Characterization and Biosynthesis of a Rare Fungal Hopane-Type

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Characterization and Biosynthesis of a Rare Fungal Hopane-Type Triterpenoid Glycoside Involved in the Antistress Property of Aspergillus fumigatus Ke Ma,†,‡ Peng Zhang,† Qiaoqiao Tao,† Nancy P. Keller,§ Yanlong Yang,∥ Wen-Bing Yin,*,†,‡ and Hongwei Liu*,†,‡

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†

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China ‡ Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Department of Medical Microbiology and Immunology, University of WisconsinMadison, Madison, Wisconsin 53706, United States ∥ College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: A rare fungal hopane-type triterpenoid glycoside fumihopaside A was identified by genome mining in combination with HPLC−MS/MS in Aspergillus f umigatus. Combining genetic deletions in A. f umigatus with heterologous reconstitutions in Aspergillus nidulans of the afum gene cluster, we identified one fungal squalene hopane cyclase AfumA charging the cyclization of the hopene skeleton, one cytochrome P450, and one UDP-glycosyltransferase. Bioassays indicated that fumihopaside A plays important roles in protecting A. f umigatus against heat or ultraviolet stress.

H

opanoid derivatives make up a group of pentacyclic triterpenes that feature an icosahydro-1H-cyclopenta[a]chrysene skeleton in structures. A number of hopanoids with diverse structures and interesting bioactivities have been identified from bacteria, fungi, lichens, ferns, and plants. Examples include 3β-acetoxy-7β,15α,22-trihydroxyhopane with strong antimalarial and antimycobacterial activities from Hypocrella sp., 3β,6β-dihydroxy-7β-[(4-hydroxybenzoyl)oxy]21αH-24-norhopa-4(23),22(29)-diene with insecticidal activity toward bruchid beetles from Zanha af ricana, and diplazioside V from the fern Diaplazium subsinuatum (Figure 1).1 Additionally, hopanoids with diverse C5 ribose-derived side chains, such as bacteriohopanetetrol, are common bacterial membrane components, which play important roles in modulating the fluidity and permeability of the membrane, regulating lipid raft formation, enhancing stress tolerance, and promoting plant−bacterium interactions.2 It has been reported that the squalene hopene cyclases (SHCs) catalyze the formation of the hopene skeleton from squalene or 2,3-oxidosqualene.3 Several enzymes (HpnF2, HpnF3, HpnG, HpnH, HpnI, HpnJ, and HpnK) involved in the biosynthesis of the hopanoids have been defined in the bacterium Burkholderia cenocepacia by gene mutagenesis and end product characterization.4 However, the genetic basis and © XXXX American Chemical Society

Figure 1. Representative hopane-type triterpenes in nature.

biosynthetic mechanism of hopanoids are not fully understood, especially in the eukaryotic organisms. As a ubiquitous and opportunistic human pathogen, Aspergillus fumigatus has been reported to produce a variety of secondary metabolites (SMs).5 However, no secondary metabolites with glycosyl modifications have been detected in Received: March 20, 2019

A

DOI: 10.1021/acs.orglett.9b00984 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters A. f umigatus, and rarely have they been detected in fungi.6 The genome sequencing of A. f umigatus and further bioinformatics analysis revealed a potential gene cluster (AFUB_071550− AFUB_071570, named afum) for the putative biosynthesis of hopane-type glycosides, which contains four genes, including an annotated squalene hopane cyclase (SHC, afumA, including a conservative DXDD motif),7 one cytochrome P450 (P450, afumB), one glycosyltransferase (GT, af umC), and a transcription factor (afumD) as shown in Figure 2A and Table S1.

