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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
Correspondence
Supapon Cheevadhanarak
supaponche{at}gmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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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 13C NMR data for substance A in CDCl3, and three supplementary figures showing 1H NMR and 13C NMR spectra of the substance A molecule purified from Xylaria sp. BCC1067 in CDCl3 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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). 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, beauvericin and valinomycin (Scherkenbeck et al., 2002; Sarabia et al., 2004).
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; 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 anticancer drugs (Konz & Marahiel, 1999; von Döhren & Grafe, 1997).
So far, the majority 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 experimentally. These genes include acvA from Aspergillus nidulans and Penicillium chrysogenum, which controls the production of the precursor of β-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 N',N',N'''-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 been 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 genes 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 gliotoxin production (Cramer et al., 2006b). However, the functions of most NRPS genes in fungi remain unknown, requiring further investigation 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 polyketide 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 biosynthesis by insertional 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.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(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 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 oligonucleotides used were as follows: forward primer A3, 5'-TA(C/T) AC(T/C/G) TC(A/T/C) GGI (A/T)CI AA(G/A) GC-3'; and reverse primer A5, 5'-(C/T)TC (T/C/G)GT IGG (T/C/G)CC (A/G)TA (T/G)GC-3'. The NRPS PCR product (EN11) labelled with [32P]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 
XyENRPS 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).
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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 °C 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–1. Separation of metabolites from 20 µl of extract was performed on an Inertsil ODS-3 reverse-phase column. Analysis was done at a flow rate of 1 ml min–1 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 (1H NMR) and carbon-13 NMR (13C NMR) spectra were measured in CDCl3 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 °C for 15 h. After concentration to dryness, the residue was dissolved in 100 µl methanol. Twenty microlitres of hydrolysed substance A was subjected to HPLC using a ligand-exchange-type chiral column (SUMICHIRAL OA-5000, 5 µm bead size, 4.6x150 mm internal diameterxlength; Sumika Chemical Analysis Service) with 5 % CH3OH in 2 mM CuSO4 as the system eluent (flow rate 1 ml min–1, UV wavelength 235 nm). D and L configurations of -hydroxyisovaleric 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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400 bp), EN11, showed the highly conserved core motifs A3–A5 of typical NRPS adenylation domains. Probing the genomic library of Xylaria sp. BCC1067 with EN11 allowed us to identify two overlapping phages, XyENRPS I and XyEN9.1, which covered 7.24 kb corresponding to a partial NRPS gene lacking its 3'-terminal region. To identify the remaining part of this gene, an additional step of chromosomal walking was performed. A SalI fragment of the 3'-terminal end of XyEN9.1, namely EN643, was used as a probe to screen the genomic library (Fig. 2a). One positive phage, EN643T113, 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 3' 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.
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). The predicted protein encoded by the nrpsxy gene contained 3546 amino acids with 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 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_C of Magnaporthe grisea 70-15 (XP_363700; 45 % identity and 66 % similarity), 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 condensation (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 residues are indicated in bold type in Table 1.
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. 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 % similarity), 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 cyclosporin synthetase of T. inflatum. It also showed homology to the valine-activating domain from ESYN of F. equiseti (54 % identity and 71 % similarity) and from 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 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 fourth module of CssA of T. inflatum (50 %), and the leucine-activating domain 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-hydroxyisovaleric acid, while the second A domain activates N-methylleucine.
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), 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 gene (Fig. 3a). Transformation of Xylaria sp. BCC1067 with the linear DNA fragment and selection on phleomycin led to the isolation of 14 transformants. 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) revealed 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 5'- and 3'-flanking regions of the integration site, respectively). These primer pairs produced specific products, 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 5' 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 13C NMR, 1H NMR and MS analysis. The 1H NMR spectrum showed five N-methyl signals at 2.87, 2.90, 3.02, 3.06 and 3.27 p.p.m.; and the 13C NMR spectrum in the same solvent revealed a total of 60 signals. The NMR spectra are shown in Supplementary Fig. S1 and the 13C 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.
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). However, substance A exhibited no observable toxicity against the other tested bacteria.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; 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 substance 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--hydroxyisovaleryl-L-N-methylleucyl units, which was identical to bassianolide, a cyclooctadepsipeptide with a molecular formula of C48H84N4O12, 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-cyclooctadepsipeptide, bassianolide.
The NRPS encoded by the nrpsxy gene is an enzyme consisting of a single polypeptide chain. Its domain architecture was found to be highly similar to that of N-methyl-cyclohexadepsipeptide enniatin synthetase (ESYN) 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-hydroxyisovaleric acid and the branched-chain L-amino acid, causing the substrates to assemble into three units of dipeptidol building blocks (von Döhren et al., 1997). It has been reported that the N-methylated cyclodepsipeptides beauvericin and PF1022A are synthesized by NRPSs that are probably of similar domain architecture to ESYN (von Döhren 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, beauvericin 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 Döhren 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-hydroxyisovaleric acid precursor is synthesized from 2-ketoisovalerate (2-KIV) catalysed by the D-hydroxyisovalerate dehydrogenase 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 -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 potential of Xylaria sp. BCC1067 as a prolific resource for bioactive compounds has been demonstrated by 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.
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ACKNOWLEDGEMENTS |
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Edited by: L. N. Glass
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Received 11 October 2007;
revised 2 January 2008;
accepted 3 January 2008.
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