIRGHRIELAEVE
A9 LPxYM(IV)P LPQHSIP LP
SYMIP
A10 NGK(VL)DR TGKTDR SGKVDR
Thiolation
T (core 6) DxFFxLGG(HD)S(LI) ASFFELGGDSI
[
T2
]
: DNFFKLGGHSL
Condensation
C1 SxAQxR(LM)(WY)xL TPFQSDVMDC
[
C2
]
: SYAQGRIWFL
[
C3
]
:YPATQMQRLF
C2 RHExLRTxF RQTPALRTC RHETLRTTF QHFDMFRTV
C3 MHHxISDG(WY)xL SHALVDNVLQE MHHIISDGWSI MSHALYDGIEL
C4 YxD(FY)AVW NVQYANG YRDFAVW PPKFARY
C5 (IV)GxFVNT(QL)(~)xR
ND IGFFVNTQCMRVGPCTNTIPVR
C6 (HN)QD(YV)PFE HDDAMHE HQDVPFE MQDQYLD
C7 RDxSRNPL RDVARFLR RDTSRNPL REDQLANE
N-Methylation
M1 (SAM) VL(DE)GxGxG VLEIGTGSG
M2 NELSxYRYxAV NELSAYRYAAV
M3 VExSxARQxGxLD VEVSCARQWSQSG
Reductase
R1
[
NAD(P)H
binding site
]
V(LF)(LV)TG(AV)(TN)G(YF)LG ILGVGNLGKFVA
R2 VxxxVRA WDAAVKA
R3 GDL VDL
R4 VYPYxxLRx(PL)NVxxT VYPTSPFSIVHAGRG
R5 GYxxSKWxx GYLALQGKRL
R6 RPG
ND
R7 LExx(VI)GFLxxP IETLVNMVKQG
J. Jirakkakul and others
1000 Microbiology 154
isolation of 14 tr ansformants. All transformants were
screened by PCR and Southern blot analysis to verify
disruption of the nrpsxy locus (data not shown). Only one
positive transformant (mutant NTT4) revea led disruption
of the nrpsxy gene. Mutant NTT4 was identified on the
basis of a band shift in the mutant and the absence of
similar products in the wild-type (Fig. 3a). Insertion of the
ble cassette into the nrpsxy gene was verified using primer
pairs specific to the ble cassette (BleF and OliC) and the
region of the nrpsxy gene flanking the insertion site
(OliC+T3 and T10+BleF; T10 and T3 are specific primers
for the 59-and39-flanking regions of the integration site,
respectively). These primer pairs produced specific pro-
ducts, suggesting that the nrpsxy gene was disrupted by ble
cassette insertion. Amplification of genomic DNA from
mutant NTT4 with a T6+T7 primer pair generated a PCR
product of ~2.7 kb, which may be compared to the
nondisrupted size of genomic DNA from a wild-type strain,
which generated a PCR product of ~720 bp. OliC+T3 and
T10+BleF primer pairs showed PCR products of the
predicted sizes of ~1.9 and ~3.5 kb, respectively (Fig. 3b),
which were absent when amplified from genomic DNA from
a wild-type strain. This result was confirmed by Southern
blot analysis. DraI-digested genomic DNA from a wild-type
strain and mutant NTT4 was hybridized to a 2.5 kb probe
located at the 59 end of the gene. A homologous crossover
into the DraI fragment would yield bands of ~4.5 and
~5.2 kb and the absence of the 3.1 kb wild-type band, as
seen with mutant NTT4, indicating ble cassette insertion
into the nrpsxy gene, as shown in Fig. 3(c).
Identification of a substance encoded by the
nrpsxy gene
HPLC analysis of mycelium extracts from the wild-type
was used as a reference in comparison to the mutant NTT4.
The wild-type metabolic profile showed a visible peak
related to substance A at a retention time of 49.3 min
(Fig. 4a), whereas this peak disappeared in the profile of
mutant NTT4 under the same conditions (Fig. 4b).
Substance A was detected in the wild-type by methanol
extraction of mycelia of Xylaria sp. BCC1067 separated by
Sephadex LH20 column chromatography and purified by
HPLC. The structure of substance A was elucidated using
spectroscopic methods, especially
13
C NMR,
1
H NMR and
MS analysis. The
1
H NMR spectrum showed five N-methyl
signals at 2.87, 2.90, 3.02, 3.06 and 3.27 p.p.m.; and the
13
C
NMR spectrum in the same solvent revealed a total of 60
signals. The NMR spectra are shown in Supplem entary Fig.
