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A Putative Histone Deacetylase Modulates the Biosynthesis of Pestalotiollide B and Conidiation in Pestalotiopsis microspora
A Putative Histone Deacetylase Modulates the Biosynthesis of Pestalotiollide B and Conidiation in Pestalotiopsis microspora
Journal of Microbiology and Biotechnology. 2015. May, 25(5): 579-588
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : September 22, 2014
  • Accepted : November 12, 2014
  • Published : May 28, 2015
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About the Authors
Xueliang Niu
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
Xiaoran Hao
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, P.R. China
Zhangyong Hong
Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
Longfei Chen
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
Xi Yu
State Key Program of Microbiology and Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
Xudong Zhu
Department of Biochemistry and Molecular Biology, Beijing Normal University, Beijing 100875, P.R. China
xudong82@nankai.edu.cn

Abstract
Fungi of the genus Pestalotiopsis have drawn attention for their capability to produce an array of bioactive secondary metabolites that have potential for drug development. Here, we report the determination of a polyketide derivative compound, pestalotiollide B, in the culture of the saprophytic fungus Pestalotiopsis microspora NK17. Structural information acquired by analyses with a set of spectroscopic and chromatographic techniques suggests that pestalotiollide B has the same skeleton as the penicillide derivatives, dibenzodioxocinones, which are inhibitors of cholesterol ester transfer protein (CETP), and as purpactins A and C’, inhibitors of acyl-CoA:cholesterol acyltransferase (ACAT). Strain NK17 can make a fairly high yield of pestalotiollide B ( i.e. , up to 7.22 mg/l) in a constitutive manner in liquid culture. Moreover, we found that a putative histone deacetylase gene, designated as hid1 , played a role in the biosynthesis of pestalotiollide B. In the hid1 null mutant, the yield of pestalotiollide B increased approximately 2-fold to 15.90 mg/l. In contrast, deletion of gene hid1 led to a dramatic decrease of conidia production of the fungus. These results suggest that hid1 is a modulator, concerting secondary metabolism and development such as conidiation in P. microspora . Our work may help with the investigation into the biosynthesis of pestalotiollide B and the development for new CETP and ACAT inhibitors.
Keywords
Introduction
Filamentous fungi produce a large myriad of secondary metabolites that have long been extensively exploited for drug development. The repertoire of the secondary metabolites synthesized by a single fungus can be larger than people have considered. In the past decade, fungal genome projects have revealed that many gene clusters potentially involved in secondary metabolism are silenced in the genome. The epigenetic process is vigorously involved in regulating the silence and activation of these gene clusters for secondary metabolism [2 , 6] . Among the types of epigenetic regulation of gene expression, histone acetylation has emerged to have a major regulatory significance. Histone acetylation is a dynamic process that depends on the concerted action of histone acetyltransferases (HATs) and a second group of enzymes, histone deacetylases (HDACs). Generally, hypoacetylation of histones tends to be associated with heterochromatin and gene silencing, whereas hyperacetylation is associated with euchromatin and gene activation [4 , 21] . Exceptions that deacetylation of histones can also be responsible for direct activation of genes are also frequently reported [7 , 27] .
Fungal HDACs consist of at least three subclasses: (i) Class I includes the RPD3-type enzymes, (ii) Class II includes the HDA1-type enzymes, and (iii) the group of enzymes of human HDAC11 and HDA2 of Arabidopsis thaliana [10] . RpdA and HosA, which are the Class I RPD3-type enzymes, represent the first HDACs identified in Aspergillus nidulans [9] . After that, several HDACs have been studied in Fusarium graminearum [16] , A. fumigatus [14] , Magnaporthe oryzae [8] , and Cochliobolus carbonum [26] . It was found that HDACs were involved in regulating the genes that were important for pathogenicity, stress response, secondary metabolism, conidiation, sexual reproduction, growth, and development [8 - 10 , 14 , 16 , 26] .
Fungi in the genus Pestalotiopsis are widely distributed in tropical and temperate regions and are described as the “ E. coli of rainforest” by investigators [17 , 29] . These fungi have recently caught attention for their remarkable capability to produce a number of secondary metabolites [17 , 28 , 29] . For instance, the antitumor drug paclitaxel (taxol) has been found to be synthesized by more than ten species of Pestalotiopsis isolated from all over the world; to name a few, P. microspora [24] , P. guepinii [29] , and P. breviseta [13] . A group of novel polyketide derivatives, pestalotiollides, were lately identified in the culture of an endophytic strain of Pestalotiopsis sp. that was isolated from the mangrove tree [28] . Unfortunately, they were synthesized as the minor metabolites on solid rice substrate.
