Biosynthesis of Diphenyl Ethers in Fungi - ACS Publications

Mar 1, 2019 - Fungi are another rich source of DPEs. Plenty of OH-/. MeO-DPEs and chlorinated DPEs (chloro-DPEs) (Figure 1 and Figure S1) have been ...
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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Biosynthesis of Diphenyl Ethers in Fungi Cheng Feng, Qian Wei, Changhua Hu,* and Yi Zou* College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China

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ABSTRACT: A new and concise biosynthetic pathway of fungal diphenyl ethers (DPEs) has been elucidated and efficiently reconstructed in yeast. The pathway includes an unusual nonreducing polyketide synthase (NRPKS) responsible for the formation of a polyketide dimer with an ether bond linkage as well as two cofactor-independent enzymes that catalyze tandem regioselective decarboxylation reactions. Our discovery here opens a new window for utilizing fungal DPEs as a platform to design and synthesize analogues for the development of highly useful drug leads.

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identify. Thus, few biosynthetic gene clusters (BGCs) have been characterized.5 Recently, Moore’s group reported that marine bacteria (Pseudoalteromonas spp.) and cyanobacterial symbionts of sponges (H. spongeliae) are the main producers of BDEs.6,7 Using cyanobacteria as the heterologous host, a complete pathway of BDEs has been well documented, revealing that a cytochrome P450 (Bmp7) catalyzes the diradical coupling of a bromophenolic monomer to form the dimer (Figure 2a).6,7 The bromophenolic monomer is derived from cellular central metabolite chorismic acid through the process of decarboxylative halogenation.6,7 The discovery of BDE BGC sets up a universal model for describing the assembly mechanism of bacterial DPEs and promotes the exploration of more DPE BGCs from other natural sources. Fungi are another rich source of DPEs. Plenty of OH-/ MeO-DPEs and chlorinated DPEs (chloro-DPEs) (Figure 1 and Figure S1) have been isolated from different fungi, including Aspergillus sp., Simpilcillium sp., Pestalotiopsis sp., Phomopsis sp., and Cordyceps sp.8−12 Fungal DPEs possess broad biological activities including antimalarial, antitubercula, antisaprolegnia, antifungal, and anti-Aβ42 aggregation.11,13 They are therefore usually used as a platform to design and synthesize analogues for the development of highly useful drug leads.14,15 A distinguished example is diorcinol (1) (Figure 1), one of the most universal DPEs among fungus, the postmodifications of which such as hydroxylation, methylation, and prenylation have enlarged its structural and bioactive diversity (Figure S1). The first BGC of fungal DPE (pestheic acid, Figure 1) was recently reported and shows the Cu oxidase-catalyzed oxidative rearrangement responsible for conversion of the benzophenone intermediate to pestheic acid (Figure 2b);16 however, 1 is

iphenyl ether (DPE) and its derivatives permit a variety of extensive applications such as heat transfer media, cosmetics, pharmaceutical intermediates, and chemical products. Aside from the man-made DPEs (Figure 1), the ocean is

Figure 1. Representative DPEs from chemical syntheses and natural sources.

one of the most abundant sources of these molecules. Marinederived DPEs are usually modified by oxygenase and/or methyltransferase with halogenases to form the hydroxylated or methoxylated polybrominated DPEs (OH-/MeO-BDEs) (Figure 1).1−3 Due to their wide accumulation in various sea foods and potential teratogenicity and carcinogenicity, OH-/ MeO-BDEs are considered to be one of the most serious toxins or pollutants to humans. Consequently, they have attracted widespread attention.4 The biogenic basis for marine-derived DPE biosynthesis has lagged far behind their discovery and chemical synthesis largely because the actual producers of DPEs have proven difficult to © XXXX American Chemical Society

Received: March 1, 2019

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DOI: 10.1021/acs.orglett.9b00768 Org. Lett. XXXX, XXX, XXX−XXX

