Sequestration of Guest Intermediates by Dalesconol Bioassembly

Letter pubs.acs.org/OrgLett

Sequestration of Guest Intermediates by Dalesconol Bioassembly Lines in Daldinia eschscholzii Ai Hua Zhang,† Wen Liu,† Nan Jiang,§ Xin Lei Wang,† Gang Wang,† Qiang Xu,† and Ren Xiang Tan*,†,‡ †

State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Nanjing University, Nanjing 210046, China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China § School of Pharmacy, Nanjing Medical University, Nanjing 210029, China S Supporting Information *

ABSTRACT: Microbial constructions of secondary metabolites are generally biosynthetic gene cluster (BGC)-based, and the forging of different BGC-sourced intermediates tends to be overlooked. Here, we show that the dalesconol bioassembly lines in Daldinia eschscholzii can sequester guest intermediates (i.e., building blocks produced outside the dalesconol biosynthetic gene cluster) to form arrays of skeletally undescribed molecules such as (+)-dalescone A, a potent inhibitor against the NLRP3 inflammasome activation.

M

icrobes are a prolific source of bioactive secondary metabolites, also referred to as natural products, whose structural complexity and diversity are key to the discovery of drugs (e.g., penicillin, lovastatin, and cyclosporine A) and agrochemicals such as gibberellins. Microbial metabolites are usually assembled by the biosynthetic gene clusters (BGCs), and compounds estimated from the genome-based prediction substantially outnumber the identifiable products generated through laboratory cultivations.1 Furthermore, such estimations on the basis of stand-alone secondary-metabolite BGCs may ignore the natural products resulting from the intercluster consortium of disparate BGCs.2 Accordingly, the new chemical entities, pivotal to the drug discovery pipelines,3 might be additionally exploited by modulating the intercluster interaction of different BGCs in parallel with the silent BGC activation appraches.1 For mining metabolites resulting from the forging of intermediates assembled in different BGCs, we demonstrate that the single BGC-governed biosynthetic pathway can sequester the “guest intermediates” (GIs) sourced from other BGCs to generate natural products with unprecedented frameworks. This is potentially generic in microbes and may signify an option for enhancing the chemodiversity outcome of microbial secondary metabolisms. In Daldinia eschscholzii IFB-TL01, the dalesconol bioassembly lines are characterized by the laccase (Lac)-catalyzed couplings of three naphthol radicals (Figure 1).4 Interestingly, 1,3,6,8-tetrahydroxynaphthalene (4HN, 1), an early-stage dalesconol precursor, can be transformed simultaneously into 4-hydroxyscytalone through a bypath mediated by flaviolin. This probably resembles the fungal generation of 5 and 6 from 4HN-like precursors 7 and 8, respectively (Figure 1).5b Such a similarity in substrate structures might accommodate a possibility that the dalesconol bioassembly lines could sequester © XXXX American Chemical Society

Figure 1. GI sequestration hypothesized from fungal biosyntheses of 4-hydroxyscytalone and dalesconols A−C. In fungi, 4HN (1) is reduced by 4HNR to generate 3HN (2) after the SD-catalyzed dehydration. Under tandem catalyses of 3HNR and SD, 3HN (2) gives DHN (3) as the building block for melanin construction.

7 and 8, and likely other phenols, to construct the GIincorporated novel natural products. Laccases are widely involved in the natural product biosynthesis in microbes and plants by mediating the Received: March 16, 2017

A

DOI: 10.1021/acs.orglett.7b00786 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Figure 2. Model for the GI sequestration. (A) GI-sequestered products of unprecedented frameworks by the dalesconol A biosynthetic pathway. (B) LC−MS profiling of the GI-sequestered products by the dalesconol A bioassembly line with their abundance significantly enhanced in the procaineexposed culture (+) compared to the control without any chemical exposure (−). Reduction of A4HN might afford presumable phenolic precursors 2-(hydroxymethyl)naphthalene-1,8-diol (9) and 2-(1-hydroxyethyl)naphthalene-1,8-diol (10).