for a glucopyranose fragment (δC 101.9, 74.2, 76.0, 72.6, 75.2, and 63.4). The smaller coupling constant of 3.6 Hz confirmed the α configuration for the anomeric carbon of the sugar moiety. The presence of D-glucose was further confirmed by acid hydrolysis and derivation followed by HPLC analysis (Figure S10). Compound 1 was assigned to be a new hopanetype glucoside named fumihopaside A. The structure of 2 was definitely assigned by interpretation of MS and NMR spectral data and named fumihopaside B (Figure 2B and Figure S9), which is different from 1 due to the presence of a double bond between C-22 and C-29. The relative configurations of 1 and 2 were determined by NOESY correlations as shown in Figure S9. To experimentally validate whether the cluster af um is responsible for the biosynthesis of fumihopaside A, the SHCencoded gene afumA was deleted from A. f umigatus. As shown in Figure 3A, target product 1 appeared in the extract of the

Figure 3. HPLC−ELSD analysis of metabolites produced in A. f umigatus strains.

Figure 2. Gene cluster and proposed biosynthetic pathway of fumihopaside A. (A) Biosynthetic gene cluster af um in A. f umigatus. Abbreviations: SHC, squalene hopane cyclase; P450, cytochrome P450; GT, glycosyltransferase; TF, transcription factor. (B) Biosynthetic pathway of fumihopaside A in A. f umigatus.

wild-type strain but not in the ΔafumA strain. This result clearly demonstrated that cluster afum is responsible for the biosynthesis of fumihopaside A. To prove the functions of genes afumA, af umB, and afumC, we deleted genes afumA−C individually and conducted HPLC analysis of the corresponding culture extracts of the mutant strains. When af umC was disrupted, the resulting mutant strain ΔafumC failed to produce compound 1, but intermediate 3 did accumulate. The structure of 3 was determined by analysis of the NMR spectral data and X-ray crystallography data with a Flack value of 0.02 (Figures S9 and S11). Considering compounds 1−3 have the same biosynthetic origin, the absolute stereochemisty in the aglycone of 1 and 2 was assigned to be the same as that of 3. Structural comparisons between 1 and 3 revealed that the function of AfumC is responsible for the glycosylation at C-24. Similarly, when we disrupted af umB, the extract of resulting mutant strain ΔafumB also generated compound 4 when compared with that of strain Δaf umA (Figure 3B). The structure of compound 4 was determined to be 21βH-hopane-3β,22-diol by comparing the MS and one-dimensional NMR data with the literature data.8 Thus, AfumB was deduced to be responsible for both hydroxylation at C-24 and oxidations at C-30. On the basis of the structural features of 1−4 and

To probe for afum-dependent metabolites, we employed HPLC−MS/MS and HPLC−ELSD to detect the SMs of A. f umigatus strain A1163. The ESI-MS spectrum showed a molecular ion peak at m/z 651 [M − H]−. Furthermore, upon ESI-MS/MS of the peak at m/z 651, the fragment ion at m/z 488 [M − glycosyl − H]− was observed (Figure S1). This fragment pattern revealed that a glycoside compound was possibly produced by A. f umigatus. These results corresponded to the bioinformatics analysis of the cluster afum. Guided by the MS experiment, compound 1 and another minor product 2 were purified from the culture extract by a combination of reversed-phase C18 column chromatography and preparative thin-layer chromatography using silica gel as the adsorbent. The molecular formula of 1 was determined to be C36H60O10 on the basis of the [M − H]− ion in HRTOFMS at m/z 651.4111. The planar structure of 1 was assigned by one- and two-dimensional NMR spectral data elucidation (Figure 2B and Figure S9). HMBC correlations from H2-24 to C-1′ elucidated the linkage of a sugar moiety at C-24. The carbon spectrum of 1 indicated the characteristic resonances B

