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Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for
anthraquinone dimerization
Scott Griffiths, Carl H. Mesarich, Benedetta Saccomanno, Abraham Vaisberg, Pierre J. G. M. de Wit, Russell Cox, Jérôme Collemare
To cite this version:
Scott Griffiths, Carl H. Mesarich, Benedetta Saccomanno, Abraham Vaisberg, Pierre J. G. M. de Wit, et al.. Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 2016, 113 (25), pp.6851-6856. �10.1073/pnas.1603528113�.
�hal-02635376�
Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization
Scott Griffithsa, Carl H. Mesaricha,b, Benedetta Saccomannoa, Abraham Vaisbergc, Pierre J. G. M. De Wita,1, Russell Coxd,e,2, and Jérôme Collemarea,f,1,2
aLaboratory of Phytopathology, Wageningen University, 6708 PB Wageningen, The Netherlands;bLaboratory of Molecular Plant Pathology, Institute of Agriculture and Environment, Massey University, Palmerston North 4442, New Zealand;cFacultad de Ciencias y Filosofía, Departamento de Ciencias Celulares y Moleculares y Laboratorios de Investigación y Desarrollo, Avenida Honorio Delgado 430, Urbanización Ingeniería-San Martin de Porras, Universidad Peruana Cayetano Heredia, Lima 31, Peru;dInstitut für Organische Chemie, Leibniz Universität Hannover, 30167 Hannover, Germany;eSchool of Chemistry, University of Bristol, Bristol BS8 1TS, United Kingdom; andfUMR1345 Institut de Recherche en Horticulture et Semences (IRHS)-INRA, ACO, Université d’Angers, 49071 Beaucouzé Cedex, France
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 9, 2016 (received for review March 4, 2016) Anthraquinones are a large family of secondary metabolites (SMs) that
are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the for- mation of dimers from anthraquinone monomers. Despite their numer- ous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungusCladosporium fulvum. A gene cluster of 10 genes con- trols cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These find- ings shed light on the enzymatic mechanisms involved in anthraqui- none dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric an- thraquinones and structurally related compound families.
secondary metabolism
|
gene cluster|
cytoxicity|
emodin|
nataloe-emodinN aturally occurring anthraquinones and structurally related xanthones are particularly interesting families of secondary metabolites (SMs) that have long been exploited as vibrantly colored dyestuffs and for their excellent medicinal qualities (1–3).
They have been largely used in both traditional Asian and modern Occidental medicines. For example, emodin is one of the most in- tensively studied anthraquinones because of its ubiquity and potent anticancer, antidiabetic, antiinfective, antiinflammatory, and cathartic properties as well as its cardio-, hepato-, and neuroprotective qualities (4). Anthraquinones and xanthones contain an aromatic core that serves as a scaffold for the attachment of diverse functional groups, resulting in a wide variety of molecules with distinct biological and biochemical characteristics. This diversity is further increased by their assembly into homo- and hetero- dimers (3, 5), with activities that are distinct from those exhibited by their respective monomeric components (6). Despite the large number of identified anthraquinones and xanthones and their potential applications, very few of their biosynthetic pathways are known. Such knowledge is of great interest to academics and industrialists in pursuit of new medicines or enzymes for meta- bolic engineering.
Bacteria, fungi, lichens, and plants produce anthraquinones and xanthones, but the best characterized biosynthetic pathways are fungal, including those that produce the emodin-derived mono- dictyphenone in Aspergillus nidulans (7, 8) and the highly carcinogenic
aflatoxin in Aspergillus parasiticus (9, 10). The biosynthesis of aflatoxin begins with the production of a raw polyketide by the core polyketide synthase (PKS) PksA followed by a large number of modifications that are catalyzed by discrete tailoring/decorating enzymes (9).
