Deletion of a Histone Acetyltransferase Leads to the Pleiotropic

Letter pubs.acs.org/OrgLett

Deletion of a Histone Acetyltransferase Leads to the Pleiotropic Activation of Natural Products in Metarhizium robertsii Aili Fan,†,§,⊥ Wubin Mi,‡,⊥ Zhiguo Liu,† Guohong Zeng,‡ Peng Zhang,† Youcai Hu,∥ Weiguo Fang,‡ and Wen-Bing Yin*,† †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, China § Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China ∥ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China ‡

S Supporting Information *

ABSTRACT: Histone deacetylation normally decreases the gene expression in organisms. By genome-wide deletions of epigenetic regulators in entomopathogenic fungus Metarhizium robertsii, unexpected activations of orphan secondary metabolite genes have been found upon the disruption of a histone acetyltransferase (HAT) gene Hat1. This led to the characterization of 11 new natural products, including eight isocoumarin derivatives meromusides A−H and two nonribosomal peptides meromutides A and B. Therefore, disruption of HAT represents a new approach to mine chemical diversity from fungi.

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coumarin glycosides from Metarhizium anisopliae (No. DTH1210).17 Genome sequencing of M. robertsii revealed the genome encodes 79 core genes that are involved in SM biosynthesis, including 14 putative NRPSs, 24 PKSs, and five hybrid PKSNRPS genes.18 This large amount of biosynthetic gene clusters is in contrast to the number of isolated natural products from M. robertsii. Therefore, M. robertsii represents valuable resource for mining novel active small molecules by targeting epigenetic modification. Epigenetic modification such as histone acetylation and deacetylation occurs in different organisms ranging from yeast to plants to mammals and plays an important role in the regulation of gene expression.19 It is widely investigated as a target for curing human diseases, such as the use of KAT6HATs in regulating cell proliferation.20 Moreover, histone acetylation plays an important role in the regulation of SM in fungi. For example, overexpression of the histone 4 acetyltransferase EsaA increases SM production in Aspergillus nidulans.21 In A. f lavus, loss of the HAT AflGcnE demonstrated a significant down-regulation on aflatoxin gene expression.22 In this study, we performed a genome-wide search for genes encoding epigenetic regulators and selected 13 for gene disruption in M. robertsii based on homologous recombination as described (Tables S1 and S2 and Figure S1).23 The wild-type

ungal secondary metabolites (SMs) and their derivatives contribute significantly to drug discovery and development, exemplified by the well-known antibiotics penicillin, which started a new era in medicine. Recent genome sequencing and mining of many fungal species have revealed that most SM gene clusters are silent or expressed at low levels under standard laboratory conditions, indicating that the capacity of fungi to produce SMs is far more than we anticipated. Meanwhile, genetic regulations to produce natural products in different kinds of organisms, such as bacteria and plants, have been reported and are currently being investigated intensively.1,2 As for fungi, to explore their full biosynthetic potential, different strategies have also been developed to activate these silent pathways including microbial communication, epigenetic approaches, promoter engineering, as well as heterologous expression.3−5 Metarhizium robertsii is a broad-spectrum entomopathogenic fungus distributed worldwide and has been well used for biocontrol in agriculture.6 Various SMs of great interest have been isolated from entomopathogenic fungi in recent years.7 For M. robertsii, several natural products were identified including the nonribosomal peptide synthetase (NRPS) products destruxins,8,9 serinocylins, and hybrid polyketide synthase (PKS)-NRPS product NG-39x10 and cytochalasins, metroterpenes subglutinols A−D,11 antimicrobial terpenoid helvolic acid,12 tyrosine betaine,13 ovalicin,14 siderophores,15 metachelins,15 swainsonine,16 and eight PKS products iso© XXXX American Chemical Society

Received: February 16, 2017

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DOI: 10.1021/acs.orglett.7b00476 Org. Lett. XXXX, XXX, XXX−XXX

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Figure 1. HPLC analysis of the crude extract (A) and Western blot analysis of the histone H3 acetylation level in WT, Hat1 deletion strain, and the complemented strain (B). Detection was carried out at 210 nm. The acetylation level of histone H3 was clearly decreased in the Hat1 deletion mutant and mostly recovered in the complemented strain by Western blot analysis.

