Genetic Manipulation of the COP9 Signalosome Subunit PfCsnE

Publication Date (Web): August 24, 2017. Copyright © 2017 American ... Sambasivarao Kotha , Milind Meshram , Yuvaraj Dommaraju. The Chemical Record ...
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Genetic Manipulation of the COP9 Signalosome Subunit PfCsnE Leads to the Discovery of Pestaloficins in Pestalotiopsis fici Yanjing Zheng,†,‡ Ke Ma,†,§ Haining Lyu,† Ying Huang,# Hongwei Liu,† Ling Liu,† Yongsheng Che,∥ Xingzhong Liu,† Huixi Zou,‡ and Wen-Bing Yin*,†,§ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China ‡ Zhejiang Provincial (Wenzhou) Key Lab for Water Environment and Marine Biological Resources Protection, College of Life and Environmental Science, Wenzhou University, Wenzhou 325035, China # State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China ∥ State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China §

S Supporting Information *

ABSTRACT: By deleting the COP9 signalosome subunit PfcsnE from Pestalotiopsis f ici, seven compounds that were newly produced by the mutant could be characterized, including five new structures, pestaloficins A−E (1 and 3− 6). Pestaloficin A (1) represents a new type of dimeric cyclohexanone derivative linked through an unprecedented pentacyclic spiral ring.

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unexpectedly low. The majority of the BGCs seem to be unexpressed or lowly expressed under normal laboratory cultivation conditions.6−8 Significant effort using a variety of diverse strategies has been made to activate these clusters. The first attempts involved alterations to the cultivation conditions and media composition and the use of biotic and abiotic elicitors. Additionally, cocultivation of various microorganisms led to the activation of formerly silent gene clusters.9 Further success was achieved by interfering in the condensation state of the genomic DNA, which regulates the accessibility of a region of DNA to transcription factors. This was accomplished through the inhibition or inactivation of DNA-modifying enzymes, e.g., histone deacetylases via chemical inhibitors or disruptions of the encoding genes.10−14 Taken together, these approaches of either simulating environmental conditions or genetic modifications of chromatin structures greatly accelerated the discovery of NPs and enhanced the accessibility of new chemically diverse NPs in fungi. However, it is urgent to find new approaches to the discovery of fungal NPs. In working with the plant endophytic fungus Pestalotiopsis f ici, several regulators have been reported to be involved in both secondary metabolism and fungal develop-

atural products (NPs), mostly secondary metabolites (SMs), contribute significantly to drug discovery and development. In addition to the important producers of NPs such as Actinobacteria and Ascomycota microorganisms, fungi have become increasingly important for NP production in pharmaceutical research.1,2 In general, the genes encoding the biosynthesis of a given SM are organized in close proximity in the genome and are referred to as a biosynthetic gene cluster (BGC). Typically, the SM BGCs share common gene encoded enzymes, usually consisting of a backbone enzyme and tailoring enzymes. Nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and terpene synthase are backbone enzymes that are involved in the synthesis of the metabolite scaffold.3−5 In addition, other genes that are important for regulation of the whole gene cluster, transportation for secretion of the product, or uptake of a special substrate are also included in BGCs. Due to the ongoing development and improvement of sequencing technologies, the cost of sequencing a fungal genome has decreased. Simultaneously, the number of publications about newly discovered BGCs has increased and led to new NPs with significant pharmaceutical potential. However, mining the sequenced genomes has also revealed the largely unexplored biosynthetic potential of many microorganisms. Compared to the huge number of newly identified BGCs, the number of accumulated NPs is © 2017 American Chemical Society

Received: August 10, 2017 Published: August 24, 2017 4700

DOI: 10.1021/acs.orglett.7b02346 Org. Lett. 2017, 19, 4700−4703

Letter

Organic Letters

NMR spectroscopy, circular dichroism (CD) spectroscopy, and HR-ESI-MS.

