Letter pubs.acs.org/OrgLett
Integration of Chemical, Genetic, and Bioinformatic Approaches Delineates Fungal Polyketide−Peptide Hybrid Biosynthesis Mamoru Yokoyama, Yuichiro Hirayama, Tsuyoshi Yamamoto, Shinji Kishimoto, Yuta Tsunematsu, and Kenji Watanabe* Department of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan S Supporting Information *
ABSTRACT: To identify natural products and their associated biosynthetic genes from underutilized, difficult-to-manipulate microbes, chemical screening and bioinformatic analysis were employed to identify secondary metabolites and a potentially associated biosynthetic gene cluster. Subsequently, a heterologous expression system was used to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. This approach successfully identified the curvupallide and spirostaphylotrichin biosynthetic pathways in endophytic fungus Curvularia pallescens and the short-chain pyranonigrin biosynthetic pathway in Aspergillus niger.
fungi, such as Aspergillus nidulans and Aspergillus oryzae, are typically chosen as the heterologous hosts for expression of functional HPNs and identification of compounds those enzymes produce.10 Lastly, combining the results from the chemical screening and the heterologous expression study connects the secondary metabolites to the biosynthetic genes of unknown function, revealing the identity of the previously unassigned biosynthetic gene cluster and the possible biosynthetic mechanism involved. In the current study, we focused on a gene cluster named cpa found in the genome of an endophytic fungus Curvularia pallescens. DNA sequence analysis of this 35 kilobase (kb)-long cpa gene cluster (Figure S6) revealed the presence of 12 genes (cpaA−L) as shown in Table S2 that may be responsible for a PK−NRP biosynthesis. The cluster contains cpaA, which codes for an HPN with 9 domains: KS (ketosynthase), AT (acyltransferase), DH (dehydratase), KR (ketoreductase), ACP (acyl carrier protein), C (condensation), A (adenylation), PCP (peptidyl carrier protein), and R (reductase) as ascertained by in silico analysis. CpaA is highly homologous to PynA, the fungal HPN from Aspergillus niger responsible for the pyranonigrin biosynthesis.11 Since CpaA and PynA share 59.9%/24.5% amino acid sequence similarity/identity and a virtually identical module organization, we speculated that CpaA would synthesize a backbone structure that resembles that found in pyranonigrin J (5) or I (6) produced by PynA, including a straight polyene chain and a tetramic acid moiety (Scheme 1a).
Polyketides (PKs) and nonribosomal peptides (NRPs) have been isolated from various Streptomycetes strains and other prokaryotes, as well as eukaryotes such as filamentous fungi. In recent years, numerous biosynthetic gene clusters encoding enzymes for biosynthesis of those compounds, such as PK synthases (PKSs) and NRP synthetases (NRPSs), have been discovered in fungal genomes. Despite the presence of 30 to 40 gene clusters in a single Aspergillus genome,1 only a handful of fungal polyketide, peptide, and terpene natural products can be isolated from a culture grown under typical growth conditions, indicating that the majority of the gene clusters are cryptic. The current ultrahigh performance liquid chromatograph− high-resolution mass spectrometry (UHPLC−HRMS) allows characterization of secondary metabolites present at a minute level in the culture of a source organism.2 The expanding knowledge of natural product chemistry is making it progressively more straightforward to determine the identity of the detected metabolites. This approach enables identification of new compounds without resorting to performing large-scale fermentation or laborious bioactivity assays, thereby achieving reduction in the time and the cost of natural product discovery. On the other hand, heterologous expression of predicted biosynthetic genes encoded on the genome of the source organism is conducted to determine the type of secondary metabolites the corresponding enzymes would produce. Putative genes that are predicted to encode hybrid PKS−NRPS (HPN) enzymes are usually targeted for this type of study, because they are frequently involved in the biosynthesis of the carbon skeleton of interesting natural products as exemplified by chaetoglobosin,3−5 Sch 2109726 and pseurotins7−9 isolated from different fungal sources. Model © 2017 American Chemical Society
Received: February 23, 2017 Published: March 31, 2017 2002
DOI: 10.1021/acs.orglett.7b00559 Org. Lett. 2017, 19, 2002−2005
Letter
Organic Letters Scheme 1. Biosynthetic Pathways of Natural Products Containing a Tetramic Acid Fused γ-Pyrone Derived from Polyketide−peptide Hybrid Straight Chains: (a) Proposed Pyranonigrin Biosynthetic Pathway; (b) Proposed Curvupallide B Biosynthetic Pathway; (c) Proposed ShortChain Pyranonigrin Biosynthetic Pathwaya
Figure 1. LC−HRMS analysis of extracts from the cultures of A. nidulans A1145 strains transformed with different expression vectors. (a) The HPLC traces of the metabolic extract from (i) A. nidulans A1145 carrying an empty vector pKW2008816 as a negative control and (ii) A. nidulans A1145 carrying the cpaA expression vector pKW10031. (b) UV spectrum and (c) HRMS spectrum of 1. See Supporting Information for details. All HPLC traces recorded in this study were monitored at 280 nm. N-Boc-L-tryptophan methyl ester was used as an internal standard (IS) throughout the study.