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Organic Letters

AfumC protein was purified on a nickel-affinity chromatography column and analyzed via SDS−PAGE (Figure S4). LC− MS/MS analysis identified the target protein with the His tag sequence (score of 1150, coverage of 54%). Next, AfumC was incubated in vitro with intermediate 3 and UDP-D-glucose. HPLC analysis confirmed the production of fumihopaside A (1) in the reaction mixture (Figure 5). In the reaction with 1

bioinformatics analysis, we elucidated the biosynthetic pathway of fumihopasides A and B in A. f umigatus (Figure 2B) and demonstrated the enzymatic functions of AfumA−C. Furthermore, a heterologous system in Aspergillus nidulans was used to determine that the afum gene cluster was exactly responsible for the biosynthesis of fumihopasides without any other required enzymes, especially P450s, from A. f umigatus. The DNA fragments carrying genes af umA−C with their own promoters and terminators were amplified from genomic DNA and integrated into vector pYH-WA-pyrG by a yeast assembly method.9 The constructed plasmid (Table S4) and the empty vector were transformed into the host A. nidulans LO8030 that has been engineered and approved for fungal biosynthetic gene cluster studies.10 Transformants containing genes af umA−C were cultivated on PDA medium. HPLC analysis of the culture extract of the mutant expressing afumA−C showed compounds 1 and 3 when compared to that of the control strain (Figure 4A). Then, plasmids integrated with afumA or afumA

Figure 5. Analysis of the enzymatic reaction products of AfumC. (A) HPLC−ELSD analysis of the product catalyzed by AfumC with UDPD-glucose (Glc) as the sugar donor. (B) HPLC−MS analysis of the product catalyzed by AfumC with UDP-D-galactose (Gal) or UDP-Dglucuronic acid (GlcA) as the sugar donor. (C) Typical negative-ion MS spectra for peaks of 1a and 1b. (D) AfumC converted 3 into 1, 1a, and 1b with UDP-D-glucose, UDP-D-galactose, and UDP-Dglucuronic acid as donors, respectively. However, AfumC exhibited no deglucosylation activity.

Figure 4. HPLC−ELSD analysis of metabolites produced in A. nidulans with complete or segmental afum cluster: (i) A. nidulans LO8030 with plasmid pYH-WA-pyrG-afumABC, (ii) A. nidulans LO8030 with plasmid pYH-WA-pyrG, (iii) A. nidulans LO4389 with plasmid pRGAMA1-afumAB, (iv) A. nidulans LO4389 with plasmid pRGAMA1, (vii) A. nidulans LO8030 with plasmid pYH-WA-pyrGgpdA-afumA, and (viii) A. nidulans LO8030 with plasmid pYH-WApyrG-gpdA.

and UDP, AfumC exhibited no activity of deglucosylation (Figure 5A). The optimal pH value and temperature for the reaction were found to be around 8.0 and 37 °C, respectively. The activity of AfumC was significantly decreased by addition of divalent cations (e.g., Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, and Ni2+), while Fe2+ had little effect. AfumC showed the strongest activity with the addition of 5 mM EDTA, which indicated the nondivalent cation-dependent nature of this enzyme. The Km value and the corresponding kcat value of AfumC for 3 with UDP-D-glucose as the donor were determined to be 0.33 mM and 0.022 s−1, respectively (Figure S6). Next, the sugar nucleotide specificity test showed that AfumC accepted UDP-D-galactose and UDP-D-glucuronic acid as donors, thus converting 3 into 1a and 1b, respectively, as confirmed by HR-ESI-MS data (Figure 5). Finally, 0.7 mg of 1a was isolated from the preparative scale enzyme reaction, and its structure was determined by comparison of the 1H and 13 C NMR data between 1a and 3 (Figures S26 and S27). The α configuration for the glycoside bond in 1a was assigned from the small coupling constant between H-1′ and H-2′ (δH 5.34, d, J = 3.5 Hz).