Genes that encode these enzymes are arranged in a cluster, which is conserved across a number of other fungal species, although with different sets of tailoring genes. Such changes have been shown to drive the production of different anthraquinones, such as ster- igmatocystin in A. nidulans and dothistromin in Dothistroma septo- sporum (11). A large gene cluster is also involved in the production of monodictyphenone in A. nidulans, which consists of the core PKS gene mdpG and at least seven tailoring genes (7, 8). In the latter case, it has been shown that these genes also belong to a three-loci supercluster that produces prenyl xanthones (10, 12). Together, these examples illustrate how structurally related but functionally distinct compounds can result from the diversification of tailoring genes within a conserved biosynthetic gene cluster. Many anthraquinone and xanthone homo- and heterodimers have been isolated from natural producers (5, 13) or genetically modified Aspergillus species expressing genes related to monodictyphenone and atrochrysone biosynthesis (7, 14). Although dimerization of anthraquinone
Significance
Anthraquinones are potent secondary metabolites produced by many fungi and plants used in traditional Chinese and Indian medicine. Many display useful biological properties, including antineoplastic, antiinflammatory, antiinfective, or antiparasitic activities. The chemical structure of anthraquinones is very di- verse, with many occurring as homo- and heterodimers. Anthra- quinone biosynthetic pathways must be elucidated before novel structurally complex chemicals with new or enhanced biological activity can be engineered. In this study, we identified an enzyme involved in asymmetrical dimerization of nataloe-emodin, which results in increased cytotoxicity toward a range of cancer cell lines.
Mastering the substrate specificity of this enzyme (and other similar enzymes) could lead to the dimerization of anthraquinone- related compounds with medicinal activities.
Author contributions: S.G., P.J.G.M.D.W., R.C., and J.C. designed research; S.G., C.H.M., B.S., A.V., R.C., and J.C. performed research; S.G., C.H.M., and B.S. contributed new reagents/
analytic tools; S.G., R.C., and J.C. analyzed data; and S.G., P.J.G.M.D.W., R.C., and J.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. Email: pierre.dewit@wur.nl or j.collemare@
gmail.com.
2R.C. and J.C. contributed equally to this work.
This article contains supporting information online atwww.pnas.org/lookup/suppl/doi:10.
1073/pnas.1603528113/-/DCSupplemental.
CHEMISTRYMICROBIOLOGY
through aryl–aryl bond formation is assumed to be catalyzed enzymatically, no responsible enzymes have so far been identified in fungi.
The bianthraquinone cladofulvin
1is a homodimer composed of two nataloe-emodin moieties linked by an aryl–aryl bond, and it is the sole detectable SM produced by the fungal plant pathogen Cladosporium fulvum during growth on artificial media (15). How- ever, no biological activities or functions have so far been determined (15). From 23 predicted core SM genes identified in the genome of C. fulvum, only the PKS6 gene is consistently and highly expressed during cladofulvin production (15). Extensive synteny was reported between the PKS6 locus and the monodictyphenone gene cluster in A. nidulans but with clear signs of divergence (Fig.
1A) (15). Notably, the PKS6 locus does not contain homologs of mdpH, mdpJ, or mdpL, genes that are essential for the production of monodictyphenone (7, 15). Conversely, the PKS6 locus con- tains two genes that are not present in the monodictyphenone gene cluster, genes that encode a predicted cytochrome P450 monooxygenase and a predicted short-chain dehydrogenase/
reductase (15). A homologous gene cluster appears to be pre- sent in Aspergillus terreus (Fig. 1A and
SI Appendix, Table S1),which contains genes that encode enzymes for the production of atrochrysone
2(14).
In this study, we aimed to elucidate the cladofulvin biosynthetic pathway and identify the enzyme(s) involved in the dimerization of nataloe-emodin to yield cladofulvin. Using C. fulvum deletion mu- tants and heterologous expression of presumed cladofulvin bio- synthetic genes in Aspergillus oryzae, we propose a complete route to cladofulvin biosynthesis, including the final enzymatic dimerization step. We also report that dimerization of nataloe-emodin increases the ability of this compound to inhibit the growth of mammalian cell lines.
Results
(1) Nine Genes at the claGLocus and claHAre Coregulated During Cladofulvin Production and Define the Putative Cladofulvin Gene Cluster.
We first wanted to ascertain which genes define the pre- dicted cladofulvin gene cluster. For this purpose, we compared the expressions of the cladofulvin (cla) genes predicted to be involved in cladofulvin
1production in WT C. fulvum and genetically modified strains of this fungus in which cladofulvin
1production is abolished. Indeed, C. fulvum deletion mutants of the global transcriptional regulator Wor1 and the overexpression OE.CfWor1 transformant do not produce cladofulvin
1, consistent with down-regulation of the PKS6 gene in both genetic backgrounds (16). Deletion of the histone deacetylase gene, CfHdaA, in C. fulvum also abolished cladofulvin production (17). Available RNA-sequencing (RNA-seq) data from the
Δcfwor1deletion mu- tants showed that, relative to the WT, with the exception of claA, all of the predicted cla genes are significantly down-regulated, whereas the
β-tubulincontrol gene and clafu184401 border gene are not (Fig. 1B). In the OE.CfWor1 transformant, only genes from the predicted cladofulvin gene cluster, including claA, are signifi- cantly down-regulated at the claG locus (Fig. 1B). Similarly, in the
Δcfhdaadeletion mutants, claB, claC, claG, and claM are signifi- cantly down-regulated compared with the WT, with the other genes also showing a clear decrease in expression (Fig. 1B). In ad- dition to genes at the claG locus, the clafu190728 and clafu197287 genes were included, because their products bear high similar- ity to MdpH and MdpL, respectively, in A. nidulans (SI Appen-
dix, Table S1). The putativeclaH gene (clafu190728) is significantly down-regulated in all three genetic backgrounds compared with the WT, but the expression of clafu197287 was unchanged (Fig.