Here, we report the chemical characterization of pestalotiollide B (PB) that was purified from the culture of a saprophytic fungus, P. microspora NK17. This fungus was initially isolated as a taxol producer from South China [24] . Strain NK17 can constitutively produce a fairly high level of PB ( e.g. , up to 7.22 mg/l) in the optimized liquid culture. In the meanwhile, we noticed that PB actually shares the same skeleton with dibenzodioxocinones, which are a new class of inhibitors of cholesterol ester transfer protein (CETP) derived from fungal natural products, penicillides [3] . Still, we found that the biosynthesis of PB in this strain was subject to epigenetic modulation. Deletion of a putative histone deacetylase gene ortholog, designated as hid1 , led to a significant increase of over 2-fold in PB production (15.90 mg/l). We also found some other function for hid1 ; that is, in conidiation of this fungus. We describe the results below.
Materials and Methods
- PestalotiopsisStrains and Culture Conditions
P. microspora strain NK17 was originally isolated as a taxol producer and stored at this laboratory (China Patent ZL 2008 10152500.1). The genome of NK17 has been partially sequenced by BGI (Shenzhen, China; http://www.genomics.cn/index ). The availability of the sequence information assured the identification of the histone deacetylase-encoding gene hid1 in NK17 (GenBank Accession No. KF679829). A uracil auxotroph ( ura3 ∆) of NK17 was created by this laboratory (Chen et al. , manuscript in preparation) and was used as the recipient in the making of the hid1 deletion mutant via homologous replacement.
The fungal strains were routinely grown on PDA (20% peeled and cut potato, 2.0% dextrose, and 2% agar, pH 7.0) at 28℃, for seed preparation and storage. For PB production, fungal inoculum was prepared on a fresh PLA plate (15% peeled and cut potato, 4.0% lactose, and 2% agar, pH 6.5) at 28 ℃. Agar blocks (5 mm 2 ) were obtained from the margins of the actively growing hyphae and inoculated into 500 ml Erlenmeyer flasks with 200 ml of potato lactose broth (PLB; i.e. , PLA without agar). Cultures were incubated on a rotary shaker at 180 rpm, at 24℃ for 7 days. A concentration of 150 mg/l of uracil was supplemented in the medium if needed.
- Extraction, Purification, and Characterization of PB
Extraction and purification of PB was as described by Xu et al. [28] . Fungal mycelium was collected from PLB culture (180 rpm at 28℃ for 8 days). Mycelium and the liquid phase were separated by centrifugation, followed by vacuum filtration with 3M filter paper. The culture supernatant was extracted with an equal volume of ethyl acetate. The ethyl acetate extractions were evaporated at 50℃ and the residue was dissolved again in 2 ml of methanol. Liquid chromatography-mass spectrometry (LCMS-2020; Shimadzu, Tokyo, Japan) was adopted for detection of PB with a C 18 HPLC column (3.0 mm × 75 mm, 5u; Agela Technoogies, Tianjin, China) under the following conditions: column oven, 30℃; detected at 227 nm; flow rate, 0.4 ml/min; solvent A, 98% water and 2% methanol with 0.05% trifluoroacetic acid (TFA); solvent B, 2% water and 98% methanol with 0.05% TFA; gradient, 0 min 90% A, 2.5 min 55% A, 5 min 0% A, 8 min 0% A, 9 min 90% A, and 10 min 90% A. Further purification was achieved by a semi-preparative reversed-phase HPLC (LC-06A; Shimadzu) with a inertsil prep ODS HPLC column (10 mm × 250 mm, 10 µm; Shimadzu) under the following conditions: column oven, 30℃; detected at 227 nm; flow rate, 3 ml/min; solvent A, 98% water and 2% methanol with 0.05% TFA; solvent B, 2% water and 98% methanol with 0.05% TFA; gradient, 0 min 90% A, 5 min 90% A, 30 min 5% A, 35 min 5% A, 37 min 90% A, and 40 min 90% A.
The IR spectrum of the compound was obtained on a Bio-Rad FTs 6000 series instrument (Bio-Rad, CA, USA). Purified PB was resuspended with IR-grade KBr (1:10) that was pressed into discs using a spectrum pelletizer. The IR spectrum was recorded in the region between 400 and 4,000 cm -1 . High-resolution ESI-MS was carried out on a Varian 7.0T FTMS mass spectrometer. 1 H and 13 C NMR spectra were obtained in CD 3 OD using a Bruker AV 400 NMR spectrometer. Circular dichroism (CD) spectra were recorded with a BioLogic MOS-450 spectrometer (BioLogic Science Instruments, France).
- Quantification of PB from the Culture Liquid
About 10 6 conidia of the fungi were inoculated to 200 ml of PLB and grown at 180 rpm and 28℃ in triplicate for 8 days. After the extraction and purification process mentioned above, the quantification of PB from the culture liquid was done using an analytical HPLC (CoM 6000; CoMetro Technology, NJ, USA) with a Kromasil C 18 ODS column (4.6 mm × 250 mm, 5 µm; AKZONobel, Gland, Switzerland). Samples in 20 µl of methanol were injected and then eluted with methanol/H 2 O (70:30 (v/v); pH 7.0). The flow rate was fixed at 1 ml/min. For quantification of PB, a standard curve was created using the purified PB samples with known concentration.