S1 and the
13
C NMR spectra of substance A are
summarized in Supplementary Table S1. The ESITOF
mass spectra of substance A of m /z 931.5991 corresponded
to
[
M+Na
]
+
(Supplementary Fig. S2), giving a molecular
mass of 908.5991. HPLC analysis of its acid hydrolysate was
performed using a chiral column. These analyses confirmed
that substance A was a cyclodepsipeptide consisting of four
residues of 2-
D-hydroxyisovaleric acid (D-Hiv) and four
residues of
L-methylleucine (N-Me-L-Leu). HPLC analysis
of the acid hydrolysate of substance A is shown in
Supplementary Fig. S3. NMR spectra and mass analysis
together with the amino acid composition are shown in
Table 3.
Table 2. Signature sequences of amino acids in the putative activating domains of NRPSXY
Abbreviations: ESYN, enniatin synthetase; CssA, cyclosporin synthetase; BacA, bacitracin synthetase; SrfA, surfactin synthetase; PhsC,
phosphinotricin synthetase; Ent, enterobactin synthetase; LicD, lichenycin synthetase; CDA, calcium-dependent antibiotic synthetase; Com,
complestatin synthetase; HTS, HC-toxin synthetase. Bold or italic type indicates the closest resemblance of NRPSXY modules to others present in
the databases.
NRPS module Activated substrate (source) Amino acid residue at signature sequence position*
235 236 239 278 299 301 322 330 331 517
NRPSXY module 1
D-2-Hydroxyisovaleric acid (this work)
GAL LVVG I CK
NRPSXY module 2 N-Methylleucine (this work) D A W L V G A V M KD
ESYN module 1
D-2-Hydroxyisovaleric acid
GALHVVGI CK
ESYN module 2 N-Methylvaline D G W F I G I I I KD
CssA module 2 N-Methylleucine D A W L Y G A V M KD
CssA module 4 N-Methylvaline D A W M F A A I L KD
CssA module 9 Valine D A W M F A A V M K
BacA module 3 Leucine D A W F L G N V V K
SrfA module 2 Leucine D A F M M G M V F K
PhsC module 1 Proline D V L L V A G V L K
EntE module 1 2,3-Dihydroxybenzoate N Y S A Q G Q G L K
Com3 module 1 4-Hydroxyphenylglycine D I F H L G V E V K
HTS module 2 Alanine D A G G C A M V A K
*The residues correspond to the gramicidin S synthetase PheA numbering (Stachelhaus et al., 1999).
DThe N-methylation domain is integrated in the adenylation domain between the motifs A8 and A9.
NRPS gene in Xylaria for bassianolide synthesis
http://mic.sgmjournals.org 1001
Fig. 4. HPLC chromatogram of methanol extracts from mycelia of Xylaria sp. BCC1067 wild-type and mutant NTT4 strains. (a)
HPLC results from the wild-type extract are presented as a peak with retention time 49.3 min, designated the substance A
peak. (b) The substance A peak is missing in extracts from mutant NTT4. (c) The extracts from mutant NTT4 were spiked with
extracts from the wild-type.
J. Jirakkakul and others
1002 Microbiology 154
Biological activities of substance A
To evaluate the biological activity of substance A, pure
substance A was subjecte d to several biological assays. The
compound exhibited activity against M. tuberculosis H37Ra
(MIC 6.25
mgml
21
), strongly inhibited proliferation of the
human malaria parasite Pl. falciparum K1 (IC
50
1.65 mg
ml
21
), and also showed cytotoxic activity against Vero cells
and three other cancer cell lines, KB , BC and NCI-H187
(Table 4). However, substance A exhibited no observable
toxicity against the other tested bacteria.