screen for a more appropriate Lac expression potentiator, which is preferred to have positive, or at least no negative, effects on the polyketide biosynthesis upon its supplementation in the fungal culture. Inspired by the epigenetic regulation of gene expressions,13 we tried to identify more suitable Lac expression enhancer(s) from a collection of natural (resveratrol, curcumin, epigallocatechingallate (EGCG), and thymoquinone) and synthetic epigenetic modifiers (procaine, suberoylanilidehydroxamic acid (SAHA), suberohydroxamic acid (SBHA), and 5azacytidine) (Table S1). In agreement with our expectations, the real-time quantitative reverse transcription−PCR (qRTPCR) revealed that procaine, an analgesic drug and also a prokaryotic gene regulator,14 afforded selectively 3.2- and 20.1fold increases in the fungal expressions of polyketide synthase (pksTL) and laccase genes (lacTL) (Figure S1). As a relevant indication of the coupregulated expression of pksTL and lacTL, the abundance of the naphthol dimer (e.g., 1,1′-binaphthalene4,4′,5,5′-tetrol) and trimer (e.g., daeschol A) was ascertained to have been remarkably increased in the procaine-exposed fungal culture compared to the control with no exogenous chemical supplemented (Figure 2 and Supporting Information). The increment in the fungal production of the naphthol oligomers signified that procaine up-regulates the expression of Lac and PKS, two key enzymes involved in the carbon−carbon bond formation in the dalesconol biosynthesis.4 We inferred that the fungal exposure to procaine might help the dalesconol A biosynthetic pathway, a dominant polyketide bioassembly line in D. eschscholzii, to form new natural products by sequestering Lac-sensitive (viz., phenolic) guest intermediates originated from other BGCs. To address the assumption, the ethyl acetate extract derived from the procaine-exposed fungal culture was fractionated under the guidance of high-resolution liquid chromatography−mass spectrometry (HR-LC−MS) to isolate the aromatic polyketides with molecular weights higher than dalesconol A (molecular formula/weight: C29H18O6/462), as predicted from incorporations of side-chain-bearing naphthols into the dalesconol A biosynthetic pathway. The fractionation procedure afforded previously undescribed compounds named daeschol B and dalescones A−D (13− 16). Interestingly, the procaine exposed cultivation of the

generation of reactive species from diverse O(N)-substituted arenes at the expense of oxygen.6 This family of enzymes catalyzes typically the oxidative homo- or cross-couplings of small molecule phenols leading to di-, oligo-, and even polymeric compounds.7 Such laccase catalyzation features have been showcased by the construction of dalesconols A− C by D. eschscholzii from 4HN (1) and its desoxy congeners 1,3,8-trihydroxynaphthalene (3HN, 2) and 1,8-dihydroxynaphthalene (DHN, 3). Specifically, the fungal laccase (Lac) catalyzes both the homodimerization of 2 and 3 and the follow-up couplings of the produced dimers (4a and 4b) with the third naphthol molecules (1 or 2). Also essential is the 4HN reductase (4HNR) that initiates the transformation of 1 into 2 and 3 in collaboration with 3HN reductase (3HNR) and scytalone dehydratase (SD) (Figure 1).4 Intriguingly, the secondary metabolite profiling of D. eschscholzii showed the fungal production of other low-molecular-weight phenols such as 5 and 6,5 which resemble structurally 4-hydroxyscytalone derived from 1 after the final 4HNR-catalyzed reduction step (Figure 1).8 In view of the substrate promiscuity of 4HNR,8,9 we inferred that 5 and 6 might result from their presumed precursors 7 and 8, respectively, via a 4HNR-involved process similar to the conversion of 1 into 4-hydroxyscytalone (Figure 1). If so, the other-BGC-sourced phenols such as 7 and 8 might be sequestered as the guest intermediate by the dalesconol bioassembly line to generate the polyketides with novel frameworks, provided that the Lac-catalyzed couplings and phenolic substructure production are adequately efficient. The observation motivated us to search for a small-molecule activator that can enhance the Lac-catalyzed coupling step of the dalesconol biosynthetic pathway in D. eschscholzii. The laccase activity of some ligninolytic basidiomycete fungi can be increased by the fungal exposure to CuSO4 or to some small molecule phenols (e.g., syringaldehyde, syringol, guaiacol, sinapic acid, vanillin, and ferulic acid), but such observations need to be ascertained for each particular strain.10 Furthermore, those phenols themselves are probably transformable by laccases.5 Copper(II) can inhibit the fungal growth at higher concentrations11 and may affect the aldol reaction involved in polyketide biosyntheses.12 The rationalization enforced us to B