1B). These results show that claH and genes at the claG locus are coregulated and likely belong to the same biosynthetic pathway.
(2) Biosynthesis of Cladofulvin Depends on the Megasynthase ClaG.
To validate the hypothesis that the nonreducing polyketide synthase (nrPKS) (18) ClaG is responsible for cladofulvin
1production, we replaced the claG gene in C. fulvum WT through homologous recombination. Two confirmed independent deletion mutants and an ectopic transformant were selected for additional analysis (SI
Appendix, Fig. S1). Both Δclagdeletion mutants appeared to be grayer in color than the ectopic transformant and the WT, but otherwise, they showed no obvious phenotypic abnormalities (Fig.
2A). Ethyl acetate extracts from cultures of two independent
Δclagdeletion mutants, WT and ectopic transformant, were analyzed by HPLC alongside purified cladofulvin
1as a standard (15). Clado- fulvin
1was detected in the controls as previously reported (15) but not in the
Δclagdeletion mutants (Fig. 2B), confirming that claG, indeed, encodes the responsible PKS. The other coregulated cla genes described above might, therefore, play a role in the regulation or production of cladofulvin
1in C. fulvum.
(3) Concerted Activity of ClaG and ClaF Results in Atrochrysone Biosynthesis.
To characterize the early steps of cladofulvin
1bio- synthesis, we heterologously expressed selected cla genes in A. oryzae M-2–3, a fungal strain with a silent SM profile and a proven track record in expressing heterologous SM genes (14, 19).
Fig. 1. Definition of the cladofulvin gene cluster inC. fulvum. (A) Organiza- tion of theclaG(PKS6) gene locus in the genome ofC. fulvum(15) predicted to be involved in the biosynthesis of cladofulvin 1and of characterized homologous gene clusters inA. nidulansandA. terreusinvolved in the bio- synthesis of monodictyphenone (7) and atrochrysone2(14), respectively. Black arrows represent coregulated genes in each given species and/or shown to be involved in the production of the corresponding metabolite. Dark gray arrows represent genes potentially belonging to the depicted gene clusters; letters indicate the homologous monodictyphenone gene inA. nidulans. Light gray arrows indicate genes with functions that are not present in other gene clusters. White arrows indicate genes that are not coregulated in a given species. The dashed black square indicates genes specific toC. fulvum, and dashed gray squares indicate genes inA. nidulansandA. terreusfor which no close homolog could be found in the genome ofC. fulvum. White triangles indicate repetitive elements. Above the arrows, the functional annotation of genes fromC. fulvumis written.B,LeftandCentercharts show differential expression of genes at theclaGlocus in theΔcfwor1deletion mutant and OE.CfWor1transformant, respectively, compared with the WT as determined by RNA-seq (16).B,Rightchart shows differential expression of genes at the claGlocus in theΔcfhdaadeletion mutant (17) compared with the WT as de- termined by reverse transcription-quantitative real-time polymerase chain re- action (RT-qrtPCR). Error bars represent SD of three biological repeats. Black and gray bars show significant and nonsignificant fold changes, respectively, according to Cuffdiff analysis of three biological repeats. Statistics inRightwere performed on 2−ΔCtvalues using a two-way ANOVA with a posthoc Sidak multicomparison test at the significance level of 0.05.
6852 | www.pnas.org/cgi/doi/10.1073/pnas.1603528113 Griffiths et al.