ment. The bZIP transcription factor PfZipA was found to regulate secondary metabolism and stress responses in P. fici. Deletion of PfZipA exposed resistant phenotypes to oxidative stress and changed the chemical profile of the organism.15 In addition, genetic manipulation of epigenetic regulators such as histone methyltransferase PfCclA and deacetylase PfHdaA revealed an expanded view of chemical diversity and led to the identification of 15 new metabolites in P. f ici.16 Interestingly, deletion of histone acetyltransferase Hat1 led to the activation of BGC, which presents an unusual approach to accessing new fungal SMs in Metarhizium robertsii.17 More recently, the COP9 signalosome (CSN) has been linked to fungal development and secondary metabolism.18 CSN is a highly conserved multiprotein complex in all eukaryotes. By disrupting the fifth subunit of CSN, called CsnE in A. nidulans, a PKS gene cluster was activated, leading to the identification of the antibiotic 2,4dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA).19 This led to the hypothesis that the manipulation of fungal development is a promising approach to discovering novel SMs from fungi. To test this hypothesis, a recently certified PfcsnE deletion mutant that abolished conidia production was used.20 Chemical assessments between wild-type (WT) and ΔPfcsnE strains indicated that their SM profiles were substantially different (Figure 1). To characterize the newly produced compounds

Figure 2. Compounds isolated from ΔPfcsnE mutants.

Pestaloficin A (1) was determined to have the molecular formula C27H32O6 based on the HR-ESI-MS signal at m/z [M + Na]+ 475.2093. The 1H NMR and HSQC data of 1 (Table 1) revealed four methyls, three methylenes, seven methines (six oxymethines), two sp3 quaternary carbons (one oxygenated quaternary carbon), eight olefinic carbons including one Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for 1 in Methanol-d4 1

Figure 1. HPLC profiles of crude extracts from ΔPfcsnE strains compared to the WT strain. The ΔPfcsnE and WT strains were cultivated in PDA medium (a) and rice medium (b), respectively. Detection was carried out at 260 nm.

from the ΔPfcsnE strain, large-scale fermentation was carried out. The organic extracts were fractionated by ODS and Sephadex LH-20 column chromatography (Methods, Supporting Information). The subfractions containing the target compounds were then selected for further purification. As a result, five new compounds were isolated from ΔPfcsnE strains, pestaloficins A−E (1, 3−6), along with two known compounds, pestalotiopyrone G21 (2) and pestalolide22 (8) (Figure 2). The structures were elucidated by comprehensive analysis with 4701

position

δC

δH (J in Hz)

1 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′

23.6 121.9 128.2 88.9 86.9 54.9 56.4 65.2 52.6 67.0 206.9 18.1 26.0 136.4 119.2 34.4

1.84 (3H, s) 5.19 (1H, overlap) 5.18 (1H, overlap)

6′ 7′ 8′ 9′

66.2 63.1 68.1 29.0

10′ 11′ 12′ 13′ 14′

131.0 131.2 121.2 136.1 32.4

15′ 16′

25.0 65.9

2.19 (1H, overlap) 3.61 (1H, d, J = 3.5 Hz) 3.54 (1H, d, J = 3.5 Hz) 3.85 (1H, overlap) 1.70 (3H, s) 1.74 (3H, s) 5.13 (1H, t, J = 7.0 Hz) 2.17 (1H, overlap) 2.78 (1H, dd, J = 14.8, 7.0 Hz) 3.18 3.84 2.21 2.39

(1H, (1H, (1H, (1H,

s) overlap) overlap) dd, J = 14.1, 4.9 Hz)

6.44 (1H, s) 2.28 2.61 1.91 4.67

(1H, (1H, (3H, (1H,

d, J = 17.2 Hz) d, J = 17.2 Hz) s) s) DOI: 10.1021/acs.orglett.7b02346 Org. Lett. 2017, 19, 4700−4703