Letter

Organic Letters

products formed by the dalesconol B bioassembly line. Thus, an LC−MS guided fractionation of the extract derived from the procaine-exposed culture was performed to obain three more new compounds, named dalescones E−G (17−19, Figures 4 and 5).

fungus multiplied the production of daeschol B, whose abundance was below the detection limit in the previous work (Figure 1).4,5b The unique structures of 13−16 represent four unprecedented carbon skeletons resulting from the sequestrations of the side-chain-bearing GIs by the dalesconol A bioassembly line (Figure 2A). Dalescone A (13) was analyzed to have a molecular formula of C31H20O7 by its HR-ESI-MS spectrum exhibiting the protonated molecular ion at m/z 505.12821 (calcd 505.12818). The 1H NMR spectrum of 13 was comparable to that of dalesconol A.5a However, the H-4 and H-5 signals at δH 6.97 (d, J = 8.0 Hz) and 7.47 (t, J = 8.0 Hz) in the 1H NMR spectrum of dalesconol A were replaced by two oxymethylene groups of 13 (appeared as an overlapped four-proton multiplet at δH 5.12), indicating the presence of a 1,3-dihydrofuran residue. The structural assignment for 13 was substantiated by the single-crystal X-ray diffraction analysis with Cu Kα radiation (Figure 3). Compound 13 was afforded as a racemic mixture,

Figure 4. GI-incorporated compounds of dalesconol B biosynthetic pathway. The identification of daeschol B (the immediate precursor of dalesconol B) was enabled by the procaine-exposed fungal cultivation, upon which phenolic subunits 7−10 were shown to be sequestered by the dalesconol B bioassembly line to form the previously undescribed metabolites 17−19.

Figure 3. X-ray crystal structure of dalescone A (13, a CH3OH molecule is included).

and subsequent chiral HPLC separation gave the enantiomers (+)-13 and (−)-13 whose absolute configurations were established to be 19S,28S and 19R,28R, respectively, by comparing their experimental CD curves with the calculated ECD spectra for all possible stereoisomers (Figure S20). The structure determination of dalescones B−G (14−19) is detailed in the SI. The polyketide synthase and laccase genes are ubiquitous in plants and microbes.15 In particular, the two enzymes catalyze the construction of dalesconols B and C.4 Therefore, we were curious about whether the bioassembly lines of dalesconols B and/or C have produced new metabolites upon the aforementioned procaine-exposed fungal cultivation. Thus, the extract derived from the procaine-supplemented fungal culture was reanalyzed by the LC−MS screening for molecules with reasonable molecular weight differences from those of dalesconols B and C. The first compound obtained in such an effort was evidenced to have a molecular formula of C30H18O8, an oxygen extra over that of daeschol A,4,5b from the protonated molecular ion at m/z 507.1092 in its HR-ESI-MS. We inferred that it could be the previously unidentified immediate precursor of dalesconol B. This assumption was confirmed by a detailed interpretation of its 1D and 2D NMR spectra that accommodated the structure of the new compound, named daeschol B after its congener daeschol A.4,5b Previously, daeschol B, an immediate precursor of dalesconol B, was too scarce to be detected in the culture of D. eschscholzii.4,5b Here, it became identifiable owing to its abundance increment in response to the procaine-promoted laccase expression during the fungal cultivation. This rationaliztion enabled us to assume that the GI sequestration might be generically applicable for the exploration of new natural

Figure 5. X-ray crystal structure of dalescone E (17).