and afumB were transformed into A. nidulans (Table S4). As shown in Figure 4, the culture extract of the strain expressing the plasmid with af umA and af umB displayed one new peak corresponding to compound 3 when compared with that of the control strain. HPLC analysis of the culture extract of the strain expressing the plasmid integrated with afumA revealed another intermediate 4 (Figure 4B). Glycosylation catalyzed by glycosyltransferases (GTs) plays an essential role in the structural diversity and biological activities of natural products. A small number of α-glycosides with diverse bioactivities have been reported from the fungi Xylaria polymorpha, Hymenoscyphus f raxineus, Acremonium striatisporum, and Neurospora terricola.11 To the best of our knowledge, AfumC is the first glycosyltransferase identified in the biosynthesis of fungal secondary metabolites. Thus, we probed the AfumC activity in vitro. The coding region of afumC was amplified from cDNA of A. fumigatus and cloned into yeast expression vector pXW55 with a fused His tag. The C

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germination rate similar to that of the Δalb1 mutant strain (Figure 6B). Fungal hopanoids have been reported from several fungal strains, including Hypocrella sp., Aspergillus variecolor, Aschersonia aleyrodis, Aspergillus tubalata, Paecilomyces cinnamomeus, and Aspergillus sp.16 Bioinformatics analysis has revealed SHCs in the genomes of some filamentous fungi in the Pezizomycotina, such as A. f umigatus, Aspergillus clavatus, Aspergillus niger, Aspergillus oryzae, Ajellomyces capsulatus, Neosartorya f isheri, and Magnaporthe grisea.3 However, the connection between fungal SHCs and hopanoids is still unresolved. In our study, the isolation of fumihopasides A and B indicated that SHCs in fungi indeed catalyzed the production of hopanoids from the 2,3-epoxysqualene, which is different from those of bacterial SHCs.17 Bioinformatics analysis also revealed the homologues of the af um gene cluster in A. clavatus, Aspergillus nomius, and Penicillium camemberti (Table S2 and Figure S3). The structures and biological functions of haponoids in these fungi deserve to be investigated more thoroughly. In contrast with hopanoids in bacteria, the chemistry, biosynthesis, and physiological functions of fungal hopanoids are seldom investigated. In this study, we obtained two hopane-type glucosides, fumihopaside A (1) and B (2), from the culture of the human pathogenic fungus A. fumigatus. The biosynthetic pathway of 1 was elucidated by a combination of heterologous expression and in vivo gene deletion experiments. The AfumC enzyme responsible for the glucosidation of the haponoid produced in A. f umigatus was characterized as the retaining glycosyltransferase with a certain substrate flexibility by accepting UDP-D-glucose, UDP-D-galactose, or UDP-Dglucuronic acid as the sugar donor. In addition, the biological functions of fumihopaside A in enhancing the thermotolerance and UV resistance of A. f umigatus were demonstrated for the first time. This study provides new insights into the biosynthesis and physiological functions of hopanoids in fungi.

A few secondary metabolites from A. f umigatus have been demonstrated to be correlated with the pathogenicity and survivability of this fungus. The melanin in A. f umigatus was confirmed as a virulence factor to provide protection against reactive oxygen species and inhibit host-cell phagocytosis, cytokine production, and apoptosis.12 Hexadehydroastechrome, an iron(III) complex identified from A. f umigatus, was shown to possess important biological functions related to virulence and stress resistance.13 Recently, one study showed that the afum gene cluster was increasingly expressed with an increasing culture temperature.14 In this study, we found that fumihopaside A and ergosterol were co-located on the membrane system of A. f umigatus, as confirmed by HPLC− ELSD−DAD analysis of the crude membrane fraction (Figure S8). On the basis of analysis presented above and the structural similarity between fumihopasides and the bacterial hopanoids, we proposed that fumihopaside A shares similar biological functions with the hopanoids in bacteria. To clarify the biological functions of this unique metabolite in A. f umigatus, we tested the rate of spore germination of the wild-type and ΔafumA mutant strains under high-temperature or ultraviolet (UV) treatment. In the thermotolerance test, strains were pretreated with an extremely high temperature of 57 °C for 12 h and then incubated at 50 °C for 48 h. The spore germination rate of the wild-type strain in PDA medium was much higher than that of the ΔafumA mutant (Figure 6A). A. fumigatus is