The nrPKSs MdpG, ACAS, EncA, AptA, and AdaA involved in the first step of monodictyphenone, atrochrysone
2, endocrocin 3,asperthecin, and TAN-1612 biosynthesis, respectively, do not contain any canonical thiolesterase (TE) or Claisen cyclase domains (7, 14, 20–22). Instead, the metallo-β-lactamase–type TEs MdpF, ACTE, EncB, AptB, and AdaB, respectively, are required for closure and release of each nascent polyketide. ClaG is orthologous to these nrPKSs (15) (SI Appendix, Fig. S2) and does not contain a TE or Claisen cyclase domain, and ClaF is orthologous to these metallo-
β-lactamase–type TEs (SI Appendix, Fig. S2). Consistent with ourexpectations, A. oryzae transformants expressing claG alone did not produce any detectable polyketide (Fig. 3A and
SI Appendix, Fig. S3).In contrast, A. oryzae transformants coexpressing claG and claF genes produced several compounds,
2–8, bearing UV signatures diagnosticof aromatic polyketides (Fig. 3B and
SI Appendix, Figs. S3–S6).Product
2[retention time (RT)
=5.1 min; UV maximum
=225, 271, 320, and 397 nm; m/z (electrospray; ES
−) 273 [M−H]
−] was de- termined as atrochrysone by comparing its UV and MS data with those published (18). Its exact mass was confirmed with high-reso- lution MS analysis [m/z (ES
+) 275.0919 [M+H]
+] (SI Appendix, Fig.
S7). Product3
[RT
=5.5 min; UV maximum
=224, 287, and 442 nm;
m/z (ES
−) 313 [M−H]
−] was assigned to endocrocin by comparing its UV and MS data with those published (7, 14) (SI Appendix, Figs. S3–
S6). Products4
[RT
=8 min; UV maximum
=226 and 351 nm; m/z (ES
−) 255 [M−H]
−] and
5[RT
=8.1 min; UV maximum
=224, 288, and 440 nm; m/z (ES
−) 269 [M−H]
−] are likely emodin an- throne and emodin, respectively, by comparing their UV and MS data with those published (7, 14) (SI Appendix, Figs. S3–S6). Pre- viously, coexpression of ACAS and ACTE enzymes in A. oryzae yielded diastereomers of homodimers and heterodimers of endo- crocin anthrone and emodin anthrone in addition to products
2–4(14). Based on UV and MS data from this study, the minor products
6[RT
=5.6 min; UV maximum
=224 and 361 nm; m/z (ES
−) 597 [M−H]
−],
7[RT
=5.8 min; UV maximum
=224 and 359;
m/z (ES
−) 597 [M−H]
−], and
8[RT
=7 min; UV maximum
=255 and 361 nm; m/z (ES
−) 553 [M−H]
−] likely correspond to such dimers, but they were not further investigated. An evaporative light scat- tering detector chromatogram showed that ClaG and ClaF produced atrochrysone
2as the major product in A. oryzae (SI
Appendix, Fig. S8).(4) ClaH Is a Decarboxylase That Yields Emodin.
In A. nidulans, bio- synthesis of monodictyphenone involves atrochrysone
2and emodin
5as intermediate compounds (7). It was hypothesized that mdpH is required for the biosynthesis of these intermediates, because endocrocin alone accumulated in
Δmdphdeletion mutants (7).
In contrast, deletion of any other tailoring gene resulted in the production of emodin and other emodin-related shunt products, which lack a carboxyl group (7). In the C. fulvum genome, the claH gene is coregulated with the other cla genes (Fig. 1) and encodes a predicted protein bearing 64% identity to the N-terminal region of MdpH (SI Appendix, Fig. S9 and Table S1).
To address the role of ClaH in cladofulvin biosynthesis, we coexpressed claH with claF and claG in A. oryzae. The resulting transformants mainly produced product
5, which was confirmedto be emodin from its comparison with the RT, UV, and MS data of a commercial emodin standard (Fig. 3 C and D and
SI Ap- pendix, Figs. S3–S6). This result suggests thatclaH and mdpH are functional decarboxylase homologs and that emodin is the sec- ond stable intermediate in cladofulvin biosynthesis.
Fig. 2. The megasynthase ClaG is required for cladofulvin production.
(A) Deletion ofclaGaltered the pigmentation ofC. fulvumduring growth on potato–dextrose agar. (B) HPLC diode array chromatograms (254 nm) of ethyl acetate extracts from the WT, ectopic transformant, and deletion mutant strains showed the absence of cladofulvin1inΔclagdeletion mutants. Pure cladofulvin 1(0.5 mg) was used as a standard (retention time=12.3 min).Insetsshow the UV spectrums of cladofulvin1from the single peak in each sample.
Fig. 3. Identification of intermediates in the cladofulvin biosynthetic pathway using heterologous expression of candidate biosyntheticclagenes inA. oryzae.