The new compounds afforded in the work were screened for inhibition of the NLRP3 inflammasome activation, a process involved in the incidence of diverse diseases.16,17 Thus, the GIincorporated compounds were assayed for the inhibitory effect on the IL-1β secretion, which reflects the NLRP3 inflammasome activation in macrophages stimulated by lipopolysaccharide (LPS) plus adenosine triphosphate (ATP).16 As shown in Table S10, (+)-13, (+)-14, (+)-16a, (−)-16a, and four enantiomers of 19 significantly reduced IL-1β levels with an IC50 (half maximal inhibitory concentration) range of 3.9−9.4 μM. The bioactivity magnitude of the compounds was approximately 2−5-fold more active than andrographolide (IC50: 21.53 μM), a coassayed drug that inhibits the NLRP3 inflammasome activation.17 In conclusion, the work presents the guest intermediate sequestration through up-regulating the polyketide biosynthetic gene to produce structurally unique natural products with expanded chemodiversity and promising biological function. The protocol is of general significance in mining the divergence magnitude of described biosynthetic pathways to realize the new access to skeletally unpredictable molecules from microbes. The applicability of the approach is demonstrated by the full characterization of dalescones A−G (13−19), some of which are more potent than andrographolide, a clinically prescribed medicine capable of inhibiting NLRP3 inflammasome activation. These highlight that the strategy may hold C


Letter

Organic Letters

(10) Fonseca, M. I.; Ramos-Hryb, A. B.; Fariña, J. I.; Afanasiuk, S. S.; Villalba, L. L.; Zapata, P. D. World J. Microbiol. Biotechnol. 2014, 30, 2251−2262. (11) Baldrian, P. Enzyme Microb. Technol. 2003, 32, 78−91. (12) (a) Bae, H. Y.; Sim, J. H.; Lee, J. W.; List, B.; Song, C. E. Angew. Chem., Int. Ed. 2013, 52, 12143−12147. (b) Magdziak, D.; Lalic, G.; Lee, H. M.; Fortner, K. C.; Aloise, A. D.; Shair, M. D. J. Am. Chem. Soc. 2005, 127, 7284−7285. (13) (a) Ma, H. M.; Zhou, Q.; Tang, Y. M.; Zhang, Z.; Chen, Y. S.; He, H. Y.; Pan, H. X.; Tang, M. C.; Gao, J. F.; Zhao, S. Y.; Igarashi, Y.; Tang, G. L. Chem. Biol. 2013, 20, 796−805. (b) Du, L.; Robles, A. J.; King, J. B.; Powell, D. R.; Miller, A. N.; Mooberry, S. L.; Cichewicz, R. H. Angew. Chem., Int. Ed. 2014, 53, 804−809. (14) Lyko, F.; Brown, R. J. Natl. Cancer Inst. 2005, 97, 1498−1506. (15) (a) Wang, H.; Fewer, D. P.; Holm, L.; Rouhiainen, L.; Sivonen, K. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 9259−9264. (b) Dwivedi, U. N.; Singh, P.; Pandey, V. P.; Kumar, A. J. Mol. Catal. B: Enzym. 2011, 68, 117−128. (16) (a) Strowig, T.; Henao-Mejia, J.; Elinav, E.; Flavell, R. Nature 2012, 481, 278−286. (b) Schroder, K.; Tschopp, J. Cell 2010, 140, 821−832. (17) Guo, W.; Sun, Y.; Liu, W.; Wu, X.; Guo, L.; Cai, P.; Wu, X.; Wu, X.; Shen, Y.; Shu, Y.; Gu, Y.; Xu, Q. Autophagy 2014, 10, 972−985.