a strong structural resemblance to pyranonigrin E (8) (Scheme 1a). Considering the similarity between 1 and 5, we speculated that the HPN CpaA produces 1 as a biosynthetic backbone core intermediate that is modified by a set of auxiliary enzymes encoded by other genes found in the cpa gene cluster to form 3 (Scheme 1b). To test whether CpaA is responsible for the biosynthesis of 3, cpaA was deleted from the wild-type C. pallescens DSM62482 (see SI for details.), and the mutant was shown to lose the ability to produce 3 (Figure 2(i) vs (ii)). A
a
TE: thioesterase. PCP: peptide carrier protein.
We used A. nidulans A1145 that is deficient in the orotidine 5′-monophosphate decarboxylase gene pyrG12 as the expression host of choice. A plasmid harboring the cpaA gene under the control of a strong glaA promoter13 was introduced to A. nidulans A1145 for heterologous expression of cpaA (see Supporting Information (SI) for details.). When the cpaAcarrying strain of A. nidulans A1145 was cultured under standard conditions, it was able to produce 1 at a yield of 5.44 mg/L (Figure 1 and SI). The isolated product was characterized as shown in Table S3 and Figures S9 and S10. The absolute configuration of the amino acid residue in 1 was determined to be the L-isomer using the advanced Marfey method14 (SI and Figure S5). Next, to ascertain the intended products of the cpa biosynthetic gene cluster, we conducted a chemical screening to identify secondary metabolites having chemical structures related to 1. The search indicated curvupallides as a potential candidate. In fact, a previous study15 reported isolation of curvupallide B (3) from C. pallescens (Scheme 1b), which bears
Figure 2. HPLC traces of metabolic extracts from the cultures of (i) the wild-type (WT) and (ii) a cpaA deletion strain of Curvularia pallescens DSM 62482. The authentic reference of (iii) 3 is also shown.
sample of purified 3 isolated from C. pallescens (Table S4 and Figures S11 and S12) was used as the authentic reference in this assay (Figure 2(iii)). We also attempted to disrupt other cpa genes to look for an accumulation of potential biosynthetic pathway intermediates. However, we were not able to observe any formation of new compounds from the mutant strains, presumably due to the low production level and/or instability of such intermediates. 2003
DOI: 10.1021/acs.orglett.7b00559 Org. Lett. 2017, 19, 2002−2005
Letter
Organic Letters To determine the enzyme responsible for the formation of the dihydro-γ-pyrone ring of the curvupallides from 1, we attempted coexpression of cpaA with each of the seven genes cpaB, C, F, G, J, L, and O that are predicted to code for redox enzymes in A. nidulans. We focused on the redox enzymes, because our previous study on the elucidation of the pyranonigrin biosynthesis identified redox enzymes to be responsible for the γ-pyrone ring formation.11 In the proposed mechanism, the FAD-containing monooxygenase (FMO) PynG performs an epoxidation-mediated cyclization to form the dihydro-γ-pyrone moiety, followed by the cytochrome P450 (P450) PynD catalyzing the oxidation of the C3 alcohol to a ketone and enolization to yield the characteristic tetramic acid fused γ-pyrone core of pyranonigrins (Scheme 1a). However, none of the pairwise coexpression systems failed to produce epoxidated or other highly oxidized intermediates. As an alternate approach to examine the curvupallide biosynthetic mechanism, we prepared a ΔcpaA mutant of C. pallescens (SI and Figures S2−S4). After confirming the loss of production of 3 by ΔcpaA mutant of C. pallescens, we fed 1 to this strain to observe the recovery of the production of 2 or 3. However, formation of any curvupallide was not observed in the extract of either the cells or the culture broth. Because we were able to recover 1 from the culture medium, we concluded that the C. pallescens strain did not take up 1 from the culture medium. Previously, we isolated a pyranonigrin biosynthetic intermediate 6 having a polyene chain with a serine residue as a building block of the tetramic acid moiety from an engineered A. niger strain.11 As discussed earlier, we also identified that the curvupallides have a polyene chain and employs an aspartic acid residue to form their tetramic acid moiety as found in 1. Furthermore, yet another related compounds pyranonigrin S (7) isolated from A. niger17 have been shown to possess a shorter polyene chain and incorporate a glycine residue into their tetramic core (Scheme 1c). To distinguish between the pyranonigrin S-type compounds with a short aliphatic side chain and the family of pyranonigrins mentioned earlier, we will refer to the first group short-chain pyranonigrins. Considering the high degree of structural similarity among those natural products, we suspected that the HPNs responsible for these compounds would exhibit reasonably high sequence similarity among themselves. A BLAST18 search with the cpaA sequence identified a functionally unknown gene ANI_1_982164 along with aforementioned pynA for the pyranonigrin biosynthesis from A. niger to have high sequence similarity with cpaA. When we expressed ANI_1_982164 using our A. nidulans system, we were able to isolate a new compound 4 as a natural product (Figure 3, Table S5 and Figures S13−S17). From the structural resemblance among 4, 5, and 1, and the close chemical composition between 4 and 7, we predict that ANI_1_982164 encodes for the HPN involved in the biosynthesis of 7 (Scheme 1c) and the genes in the vicinity of ANI_1_982164 constitute the short-chain pyranonigrin biosynthetic gene cluster (Figure S8; note that pyranonigrin E described by Riko et al.17 should not be confused with pyranonigrin E (8) described earlier by others11,19). Since 8 and 7 share the same degree of oxidation in the bicyclic core, we envisioned that a very similar FMO−P450catalyzed mechanism would be used for the formation of the tetramic acid fused γ-pyrone of 8 and 7. In support of this view, the FMO PynG and the P450 PynD responsible for the formation of the bicyclic core of 8 exhibit high sequence
Figure 3. LC−HRMS analysis of extracts from the cultures of A. nidulans A1145 strains transformed with different expression vectors. (a) The HPLC traces of the metabolic extract from (i) A. nidulans A1145 carrying an empty vector pKW2008816 as a negative control and (ii) A. nidulans A1145 carrying the ANI_1_982164 expression vector pKW10045. (b) UV spectrum and (c) HRMS spectrum of 4. See Supporting Information for details.
similarity with the corresponding predicted FMO ANI_1_976164 (77%) and P450 ANI_1_978164 (81%), respectively. On the other hand, curvupallides have a less oxidized dihydro-γ-pyrone core, and the predicted FMO and P450 from the curvupallide biosynthetic gene cluster exhibit significantly lower sequence similarity with those from the two pyranonigrin biosynthetic gene clusters (44−45% for the FMO CpaG and 51−53% for the P450 CpaC). These differences suggest that the cyclization and oxidation mechanism used for the curvupallide biosynthesis may be different from that used for the pyranonigrin biosynthesis. It has also been reported that C. pallescens DSM 62482 is capable of producing another class of related compounds named spirostaphylotrichins,20−22 including spirostaphylotrichin C (9). Based on the chemical structures of curvupallides and spirostaphylotrichins, we propose that 1 produced by CpaA serves as the precursor for both curvupallides and spirostaphylotrichins. After 1 is epoxidated presumably by an FMO, a cyclization can occur between either O10 and C6 to form the tetramic acid fused dihydro-γ-pyrone of curvupallides or C9 and C4 to form the spirocyclic tetramic acid of spirostaphylotrichins (Scheme 1b). Furthermore, phaeosphaerides23 and related paraphaeosphaerides24 isolated from endophytic fungi Phaeosphaeria avenaria and Paraphaeosphaeria neglecta FT462, respectively, share the same fused γ-pyrone-γlactam core structure. It is highly plausible that the biosynthesis of these compounds also involves 1 as a key intermediate. In this study, successful elucidation of the activity of an HPN gene of unknown function from an undercharacterized fungus using an A. nidulans-based heterologous expression system, combined with sensitive chemical screening of a culture extract analyzed by UHPLC−HRMS and targeted gene disruption in the fungus, resulted in the quick identification of the curvupallide biosynthetic gene cluster in an underutilized endophytic fungus C. pallescens. Furthermore, the newly 2004
DOI: 10.1021/acs.orglett.7b00559 Org. Lett. 2017, 19, 2002−2005
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Organic Letters
