Total Biosynthesis of Brassicicenes: Identification of a Key Enzyme for

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Cite This: Org. Lett. 2018, 20, 6178−6182

Total Biosynthesis of Brassicicenes: Identification of a Key Enzyme for Skeletal Diversification Akihiro Tazawa,† Ying Ye,† Taro Ozaki,*,† Chengwei Liu,† Yasushi Ogasawara,‡ Tohru Dairi,‡ Yusuke Higuchi,¶ Nobuo Kato,¶ Katsuya Gomi,§ Atsushi Minami,† and Hideaki Oikawa*,† †

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan ¶ The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan § Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan

Org. Lett. 2018.20:6178-6182. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 10/05/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: The biosynthetic pathway of brassicicenes, derived from the phytopathogen Pseudocercospora f ijiensis, was fully reconstituted. Heterologous expression of the eight genes highly expressed in infected leaf tissues generated a new brassicicene derivative as a final product. Together with the characterization of P450 from Alternaria brassicicola, a late stage of the biosynthetic pathway, which generates remarkable structural diversity, has been proposed. Notably, a unique P450 that converts 3 to the structurally distinct 4 and 6 was identified.

Pseudocercospora f ijiensis is a causal agent of black Sigatoka, which is a major disease of bananas (Musa spp.).1 Recent transcriptomic sequencing of this fungus identified several biosynthetic gene clusters of secondary metabolites with higher expression in infected leaf tissue than in an artificial medium.2 Metabolites derived from these gene clusters may play a role in the pathogenicity of this fungus and, thus, have drawn our attention. However, the corresponding compounds have not yet been identified. In the present study, we focused on one such gene cluster (referred to as the bsc gene cluster in this study) that shows significant similarity to the previously reported brassicicene biosynthetic gene cluster.3 Brassicicenes are a series of diterpenes that have been isolated from Alternaria brassicicola, a phytopathogenic fungus that causes dark leaf spots in Brassica sp. To date, over 10 derivatives have been isolated from this species.4 Brassicicenes, such as brassicicene A (BC-A), have a fused 5−8−5 carbocyclic ring system, which is related to other diterpenes, such as fusicoccin A and cotylenin A (Figure 1). Both fusicoccin A and cotylenin A are fungal metabolites that show phytohormone-like activity.5 Cotylenin A is also known as a differentiation-inducing agent against human myeloid leukemia cells and is a potential anticancer drug.6 Although all of the brassicicenes were originally proposed to have 5−8−5 ring systems, the structure of brassicicene D was recently revised to a tricyclo[9.2.1.0]tetradecane core skeleton that contains a bridgehead double bond (Figure 1).4c 13C chemical shifts of other related derivatives were calculated by employing a DFT method, and their revised structures were also proposed to have the same skeleton. Despite these extensive studies on the isolation and structural elucidation, the biosynthetic pathway © 2018 American Chemical Society

Figure 1. Brassicicenes and related diterpenes.

that generates such structural diversity is not yet fully understood. In a previous study, the biosynthetic gene cluster of brassicicene C (BC-C), which contains 11 genes (orf1 to orf11, Figure 2), was identified from A. brassicicola ATCC 96836.3 Orf 8 (bscAAb) encodes the fusicocca-2,10(14)-diene (FD) synthase that consists of an N-terminal terpene cyclase (TC) domain and a C-terminal prenyltransferase (PT) domain; the PT domain synthesizes geranylgeranyl diphosReceived: August 20, 2018 Published: September 19, 2018 6178

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Figure 2. (A) Biosynthetic gene clusters of the brassicicenes in P. f ijiensis and A. brassicicola. (B) Proposed biosynthetic pathway for brassicicenes. Key: FPP, farnesyl diphosphate; IPP, isopentenyl diphosphate; MT, methyltransferase. Solid arrows indicate the reactions experimentally verified in this study, and dashed arrows are those deduced from expected protein function.