A. oryzaeM-2–3 was transformed with expression vectors carrying the PKSclaG, β-lactamase thiolesteraseclaF,putative decarboxylaseclaH, scytalone dehydratase claB,and trihydroxynaphthalene reductaseclaCgenes. Ethyl acetate extracts were analyzed by LC-MS. Diode array chromatograms are shown. (A) The transformants expressingclaGyielded no polyketides. (B) The transformants expressingclaFand claGproduced eight compounds assigned to atrochrysone2, endocrocin3, em- odin anthrone4, emodin5, homodimers of endocrocin anthrone6and7, and heterodimers of endocrocin anthrone and emodin anthrone8. (C) The trans- formants expressingclaF,claG, andclaHproduced emodin5only. (D) Cultures of A. oryzaeM-2–3 supplemented with pure commercial emodin5standard pro- duced no new compound. (E) Cultures of the transformants expressingclaBand claCsupplemented with emodin5produced traces of chrysophanol9. (F) Pure commercial chrysophanol9was used as a standard.
CHEMISTRYMICROBIOLOGY
MdpH shares homology with HypC and EncC, which are involved in the production of aflatoxin and endocrocin
3, respectively (10, 22).HypC is an anthrone oxidase that converts norsolorinic acid anthrone to norsolorinic acid (23). EncC was also hypothesized to catalyze the oxidation of endocrocin anthrone to endocrocin
3(22). The three enzymes contain the conserved domain DUF1772, but MdpH also contains the additional conserved domain EthD (SI Appendix, Fig.
S9). ClaH contains this EthD domain only and does not share any
similarity with HypC or EncC (SI Appendix, Fig. S9). The results of the work by Chiang et al. (7) and our study suggest that MdpH and ClaH are decarboxylases. In contrast, HypC and EncC clearly do not have such activity as shown by the oxidation of norsolorinic acid anthrone (23) and the absence of emodin
5in Aspergillus fumigatus (22), respectively. Altogether, it suggests that the EthD domain is responsible for the observed decarboxylase activity. A gene encoding a protein that carries an EthD domain is present at the ACAS-ACTE locus and should be tested for a similar decarboxylation activity (SI
Appendix, Table S1).(5) ClaC and ClaB Are Responsible for the Conversion of Emodin to Chrysophanol.
Simpson (8) proposed that, during monodictyphenone biosynthesis, emodin
5is converted to chrysophanol
9by reduction and dehydration and catalyzed successively by the trihydroxynaph- thalene reductase MdpC and scytalone dehydratase MdpB. It was subsequently shown that MdpC is, indeed, capable of reducing emodin hydroquinone to 3-hydroxy-3,4-dihydroanthracen-1(2H)- one (24). To test the involvement of claC and claB in converting emodin
5to chrysophanol
9, both genes were coexpressed inA. oryzae. Induced cultures of these transformants, in addition to
untransformed A. oryzae, were supplemented with emodin
5. Liq-uid chromatography (LC)
–MS examination of organic extractsfrom the untransformed A. oryzae contained emodin
5only (Fig.
3D). Extracts from transformants coexpressing claB and claC contained not only emodin
5but also, traces of product
9[RT
=9.3 min; UV maximum
=225, 258, and 428 nm; m/z (ES
+) 255 [M+H]
+], which was assigned to chrysophanol by comparing its RT, UV, and MS spectra with those of a commercial standard (Fig.
3 E and F and
SI Appendix, Figs. S3–S6). These results suggest thatClaC and ClaB convert emodin
5to chrysophanol
9, presumablythrough sequential reduction and dehydratation.
(6) Cytochrome P450 ClaM Is Responsible for Dimerization of Nataloe-Emodin.
Targeted deletion of claM was performed in C. fulvum to determine the role of this cytochrome P450 in cladofulvin
1biosynthesis. Two confirmed independent deletion mutants and an ectopic transformant were selected for additional analysis (SI Appendix, Fig. S1). The
Δclamdeletion mutants were more gray-brown in color compared with WT and ectopic controls (Fig. 4A). A dark brown compound accumulated in the cytoplasm of fungal cells and diffused into the agar (Fig. 4 A and B). LC-MS analysis of ethyl acetate extracts confirmed that cladofulvin
1was present in the controls but not in the
Δclamdeletion mutants (Fig.