- Agrobacterium-Mediated Disruption ofhid1
Disruption of hid1 was achieved by targeted gene replacement mediated by Agrobacterium tumefaciens T-DNA transfer. To finish this, the disruption cassette was constructed on the plasmid pOSCAR [20] by a method called one-step ligation, as described by the manufacturer’s instruction for the CloneEZ PCR Cloning Kit ( http://www.genscript.com.cn/ ). Briefly, pOSCAR was digested with Hin dIII and Eco RI, and the longer fragment was extracted. Based on the sequences of hid1 (ORF: 2,355 bp) and ura3 (ORF: 1,167 bp), about a 0.8kb upstream fragment (Up) and a 0.55 kb downstream fragment (Down) of hid1 and a 2.3 kb functional ura3 gene from NK17 (as selection marker) were obtained by PCR with the use of primers Hid1UF/Hid1UR, Hid1DF/Hid1DR, and URA3F/URA3R ( Table 1 ), respectively. NK17 genomic DNA served as the template in the PCRs. The four DNA fragments were joined together by using the CloneEZ PCR Cloning Kit via homologous recombination to form the vector pOSCAR-Hid1 carrying the hid1 deletion construct.
PCR primers used in this study.
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PCR primers used in this study.
By CaCl 2 -mediated transformation, pOSCAR-Hid1 was then introduced into A. tumefaciens LBA4404, which carried the T-DNA transfer machinery [5] . The plasmid pOSCAR-Hid1 extracted from LBA4404 transformants was verified by PCR with the use of primers Hid1UF/Hid1UR and Hid1DF/Hid1DR. LBA4404 carrying the pOSCAR-Hid1 was grown at 28℃ overnight in 5 ml of LB medium supplemented with 100 mg/l streptomycin and 25 mg/l rifampicin. Bacterial cell suspensions were subsequently diluted to an optical density of OD 0.15 at 600 nm in induction medium. The cells were grown for an additional 6 h. Co-cultivation between A. tumefaciens and conidia of the uracil auxotroph of NK17 was performed as follows: 100 µl of bacterial culture (10 8 CFU) was mixed with an equal volume of uracil auxotroph conidia (10 7 ) and spread on nitrocellulose filter. The filter was incubated on LB agar in the presence of 200 µM acetosyringone (Sigma, USA), at 24℃, 48h. After co-cultivation, the filter was transferred to PLA supplemented with 200 µg/ml cefotaxime to counter-select Agrobacterium cells. Each fungal transformant was subsequently transferred to PLA. After 7 days of incubation, transformants were purified by single spore purification and were allowed to grow on PLA.
- Characterization forhid1∆ Mutants by PCR and Southern Blotting
PCR amplification and Southern blotting were conducted for screening and verification of hid1 replacement. Genomic DNA was prepared as described previously [11] . The hid1 null mutants were screened by PCR with a pair of primers, Hid1-v-up-F/Hid1-v-up-R ( Table 1 ). Hid1-v-up-F was located at 822 bp upstream of the disruption cassette, and Hid1-v-up-R was located in the open reading frame (ORF) of ura3 . A 2.1 kb PCR product was expected in the positive candidates, whereas no product would be seen in the uracil auxotroph. The PCR product was then subjected to sequencing for confirmation. With this approach, one positive candidate, designated as hid1 ∆, was chosen for Southern blotting.
Genomic DNA of the hid1 ∆ mutant and the uracil auxotrophic strain (as control) was digested with Nde I and Bam HI and separated by electrophoresis on 0.8% agarose gel, and transferred onto a Magmaprobe Nylon Transfer Membrane-N + (Osmonics, Minnetonka, MN, USA). DNA labeling, hybridization, and detection procedures were carried out by following the DIG High Prime DNA Labeling and Detection Starter Kit II protocols (Roche China, Shanghai, China). The membrane was hybridized with two DIG-labeled probes, hid1 (577 bp) and ura3 (2.3 kb) fragments ( Fig. 6 A).
To construct and confirm the complemented strains, a 3.6 kb PCR fragment of the wild-type hid1 with the primers hid1∆-c-up-F/hid1∆-c-up-R ( Table 1 ), and a 2.3 kb PCR fragment of the ura3 marker with primers URA3F/URA3R were amplified and ligated together with the method of One Step Construction of Agrobacterium -Recombination-ready-plasmids (OSCAR) [20] . The final vector pOSCAR-Hid1-c that contained the whole hid1 and the marker ura3 was then introduced into A. tumefaciens LBA4404, and fungal complements were obtained in the way described earlier. The complement strains exhibiting hygromycin B resistance were subjected to PCR confirmation and sequencing.