metabolite with m/z 317 [M + H − H2O]+ (Figure 3A). The H and 13C NMR data for the isolated compound were in good agreement with those of 3.4b Transformation by pUSA2-bscDE also gave two other types of transformants, AO-bscABCDETF1 and AO-bscABCDE-TF2. TF1 showed efficient conversion of 1, giving 3 with the putative intermediate Me-ether 1a (Figure S3A). On the other hand, TF2 provided 2, as well as its precursor 1, but did not produce 3 (Figure S3A). This observation indicated that BscE initially transformed 1 into 1a, and then BscD catalyzed the oxidative rearrangement of 1a, as reported previously.8 This was further supported by the successful in vitro conversion of 1 into 1a with the recombinant BscEAb (Figure S3B). In contrast, methylation of 2 did not occur under the same conditions. Based on the above results, we propose that BscE-catalyzed methylation precedes BscD-catalyzed oxidation in the biosynthesis of brassicicenes. Using AO-bscABCDE as a host, we characterized the remaining three genes; bscF and bscG both encode a cytochrome P450, and bscH encodes a functionally unknown protein with weak homology to NmrA. First, we introduced bscF and bscG into AO-bscABCDE with pUNA2-bscFG. The constructed AO-bscABCDEFG produced four new metabolites (Figure 3A). The NMR spectra and specific rotation of 4 were

phate (GGPP), which is subsequently cyclized to FD by the TC domain. In this gene cluster, five genes (orf1, orf 2, orf5, orf 7, and orf11) encode cytochrome P450s. Among these P450s, Orf1 (BscBAb) is responsible for the 8β-hydroxylation of FD, while Orf7 (BscCAb) catalyzes 16-hydroxylation.7 Based on an in vitro analysis of Orf9 (BscDAb) and Orf6 (BscEAb), FD-8β,16-diol (1) might be converted into brassicicene I (3) via fusicocca-1,10(14)-diene-3,8β,16-triol (2). However, conclusive evidence of this conversion is still missing, because neither conversion proceeded smoothly.3,8 The absence of orthologues of orf 2, orf 3, and orf4 suggests that P. f ijiensis produces brassicicene derivatives with simpler structures than those isolated from A. brassicicola. In the present study, we employed an Aspergillus oryzae expression system to reconstruct the biosynthetic pathway. First, we introduced bscA, bscB, and bscC into A. oryzae NSAR1 with the plasmids pTAex3-bscA and pAdeA2-bscBC to reconstitute the first three steps of the biosynthesis. LC-MS analysis of the extracts from the resulting AO-bscABC showed a new metabolite. By comparison with the synthetic sample,7,8 this compound was readily identified as 1 (Figure 3A, Figure S2).3 Subsequently, we introduced two additional genes, bscD and bscE, into AO-bscABC with the plasmid pUSA2-bscDE. LC−MS analysis showed that AO-bscABCDE produced a new

