Letter Cite This: Org. Lett. 2018, 20, 7197−7200
pubs.acs.org/OrgLett
Genetic Characterization of Neosartorin Biosynthesis Provides Insight into Heterodimeric Natural Product Generation Yudai Matsuda,*,†,‡ Charlotte H. Gotfredsen,§ and Thomas O. Larsen*,† †
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, 2800 Kongens Lyngby, Denmark Department of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China § Department of Chemistry, Technical University of Denmark, Kemitorvet, 2800 Kongens Lyngby, Denmark ‡
Org. Lett. 2018.20:7197-7200. Downloaded from pubs.acs.org by UNIV OF NORTH DAKOTA on 11/16/18. For personal use only.
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ABSTRACT: A biosynthetic gene cluster of the fungal xanthone heterodimer neosartorin (1) was discovered in Aspergillus novof umigatus, and its biosynthesis was investigated by a series of gene-deletion experiments. The results indicate that the two monomeric units of 1 are synthesized by the same set of enzymes, with chrysophanol (5) as a common precursor. Furthermore, the P450 monooxygenase NsrP for the heterodimerization was discovered, which also accepts nonnative substrates to afford novel xanthone dimers.
D
imerization is a commonly seen event in natural product biogenesis1 that contributes to the structural diversification and complexification of natural products. Some dimeric molecules are produced by enzymatic processes, as represented by the biosynthesis of the lignans,2 while dimerization reactions also occur nonenzymatically as a result of the high reactivity of the monomeric units.3 In either case, the mechanisms for the dimerizations have been investigated for selected natural products, but since a variety of reactions, such as radical couplings, esterifications, and pericyclic reactions, are used to generate dimeric compounds, the dimerization mechanisms for most natural product dimers still remain poorly understood. Thus, biosynthetic studies on dimeric natural products and characterization of the dimerization enzymes would facilitate our bioengineering effort to provide new dimeric compounds with unnatural monomeric units or dimerization patterns. Neosartorin (1), isolated from several different fungal species, is a xanthone dimer and reportedly exhibits substantial antibacterial activity against several Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA).4 Although xanthones and xanthone dimers are widespread in nature,5 1 features a characteristic molecular skeleton that consists of two different monomeric units (Figure 1A), and such a heterodimeric nature is relatively rare for xanthone dimers. The biosynthesis of fungal xanthones has been intensively studied by isotope-incorporation studies followed by genetic characterization of the biosynthesis.6 It is well-known that a nonreducing polyketide synthase (PKS) is responsible for the first committed step of the biosynthesis. However, the molecular basis for the xanthone biogenesis has not been fully elucidated, despite the wide occurrence and biological importance of xanthone natural products. Furthermore, no enzyme has been identified and characterized to catalyze the dimerization of © 2018 American Chemical Society
Figure 1. (A) Structure of neosartorin (1). Each monomeric unit is shown in red and blue. (B) Schematic representation of the nsr cluster from A. novof umigatus IBT 16806. (C) HPLC analysis of the metabolites from A. novofumigatus mutants. Chromatograms were monitored at 280−330 nm.
xanthones. Thus, biosynthetic study of 1 would provide not only new insights into the occurrence of xanthones and their dimers but also genetic tools to afford an opportunity to synthesize “unnatural” xanthone dimers with a more potent biological activity. To identify the key enzymes responsible for the construction of the unique chemical architecture of neosartorin (1), we initially sought to find the biosynthetic gene cluster of 1. To this end, we investigated the genome sequence of Aspergillus novof umigatus IBT 16806,7 which is one of the known producers of 1.8 We first searched for a gene encoding a close homologue of MdpG, which is the PKS involved in the biosynthesis of another Received: October 1, 2018 Published: November 5, 2018 7197
DOI: 10.1021/acs.orglett.8b03123 Org. Lett. 2018, 20, 7197−7200
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Organic Letters
Figure 2. Proposed early-stage biosynthetic pathway of neosartorin (1) based on bioinformatic analysis.