4C). The
Δclamdeletion mutants produced several compounds, and the major species were isolated by mass-directed HPLC and examined by NMR. The first eluted compound
10[RT
=7.3 min;
UV maximum
=231, 259, and 430 min; m/z (ES
−) 269 [M−H]
−] was purified, and comparison of its
1H NMR data with a litera- ture standard proved it to be nataloe-emodin (SI Appendix,
Figs. S3–S5 and S10 and Table S3), an SM previously unknownin fungi. The next eluted product was emodin
5,also identified by
1H NMR (SI Appendix, Figs. S3–S5 and S10 and Table S2).
Exact masses of both compounds were also confirmed with high-resolution MS analysis [product
5:m/z (ES
−) 269.0449 [M−H]
−; product
10:m/z (ES
−) 269.0448 [M−H]
−] (SI Appendix,
Fig. S7). Later eluted products were not further investigated,because they were present in traces and rapidly degraded on purification, which prevented structural elucidation. These re- sults confirm that nataloe-emodin
10is, indeed, the immediate precursor to cladofulvin and most importantly, that its dimer- ization involves the cytochrome P450 ClaM.
(7) Cytotoxicity of Cladofulvin and Its Precursors.
Nataloe-emodin
10potently inhibits the growth of immortalized mammalian cell lines (25), but no biological activities have been reported for cladofulvin
1so far (15). To test if it exhibits a similar cytotoxic activity, cladofulvin
1was applied to diverse nataloe-emodin–sensitive mammalian cell lines (25), including nontumorogenic 3T3 mouse embryo cells and diverse human tumor cell lines (Table 1). In addition, emodin
5and chrysophanol
9were also tested on the same cell lines. Emodin
5inhibited the growth of cell lines with significantly lower potency than nataloe-emodin
10[multiple t tests corrected for multiple compari- sons with the Holm–Sidak method;
α=0.025; P values ranging from 7.5e-6 (H460) to 0.012 (3T3)], except for M-14 and K562 cell lines, against which emodin
5showed higher (P value
=0.005) and equal
Fig. 4. Accumulation of nataloe-emodin inC. fulvumΔclamdeletion mutants.
(A) The deletion ofclaMaltered the pigmentation ofC. fulvumduring growth on potato–dextrose agar. (B) Spores ofΔclamdeletion mutants accumulate a pigment in the cytoplasm compared with the WT. (C) Diode array chromato- grams of ethyl acetate extracts fromC. fulvumWT, ectopic transformant, andΔclamdeletion mutants. WT and ectopic transformant produced only cladofulvin1.Δclamdeletion mutants produced emodin5and nataloe-em- odin10. The two other later eluted products were highly unstable and could not be identified.
Table 1. Toxicity of cladofulvin and its precursors against animal cell lines
Compound 3T3 H460 HuTu80 DU145 MCF-7 M-14 HT-29 K562
Emodin 15.329 (0.193) 9.928 (0.182) 7.831 (0.308) 13.973 (0.831) 8.178 (0.138) 11.802 (0.072) 17.007 (0.293) 8.094 (0.197) Chrysophanol* >200 >200 46.972 (1.153) >200 >200 >200 >200 132.042 (26.754) Nataloe-emodin† 10.345 (1.975) 4.800 (0.236) na 7.928 (0.608) 7.324 (0.087) 13.640 (0.557) 6.545 (1.194) 12.499 (3.887) Cladofulvin 0.060 (0.002) 0.037 (0.001) 0.097 (0.005) 0.055 (0.002) 0.036 (0.001) 0.097 (0.003) 0.275 (0.015) 0.039 (0.001) GI50(50% growth inhibition) (micrograms per milliliter−1) of each compound against different cell lines was determined. Numbers in parentheses indicate the SD of at least three technical replicates.
*The maximum concentration tested was 200μg mL−1.
†Data published in ref. 32.
6854 | www.pnas.org/cgi/doi/10.1073/pnas.1603528113 Griffiths et al.
(P value
=0.121) inhibition, respectively (Table 1). In contrast, chrysophanol
9weakly inhibited the growth of HuTu-80 and K562 cells only [GI
50(50% growth inhibition)
=47 and 132
μg mL−1, respectively; P values
=5.7e-7 and 0.001 compared with emodin, respectively] (Table 1). These results suggest that the C3 or C2 hydroxyl group in emodin
5and nataloe-emodin
10, respectively, isessential for the cytotoxic activity of these compounds, but the C2 position seems to provide significantly higher potency. Strikingly, compared with nataloe-emodin
10, cladofulvin1was significantly more active toward each target cell line, ranging from a 24-fold difference for HT-29 to a 320-fold increase for K562 [P values ranging from 1.2e-10 (MCF-7) to 0.001 (K562)] (Table 1). These results show that dimerization of nataloe-emodin dramatically enhanced its cytotoxicity.