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the signal for C12 was observed at δC 81.44. In addition, other spectral data revealed that the structure of 7 is the 12-hydroxy derivative of 6 (Figure S6). Given the above results, either BscF or BscG was assumed to be responsible for the oxidative rearrangement of 3 to yield 6. To determine which enzyme catalyzes this reaction, we constructed AO-bscF and conducted the biotransformation of 3. As a result, AO-bscF converted 3 into 4 and 6, whereas A. oryzae NSAR1 did not give any new metabolites (Figure 3B). This result clearly indicates BscF catalyzed the oxidation of 3 to give both 4 and 6, thus suggesting that BscG catalyzes the 12-hydroxylation of 6. Finally, we introduced bscH into AO-bscABCDEFG with pPTR1-2p-bscH. The resulting AO-bscABCDEFGH produced 8, in addition to the four metabolites produced by AObscABCDEFG (Figure 3A). HR-MS revealed the molecular formula to be C21H34O4 (8: m/z 373.2350 [M + Na]+). In the 1 H and 13C NMR spectra of 8, the signals corresponding to C18 of an exo-olefin disappeared. Instead, several new signals were observed [methyl (δH 1.10 (d); δC 11.25) and methine (δH 2.84 (q); δC 53.88)], suggesting the reduction of an exoolefin. Further analysis with HSQC, HMBC, and 1H−1H COSY confirmed the planar structure of 8, as shown in Figure 2 (Figure S6). The NOEs between H13α and H6, H13β and H11, and 18-Me and H1 indicated the absolute configuration as 3R,6S,7R,8S,11R,12S. This result clearly indicates that BscH catalyzes the reduction of the C11−C18 double bond. Similar NmrA domain (Pfam entry: PF05368)-containing reductases have been recently reported to be involved in the biosynthesis of a bacterial aromatic polyketide.9 Compound 8 (brassicicene O) would be a final metabolite of this pathway, which requires all eight genes that showed higher expression in the infected leaf tissues. In the biosynthetic gene cluster of A. brassicicola, one additional P450, Orf2 (BscIAb), is encoded. Considering that A. brassicicola produces brassicicene derivatives that undergo oxidation at C11 and/or C13 rather than 8, BscIAb would be responsible for these oxidations. To elucidate the function of BscIAb, we constructed a transformant harboring bscEAb, bscFAb, bscGAb, and bscIAb and conducted the biotransformation of 3 with this strain. In addition to the four metabolites produced by AO-bscABCDEFG, 9 and 10 were also produced by the conversion of 3 (Figure S7). This result indicates that BscFAb and BscGAb have the same function compared that of the homologous enzymes, BscF and BscG, in P. f ijiensis. BscIAb is probably involved in the formation of 9, while 10 seems to be a shunt metabolite as it was also produced by AO-bscABCDEFG (Figure S7). HR-MS analysis revealed that the molecular formulas of both compounds were C21H32O4. Compared to 6, a new oxymethine signal (δH 4.94 (d, J = 6.0 Hz)) was observed in the 1H NMR of 9. This signal exhibited correlation with H12 in the COSY spectrum. Detailed analysis of the NMR data enabled us to determine the structure of 9 as the 13-hydroxylated derivative of 6, suggesting that BscIAb catalyzes 13-hydroxylation of 6 (Figure S6). The structure of 10 was also analyzed by comparing the 1H NMR with that of 6. The methine signal of H15 (δH 2.99 (hept, J = 6.8 Hz) for 6) was not observed, and H19 and H20 were observed as singlets (δH 1.35 and δH 1.48, respectively). Extensive NMR analysis showed that 10 is a 15-hydroxylated derivative of 6 (Figure S6). These experimental results led us to elucidate the biosynthetic pathway for brassicicenes, as shown in Figure

Figure 3. LC−MS profiles of the metabolites produced by the transformants. (A) Heterologous expression of the bsc genes. (B) Feeding experiment of 3 against A. oryzae NSAR1 or AO-bscF.

in good agreement with those of brassicicene B.4a In the 1H NMR of 5, an oxymethine signal for H12 (δH 3.70 (dd, J = 7.0, 9.2 Hz, 1H) for 4) was missing, indicating that 5 was an oxidized derivative of 4. Oxidation of both 4 and 5 with Dess− Martin periodinane (DMP) gave the same diketone, 14 (Figure S4), thus confirming the structure of 5, as shown in Figure 2. Biotransformation of 4 using A. oryzae WT gave a small amount of 5, suggesting that the oxidation of 4 to 5 is catalyzed by an adventitious enzyme derived from the host strain. This oxidation might be catalyzed by Orf3, which is short chain dehydrogenase/reductase (SDR) in A. brassicicola, giving 5 as a possible precursor of brassicicene L (Figure S5).4c On the basis of the HR-MS data (6: m/z 355.2245 [M + Na]+), the molecular formula of 6 was determined to be C21H32O3. In the 1H NMR of 6, the methyl signal of H18 was missing. Instead, new signals were observed at δH 4.67 (s, 1H) and δH 4.72 (s, 1H). In the HSQC spectrum, these 1H signals showed correlations to the same 13C signal at δC 100.52, indicating the presence of an exo-olefin. In the HMBC spectrum, correlations between H18 and C12 and H12 and C2 were observed. Furthermore, in the 1H−1H COSY, correlations between H1 and H12 and H12 and H13 were observed (Figure S6). These observations suggested that 6 has the unique tricyclo[9.2.1.0]tetradecane core skeleton shown in Figure 2. The molecular formula of 7 was determined to be C21H32O4 by HR-MS (7: m/z 371.2192 [M + Na]+), suggesting that 7 is a hydroxylated derivative of 6. In the 1H and 13C NMR spectra of 7, the signal corresponding to H12 was not observed, and 6180