(WT) and mutants were cultured on PDA at 25 °C for phenotype observation and SM analysis (Figure 1A and Figure S2). Most noticeably, the deletion of MAA_02282, a putative histone acetyltransferase (HAT), exhibited slow growth phenotype and clear color change on the plate (Figure S2), indicating that chemical profiles of mutants might be changed. In contrast, complementation of this gene to the MAA_02282 deletion mutant restored the strain morphology and metabolite production (Figure 1A). In general, HAT increases gene expression in organisms. Considering the few HATs reported for SM activation, we performed Western blot for the function confirmation of MAA_02282. The result showed that the mutants had a lower histone (H3) acetylation level than WT, thus indicating it is a HAT (named Hat1) (Figure 1B). HPLC analysis of the organic extracts from Hat1 mutants verified huge changes in chemical profiles in comparison to WT and Hat1-complemented strains (Figure 1A). Specifically, some new significant peaks appeared in the extract of ΔHat1 strain with the retention time ranging from 6 to 17 min (Figure 1A). Therefore, the histone acetylation by HAT1 unexpectedly suppressed the biosynthetic genes, and its deletion led to the activation of secondary metabolism. To characterize the newly produced compounds in the ΔHat1 strain, we conducted scale-up fermentation. Then, the ethyl acetate extracts were subjected to vacuum liquid chromatography over an ODS column using a gradient elution with methanol−H2O to give five fractions (Fractions A−E). Fraction A was rich in new peaks and selected for repeated column chromatography and semipreparative HPLC purification, which yielded 24 compounds, including 18 polyketides (1−15 and 21−23), five peptides (16−20) (Figure 2), and indole-3-acetic acid. Among them, meromusides A−I (1−8 and 15) and meromutides A and B (16 and 17) were identified as new compounds (Figure 2). Meromuside A (1) was obtained as yellow amorphous powder. The molecular formula of 1 was deduced by HRMS at m/z 427.1991 [M + H]+ (calcd for C21H30O9, 426.1890) (Table S4). Only one aromatic proton (6.18, 1H, s) was identified in the 1H NMR, which indicated the multisubstitution of benzene ring. The signal of 5.48 (2H, m) suggested the existence of one double bond. The aliphatic chain was identified and connected to the C-3 of the isocoumarin moiety confirmed by the 1H−1H COSY and HMBC spectra (Figure 3 and see Table S5 and Figures S3−S7). The classic

proton signals between 3.24−4.47 ppm indicated the appearance of the 4′-O-methylglucopyranose moiety, which was confirmed by comparison the 1H and 13C NMR spectra with the published data.17 The large coupling constant (J = 7.6 Hz) for H-1′ indicated its β configuration (Table S5 and Figure S3). The D-configuration of the sugar moiety was determined via acid hydrolysis and derivatization of the resulting sugar according to the method reported previously (Supporting Methods and Figure S88).17 CD spectra of 1 showed the positive Cotton effect at 268 nm, which indicates the configuration of C-3 of 1 is S (Figure 3 and Figure S81).17 Compared to the known compound 10, 1 has the methylene instead of carbonyl group at the C-1 position. Therefore, the configuration of C-3 was further confirmed by comparison of the CD spectrum of 1 with the C-1-reduced product of 10 (Supporting Methods and Figure S89). Taken together, the absolute structure of 1 was assigned as 3S (Figure 2). Meromuside B (2) was obtained as yellow amorphous powder. The molecular formula of 2 was deduced by HRMS at m/z 427.1778 [M + H]+ (calcd for C21H30O9, 426.1890) (Table S4), which is an isomer of compound 1. Compared the NMR data of compound 2 to 1, the substitution position of the 4′-O-methylglucopyranose moiety was determined to be O-8 (Figure 2). Similarly, 2 had the 3S configuration as well, according to the positive Cotton effect at 268 nm in the CD spectrum (Figure S82), and the relative and absolute configurations of the sugar moiety are the same as those in 1 (Figure S88). Meromuside C (3) was obtained as a yellow amorphous powder. According to its HRMS at m/z 279.1236 [M + H]+, the formula for 3 is C15H18O5. On the basis of the NMR spectra, 3 shared a similar structure as compound 1 with a missing sugar moiety. In the 13C NMR of 3, the signal for the carbonyl group (171.8 ppm) showed up, while the signal for methylene (65.4 ppm, C-1, Figure 3 and Table S5) of compound 1 is missing, indicating that the methylene was oxidized to the carbonyl group (Table S7 and Figures S13− S17). Compared to a previously reported compound (10 in this study, Table S14 and Figures S47 and S48), the elucidated structure has a methoxyl group with a proton signal at 3.82 (s, 3H) instead of the 4′-O-methylglucopyranose moiety (Table S7 and Figures S13−S17).17 The rest of the NMR data of 3 corresponded well to the previously published data (Table S14 B

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Figure 3. Structure elucidation of compound 1. (A) Key HMBC correlations (H to C) and 1H−1H COSY (−). (B) CD spectra of 1.