fungal xanthone, shamixanthone, in Aspergillus nidulans.6d,e Consequently, one PKS gene, whose product shows 62% sequence identity with MdpG, was discovered. Further investigation of the flanking region of the PKS gene led to the discovery of several genes encoding homologous proteins engaged in the shamixanthone pathway as well as in the biosynthesis of an anthraquinone dimer, cladofulvin9 (Figure 1B, Figure S1, and Table S1). Thus, we reasoned that this gene cluster is responsible for the neosartorin biosynthesis in A. novofumigatus, and it was designated the nsr cluster, consisting of 19 genes altogether. To confirm the involvement of the nsr cluster in neosartorin biosynthesis, we then sought to delete the PKS gene nsrB. To facilitate the following experiments, we initially created a mutant A. novof umigatus strain suitable for the gene deletion experiments, in which the core synth(et)ases for two major secondary metabolites of the fungus, novofumigatonin and epi-aszonalenin C,10 were disrupted. Expectedly, the deletion of nsrB completely abolished the production of 1 (Figure 1C). On the basis of the homologies between some proteins encoded by the nsr cluster and their homologues in the shamixanthone and cladofulvin pathways,6e,f,9 early-stage biosynthesis of neosartorin (1) can be proposed as follows (Figure 2). In this predicted pathway, chrysophanol (5) is generated as a key pathway intermediate, which is thought to be a common precursor for the fungal xanthones.6c,f Initially, the PKS NsrB, together with the trans-acting thioesterase NsrC, produces atrochrysone carboxylic acid, which then undergoes NsrE-catalyzed decarboxylation to yield atrochrysone. The subsequent dehydration and enolization yields emodin anthrone, which is oxidized to emodin by the anthrone oxygenase NsrD. In the following steps, emodin needs to be reduced to 2 or 3 for the further reduction and dehydration by NsrJ and NsrI, respectively, to form 5, since the in vitro enzymatic reaction of MdpC, a close homologue of NsrJ (75% protein sequence identity), revealed that MdpC does not accept emodin as its substrate and that in situ reduction of emodin by sodium dithionite is required for MdpC to generate 4 as a product.11 The in vivo mechanism for this initial reduction has yet to be clarified, but it could be catalyzed by the short-chain dehydrogenase/reductase (SDR) NsrR, since this is the only reductase with an unknown function that is conserved among the three biosynthetic gene clusters. This hypothesis is further supported by the fact that two SDRs are required for the deoxygenation of the aromatic ring in the biosynthesis of bacterial anthraquinone, kosinostatin, although no dehydratase like NsrJ is involved in this transformation.12
We then focused on the late-stage biosynthesis of neosartorin (1) and performed a series of gene deletion experiments on selected genes that are either characteristic to the nsr cluster (nsrG, nsrL, and nsrO) or expected to have a distinct function from the corresponding homologue in the other pathways (nsrF, nsrK, and nsrP). Among the six gene deletion mutants, those lacking the methyltransferase gene nsrG or the SDR gene nsrO unfortunately accumulated no metabolites related to the neosartorin pathway (Figure 3A,B, lanes iv and vii). On the
Figure 3. (A) HPLC analysis of the metabolites from gene-deletion mutants. Chromatograms were monitored at 280−330 nm. (B) LC− MS analysis of metabolites from gene-deletion mutants. Extracted ion chromatograms (EICs) at m/z 255.065 (for 5), 321.096 (for 8), 325.068 (for 6 and 7), 363.107 (for 9), 639.171 (for 10 and 11), and 681.181 (for 1) are shown. (C) Structures of the compounds isolated in this study. 7198
DOI: 10.1021/acs.orglett.8b03123 Org. Lett. 2018, 20, 7197−7200
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Figure 4. Proposed branched biosynthetic pathway for blennolide C (8) and 5-acetylblennolide A (9) from chrysophanol (5) as a common precursor. Boxed compounds were isolated or detected in this study.
other hand, the deletion of the putative Baeyer−Villiger monooxygenase (BVMO) NsrF did not completely abolish the neosartorin synthesis, but the mutant accumulated compound 5, which was identified as chrysophanol by comparison with a commercially available authentic sample (Figure 3A,B, lanes iii and ix). The observation that 1 was still produced by this mutant could be attributed to the fact that the genome of A. novofumigatus encodes a homologous protein of NsrF (∼40% protein sequence identity) and that this protein might compensate for the function of NsrF. Interestingly, the mutant lacking nsrK, which encodes a flavin-dependent monooxygenase (FMO), accumulated two isomeric metabolites 6 and 7 (Figure 3A,B, lane (v), which were identified as moniliphenone13 and 2,2′,6′-trihydroxy-4-methyl-6-methoxyacyldiphenylmethanone,14 respectively (Figure 3C). Similarly, the deletion of the P450 gene nsrP led to the production of two analogous products 8 and 9 (Figure 3A,B, lane viii). While 8 was found to be a known xanthone, blennolide C,15 9 was determined to be an acetylated analogue of blennolide A15 (Figure 3C), which was therefore named 5-acetylblennolide A. Finally, the mutant without the acetyltransferase gene nsrL yielded one major product 10 as well as a minor product 11 (Figures 3A,B, lane vi). After isolation and NMR analyses of these compounds, it was revealed that 10 is a deacetylated analogue of 1 and that 11 is a homodimer of 8, which is a stereoisomer of rugulotrosin B.16 The axial chirality of both 10 and 11 was determined by NOESY analysis and was found to be the same as that of 1 (Figure 3C). Additionally, the absolute configurations of 10 and 11 were also deduced based on that of 1. The new xanthone dimers 10 and 11 were hereby designated as deacetylneosartorin and novofumigatin A, respectively. On the basis of the structures of the isolated