- RNA Extraction and Reverse Transcription PCR (RT-PCR) forhid1mRNA Detection
Fungal mycelia were harvested at 2, 4, 6, and 8days (PLB, at 28℃). Mycelia were collected, lyophilized, and ground to a fine powder. Total RNA was extracted using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The firststrand cDNA was synthesized by M-MLV RTase with 1 µg of total RNA as the template and oligo (dT) as the primer (TaKaRa, Dalian, China). The actin-encoding gene act1 of NK17 was used as the endogenous reference.
- Conidia Production
Conidia were obtained from fungal cultures inoculated on PLA for 8days at 28℃. Washed with sterile distilled water twice, the conidia were stripped from the plates and suspended in ddH 2 O. Counting was proceeded by hemocytometry. Concentrations of the conidia were calculated in triplicate. Errors were expressed as standard deviation.
Results
- Structural Characterization of the Putative Pestalotiollide B by Strain NK17
The result of detecting PB by LC-MS (ESI) is shown in Fig. 1 . The peak of PB eluting at 3.9 min on the HPLC chromatogram gave two pseudomolecular ion peaks at m/z = 408.6 [M + Na] + and m/z = 795.0 [2M + Na] + .
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LC-MS analysis for the putative pestalotiollide B purified from strain NK17. For a detailed description, see the Results section.
The absorption pattern of bands of the compound in the IR spectra was obtained to record the chemical nature of the extracted metabolite from the fungus ( Fig. 2 ). A broad peak at 3,293 cm -1 was observed, due to hydroxyl (-OH) group stretch. The aliphatic CH stretch was observed at 2,935 cm -1 . The C=O stretching frequency was seen at 1,728 cm -1 . The two peaks at 1,593 and 1,461 cm -1 were putatively due to the aromatic ring (C=C) stretch. The two peaks at 1,201 and 1,045 cm -1 were attributed to the C-O-C stretch.
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IR spectrum of the sample pestalotiollide B. For a detailed description, see the Results section.
The molecular weight and chemical formula were acquired by high-resolution ESI-MS ( Fig. 3 ). Characteristically, the compound generated molecular ion peaks [M + Na] + at m/z 409.1254 (calculated for C 21 H 22 O 7 Na, 409.1263), and [2M + Na] + at m/z 795.2630, but no [M + H] + . This result is consistent with the m/z data of PB reported by Xu et al. [28] .
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High-resolution ESI-MS spectrum of pestalotiollide B of strain NK17. Pestalotiollide B had an [M+Na]+ peak at a molecular weight of 409.1254 and a [2M+Na]+ peak at a molecular weight of 795.2630.
In 1 H NMR and 13 C NMR spectroscopic analyses ( Figs. 4 A and 4 B), the data of PB suggested the presence of a methoxy group (δ H 3.94, s, δ C 63.6, s, 4 -OCH 3 ), a terminal double bond [δ C 114.3, s, CH 2 -4’; δ C 146.7, s, C-3’], two methyl substituents (δ H 1.81, s, δ C 18.6, s, 5’-CH 3 ; δ H 2.21, s, δ C 21.0, s, 9-CH 3 ), two ortho -coupled aromatic protons [δ H 7.01 (d, J = 8.5 Hz), δ C 119.0, d, CH-1; δ H 7.72 (d, J = 8.5 Hz), δ C 134.1, s, CH-2], two aromatic methines [δ H 6.42, s , δ C 121.4, s, CH-8; δ H 6.78, s, δ C 119.4, s, CH-10], an oxygenated methylene group [δ C 70.6 s, CH 2 -7], and two oxygenated methines [δ H 5.04 (d, J = 7.25 Hz), δ C 69.4, d, CH-1’; δ H 4.25 (d, J = 7.25 Hz), δ C 79.7, d, CH-2’]. These spectral results were consistent with the former data for PB described by Xu et al. [28] .
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1H NMR (A) and 13C NMR (B) spectra of the putative pestalotiollide B produced by strain NK17 in CD3OD at 400 mHz.
The absolute configuration of PB was confirmed by its CD spectrum. As shown in Fig. 5 , the CD spectrum of PB showed a negative (∆ε = -11.94, 280 nm) Cotton effect, which was similar to the results of Li et al. [15] , suggesting the 1’ S absolute configuration [12] . Put together, the chemical structure of PB produced by NK17 is shown in the insert panel of Figs. 4 A, 4 B, and 5 .
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CD spectrum of pestalotiollide B in methanol (25℃).