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Organic Letters

(Figure S7),23 the structure of 3 was assigned as shown in Figure 2, and the configuration at C-8 was R. The same molecular formula and the similar NMR data of pestaloficin C (4) and 3 (Tables S2, S3, and S7) suggested that 4 is a congener of 3. Further 2D NMR analysis (Table S3) revealed that a tetrahydropyran ring was fused to the benzene unit of 4 instead of a tetrahydrofuran ring in 3, but the rest of the structure remained the same. Therefore, the structure of 4 was finally assigned. Comparison of the NMR spectroscopic data of pestaloficin D (5) with those of pestaloficiol J24 indicated that a methyl group in pestaloficiol J had been replaced by a hydroxymethyl group (Table S4). Hence, after analysis of the confirmatory HMBC correlations (from H2-3 to C-4 and C-4a, from H-5 to C-4, from H2-9 to C-7, C-8, and C-8a, and from H3-13 to C-10, C-11, and C-12), the structure of 5 was determined. The molecular formula of pestaloficin E (6) was determined to be C12H20O4 based on the HR-ESI-MS signal at m/z 229.1439 (calcd 229.1434) [M + H]+ (Table S7). Its NMR spectra contained resonances reminiscent of a 5-hydroxy2(5H)-furanone skeleton carrying an alkane moiety. By comparing the 1H and 13C NMR data with those of 5hydroxy-4-(1-hydroxyethyl)-3-methylfuran-2(5H)-one25 and analyzing the key HMBC correlations (from H-5 to C-3 and C-4, from H-1′ and H2-2′ to C-4, from H3-8′ to C-2, C-3, and C-4), the structure of 6 was thus assigned as shown. Even though many approaches have been used for the discovery of fungal NPs, reports on the manipulation of regulators related to fungal development are limited. In this study, we identified that the CSN subunit PfCsnE played essential roles in the regulation of asexual development and secondary metabolism in P. f ici. The genetic disruption of PfcsnE greatly altered the SM profile of the organism, which led to the identification of seven newly produced compounds. We elucidated five new structures that represent new achievements in the discovery of fungal NPs. Our results present an efficient approach for finding novel fungal SMs.

terminal olefinic carbon, two quaternary sp carbons (δC 88.9 and 86.9), and a carbonyl carbon. Detailed analysis of the HMBC data for 1 established the partial structures for an epoxycyclohexanone (ring A) and an epoxycyclohexane (ring C). The HMBC correlations from H3-15′ to C-11′, C-12′, C13′, and C-14′; from H-12′ to C-9 and C-10′; and from H-9′ to C-7′, C-8′, C-10′, C-11′, and C-16′ allowed assignment of the cyclopentene moiety (ring B) that was joined spirally to ring A at C-9 and linked to ring C via the C-10′/C-11′ double bond. Further inspection of the HMBC correlations from H2-2 to C1, C-3, and C-4; from H-7 to C-5; from H3-1′ to C-2′, C-3′, and C-4′; from H2-5′ to C-3′, C-6′, and C-7′; from HO-8′ (δH 5.01, in DMSO) to C-8′; and from HO-16′ (δH 4.90, in DMSO) to C-16′ established the planar structure and satisfied the unsaturation requirement (Figure 3). The relative

Figure 3. Key COSY, HMBC, and NOESY correlations of 1.

configuration of 1 was determined as shown by NOESY correlations of H-10 with H-6 and H-14′; H-8′ with H-7 and H-8; H-16′ with H-12′; H2-5′ with H-7′ and H-16′, and H-4′ with H-7′ and H-16′ (Figure 3). The absolute configuration of 1 was assigned by comparison of the experimental and simulated electronic circular dichroism (ECD) curves generated by time-dependent density functional theory (TDDFT) at the B3LYP/6-31G(d,p) level. By comparison of the experimental and simulated ECD curves (Figure 4), the absolute configuration of 1 was finally determined to be 6R,7R,8S,9R,10S,6′R,7′S,8′S,16′R.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b02346. Experimental methods, figures, tables, full spectroscopic data, and NMR spectra of new compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID Figure 4. Comparison of the experimental ECD spectrum of 1 (black) with calculated ECD spectra for 1a (blue) and 1b (red).

Hongwei Liu: 0000-0001-6471-131X Wen-Bing Yin: 0000-0002-9184-3198 Notes

The NMR data (Table S2) of pestaloficin B (3) closely resembled those of truncateol L,23 except for the replacement of two aromatic singlets in truncateol L with two aromatic doublets in 3. HR-ESI-MS of 3 at m/z 281.1169 (calcd 281.1148) [M + Na]+ revealed a molecular formula of C16H18O3 (Table S7). In combination with the relevant HMBC correlations from H2-7 to C-1, C-2, C-3, and C-8 and from H3-10 to C-8 and C-9, along with the ECD patterns

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Key Research and Development Program (2016YFD0400105) and the National Natural Science Foundation of China (21502220). We thank Drs. Jinwei Ren and Wenzhao Wang (Institute of Microbiology, 4702

DOI: 10.1021/acs.orglett.7b02346 Org. Lett. 2017, 19, 4700−4703

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Organic Letters CAS) for MS and NMR data collection. W.B.Y. is a scholar of “the 100 Talents Project” of CAS.

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DOI: 10.1021/acs.orglett.7b02346 Org. Lett. 2017, 19, 4700−4703