DISCUSSION
Xylaria sp. BCC1067 has been shown to be a potential
resource of bioactive secondary metabolites
(Amnuaykanjanasin et al., 2005; Isaka et al., 2000). We
have attempted to clone and analyse NRPS genes, which
are expected to be involved in bioactive compound
formation in Xylaria sp. BCC1067. In this study, we
identified an NRPS gene, nrpsxy, and showed that
disruption of this gene resulted in the elimination of
substance A production. Structural elucidation of sub-
stance A was carried out by NMR and MS. Finally,
confirmation by a literature data comparison indicated that
the structure of substance A was a cyclodepsipeptide
consisting of four
D-a-hydroxyisovaleryl- L-N-methylleucyl
units, which was identical to bassianolide, a cyclooctadep-
sipeptide with a molecular formula of C
48
H
84
N
4
O
12
,
previously isolated from B. bassiana and V. lecanii
(Suzuki et al., 1977). The results of gene disruption and
metabolite structure elucidation confirmed the role of
nrpsxy in the biosynthesis of an N-methyl-cyclooctadepsi-
peptide, bassianolide.
The NRPS encoded by the nrpsxy gene is an enzyme
consisting of a single polypeptide chain. Its domain
architecture w as found to be highly similar to that of N-
methyl-cyclohexadepsipeptide enniatin synthetase (ESYN)
Table 3. Comparison of chemical characteristics of substance A and bassianolide from Suzuki et al. (1977)
Characteristic or analytical result Substance A (this work) Cyclodepsipeptide bassianolide (Suzuki et al., 1977)
Molecular mass (m/z) 908.5991 908
Total signal in
13
C NMR spectrum 60 60
N-Methyl signal in
1
H NMR spectrum
(chemical shift in p.p.m.)
Five N-methyl signals
(2.87, 2.90, 3.02, 3.06 and 3.27)
Five N-methyl signals
(2.86, 2.89, 3.01, 3.05 and 3.25)
Acid hydrolysis analysis N-Methylleucine and
D-hydroxyisovaleric acid
N-Methylleucine and
D-hydroxyisovaleric acid
NRPS gene in Xylaria for bassianolide synthesis
http://mic.sgmjournals.org 1003
from Fusarium species (Haese et al., 1993, 1994). However,
ESYN consists of two activation modules, which contain
the catalytic binding sites for the substrates
D-2-hydroxy-
isovaleric acid and the branched-chain L-amino acid,
causing the substrates to assemb le into three units of
dipeptidol building blocks (von Do
¨
hren et al., 1997). It has
been reported that the N-methylated cyclodepsipeptides
beauvericin and PF1022A are synthesized by NRPSs that
are probably of similar domai n architecture to ESYN (von
Do
¨
hren et al., 1997; Weckwerth et al., 2000).
The domain arrangement of NRPSXY revealed a distinct
characteristic, which was composed of two tandem T
domains at the C-terminal module. The presence of the
second T domain possibly represents the waiting position for
repeating the enzymic reaction. Based on the domain/
module organization of NRPSXY and the structure of
substance A, the hypothetical mechanism of bassianolide
biosynthesis by Xylaria sp. BCC1067 can be postulated to
resemble the biosynthesis mechanism of enniatin, beauver-
icin and PF1022A. It appears likely that NRPSXY use their
modules or domains more than once in the assembly of a
single product and could be categorized as an iterative NRPS,
as is the case for enterobactin synthetase and gramicidin S
synthetase (von Do
¨
hren et al., 1997; Mootz et al.,2002).
The main difference between ESYN and NRPSXY is an
additional putative KPR (EC 1.1.1.169) located at the C-
terminal end of NRPSXY. Interestingly, in enniatin
biosynthesis of Fusarium sambucinum, its 2-
D-hydroxyiso-
valeric acid precursor is synthesized from 2-ketoisovalerate
(2-KIV) catalysed by the
D-hydroxyisovalerate dehydro-
genase enzyme (Lee et al., 1992), and it has been reported
that the KPR enzyme from Pseudomonas maltophilia 845
can catalyse the biosynthesis of 2-
D-hydroxyisovaleric acid
from 2-KIV as an alternative substrate (Shimizu et al.,
1988). Furthermore, in the study of genes involved in AF-
toxin biosynthesis in the plant-pathogenic fungus
Alternaria alternate, a gene designated AFTS1, which
encodes a protein with similarity to enzymes of the aldo-
ketoreductase superfamily, has been proposed to be the
gene encoding an enzyme that catalyses the conversion of
2-KIV to 2-
D-hydroxyisovaleric acid (Ito et al., 2004). For
cereulide biosynthesis in B. cereus, it has been shown that
the A domain of cereulide synthetase B peptide specifically
activates the
a-keto acid (in this case 2-KIV), and is
subsequently reduced to 2-
D-hydroxyisovaleric acid by the
keto-reductase domain embedded in its A domain before
condensing to an adjacent amino acid precursor by the
condensation domain (Magarvey et al., 2006). However,
the function of the R domain at the C-terminal of the
NRPSXY peptide still warrants further analysis.
