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Heterologous Biosynthesis of Nodulisporic Acid F Kyle C. Van de Bittner,†,‡ Matthew J. Nicholson,*,†,‡ Leyla Y. Bustamante,†,‡ Sarah A. Kessans,‡ Arvina Ram,§ Craig J. van Dolleweerd,∥ Barry Scott,§ and Emily J. Parker*,†,‡ †

Ferrier Research Institute, Victoria University of Wellington, Wellington 6012, New Zealand Biomolecular Interaction Centre, University of Canterbury, 20 Kirkwood Avenue, Christchurch 8041, New Zealand § Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand ∥ Protein Science & Engineering, Callaghan Innovation, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand ‡

S Supporting Information *

Chart 1. Major Products for the Confirmed IDT Pathways

ABSTRACT: Nodulisporic acids comprise a group of valuable indole diterpenes that exhibit potent insecticidal activities. We report the identification of a gene cluster in the genome of the filamentous fungus Hypoxylon pulicicidum (Nodulisporium sp.) that contains genes responsible for the biosynthesis of nodulisporic acids. Using Penicillium paxilli as a heterologous host, and through pathway reconstitution experiments, we identified the function of four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F (NAF). Two of these genes (nodM and nodW) are especially significant as they encode enzymes with previously unreported functionality: nodM encodes a 3geranylgeranylindole epoxidase capable of catalyzing only a single epoxidation step to prime formation of the distinctive ring structure of nodulisporic acids, and nodW encodes the first reported gene product capable of introducing a carboxylic acid moiety to an indole diterpene core structure that acts as a reactive handle for further modification. Here, we present the enzymatic basis for the biosynthetic branch point that gives rise to nodulisporic acids.

(GGT) catalyzes the indole condensation of GGPP 2 and indole3-glycerol phosphate 3 to make 3-geranylgeranyl indole (GGI) 4; and (III) a regioselective flavin adenine dinucleotide (FAD) dependent epoxidase creates the single and/or double epoxidized-GGI products 5a/5b/5c. At the fourth enzymatic step involving IDT cyclization (Scheme S1), the pathways diverge into the key branches (Figure S2, pink arrows) that establish a library of mono/dioxygenated Markovnikov/antiMarkovnikov-derived cyclic cores like emindole SB 6a, paspaline 6b, and aflavinine. These cyclic cores are often further modified by decorative enzymes that create the bioactive diversity of IDTs. Nodulisporic acids (NAs) (Chart S1) are a group of notably bioactive emindole SB-like IDTs produced by Hypoxylon pulicicidum, formerly classified as Nodulisporium sp.8 Nodulisporic acid A (NAA) and its chemically modified derivatives are of particular significance because of their highly potent insecticidal activity against blood-feeding arthropods and lack of observable adverse effects on mammals, in particular the tremogenicity associated with the paspaline-derived IDTs is not observed.4,5,9 Low production of NAs from H. pulicicidum10 and complexity of chemical synthetic approaches11 hinder commercialization of these valuable compounds. Therefore, elucidation of the genes involved in the biosynthetic production of NAs and subsequent expression of the genes in a more robust host

F

ilamentous fungi produce a diverse repertoire of interesting and useful chemical compounds. One such class of compounds, the indole diterpenes (IDTs), is of particular interest due to its wide range of chemical diversity and concomitant bioactivities, which include anti-MRSA,1 anticancer,2 anti-H1N1,3 insecticidal,4 and tremorgenic5 activities. Identifying enzymes from biosynthetic pathways can enable enhanced production of compounds that are difficult or impossible to access from natural sources, and provides the opportunity to engineer efficiently novel compounds with enhanced bioactive properties. Since the identification of the biosynthetic pathway for the IDT paxilline 16 in Penicillium paxilli (Figure S1), gene functionality in seven other IDT biosynthetic pathways has been elucidated (Chart 1).7 These IDT pathways share homologous genes that encode enzymes for the first three steps in IDT biosynthesis (Figure S2): (I) a geranylgeranyl pyrophosphate synthase (GGPPS) converts farnesyl pyrophosphate and isopentyl pyrophosphate into geranylgeranyl pyrophosphate (GGPP) 2; (II) a geranylgeranyl transferase © 2017 American Chemical Society

Received: October 21, 2017 Published: December 28, 2017 582

DOI: 10.1021/jacs.7b10909 J. Am. Chem. Soc. 2018, 140, 582−585

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Figure 1. Predicted NA gene cluster (NOD) from H. pulicicidum (A) and the NAF 7 Biosynthetic Pathway (B). Arrows represent individual genes, and arrow decorations represent gene function.