Meromuside E (5) shares the same isocoumarin moiety as compound 3. HRMS at m/z 277.1092 [M + H]+ determined the formula to be C15H16O5 (Table S4). The only difference between 5 and 3 is that 5 has two double bonds on the aliphatic chain, which can be clearly seen in the 1H NMR spectrum with four alkenyl protons (5.84−6.39 ppm) (Table S9 and Figure S23). Compared to 3, meromuside F (6) has one more double bond next to the ester bond, which consists of the isocoumarin moiety (Table S10 and Figures S28−S32). Meromuside G (7) has a structure similar to that of 6, with the difference on the substitution position of methoxyl group on the benzene ring, which can be deducted by HSQC and HMBC spectra (Table S11 and Figures S33−S37). Meromuside H (8) has the same skeleton as 6 with the substitution of 4′-O-methylglucopyranose moiety instead of the methyl group (Table S12 and Figures S39−S42 and S88). Meromuside I (15) was isolated as a white powder. HRMS of 15 at m/z 303.0989 [M + H]+ revealed a molecular formula of C16H14O6 accounting for 10° of unsaturation (Table S4). The signals of protons at 2.17 (3H, s) and 3.77 (3H, s) indicated the substitution of aryl methyl and −OMe groups in 15 (Table S19 and Figures S61−S64). Four broad singlet or doublet (J = 1.5 Hz) aryl protons suggested the substitution position. Taking the 13C NMR into consideration, two benzene rings with hydroxyl groups and one carbonyl group are confirmed in the structure (Table S19 and Figure S62). HSQC and HMBC signals confirmed the connection of the two benzene rings by a carbon and the substitution positions (Figures S63 and S64). As a result, 15 was identified as a new isobenzofuranone with a substituted benzene ring attached at the C-3 position of the furanone ring. The zero optical rotation observed for 15 indicated its racemic feature.24 Two new cyclic peptides, meromutides A and B (16 and 17), were isolated as the respective hydroxyl derivatives of the known compound Sch 54794 (18) and Sch 54796 (19)25 by NMR spectroscopy and HR-MS analysis and also isolated from ΔHat1 strain (Tables S4, S20−S23 and Figures S65−S72). The stereochemistry of 16 and 17 shown in Figure 2 is drawn based on those established for the Sch 54794 and Sch 54796 by comparing their optical rotations. Compounds 9−14 and 20−

Figure 2. Compounds isolated from ΔHat1 mutant. Compounds 1−8 and 15−17 are new compounds.

and Figures S47 and S48). The absolute configuration of 3 was determined as described for compound 1 (Figure S83). Meromuside D (4) was obtained as a yellow amorphous powder. The formula was determined as C21H26O10 by HRESI-MS at m/z 439.1664 [M + H]+ (Table S4). Based on the NMR data, 4 was an isocoumarin derivative as well. Compared to 1, 4 has one more double bond in the aliphatic chain which can be clearly seen in the 1H NMR spectrum with four alkenyl protons (5.71−6.35 ppm) and in the 13C NMR with four alkenyl carbons (127.0−133.4 ppm) (Table S8 and Figures S18−S22), and the C-1 is a carbonyl group instead of methylene. The absolute structure of 4 was determined as described for 1 (Figures S84 and S88). C

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ACKNOWLEDGMENTS We thank Prof. Dr. Xuebing Li for providing the chemicals and equipment for the chemical reactions. We thank Drs. Jinwei Ren and Wenzhao Wang (Institute of Microbiology, CAS) for NMR and MS data collection. W.-B.Y. is a scholar of “the 100 Talents Project” of CAS. W.F. is funded by the National Natural Science Foundation of China (31672078).

23 were elucidated by comparison of their NMR data and CD spectra with published data17,26,27 (SI). Since most of the isolated compounds are polyketide products, we further determined the relative expressions of 12 PKS genes between WT and ΔHat1 strain by qRT-PCR analysis. Six PKS genes are significantly up-regulated (Figure 4). Therefore, this confirmed that HAT1 plays a negative regulation role in the gene expression as a seldom case.

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In summary, we identified a HAT by genome-wide deletion of 13 epigenetic regulators in M. robertsii. Deletion of Hat1 led to unusual activation of secondary metabolism and the elucidation of 11 new natural products. Our study provides a new approach for the discovery of novel small molecules by manipulation of HAT in fungi.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00476. Supplementary methods, figures, tables, full spectroscopic data, and NMR spectra of new compounds (NMR, MS, and CD) (PDF)

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REFERENCES

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Figure 4. Quantitative RT-PCR analysis of relative expression of 12 PKS genes in WT and ΔHat1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Youcai Hu: 0000-0002-3752-7485 Wen-Bing Yin: 0000-0002-9184-3198 Author Contributions ⊥

A.F. and W.M. contributed equally.

Notes

The authors declare no competing financial interest. D

DOI: 10.1021/acs.orglett.7b00476 Org. Lett. XXXX, XXX, XXX−XXX