Discussion
(1) Atrochrysone Carboxylic Acid Is Likely the Raw Polyketide Produced by ClaF and ClaG.
Based on the metabolic profiles of C. fulvum deletion mutants and A. oryzae transformants expressing putative cladofulvin
1biosynthetic genes, we propose a biosynthetic route to cladofulvin
1production (Fig. 5). Consistent with the SM profile of A. oryzae expressing ACAS and ACTE (14), the coex- pression of claF and claG in the same host yielded the same com- pounds, apart from endocrocin anthrone. Spontaneous conversion of endocrocin
3to emodin
5is considered to be thermodynamically unfavorable (7, 14). Thus, to account for the codetection of endo- crocin
3, emodin5,and atrochrysone
2, the highly unstable com-pound atrochrysone carboxylic acid was proposed as the raw polyketide released from ACAS by ACTE through a hydrolysis mechanism (14). We propose the same mechanism in the clado- fulvin
1biosynthetic pathway with atrochrysone carboxylic acid as the first unstable intermediate, which preferentially degrades to form atrochrysone
2and endocrocin
3(Fig. 5). Similarly, atrochrysone carboxylic acid is likely the first intermediate produced by any other orthologous pairs, including MdpG/MdpF (7) and EncA/EncB (22).
The sole presence of emodin
5in transformants coexpressing claH, claG, and claF suggests that ClaH significantly accelerates decarboxylation of atrochrysone carboxylic acid, consistent with the absence of endocrocin
3. When transformants expressingclaG and claF only were grown for 2 additional days, endocrocin
3became the major product produced instead of atrochrysone
2(SI Appendix,
Fig. S8). This result is consistent with the decarboxylase activity ofClaH and suggests that spontaneous decarboxylation of atrochrysone
carboxylic acid is slower than its spontaneous dehydration. The trace amounts of emodin anthrone
4and emodin
5detected in A. oryzae transformants expressing claF and claG are consistent with succes- sive spontaneous dehydration and oxidation of atrochrysone
2and emodin anthrone
4, respectively. The latter step yielding emodin5was, indeed, shown to occur spontaneously and may bypass the ac- tivity of anthrone oxidases (7, 14, 23).
(2) Conversion of Emodin into Nataloe-Emodin Likely Involves Chrysophanol Hydroquinone as an Intermediate.
Heterologous ex- pression of claC and claB in A. oryzae yielded traces of chrysophanol
9after the respective cultures were supplemented with emodin
5.The very limited yield of this reaction might be explained by the substrate provided to ClaC and ClaB. Indeed, it was shown that the substrate of MdpC is emodin hydroquinone, which is expected to be the prevalent form in vivo (24). Thus, we propose that ClaC may preferentially use emodin hydroquinone as a substrate, which implies that the product of ClaB might be chrysophanol hydroquinone (Fig.
5). We hypothesized that ClaK and ClaN might be involved in the next step toward the formation of nataloe-emodin
10. However,coexpression of claK and claN in A. oryzae did not yield any new compound when cultures of the respective transformants were sup- plemented with chrysophanol
9. This result suggests that ClaK/ClaNmight oxidize the intermediate chrysophanol hydroquinone rather than chrysophanol
9. Such a route between emodin5and nataloe- emodin
10is in agreement with findings by Schätzle et al. (24) and the absence of chrysophanol
9in
Δclamdeletion mutants. Additional investigations are needed to ascertain this part of the proposed route to cladofulvin
1.(3) Dimerization of Nataloe-Emodin by the Cytochrome P450 ClaM.
Despite the large number of known dimeric anthranoids and xan- thonoids occurring in nature (5, 13), only one dimerizing enzyme from Streptomyces coelicolor A3(2), ActVA-ORF4, has been reported (26). This enzyme, which shares similarities with dihy- droflavonol-4-reductases of plants (27), catalyzes the dimerization of hydroxylated dihydrokalafungin to produce the benzoisochro- manequinone actinorhodin (26). A number of dimeric anthrones have been isolated from fungi (7, 14, 28), with strong evidence that their formation is also enzymatically catalyzed (14). However, no fungal enzyme involved in catalyzing their formation has yet been reported. Our results show for the first time, to our knowledge, that a cytochrome P450 is involved in anthraquinone dimerization.