Figure 2. HPLC analysis (271 nm) of extracts of P. paxilli knockout strains (in gray ()) expressing different H. pulicicidum (Nod) enzymes and/or P. paxilli (Pax) enzymes (in black ()). Note: a peak appears at the same retention time as emindole SB 6a, but 6a is only present in three traces (ii.b, iii.b, and v.b) as confirmed by corresponding 406.31 ± 0.01 m/z EICs (Figure S10).

By analyzing the genomic sequence of H. pulicicidum,13 we identified six genes (nodM, nodB, nodO, nodC, nodD2, and nodD1) that are homologous to genes found in the other functionally confirmed IDT biosynthetic gene clusters (Table S1−S5, Figure S3−S7). These six genes are nested in a gene cluster of 13 genes (Figure 1A) that we propose encode the enzymes necessary for the biosynthesis of NAA. Details of these predicted genes and their proposed function are shown in Table S6. Designation of the cluster boundaries was supported by identification of flanking genes that have highly conserved homologues in the genome of a closely related species of Hypoxylon (CO27−5),14 most of which were found at a single locus in the related genome (Table S6). The protein products of

provides a potential route to access these valuable compounds and their chemical derivatives. Here, we report identification of a gene cluster responsible for the production of NAs, and using a recently described method for multigene assembly,12 we have partially reconstituted the cluster in a heterologous host and have identified the genes responsible for delivering the NA core compound, NAF 7. Our results demonstrate that the action of the third enzyme in the pathway, NodM, leads to a single-epoxidized-GGI product 5a instead of a double-epoxidized-GGI product 5b, giving rise to emindole SB 6a rather than paspaline 6b, delineating the enzymatic basis for the branch point that is unique to nodulisporic acid biosynthesis (Figure S2). 583

DOI: 10.1021/jacs.7b10909 J. Am. Chem. Soc. 2018, 140, 582−585

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biosynthesis (Figure 2 trace ii.b); rather, LC−MS analysis revealed that emindole SB 6a (HRMS 406.3109 m/z), which shares the same IDT scaffold as the NAs, was the only cyclized IDT product detected (Figure S10). Heterologous expression of paxilline 1 biosynthesis genes in Aspergillus oryzae has shown that the IDT cyclase, PaxB, is capable of catalyzing the cyclization of both the monoepoxidizedGGI 5a to form emindole SB 6a and the diepoxidized-GGI 5b to form paspaline 6b.6d The complete absence of paspaline 6b and paxilline 1 when nodM was expressed in the P. paxilli paxM knockout strain (Figure 2, trace ii.b; Figures S9 and S11) confirmed that NodM is a GGI 4 monoepoxidase and PaxB can cyclize the monoepoxide product 5a. Notably, NodM is the first confirmed GGI 4 epoxidase that does not have the ability to carry out multiple epoxidations. To test that emindole SB 6a was indeed the pentacyclic skeleton of NA synthesis, we needed to confirm that the potential IDT cyclase, NodB (63% identical to PaxB), could cyclize the monoepoxidized-GGI product 5a. Thus, P. paxilli strain PN2250 (CY2), which contains a deletion of the entire PAX cluster preventing production of any IDTs6c (Figure 2, trace iii.a, Figure S9−12), was transformed with a multigene plasmid (pSK66) encoding paxG driven by the paxG promoter and nodC, nodM, and nodB, all driven by the respective heterologous PAX promoters. Expression of the four genes resulted in production of emindole SB 6a, but not paspaline 6b (Figure 2, trace iii.b, Figure S10−11), indicating that NodB is indeed the IDT cyclase capable of assembling the NA skeleton. We were curious to explore the versatility of NodB and probe the potential functional differences between NodB and PaxB. To this end, we assembled another four-gene plasmid (pKV74) containing paxG, paxC, paxM, and nodB to see if NodB could cyclize the diepoxidizedGGI product 5b. Expression of these four genes in P. paxilli strain PN2250 (CY2) restored paspaline 6b biosynthesis (Figure 2, trace iv.b; Figure S11) confirming that NodB cyclizes the diepoxidized-GGI product 5b, and thus, nodB is a full functional ortholog of paxB. The dedicated NAA core is NAF 7, generated by oxidation of the terminal methyl carbon, C28, of emindole SB 6a (Figure 1B). Thus, we turned our attention to confirming the identity of this oxidase, which catalyzes the fifth step in the pathway. The oxidation of a nonfunctionalized carbon to give a carboxylic acid moiety is consistent with the involvement of a P450 oxygenase. In common with other identified IDT clusters, the NA cluster is rich with genes that are predicted to encode P450 oxygenases. Of the 13 genes in the cluster, five are predicted to encode P450 oxygenases (nodW, nodR, nodX, nodJ, and nodZ), and only two of these, nodR and nodJ, encode proteins that share moderate identity to other IDT P450s (36% and 20% amino acid identity to PtmU (Table S6) and JanJ (Figure S13), respectively). As previously shown, introduction of nodM in a paxM deletion background produces emindole SB 6a (Figure 2, trace ii.b, Figure S10). Therefore, we sequentially tested the five candidate P450 genes (nodW, nodR, nodJ, nodX, and nodZ) by their coexpression with nodM into a paxM deletion background (strain PN2257). Of the five putative P450s that we coexpressed with nodM, only nodW (pKV64) produced a new IDT product (Figure 2, trace v.b; Figure S12; Figure S14, trace iii−vii). This new product was identified by LC−MS and NMR (Table S10, Figures S15−18) as NAF 7 (HR-MS 436.2870 m/z), indicating that a single P450 drives the oxidation of emindole SB 6a to NAF 7 (Figure 2B). Previous studies of the NA pharmacophore established that the carboxylic acid handle of NAs is readily modified to enhance the