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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.9b00984. Experimental details and characterization data (PDF) Accession Codes

CCDC 1854430 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Figure 6. Spore germination of A. fumigatus strains under heat or UV stress. (A) Rate of spore germination of the wild-type and ΔafumA strains under heat stress. (B) Rate of spore germination of the wildtype, Δaf umA, and Δalb1 mutant strains under UV exposure. **p < 0.01; ****p < 0.0001.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

known to produce a bluish-green pigment through the dihydroxynaphthalene (DHN)−melanin pathway to protect itself from UV damage.15 The non-melanin (Δalb1) mutant strain of A. f umigatus was reported to have lower germination rates under the UV treatment. At UV exposures of 50, 75, and 100 J/m2, the ΔafumA strain was found to show a germination rate much lower than that of the wild-type strain and a

ORCID

Nancy P. Keller: 0000-0002-4386-9473 Wen-Bing Yin: 0000-0002-9184-3198 Hongwei Liu: 0000-0001-6471-131X D

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Sugui, J.; Vonk, A. G.; van de Sande, W. W.; Warris, A.; Kwon-Chung, K. J.; Jan Kullberg, B. Immunobiology 2010, 215, 915−920. (13) (a) Yin, W. B.; Baccile, J. A.; Bok, J. W.; Chen, Y.; Keller, N. P.; Schroeder, F. C. J. Am. Chem. Soc. 2013, 135, 2064−2067. (b) Schrettl, M.; Kim, H. S.; Eisendle, M.; Kragl, C.; Nierman, W. C.; Heinekamp, T.; Werner, E. R.; Jacobsen, I.; Illmer, P.; Yi, H.; Brakhage, A. A.; Haas, H. Mol. Microbiol. 2008, 70, 27−43. (14) Lind, A. L.; Smith, T. D.; Saterlee, T.; Calvo, A. M.; Rokas, A. G3: Genes, Genomes, Genet. 2016, 6, 4023−4033. (15) Allam, N. G.; El-Zaher, E. A. Afr. J. Biotechnol. 2012, 11, 666− 677. (16) (a) Wang, W. L.; Liu, P. P.; Zhang, Y. P.; Li, J.; Tao, H. W.; Gu, Q. Q.; Zhu, W. M. Arch. Pharmacal Res. 2009, 32, 1211−1214. (b) Van Eijk, G. W.; Roeijmans, H. J.; Seykens, D. Tetrahedron Lett. 1986, 27, 2533−2534. (c) Boonphong, S.; Kittakoop, P.; Isaka, M.; Palittapongarnpim, P.; Jaturapat, A.; Danwisetkanjana, K.; Tanticharoen, M.; Thebtaranonth, Y. Planta Med. 2001, 67, 279− 281. (d) Isaka, M.; Palasarn, S.; Kocharin, K.; Hywel-Jones, N. L. J. Antibiot. 2007, 60, 577. (e) Tsuda, Y.; Isobe, K. Tetrahedron Lett. 1965, 6, 3337−3343. (17) Hammer, S. C.; Syrén, P. O.; Seitz, M.; Nestl, B. M.; Hauer, B. Curr. Opin. Chem. Biol. 2013, 17, 293−300.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Drs. Jinwei Ren, Wenzhao Wang, and Hao Qin (Institute of Microbiology and Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for NMR and MS data collection. This work was supported by grants from the National Special Project for Key Science and Technology of Food Safety (Grant 2017YFC1601302), the National Natural Science Foundation (Grants 81673334 and 31470178), and the Youth Innovation Promotion Association of CAS (Grant 2014074). W.-B.Y. is a scholar of “the 100 Talents Project” of CAS.

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