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the two molecules of monomer 5 to dimer 1 is possibly catalyzed by the last candidate gene AN7910 (previously undefined) (Figure 2c), and the process may be similar to Bmp7-mediated diradical coupling (Figure 2a). Bioinformatic analysis of AN7911 shows that it belongs to the amidohydrolase superfamily of proteins and contains a sturdy and versatile triosephosphate isomerase (TIM)-like βbarrel fold in the catalytic domain.21 Aside from the classical hydrolytic reactions, amidohydrolase can also catalyze diverse nonhydrolytic reactions, such as the cleavage of C−N, C−C, or C−O bonds of organic compounds.21 To uncover the possible AN7911-catalyzed decarboxylation of 4 to 5, intron-free AN7911 was cloned from the cDNA of A. nidulans, heterologously expressed in Escherichia coli, and purified to homogeneity (Supporting Information, SI and Figure S3). Following the incubation of 10 μM AN7911 with 500 μM 4 for 2 h (SI), a single and clear product was detected, of which the UV absorption and retention time were fully consistent with the 5 standard (Figure 3, (i), (ii), and (v)). When 10 mM Figure 2. Gene cluster and biosynthesis of DPEs from natural sources. (a) P450-catalyzed diradical coupling in bacterial DPEs. (b) Cu oxidase-catalyzed oxidative rearrangement in pestheic acid. (c) The proposed pathway of 1 in A. nidulans.

the simplest fungal DPE and the ideal model for the investigation of fungal DPE biosynthesis, especially in favor of identifying the new eukaryotic enzyme for the key ether bond formation, and the biosynthetic mechanism remains elusive. Here, we identified three enzymes from A. nidulans and clarified their functions through in vitro biochemical analyses as well as reconstruction of the 1 pathway in the heterologous host Saccharomyces cerevisiae. The biosynthetic mechanism of 1 is entirely different from BDEs and pestheic acid, which includes (i) an unusual nonreducing polyketide synthase (NRPKS) AN7909 that is responsible for the production of diorcinolic acid (2, Figure 1) and (ii) two enzymes (AN7910 and AN7911) that catalyze tandem regioselective decarboxylations on 2 to furnish 1. Previous reports showed that 1 and other DPE derivatives, such as 2, gerfelin, C10-deoxy gerfelin (3), cordyol C, and violaceol I/II (Figure 1 and Figure S2), are the cometabolites of orsellinic acid (4) in A. nidulans, all of which are linked to the ors gene cluster (AN7909−AN7914, Figure 2c).17,18 Moreover, in vivo gene knockout results demonstrated that (i) the yields of 4 and 1 were abolished in the ΔorsA (AN7909, NRPKS) mutant; (ii) gerfelin and 3 accumulated in the ΔorsB (AN7911, decarboxylase) mutant, whereas 1 was found in high amounts in the ΔorsC (AN7912, tyrosinase) mutant; and (iii) ΔorsD (AN7913, transcriptional regulation factor) and ΔorsE (AN7914, dehydrogenase) mutants did not affect the production of 4 and 1.17 Based on the results from these studies, three genes (AN7912−AN7914) of the ors gene cluster are clearly not involved in 4 and 1 production. The biosynthetic pathway of 1 as well as other DPE derivatives in A. nidulans has been proposed (Figure 2c and Figure S2).19,20 One acetyl-CoA and three malonyl-CoA units are utilized by AN7909 to yield 4. The decarboxylation of 4 by AN7911 generates orcinol (5). The oxidation of 5 in the para position then leads to 5-methyl-benzene-1,2,3-triol, which is believed to dimerize with the loss of water to give violaceol I/II (Figure S2). On the other hand, 1, gerfelin, 3, and cordyol C are all dimers built up of two of the three suggested monomer units 4, 5, and 5-methyl-benzene-1,2,3-triol. The pivotal conversion of

Figure 3. Biochemical analyses of AN7911 with 4 in vitro.

EDTA was added, the complete conversion of 4 to 5 was still detected (Figure 3, (iii), which suggests that AN7911 possibly does not require a divalent metal ion for catalytic activity. AN7911 differs from previously identified amidohydrolase family decarboxylases such as γ-resorcylate decarboxylase, which requires Mn2+ as the cofactor.22 Further investigations of benzoate monomers reacting with AN7911 show that the substrate tolerance of AN7911 is relatively specific, except for 4, and only 2,6-dihydroxybenzoic acid can be accepted (Figure S4). AN7910 belongs to the nuclear transport factor 2 (NTF2)like superfamily of proteins, which mediates the nuclear import of RanGDP and binds to both RanGDP and FxFG repeatcontaining nucleoporins.23 Careful domain and phylogenetic tree analyses show that AN7910 shares an individual clade that contains fungal SnoaL_4 fold (Figure S5a). Bacterial SnoaL or SnoaL_4 fold enzymes as well as fungal SnoaL_2 fold enzymes (Figure S5b) have been biochemically identified as polyketide cyclase, hydroxylase, aldol, and Claisen condensation synthase in the biosynthesis of natural products, respectively;24−28 however, the biochemical function of the fungal SnoaL_4 enzyme including AN7910 remains unknown. To reveal the function of AN7910, the recombined AN7910 was expressed and purified from E. coli (SI and Figure S3). After an incubation of AN7910 with 5, the expectant conversion of 5 to 1 was not detected in an in vitro assay (Figure 3, (iv)). These results confirm that 5 is not the natural substrate of AN7910. B