- Identification and Disruption of the Putative Histone Deacetylase Genehid1
The full-length genomic DNA of hid1 was cloned from P. microspora NK17 and sequenced. The hid1 gene is composed of three introns and four exons and encodes a putative polypeptide of 730 amino acids. The putative Hid1 protein sequence was compared with other HDAC protein sequences in GenBank. Hid1 shared a high identity at the amino acid level with A. nidulans HdaA (GenBank Accession No. AF306859, 47.1%), C. carbonum HDC3 (AF307341, 45.0%), and Saccharomyces cerevisiae HDA1 (Z71297, 38.5%). All the three proteins belongs to the group of Class II HDACs. In the meanwhile, Hid1 displayed relatively lower sequence identity to S. cerevisiae RPD3 (P32561, 22.3%), A. nidulans RpdA (AF163862, 20.7%), A. nidulans HosA (AF164342, 19.3%), and C. carbonum HDC1 (AF306507, 18.3%), the Class I enzymes. Thus, our Hid1 should be sorted into Class II. Reverse transcription PCR demonstrated that hid1 was constitutively expressed and was independent of culture time in NK17 (Fig. S1).
A portion of the hid1 ORF was replaced by ura3 via homologous recombination as a routine practice in uracil auxotroph of NK17 ( Fig. 6 A; see Materials and Methods). By PCR screening, one hid1 disruption candidate, hid1 ∆, was obtained (data not shown). hid1 ∆ was complemented with a 3.6kb wild-type copy of gene hid1 and the complement strain hid1 ∆- c was created. The strains hid1 ∆ and hid1 ∆- c were verified by Southern blotting ( Fig. 6 B) and PCR amplification (Fig. S2).
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Restriction map and Southern blot of the hid1 null mutant. (A) Schematic diagram for the disruption of hid1 in NK17 with the construct cassette on the plasmid pOSCAR-Hid1. The flanking homologous fragments of hid1 are indicated. The ura3 gene of P. microspora NK17 served as the selection marker in the transformation of a uracil auxotrophic mutant of NK17. Restriction sites of NdeI and BamHI in Southern blotting are shown. (B) Southern blotting analysis of a null mutant of hid1, probing with the ura3 fragment (left panel), or with the hid1 fragment (right panel), respectively. In the probing with ura3, no band was acquired for the uracil auxotroph of NK17 (UA); one band, 6.6 kb, was detected for the mutant hid1∆ (∆). In the probing with hid1 fragment, a single 5.5 kb band was detected for the uracil auxotroph, but no band was detected in hid1∆. Approximately 5 µg of genomic DNA from the hid1∆ and the uracil auxotroph of NK17 was digested with NdeI and BamHI.
In Southern blotting ( Fig. 6 B), when probing with the fragment of ura3 sequence, a 6.6 kb band was detected in the hid1 ∆, but no hybridization was observed in the uracil auxotroph (left panel). When probing with the fragment of hid1 ORF, a sole band (5.3 kb) was detected in the uracil auxotroph, but no hybridization was seen in the mutant strain hid1 ∆, as anticipated (right panel). Therefore, the disruption of hid1 occurred as expected in the mutant strain hid1 ∆.
The hid1 ∆- c strain was verified by two diagnostic PCRs. In the first PCR using the pair of primers hid1∆-c-up-F/hid1∆-c-up-R, a 3.6kb band was obtained in the wild type and the complement hid1 ∆- c , and a 5.0 kb band in hid1 ∆. In the second PCR with pair of primers Hid1-ORF-F/URA3R ( Table 1 ), a 5.5 kb band was obtained in hid1 ∆- c , but no band was seen in the wild type and the mutant hid1 ∆ (Fig. S2). All the PCR products were sequenced for confirmation. Moreover, an RT-PCR demonstrated that the mRNA transcripts of hid1 were detected again in the complemented strain hid1 ∆- c (data not shown).
- Effects ofhid1Disruption on Conidiation
To analyze the phenotypic effects from the disruption of hid1 , strains were compared in respects of vegetative growth and conidiation. The mutant strain hid1 ∆ showed little significant change in vegetative growth either on PLA plate ( Fig. 7 B) or in liquid culture ( Fig. 7 A, right Y-axis). However, the mutant strain generated decreased number of conidia verse the wild type. After 8 days, the number of conidia on PLA plates was approximately 1.34 × 10 6 /ml in hid1 ∆, whereas the numbers were 3.22 × 10 6 /ml and 2.89 × 10 6 /ml for the wild type and hid1 ∆- c , respectively. These results suggest that hid1 is required for conidia production in P. microspora .
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Effect of hid1 disruption on pestalotiollide B production and strain growth. (A) Quantity of pestalotiollide B and dry weight of mycelia in the wild type, hid1∆, and hid1∆-c. Values are the mean of three replicates and error bars represent standard deviation. (B) Colonies of the strains on PLA plates (28℃ for 8 days).
- Increased Production of PB inhid1∆
We had tried several different media, including synthetic ones. We found that the semi-synthetic medium PLB is by far the optimal medium for the biosynthesis of PB and the biomass production. To see whether PB was accumulated by NK17, we determined the quantity in the culture versus cultural time (Fig. S3). The mycelium was harvested, and dry weighed at the time points. As shown in Fig. S3, the maximal quantity of PB was obtained on day 8 to (6.69 ± 0.36 mg/l). Biosynthesis of the compound then declined slightly with longer cultivation.