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modification enzymes generate the structural diversity at the C-ring oxidation level (Figure S5). Although compounds 4−8 were produced by the heterologous host, the variety and ratio of the products might be different in P. f ijiensis. During our careful study, we found that the host strain A. oryzae also converted late-stage intermediates to their corresponding oxidation products, such as 5 and 10 (Figure S7). We have also encountered undesired oxidation in the heterologous production of other fungal metabolites.13 In summary, we describe the heterologous expression of the eight genes, bscA-bscH, that show higher expression during infection with P. f ijiensis. The resulting transformant successfully produced novel brassicicene derivatives. To date, several phytotoxic metabolites, including fijiensin, have been identified from this fungus.14 However, the function of these metabolites in the process is unclear, because the expression of the biosynthetic genes did not correlate with the infection of host plants. As exemplified in the present study and a previous report,15 reconstitution of the biosynthetic pathway provides a promising way to identify the metabolites that are involved in pathogenicity of various fungi. We hope that the role of the metabolites we isolated in infectious events will be elucidated.

2B. Based on previous studies, we reconstituted the early steps of the biosynthesis to yield 3, a possible intermediate of the biosynthesis. The resulting transformant that could produce 3 enabled us to characterize the late biosynthetic stages for brassicicenes. BscF catalyzes the oxidation of 3, giving the structurally different products 4 and 6. This observation suggested that BscF abstracts H12, and the subsequent [HO•] rebound to the radical intermediate 11 generates 4 (Scheme 1). Alternatively, 11 can also provide the rearranged product 6 Scheme 1. Proposed Mechanism for the BscF-Catalyzed Reaction

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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.8b02654. Experimental procedures, NMR data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Taro Ozaki: 0000-0002-4673-4478 Tohru Dairi: 0000-0002-3406-7970 Hideaki Oikawa: 0000-0002-6947-3397

via a 1,2-shift, followed by H18 abstraction, to generate a BC-C skeleton. Recently, P450-catalyzed cationic rearrangements have often been proposed in the literature.10 Applying this mechanism, the corresponding cationic intermediate 12 can be generated by the further, one electron oxidation of 11. Preferential migration of the alkenyl group may occur to afford 13, which is quenched by H18 deprotonation. A similar P450-catalyzed oxidative rearrangement, which led to the formation of an exo-olefin, was recently found in the biosynthesis of penitrem.11 Skeletal rearrangement by P450s is known in natural product biosynthesis.10,12 In contrast to these enzymes that usually produce a single product, BscF is a unique enzyme that generates two structurally different products from the single substrate 3. Therefore, our findings not only add a new example to the collection of P450-catalyzed rearrangements, but also propose an intriguing strategy for the structural diversification of natural products. Brassicicenes from a producing strain have significant structural diversity at the C-ring oxidation level. In this study, we determined the function of the modification enzymes, BscF (12-hydroxylation of the C-ring of the BC-A skeleton), G (12-hydroxylation of the BC-C skeleton), I (13hydroxylation of the BC-C skeleton), and H (reduction of exoolefin of the BC-C skeleton). Considering the structural diversity of brassicicenes, we can readily speculate that the accumulation of late stage intermediates and the promiscuity of

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Nos. 15H01835 (H.O.), 16H06446 (A.M.), and 17H05425 (T.O.)). We are grateful to Prof. Dr. Margaret E. Daub and Dr. Elizabeth Thomas at North Carolina State University for providing the genomic DNA of P. f ijiensis.

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