metabolites and the predicted functions of the deleted genes, a biosynthetic route to neosartorin (1) could now be proposed (Figure 4). In a plausible scenario, the BVMO NsrF accepts chrysophanol (5) as a substrate to insert one oxygen atom at two different positions of 5 to yield 12 or 13. Importantly, this indicates that NsrF is promiscuous/flexible in interacting with the two (nonmethylated and methylated) aromatic rings of 5, thus diverging the biosynthetic pathway at this point. After the hydrolysis of the lactones, the resulting ring-opening forms 14 and 15 undergo methylesterification by the methyltransferase NsrG to, respectively, yield 6 and 7, which were isolated from the nsrKΔ strain. Given the structures of 8 and 9, it is most likely that the FMO NsrK oxidizes the C-3 or C-5 position of 6 and 7, respectively; however, the function of NsrK remains enigmatic, since we were unable to isolate the products of the enzyme. One possible
explanation would be that NsrK serves as an epoxidase and that the hydroxyl groups are formed as a consequence of the ring opening of the generated epoxides, as previously proposed for the biosynthesis of structurally related fungal xanthones.17 Thus, 6 and 7 are transformed into the epoxides 16 and 17, respectively, which then undergo nucleophilic attack by the hydroxyl group on the other aromatic ring to yield the tricyclic products 18 and 19. In the following step, the SDR NsrO should function as a reductase to afford the cyclohexene moiety of 8 and 9. Contrary to the other tailoring enzymes, the acetyltransferase NsrL seems to have a strict substrate specificity, only accepting 20, but not 8, as a substrate to yield 9 as the single-acetylated product. In the final step of the biosynthesis, the heterodimerization of the two xanthones, 8 and 9, is catalyzed by the P450 monooxygenase NsrP (Figure 5). Considering that two new
Figure 5. Proposed function of the xanthone-dimerizing P450 NsrP.
xanthone dimers, 10 and 11, were obtained upon the deletion of the acetyltransferase gene nsrL, it is suggested that NsrP can utilize at least three different xanthones as its substrates to perform the dimerization reaction (Figure 5). It is also possible that the acetylation by NsrL occurs after the formation of 10, but given that 9, not its deacetylated analogue 20, was obtained in the absence of the P450 gene, it is likely that NsrP can accept 9 as its substrate. Interestingly, given the structures of 1, 10, and 11, the regiospecificity and stereochemistry of the dimerization by NsrP appears to be strictly controlled, regardless of the monomeric units used for the reaction. The P450 NsrP is thus the first reported enzyme that catalyzes the dimerization, including heterodimerization, of xanthone derivatives. NsrP is homologous to ClaM (42% protein sequence identity), which is responsible for the anthraquinone dimerization in the cladofulvin pathway.9 NsrP also exhibits sequence similarity 7199
DOI: 10.1021/acs.orglett.8b03123 Org. Lett. 2018, 20, 7197−7200
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with a P450 encoded by a gene located upstream of the predicted biosynthetic gene cluster of other fungal xanthone dimers, ergochromes (CPUR_05419; 42% protein sequence identity), although the P450 gene was not included in the predicted cluster.18 On the other hand, NsrP does not show significant sequence similarity with DesC or KtnC, which are the P450s for the dimerization of the coumarin derivative 7demethylsiderin.19 Further bioengineering of these P450s will have to await the structural characterization and mutational experiments of the enzymes. Finally, we investigated the biological activity of neosartorin (1) and the new xanthone dimers; as previously reported,4b,c 1 exhibited antibacterial activity against MRSA (MB5393) with a minimum inhibitory concentration (MIC) value of 128 μg/mL. Although 11 was not subjected to the bioassay due to the low yield, 10, the deacetylated analogue of 1 interestingly showed better activity against MRSA with an MIC value of 64 μg/mL. In conclusion, we have identified the biosynthetic gene cluster of the natural xanthone heterodimer neosartorin (1) and partially elucidated its biosynthetic pathway by gene deletion experiments. Intriguingly, the two different monomeric units of 1 appear to be synthesized by the same set of enzymes, among which the BVMO NsrF is the key enzyme for the divergence of the biosynthetic routes. Furthermore, we have identified the first enzyme that can catalyze the heterodimerization of xanthones, and the P450 for the reaction, NsrP, seems to be capable of accepting several monomeric units to generate xanthone dimers. Interestingly, one of the new xanthone dimers displayed a better antibacterial activity than the original natural product 1. Collectively, the discovery of a series of multifunctional enzymes for fungal xanthone biosynthesis suggests their potential as genetic tools to create novel xanthone dimers with “unnatural” monomeric units or dimerization modes. We believe that this study facilitates future biosynthetic studies and bioengineering efforts on fungal xanthones and their dimers.
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03123. Experimental details and supplementary tables and figures (PDF)
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Corresponding Authors
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Yudai Matsuda: 0000-0001-5650-4732 Thomas O. Larsen: 0000-0002-3362-5707 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The NMR Center·DTU and the Villum Foundation are acknowledged for access to the 800 MHz NMR spectrometers. This work was supported by a postdoctoral fellowship from the Novo Nordisk Foundation (NNF15OC0015172) and by grants from City University of Hong Kong (Project No. 7200579 and 9610412) to Y.M. 7200
DOI: 10.1021/acs.orglett.8b03123 Org. Lett. 2018, 20, 7197−7200