DOI: 10.1021/acs.orglett.9b00768 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters The actual function of AN7910 as well as the formation of the ether bond in 1 are still unsolved. The unsuccessful reconstitution of the enzymatic activity of AN7910 in vitro prompts us to reconsider the possible products yielded by AN7909. AN7909 shows 48%/33% (similarity/identity) to PKS14 from Fusarium graminearum, 48%/32% to PKS1 from Stereum sp. BY1, and 44%/27% to StbA from Stachybotrys bisbyi PYH05−7. These three NRPKSs have been demonstrated to be specifically involved in 4 production;29−31 however, compared to PKS14, PKS1, and StbA, the KS domain phylogenetic tree analysis shows that AN7909 represents an individual clade (Figure S6), indicating that the metabolites produced by AN7909 and the other three NRPKSs are different. We initially attempted to carry out the in vitro assay using the purified AN7909 (225.5 kDa) with acetyl-CoA and malonyl-CoA; however, many attempts to express AN7909 as a soluble protein from E. coli BAP1 were not successful, thereby precluding the direct assay of this reaction using purified enzyme. Alternatively, the yeast was selected as the heterologous host, and the intron-free AN7909 was introduced into S. cerevisiae BJ5464-NpgA (BJ-AN7909) under the control of the ADH2 promoter. To our surprise, 4 was produced in low yields, while BJ-AN7909 generated the other four compounds including two minor compounds with m/z 317.0671 [M-H]− and m/z 195.0664 [M-H]− and two major compounds with m/z 317.0668 [M-H]− and m/z 345.0977 [M-H]− (Figure 4a, (i) and (v), Table S11, Figures S15 and S17−S19). Subsequent large-scale fermentation of BJ-AN7909 and isolation of the four compounds were carried out (SI). Structural characterization by complete 1D and 2D NMR (Tables S4−S5 and S7−S9 and Figures S23−S29 and S32− S40) identified the two minor compounds as lecanoric acid (6) and 7; the two major compounds are 2 and 8 (Figure 4b). 7 and 8 are likely the spontaneous or artificial ethyl esterification derivatives of 4 and 2, respectively, generated during the processes of fermentation, extraction, and isolation. The production of 2 as the major product in BJ-AN7909 suggests that AN7909 is an unusual fungal NRPKS that can catalyze the formation of an ether bond as well as release the protein-tethered polyketide dimer intermediate (Figure 5). The overexpression of AN7909 back to its original host A. nidulans (Δors-BGC strain) also confirms that the major product produced by AN7909 is indeed 2 (Figure S7a). Upon further feeding of the free monomer 4 to the BJ strain, the conversion of 4 to 2 was not detected even after 24 h (Figure S7b). These results suggest that the coupling of the two molecules of 4 to form 2 is catalyzed by AN7909 itself, and the formation of the ether bond is possibly via in-line assembly. This process is completely different from the pathway of bacterial DPEs and previously identified fungal DPEs, of which the formation of the ether bond is catalyzed by an additional enzyme such as P450 or Cu oxidase.6,7,16 Further, domain organization analysis shows that PKS14 and PKS1 have one ACP domain, whereas AN7909 has two (Figure S8). Moreover, the heterologous expression of the StbA homologue gene AcsA from Acremonium sp. Mcr (SI) in BJ (BJ-AcsA) only produces 4 (Figure S9), which suggests that the two tandem ACP domains might be critical for the formation of 2 by AN7909 (Figure 5). Respective ACP domain mutation of AN7909 identified that two ACP domains are indeed important for 2 production, of which the ACP1 mutant (S1676A) observably decreased the yields of 2. The ratio of 2

Figure 4. Identification of the functions of AN7909−AN7911 in yeast and in vitro. (a) Reconstruction of 1 pathway in yeast. LC-MS analyses of the metabolites produced by BJ and its transformants. (b) Compounds identified from BJ transformants. The asterisk-labeled compound is produced by BJ with a retention time close to 2. (c) In vitro biochemical assays of AN7910 and AN7911.