ClaM is required for the production of the bianthraquinone
Fig. 5. Proposed route for cladofulvin1biosynthesis. The formation of endocrocin3and emodin5might occur spontaneously. All oxidation ([O]) steps could be spontaneous in air. Conversion of chrysophanol9to nataloe-emodin10is the only step without any support and might involve ClaN and ClaK. Compounds in between brackets were not identified in this study. ACP, acyl carrier protein; AT, acyl transferase; KS, keto synthase; PT, product template; SAT, starter unit acyl carrier protetransacylase.
CHEMISTRYMICROBIOLOGY
cladofulvin
1through the C5–C7′ linkage of nataloe-emodin
10(Fig. 5). However, cladofulvin
1was not produced when A. oryzae transformants expressing claM were supplemented with nataloe- emodin
10. Therefore, it cannot be excluded that the reducedform of nataloe-emodin
10is the actual substrate for dimeri- zation by ClaM.
The involvement of cytochrome P450s in the dimerization of napthoquinones, coumarins, and diketopiperazine alkaloid was reported in bacteria, Aspergillus niger, and Aspergillus flavus, respectively (29–31). In S. coelicolor A3(2), flaviolin is dimerized by a cytochrome P450 (30), and a similar enzyme in Streptomyces griseus dimerizes tetrahydroxynapthalene into 1,4,6,7,9,12-hexahydroxyperylene-3,10- quinone (32). In A. niger, the cytochrome P450 KtnC dimerizes di- methyl siderin into kotanin (29). The cytochrome P450 DtpC in A. flavus is responsible for the concomitant pyrroloindole ring for- mation and homo- or heterodimerization of the diketopiperazine alkaloids ditryptophenaline, dibrevianamide F, and tryptophenaline (31). Nataloe-emodin
10dimers with alternative aryl–aryl bonds have not been observed in C. fulvum, and they do not have dimeric forms of emodin
5, which suggests that ClaM might be highly se-lective for both substrate and type of linkage that it catalyzes. In contrast, Alternaria species produce a large variety of alterporriol compounds, which are heterodimers of macrosporin and altersolanol exhibiting various couplings, including C5–C5′, C1–C7′, C7–C5′, and C2–C2′ bonds (28, 33). Homodimers of macrosporin or altersolanol were also isolated and show C4–C6′ and C4–C8′ bonds (28). The diversity of dimers for a given anthraquinone likely depends on both substrates and specificity of enzymes involved in dimerization. The former will determine the positions at which radicals or cations can be generated (30). Combined with substrate specificity of cyto- chrome P450s, diverse aryl–aryl bond formations will be catalyzed.
The production of so many different alterporriols by Alternaria
species may be caused by multiple dimerizing enzymes or fewer but less selective enzymes that catalyze multiple reactions.
Dimerization and the type of bond that links monomers profoundly affect the biological activities of SMs. As we showed for nataloe-emodin
10and cladofulvin
1, dimerization can strengthenan existing activity. Additional investigation is needed to determine the exact dimerization mechanism catalyzed by ClaM and assess the substrate specificity of this enzyme. The identification of ClaM ought to accelerate the in silico discovery of functionally similar enzymes in other fungal species, although only a limited number of close homologs were found by BLAST analysis using current publically available databases (SI Appendix, Fig. S11). A future challenge will be to assess the potential utility of ClaM and related cytochrome P450s as biocatalysts for the conversion of natural monomers into novel dimeric compounds with enhanced or novel biological activities.
Materials and Methods
Heterologous expression inA. oryzaeand construction ofC. fulvumdeletion mutants were conducted as previously described (14, 16, 17, 19). Strains were grown in standard media, and SMs were extracted using ethyl acetate. Extracts were analyzed by UV-HPLC and LC-MS. Standard chromatographic methods were run, and peaks were detected either between 200 and 800 nm or be- tween 200 and 400 nm. Analyses were performed simultaneously in ES+and ES−modes between 100 and 650m/z. NMR analyses were performed using a mass-directed chromatography system (15). Mammalian cell cytotoxicity bio- assays were performed as previously described (25). Full experimental proce- dures are described inSI Appendix.
ACKNOWLEDGMENTS.We thank Dr. Colin Lazarus (School of Biology, University of Bristol) for technical advice and the gift of vectors. We also thank Doug Roberts for assistance submitting samples for NMR analysis. S.G., C.H.M., P.J.G.M.D.W., and J.C. were financially supported by a grant from the Royal Netherlands Academy of Sciences.
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