In A. nidulans , HdaA, a major Class II histone deacetylase, is involved in the regulation of telomere-proximal secondary metabolism gene clusters. The production of sterigmatocystin and penicillin increased substantially after the deletion of hdaA [23] . Thus, we determined whether the biosynthesis of PB in the hid1 ∆ mutant of NK17 had been affected. We found that PB production indeed increased significantly in the mutant strain hid1 ∆, comparing with the wild type and the complement strain hid1 ∆- c ( Fig. 7 A, left Y-axis). The concentration of PB was 15.90 mg/l by hid1 ∆, whereas the concentration for the wild type and the hid1 ∆- c strain were 7.22 mg/l and 9.12 mg/l, respectively. Thus, this result clearly demonstrates that PB biosynthesis is negatively regulated by hid1 .
Discussion
A secondary compound was tentatively characterized to be pestalotiollide B (PB) produced by P. microspora NK17 (the insert panel of Figs. 4 A, 4 B, and 5 ). Chromatographic and spectroscopic analyses, including LC-MS, IR, highresolution ESI-MS, CD, 1 H NMR, and 13 C NMR, were conducted for the structural characterization. PB is one of the pestalotiollide derivatives that were first described in the solid rice substrate of an endophytic Pestalotiopsis sp. isolated from the plant mangrove [28] . Making a strict comparison for the chemical structure, we realized that pestalotiollides actually have the same dibenzodioxocinone skeleton with other groups of fungal natural products such as penicillides that are found in a Penicillium sp. [18] . This core structure is also present in the compounds purpactins A and C’ that were isolated in the culture of Penicillium purpurogenum [18 , 25] . The structural similarity suggests that penicillides, pestalotiollides, and purpactins are perhaps synthesized via similar biochemical routes that should correspond to related genetic backgrounds. Constitutive production of PB in a bunch quantity in the saprophytic fungus NK17 indicates PB may have important physiological and ecological significance for fungi.
A group of the drug candidates, dibenzodioxocinones, in clinical study for the treatment of coronary heart disease (CHD) have been created from penicillides [3] . Inhibition of CETP by dibenzodioxocinones raises the high-density lipoprotein level, which promises an effective strategy in the treatment of CHD [3] . Other studies have discovered that penicillide derivatives also possess activities of oxytocin antagonism [22] and antihypertension (European Patent EP. 0411268). Purpactins, sharing the same skeleton, have an inhibitory activity on acyl-CoA:cholesterol acyltransferase (ACAT) [18 , 25] . Through efforts in the study of structure-activity relationship, new relative molecules with improved stability and inhibition effects against CETP have been generated. For instance, penicillide 13 has an IC 50 of 15 nmol/l on CETP and is stable in rat plasma [3 , 30] . Given the structure similarity, it is intriguing to believe that PB is perhaps applicable for the development of novel CETP/ACAT inhibitors, for example, by chemical modification. Thus, high-yield and constant production of PB, such as in the fungus strain NK17, will promise benefits for this purpose.
To clarify the ecological significance of fungal secondary metabolites is a challenging theme. PB was first identified as a minor metabolite from the endophytic Pestalotiopsis sp., when the fungus was grown on solid rice medium [28] . In contrast, P. microspora NK17 was originally isolated as a saprophyte from the soil and it synthesizes PB in the liquid culture as a major secondary product (China Patent ZL 2008 10152500.1). This discrepancy in the biosynthesis of pestalotiollides between saprophytic and endophytic Pestalotiopsis strains implicate that the biosynthetic genes (or clusters) are subject to regulation by environmental elements or physiological conditions. One plausible explanation is that PB is dispensable for the endophytic strain since PB may be toxic to its symbiotic host plant. Thus, genes for the biosynthesis of PB in endophytes are most likely silenced. It has been quite well established that many genes/clusters in filamentous fungal genomes that are putatively involved in secondary metabolism remain silenced [2 , 6] . On the other side, constitutive synthesis of PB should favor the saprophytic strains in competition against competitors and predators. Thus, the variation of PB production in different Pestalotiopsis strains represents an example of complement of fungal secondary metabolism to their environmental niches or hosts.
In this report, we demonstrated that the biosynthesis of PB was regulated by an epigenetic process in P. microspora NK17. A Class II histone deacetylase homolog, hid1 , was identified to play a negative role in PB production. The hid1 deletion resulted in a significant increase in PB yield compared with the wild type and hid1 ∆- c strain ( Fig. 7 A, left Y-axis). Despite that hypoacetylation of histones (deacetylated status) tends to be associated with heterochromatin and gene silencing [4 , 21] , in many cases, deacetylation of histones can lead to activation of genes/clusters of secondary metabolites [7 , 26 , 27] . This is exemplified in the case of gene hdaA in A. nidulans , where disruption of the hdaA gene led to transcriptional activation of the gene clusters for the biosynthesis of sterigmatocystin and penicillin [23] , although the production of gliotoxinin decreased, caused by the deletion of gene hdaA in A. fumigatus [14] . An explanation for this differential effect of histone deacetylase could be that the gene clusters for the production of sterigmatocystin or penicillin are located proximal to the telomere, but not the clusters for gliotoxinin [23] . Based on this theory, genes/clusters responsible for the biosynthesis of PB are likely located in the telomere-proximal loci.