In conclusion, NRPSXY most resembles ESYN with respect
to its domain organization. However, the end product of
NRPSXY is a cyclooctadepsipeptide, a compound consisting
of eight residues instead of the six residues synthesized by
ESYN. In our case, comparative study of the amino acid
specificity of the modules of NRPSXY, by predicting the
signature sequence of the substrate-binding pocket together
with chemical structure analysis of the NRPSXY product,
revealed the specificity of 2-
D-hydroxyisovaleric acid for the
first A domain and of N-methylleucine for the second A
domain. These results confirmed the hypothesis proposed
by Stachelhaus et al. (1999). This finding may be useful for
future prediction of amino acid-activating domains and
could help as a guide to the characterization of newly
discovered NRPSs.
A second ORF, the efxy gene, was located 5.7 kb upstream of
the nrpsxy gene. This gene putatively encodes a transporter
protein of the MFS. These proteins are usually of a
membrane-bound type that may help prevent accumulation
of toxic compounds in cells (Hayashi et al., 2002). In
filamentous fungi, a number of MFS transporters are known
to mediate the secretion of endogenously produced toxins
(Pitkin et al., 1996), including cercosporin, HC-toxin and
trichothecenes. These MFS genes are located in gene clusters
together with genes that encode the enzymes involved in the
biosynthesis of these toxins (Hayashi et al., 2002; Pitkin et
al., 1996). Therefore, the protein encoded by efxy may serve
as an efflux pump that transports bassianolide out of the
cells of Xylaria sp. BCC1067.
We report here what is believed to be the first evidence of a
biosynthetic gene for the NRP bassianolide. In this study,
we have shown an in vitro bioassay of substance A, which
exhibited antiplasmodial, antimycobacterial and antitumor
activities beyond the previously reported insecticidal
activity of bassianolide (Suzuki et al., 1977; Champlin &
Grula, 1979). These activities may be related to its cytotoxic
actions; however, substance A showed no observable
toxicity against bacteria such as Staph. aureus ATCC
29213, B. subtilis ATCC 6633, E. coli ACTT 25922 and Ps.
aeruginosa ATCC 27853.
The po tential of Xylaria sp. BCC1067 as a prolific resource
for bioactive compounds has been demonstrated by
Table 4. Antimycobacterial, antiplasmodial and cytotoxic
activities of substance A
Target cells Antimycobacterial, antiplasmodial or
cytotoxic activity (
mgml
”1
)
M. tuberculosis* (MIC) 6.25
Pl. falciparumD (IC
50
) 1.65
KB cellsd (IC
50
) 3.64
BC cellsd (IC
50
) 2.49
NCI-H187 cellsd (IC
50
) 1.10
Vero cellsd (IC
50
) 4.8
*MIC values of the standard drugs rifampicin, kanamycin and
isoniazide were 0.0047, 2.5 and 0.05
mgml
21
, respectively.
DThe IC
50
value of the standard antimalarial compound dihydroarte-
misinine was 3.9 nM.
dIC
50
values of the standard compound ellipticine were 0.670 mg
ml
21
for KB cells, 0.129 mgml
21
for BC cells, 0.273 mgml
21
for NCI-
H187 cells and 0.5
mgml
21
for Vero cells.
J. Jirakkakul and others
1004 Microbiology 154
chemical and genetic approaches, and future identification
and characterization of new natural products are in the
pipeline. We expect that using genetic knowledge as a basic
tool for further modification of biosynthetic genes in this
micro-organism will result in novel compounds with
biotechnological value for medical, agricultural and
industrial exploitation in the near future.
ACKNOWLEDGEMENTS
We thank Dr Masahiko Isaka for chemical identification of substance
A (bassianolide). We also thank Dr Vanida Bhavakul for helpful
discussion and suggestions, Dr Alongkorn Amnuaykanjanasin,
Amporn Rungrod and Suranat Phonghanpot for their kind advice
and support, and Pitchapa Berkaew for acid hydrolysis of substance A
and HPLC analysis of the hydrolysate. This work was financially
supported by a research grant from King Mongkut’s University of
Technology Thonburi.
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Edited by: L. N. Glass
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1006 Microbiology 154