(6) Sato, M.; Yagishita, F.; Mino, T.; Uchiyama, N.; Patel, A.; Chooi, Y. H.; Goda, Y.; Xu, W.; Noguchi, H.; Yamamoto, T.; Hotta, K.; Houk, K. N.; Tang, Y.; Watanabe, K. ChemBioChem 2015, 16, 2294. (7) 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. (8) Tsunematsu, Y.; Fukutomi, M.; Saruwatari, T.; Noguchi, H.; Hotta, K.; Tang, Y.; Watanabe, K. Angew. Chem., Int. Ed. 2014, 53, 8475. (9) Yamamoto, T.; Tsunematsu, Y.; Hara, K.; Suzuki, T.; Kishimoto, S.; Kawagishi, H.; Noguchi, H.; Hashimoto, H.; Tang, Y.; Hotta, K.; Watanabe, K. Angew. Chem., Int. Ed. 2016, 55, 6207. (10) Lazarus, C. M.; Williams, K.; Bailey, A. M. Nat. Prod. Rep. 2014, 31, 1339. (11) Yamamoto, T.; Tsunematsu, Y.; Noguchi, H.; Hotta, K.; Watanabe, K. Org. Lett. 2015, 17, 4992. (12) Nayak, T.; Szewczyk, E.; Oakley, C. E.; Osmani, A.; Ukil, L.; Murray, S. L.; Hynes, M. J.; Osmani, S. A.; Oakley, B. R. Genetics 2005, 172, 1557. (13) Ganzlin, M.; Rinas, U. J. Biotechnol. 2008, 135, 266. (14) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 5146. (15) Abraham, W.-R.; Meyer, H.; Abate, D. Tetrahedron 1995, 51, 4947. (16) Tsunematsu, Y.; Ishikawa, N.; Wakana, D.; Goda, Y.; Noguchi, H.; Moriya, H.; Hotta, K.; Watanabe, K. Nat. Chem. Biol. 2013, 9, 818. (17) Riko, R.; Nakamura, H.; Shindo, K. J. Antibiot. 2014, 67, 179. (18) Johnson, M.; Zaretskaya, I.; Raytselis, Y.; Merezhuk, Y.; McGinnis, S.; Madden, T. L. Nucleic Acids Res. 2008, 36, W5. (19) Awakawa, T.; Yang, X. L.; Wakimoto, T.; Abe, I. ChemBioChem 2013, 14, 2095. (20) Abraham, W.-R.; Hanssen, H.-P.; Arfmann, H.-A. Phytochemistry 1995, 38, 843. (21) Sandmeier, P.; Tamm, C. Helv. Chim. Acta 1989, 72, 784. (22) Masi, M.; Meyer, S.; Cimmino, A.; Clement, S.; Black, B.; Evidente, A. J. Agric. Food Chem. 2014, 62, 10304. (23) Maloney, K. N.; Hao, W.; Xu, J.; Gibbons, J.; Hucul, J.; Roll, D.; Brady, S. F.; Schroeder, F. C.; Clardy, J. Org. Lett. 2006, 8, 4067. (24) Li, C. S.; Ding, Y.; Yang, B. J.; Miklossy, G.; Yin, H. Q.; Walker, L. A.; Turkson, J.; Cao, S. Org. Lett. 2015, 17, 3556.
acquired sequence information on the curvupallide biosynthetic enzymes combined with the knowledge obtained from our previous study on the pyranonigrin biosynthesis allowed us to assign a function to another uncharacterized gene cluster from A. niger as a cluster encoding enzyme that biosynthesizes shortchain pyranonigrins, such as pyranonigrin S and A. Finally, comparative sequence analysis of the two groups of pyranonigrin biosynthetic gene clusters from A. niger and the curvupallide biosynthetic gene cluster from C. pallescens permitted us to propose the biosynthetic mechanisms leading to the formation of a wide range of natural products covering pyranonigrins, curvupallides, and spirostaphylotrichins. Effective use of heterologous expression of HPNs, chemical screening of metabolites, and gene disruption studies supplemented with the accumulated knowledge of the mechanism of natural product biosynthesis can lead to rapid annotation of uncharacterized gene clusters and deduction of corresponding biosynthetic pathways. This approach should be useful in quickly examining the function of numerous uncharacterized gene clusters that continue to be discovered through the ongoing genome sequencing efforts.
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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.7b00559. Data from the NMR and MS determination of the compounds, and additional experimental information (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Kenji Watanabe: 0000-0002-0463-4831 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are thankful for the financial support from the Japan Society for the Promotion of Science (JSPS) Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers (No. G2604) (K.W.). This work was also supported in part by the Japan Society for the Promotion of Science (JSPS) (K.W., 15KT0068, 26560450), Innovative Areas from MEXT, Japan (K.W., 16H06449), the Takeda Science Foundation (K.W.), the Institution of Fermentation at Osaka (K.W.), the Japan Antibiotics Research Association (K.W.), the Uehara Memorial Foundation (K.W.), and the Tokyo Biochemical Research Foundation (K.W.).
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REFERENCES
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DOI: 10.1021/acs.orglett.7b00559 Org. Lett. 2017, 19, 2002−2005