Last but not the least, studies in other fungi have revealed that histone deacetylases modulate the development of asexual conidiation in many fungi. For instance, in Fusarium graminearum , disruption of gene hdf1 led to diminished conidiation [16] . In Magnaporthe oryzae , deletion of mohos2 also resulted in a similar phenotype [8] . Similarly, the hid1 mutant hid1 ∆ displayed decreased conidiation in our fungus. The number of conidia formed in hid1 ∆ dropped by approximately 60% of the number for the wild type (see Results section). Some studies have shown that histone deacetylation may affect the expression of the regulators for conidiophore development, including brlA, abaA , and wetA [1 , 19] .
In conclusion, we have identified and characterized a natural compound, PB, which shares a similar structure to the CETP and ACAT inhibitors. P. microspora NK17 can synthesize a relatively high level of PB (over 7.0 mg/l). We also demonstrated a Class II histone deacetylase ortholog, hid1 , to have a negative modulating effect on PB biosynthesis. This work may facilitate the elucidation of the biosynthesis of PB in P. microspora . PB may help with the development of novel CETP and ACAT inhibitors. The mutant strain hid1 ∆ can be used as a starter in lead preparation on the large scale.
Acknowledgements
This work was supported by the National High Technology Research and Development Program (“863” Program #2012AA023405).
References
Adams TH , Wieser JK , Yu JH 1998 Asexual sporulation inAspergillus nidulans. Microbiol. Mol. Biol. Rev. 62 35 - 54
Brakhage AA , Schroeckh V 2011 Fungal secondary metabolites - Strategies to activate silent gene clusters. Fungal Genet. Biol. 48 15 - 22    DOI : 10.1016/j.fgb.2010.04.004
Bruckner D , Hafner FT , Li V , Schmeck C , Telser J , Vakalopoulos A , Wirtz G 2005 Dibenzodioxocinones - a new class of CETP inhibitors. Bioorg. Med. Chem. Lett. 15 3611 - 3614    DOI : 10.1016/j.bmcl.2005.05.073
Bulger M 2005 Hyperacetylated chromatin domains: Lessons from heterochromatin. J. Biol. Chem. 280 21689 - 21692    DOI : 10.1074/jbc.R500004200
Bundock P , den Dulk-Ras A , Beijersbergen A , Hooykaas PJ 1995 Trans-kingdom T-DNA transfer fromAgrobacterium tumefacienstoSaccharomyces cerevisiae. EMBO J. 14 3206 - 3214
Cichewicz RH 2010 Epigenome manipulation as a pathway to new natural product scaffolds and their congeners. Nat. Prod. Rep. 27 11 - 22    DOI : 10.1039/B920860G
de Nadal E , Zapater M , Alepuz PM , Sumoy L , Mas G , Posas F 2004 The MAPK Hog1 recruits Rpd3 histone deacetylase to activate osmoresponsive genes. Nature 427 370 - 374    DOI : 10.1038/nature02258
Ding SL , Liu WD , Iliuk A , Ribot C , Vallet J , Tao A 2010 The Tig1 histone deacetylase complex regulates infectious growth in the rice blast fungusMagnaporthe oryzae. Plant Cell 22 2495 - 2508    DOI : 10.1105/tpc.110.074302
Graessle S , Dangl M , Haas H , Mair K , Trojer P , Brandtner EM 2000 Characterization of two putative histone deacetylase genes fromAspergillus nidulans. Biochim. Biophys. Acta 1492 120 - 126    DOI : 10.1016/S0167-4781(00)00093-2
Gregoretti IV , Lee YM , Goodson HV 2004 Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J. Mol. Biol. 338 17 - 31    DOI : 10.1016/j.jmb.2004.02.006
Hao X , Ji Y , Liu S , Bi J , Bi Q , Pan J , Zhu X 2012 Optimized integration of T-DNA in the taxol-producing fungusPestalotiopsis malicola. Afr. J. Biotechnol. 11 771 - 776
Ishida T , Bounds SVJ , Caldwell J , Drake A , Takeshita M 1996 The absolute configuration of the four stereoisomers oftrans-anethole diol (1-(4’-methoxyphenyl)-1,2-propanediol), a metabolite of anethole in the rat. Tetrahedron Asymmetry 7 3113 - 3118    DOI : 10.1016/0957-4166(96)00411-9