the six predicted genes that are homologous to IDT biosynthesis genes have at least 40% amino acid identity to their homologues in the PAX cluster of P. paxilli and include a GGT (NodC), a regioselective FAD-dependent epoxidase (NodM), an IDT cyclase (NodB), two paralogous prenyl transferases (NodD2 and NodD1), and one FAD-dependent cyclo-oxidase (NodO). The other seven genes encode five cytochrome P450 oxygenases (NodW, NodR, NodX, NodJ, and NodZ), and a pair of paralogous FAD-dependent oxygenases (NodY1 and NodY2, Table S7). Similar to the TER gene cluster from Chaunopycnis alba (Tolypocladium album) responsible for terpendole biosynthesis,11 the NOD cluster does not appear to contain a secondary metabolite-specific GGPPS gene. Notably, we identified only one GGPPS-encoding gene in the genome of H. pulicicidum. Analysis of the amino acid sequence of the predicted protein product of this gene (Nod ggs1, Table S8, Figure S8) and its location outside of the identified cluster strongly, suggest that it is responsible for primary metabolic function similar to ggs1 in P. paxilli.15 To confirm the function of gene products and directly establish their respective roles in NAA biosynthesis, we used the recently described MIDAS platform12 to construct a series of plasmids harboring various combinations of these genes, which we then transformed into appropriate P. paxilli hosts (Table S9) for heterologous production of NAA precursors. Using our repertoire of P. paxilli knockout strains, we used functional complementation and pathway reconstitution to determine the functions of genes in the NOD cluster. Following transformation of P. paxilli hosts with multigene plasmids, chemical phenotypes of the transformants were identified by reversed phase liquid chromatography−mass spectrometry (LC−MS) analysis of fungal extracts. Newly expressed metabolites, as determined by high-resolution mass spectrometry (HRMS), were purified by semipreparative high-performance liquid chromatography (HPLC) and subjected to nuclear magnetic resonance (NMR) analysis (1H, 13C, and heteronuclear single quantum coherence (HSQC)) for compound identification. Since the NOD gene cluster does not appear to contain a GGPPS-encoding gene, we began by analyzing the gene that we predicted was responsible for the production of GGI 4, the second secondary-metabolic step in IDT biosynthesis. For paxilline 1 production in P. paxilli, this step is catalyzed by the GGT, PaxC,6d which shares 52.3% amino acid identity to NodC. A P. paxilli paxC knockout strain (PN2290), which does not produce paxilline (Figure 2, trace i.a; Figure S9), was transformed with a plasmid (pKV27) harboring nodC under control of the trpC promoter. LC−MS analysis of transformants identified a new compound (Figure 2, trace i.b; Figure S9), whose retention time (5.2 min) and HR-MS (436.2485 m/z) corresponded to that of paxilline 1, thereby revealing that nodC is a functional ortholog of paxC. For IDT biosynthesis, the geranylgeranyl tail of GGI 4 is primed for cyclization by epoxidation (Figure 1B). Whereas the hexacyclic structure of paspaline 6b requires diepoxidation, the pentacyclic structure of NAF 7 implies only a single epoxidation takes place. In paxilline 1 biosynthesis, the epoxidation of GGI 4 is catalyzed by PaxM,6d which shares 48.6% amino acid identity to NodM. Hence, nodM was tested for its role in the third enzymatic step of NA production. A P. paxilli paxM knockout strain (PN2257), which does not produce emindole SB 6a or paspaline 6b (Figure 2, trace ii.a; Figure S11), was transformed with a plasmid (pKV63) containing nodM regulated by the paxM promoter. Intriguingly, nodM did not restore paxilline 1 584