promise for the rational re-exploitation of new bioactive molecules from clarified bioassembly lines.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00786. General methods and details of isolation of metabolite, 1D and 2D NMR spectra, and crystallographic data (PDF) X-ray data for 13 (CIF) X-ray data for 16a (CIF) X-ray data for 16b (CIF) X-ray data for 17 (CIF) X-ray data for 14-r (CIF) X-ray data for 15-r (CIF) X-ray data for 19-r (CIF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ren Xiang Tan: 0000-0001-6532-6261 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The work was cofinanced by the NSFC grants (81530089, 81421091, 21402090, 21672101, 21472091, 81330079, 81673333, and 21661140001).

■

REFERENCES

(1) Brakhage, A. A. Nat. Rev. Microbiol. 2013, 11, 21−32. (2) (a) Chiang, Y. M.; Ahuja, M.; Oakley, C. E.; Entwistle, R.; Asokan, A.; Zutz, C.; Wang, C. C.; Oakley, B. R. Angew. Chem., Int. Ed. 2016, 55, 1662−1665. (b) Wiemann, P.; Guo, C. J.; Palmer, J. M.; Sekonyela, R.; Wang, C. C.; Keller, N. P. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17065−17070. (c) Lo, H. C.; Entwistle, R.; Guo, C. J.; Ahuja, M.; Szewczyk, E.; Hung, J. H.; Chiang, Y. M.; Oakley, B. R.; Wang, C. C. J. Am. Chem. Soc. 2012, 134, 4709−4720. (3) Harvey, A. L.; Edrada-Ebel, R.; Quinn, R. J. Nat. Rev. Drug Discovery 2015, 14, 111−129. (4) Fang, W.; Ji, S.; Jiang, N.; Wang, W.; Zhao, G. Y.; Zhang, S.; Ge, H. M.; Xu, Q.; Zhang, A. H.; Zhang, Y. L.; Song, Y. C.; Zhang, J.; Tan, R. X. Nat. Commun. 2012, 3, 1039. (5) (a) Zhang, Y. L.; Ge, H. M.; Zhao, W.; Dong, H.; Xu, Q.; Li, S. H.; Li, J.; Zhang, J.; Song, Y. C.; Tan, R. X. Angew. Chem., Int. Ed. 2008, 47, 5823−5826. (b) Zhang, Y. L.; Zhang, J.; Jiang, N.; Lu, Y. H.; Wang, L.; Xu, S. H.; Wang, W.; Zhang, G. F.; Xu, Q.; Ge, H. M.; Ma, J.; Song, Y. C.; Tan, R. X. J. Am. Chem. Soc. 2011, 133, 5931−5940. (6) Resch, V.; Schrittwieser, J. H.; Siirola, E.; Kroutil, W. Curr. Opin. Biotechnol. 2011, 22, 793−799. (7) Witayakran, S.; Ragauskas, A. Adv. Synth. Catal. 2009, 351, 1187− 1209. (8) (a) Conradt, D.; Schätzle, M. A.; Husain, S. M.; Müller, M. ChemCatChem 2015, 7, 3116−3120. (b) Husain, S. M.; Schätzle, M. A.; Lüdeke, S.; Müller, M. Angew. Chem., Int. Ed. 2014, 53, 9806−9811. (9) (a) Husain, S. M.; Schätzle, M. A.; Röhr, C.; Lüdeke, S.; Müller, M. Org. Lett. 2012, 14, 3600−3603. (b) Schätzle, M. A.; Flemming, S.; Husain, S. M.; Richter, M.; Günther, S.; Müller, M. Angew. Chem., Int. Ed. 2012, 51, 2643−2646. D


Sequestration of Guest Intermediates by Dalesconol Bioassembly

Recommend Documents