Kathiravan G , Raman VS 2010 In vitroTAXOL production, byPestalotiopsis breviseta- A first report. Fitoterapia 81 557 - 564    DOI : 10.1016/j.fitote.2010.01.021
Lee I , Oh JH , Shwab EK , Dagenais TR , Andes D , Keller NP 2009 HdaA, a class 2 histone deacetylase ofAspergillus fumigatus, affects germination and secondary metabolite production. Fungal Genet. Biol. 46 782 - 790    DOI : 10.1016/j.fgb.2009.06.007
Li J , Wu XF , Ding G , Feng Y , Jiang XJ , Guo LD , Che YS 2012 α-Pyrones and pyranes from the plant pathogenic fungusPestalotiopsis scirpina. Eur. J. Org. Chem. 2012 2445 - 2452    DOI : 10.1002/ejoc.201200007
Li Y , Wang C , Liu W , Wang G , Kang Z , Kistler HC , Xu JR 2011 TheHDF1histone deacetylase gene is important for conidiation, sexual reproduction, and pathogenesis inFusarium graminearum. Mol. Plant Microbe Interact. 24 487 - 496    DOI : 10.1094/MPMI-10-10-0233
Maharachchikumbura SSN , Guo LD , Chukeatirote E , Bahkali AH , Hyde KD 2011 Pestalotiopsis- morphology, phylogeny, biochemistry and diversity. Fungal Divers. 50 167 - 187    DOI : 10.1007/s13225-011-0125-x
Nishida H , Tomoda H , Cao J , Okuda S , Omura S 1991 Purpactins, new inhibitors of acyl-CoA: cholesterol acyltrasferase produced byPenicillium purpurogenumII. J. Antibiot. 44 144 - 151    DOI : 10.7164/antibiotics.44.144
Palmer JM , Perrin RM , Dagenais TR , Keller NP 2008 H3K9 methylation regulates growth and development inAspergillus fumigatus. Eukaryot. Cell 7 2052 - 2060    DOI : 10.1128/EC.00224-08
Paz Z , Garcia-Pedrajas MD , Andrews DL , Klosterman SJ , Baeza-Montanez L , Gold SE 2011 One Step Construction ofAgrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genet. Biol. 48 677 - 684    DOI : 10.1016/j.fgb.2011.02.003
Robyr D , Suka Y , Xenarios I , Kurdistani SK , Wang A , Suka N , Grunstein M 2002 Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases. Cell 109 437 - 446    DOI : 10.1016/S0092-8674(02)00746-8
Salituro GM , Pettibone DJ , Clineschmidt BV , Williamson JM , Zink DL 1993 Potent, non-peptidic oxytocin receptor antagonists from a natural source. Bioorg. Med. Chem. Lett. 3 337 - 340    DOI : 10.1016/S0960-894X(01)80905-7
Shwab EK , Bok JW , Tribus M , Galehr J , Graessle S , Keller NP 2007 Histone deacetylase activity regulates chemical diversity inAspergillus. Eukaryot. Cell 6 1656 - 1664    DOI : 10.1128/EC.00186-07
Strobel G , Yang X , Sears J , Kramer R , Sidhu RS , Hess WM 1996 Taxol fromPestalotiopsis microspora, an endophytic fungus ofTaxus wallachiana. Microbiology 142 435 - 440    DOI : 10.1099/13500872-142-2-435
Tomoda H , Nishida H , Masuma R , Cao J , Okuda S , Omura S 1991 Purpactins, new inhibitors of acyl-CoA: Cholesterol acyltrasferase produced byPenicillium purpurogenumI. J. Antibiot. 44 136 - 143    DOI : 10.7164/antibiotics.44.136
Trojer P , Brandtner EM , Brosch G , Loidl P , Galehr J , Linzmaier R 2003 Histone deacetylases in fungi: novel members, new facts. Nucleic Acids Res. 31 3971 - 3981    DOI : 10.1093/nar/gkg473
Wang A , Kurdistani SK , Grunstein M 2002 Requirement of Hos2 histone deacetylase for gene activity in yeast. Science 298 1412 - 1414    DOI : 10.1126/science.1077790
Xu J , Aly AH , Wray V , Proksch P 2011 Polyketide derivatives of endophytic fungusPestalotiopsissp. isolated from the Chinese mangrove plantRhizophora mucronata. Tetrahedron Lett. 52 21 - 25    DOI : 10.1016/j.tetlet.2010.10.131
Yang XL , Zhang JZ , Luo DQ 2012 The taxonomy, biology and chemistry of the fungalPestalotiopsisgenus. Nat. Prod. Rep. 29 622 - 641    DOI : 10.1039/c2np00073c
Zhang Q , Deng CL , Fang LS , Xu WW , Zhao Q , Zhang JG 2013 Synthesis and evaluation of the analogues of penicillide against cholesterol ester transfer protein. Chin. J. Chem. 31 355 - 370    DOI : 10.1002/cjoc.201200977