DOI: 10.1021/jacs.7b10909 J. Am. Chem. Soc. 2018, 140, 582−585

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bioactivity and insecticidal specificity of the natural compounds.4 Thus, the identification of nodW provides access to a key modifiable core structure, NAF 7, for exploration of novel IDTs. Additionally, the discovery of the nodW sequence establishes a new subclass of IDT P450 oxidases and will facilitate the identification of other oxidases that may modify the IDT cores in a similar but discrete manner. To confirm that only five genes are essential for the production of NAF 7 and to establish that no other P. paxilli genes from the PAX gene cluster contributed to NAF 7 production, we assembled a five gene construct (pSK68) containing paxG, nodC, nodM, nodB, and nodW and transformed it into the P. paxilli strain containing the deletion of the entire PAX cluster, PN2250 (CY2). As expected, LC−MS analysis revealed that expression of the five genes resulted in NAF 7 biosynthesis (Figure S12), confirming that five secondary-metabolic genes (paxG, nodC, nodM, nodB, and nodW) are required to produce NAF 7. In summary, we have used heterologous expression to identify the first five steps that deliver the simplest nodulisporic acid, NAF 7. In particular, we have confirmed the function of four new genes from H. pulicicidum: nodC, nodM, nodB, and nodW. We have discovered that the H. pulicicidum genome lacks a secondary metabolic GGPPS-encoding gene providing a possible explanation as to why such strict growth conditions are required to produce detectable quantities of NAs. The low quantities of NAs produced by H. pulicicidum is a challenge for both resolving the biosynthetic details and for usage of the compounds or their derivatives. Using the efficient gene reassembly of MIDAS and heterologous expression in P. paxilli, we have overcome both these issues. Elucidation of the biosynthetic routes for production of NAF 7 now allows straightforward identification of the “decoration” steps to make fully functionalized NAA. Genes predicted to catalyze the required prenylation (nodD1 or nodD2), to form nodulisporic acid E, and oxidations (nodO plus a P450 (nodR, nodX, or nodZ)), to form nodulisporic acid D, have been identified in the cluster (Figure S19), and we are in the process of confirming function. Overall, this work demonstrates that heterologous expression of IDT genes is a viable method that can enable access to a nearly infinite pool of natural products that could be useful across many industries.

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ACKNOWLEDGMENTS This work was supported by Fulbright New Zealand, University of Canterbury and Victoria University of Wellington, for doctoral support, and the New Zealand Ministry of Business, Innovation, and Employment (UOCX1405). We thank Dr. Amelia Albrett and Jan Vorster for assistance with spectra collection, and Dr. Jan Tkacz for insightful discussions.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b10909. Experimental details and spectroscopic data (PDF)

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*[email protected] *[email protected] ORCID

Craig J. van Dolleweerd: 0000-0002-1380-6930 Emily J. Parker: 0000-0002-9571-9987 Notes

The authors declare the following competing financial interest(s): We have filed a provisional patent on this work. 585

DOI: 10.1021/jacs.7b10909 J. Am. Chem. Soc. 2018, 140, 582−585