Figure 5. New and concise biosynthetic pathway of 1 in A. nidulans.

and 8 (dimers) to 4 and 7 (monomers) in wild-type AN7909 is 9.95 ± 0.33, while the ACP1 mutant is 3.22 ± 0.04 (Figure S10), which is more than a 3-fold decrease. The TE domain mutant (S1907A, BJ-AN7909-TEm, SI) abolished the production of 2 and 4; however, they can be complementary by trans-TE itself (Figure S11a). Alkaline hydrolysis of the BJAN7909-TEm strain showed that only 4 could be detected (Figure S11b). These results therefore confirm that the TE domain is also involved in 2 and 4 formation, and it possibly catalyzes the ether bond formation as well as releases the ACP or TE-tethered products. C

DOI: 10.1021/acs.orglett.9b00768 Org. Lett. XXXX, XXX, XXX−XXX

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The pathway from 2 to 1 requires two steps of regioselective decarboxylation. The coexpression of AN7909 and the previously identified decarboxylase gene AN7911 in BJ (BJAN7909−11, SI), however, did not generate the expectant decarboxyl product of 2 (Figure 4a, (ii)), which suggests that AN7911 cannot directly modify 2. We then coexpressed AN7909 and AN7910 in BJ (BJ-AN7909−10, SI), and accompanied by the disappearance of 2 and 8, two new products with m/z 273.0762 [M-H]− and m/z 301.1081 [MH]− were generated (Figure 4a, (iii), Table S11 and Figures S16 and S20). Based on the NMR analyses (Tables S6 and S10 and Figures S30−S31 and S41−S42), the two products were identified as 3 and 9, respectively (Figure 1 and Figure 4b). Further in vitro biochemical assays confirmed that AN7910 is a cofactor-independent decarboxylase that catalyzes the regioselective nonoxidative decarboxylation of 2 and 8 at C2′ to form 3 and 9 (Figure 4c (i), (ii) and Figure S12a). Our result here is the first biochemical evidence demonstrating that fungal SnoaL_4 superfamily protein function as decarboxylase. The His68 and Arg16 were further identified as the key amino residues required for the activity (Figure S12b). Clarification of the function of AN7910 as well as the identification of the structure of 3 bridges the key biosynthetic transformation between 2 and 1 (Figure 5). Compared to BJAN7909−10, the next three-gene-coexpression strain (BJAN7909−10−11, SI) led to the full conversion of 3 to 1 (m/z 229.0868 [M-H]−) (Figure 4a, (iv), Tables S3 and S11 and Figures S14 and S21−S22). This transformation was further confirmed by an in vitro biochemical assay of AN7911 incubated with 3 (Figure 4c, (iii) and (iv)). Although AN7911 previously exhibited substrate tolerance (Figure 3 and Figure S4), the results reported here confirm that the actual role of AN7911 is to catalyze another regioselective nonoxidative decarboxylation of 3 at C4 to finish the last biosynthetic step of 1 (Figure 5). In conclusion, a new and concise pathway of fungal DPE (1) has been elucidated from the type strain A. nidulans and successfully reconstructed in yeast. The entire pathway includes an unusual NRPKS responsible for the formation of a polyketide dimer (2) with an ether bond linkage as well as two cofactor-independent, enzyme-catalyzed tandem regioselective decarboxylations (Figure 5). Genome mining of many fungal strains shows that BGCs containing the AN7909− AN7910−AN7911 homologue are widely distributed (Figure S13), suggesting that the biosynthetic strategies of fungal DPEs are conserved. Our discovery here therefore opens a new window for mining and combinational biosynthesis of structurally related fungal DPEs.

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Yi Zou: 0000-0002-1742-9650 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Wenbing Yin from the Institute of Microbiology, CAS, for providing A. nidulans (Δors-BGC) as the heterologous host. This work is supported by the National Natural Science Foundation of China (31870022), the National Key Research and Development Program of China for Traditional Chinese Medicine Modernization (2017YFC1702601), the Fundamental Research Funds for the Central Universities (XDJK2018B035), and the Scientific Research Starting Foundation of Southwest University (SWU117034). Y.Z. is supported by the Venture & Innovation Support Program for Chongqing Overseas Returnees and the Thousand Young Talents Program of China.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00768. Full experimental details and spectroscopic data (PDF)

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DOI: 10.1021/acs.orglett.9b00768 Org. Lett. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.orglett.9b00768 Org. Lett. XXXX, XXX, XXX−XXX