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Noda Institute for Scientific Research GRANT2013 Young Investigator Research GrantOutline of Research Result

Structural diversity of fungal secondary metabolites inducedby chemical epigenetic manipulationTeigo AsaiGraduate School of Pharmaceutical Sciences, Tohoku University

Research aimsFilamentous fungi produce a variety of secondary metabolites, many of which possess medically relevant biologicalactivities. Since fungal genome sequencing has progressed,it has become apparent that fungi possess far more biosynthetic gene clusters than was evident from previous chemical studies. This observation suggests that a vast number ofsecondary metabolites have yet to be identified. A promising approach for identifying novel metabolites is to activatesilenced genes by manipulating the chromatin-based epigenetic regulation of the genes involved in secondary metabolism.1) Recently, the use of chemical epigenetic modifiers, such as histone deacetylase (HDAC) and/or DNAmethyltransferase inhibitors, was introduced as a promisingapproach for identifying cryptic fungal secondary metabolites.2) Here, we adopted this approach to examine the secondary metabolite profiles of a wide range of fungal species, and identified novel metabolites, encoded by silentbiosynthetic gene clusters, in three fungal species.
MethodsTo identify the optimal conditions for HDAC and/orDNA methyltransferase inhibitor treatment, several fungalspecies were cultivated under various inhibitor concentrations. Ethyl acetate extracts of culture media were harvested and HPLC was utilized to evaluate the efficacy ofthe treatments by assaying for the appearance of new peaksand/or an enhanced accumulation of constitutive secondarymetabolites. Entomopathogenic fungi of the genus Chaetomium, and fungi isolated from medicinal plants and insectswere used as microbial resources. The structures of newcompounds were elucidated by spectral analyses. To ana-lyze the histone acetylation state around the polyketide synthase gene pksCH-2, fungal cultures were treated with anHDAC inhibitor, and chromatin immunoprecipitation(ChIP), using an anti-acetyl histone 4 (H4Ac) antibody, wasperformed. To assess pksCH-2 gene expression, treated anduntreated cultures were analyzed by quantitative real-timePCR.
ResultsUpon treatment with a number of HDAC and DNAmethyltransferase inhibitors, suberoyl bis-hydroxamic acid(SBHA, 100–500 μM), a Zn2+ type HDAC inhibitor, andnicotinamide (NA, 10–100 μM), a NAD+ dependent HDACinhibitor, were found to enhance production of fungal secondary metabolites in a wide range of fungal species. Specifically, treatment with SBHA or NA resulted in dramaticchanges in the secondary metabolite profile in the culturemedium from Pestalotiopsis sp. isolated from Taxus cuspidata var. nana (SBHA 500 μM), Chaetomium indicum(SBHA 500 μM) and Chaetomium cancroideum (NA50 μM), compared to those of the negative controls (Fig. 1).
After scaling-up the fungal cultivations, we utilized spectral analyses to successfully identify a variety of novel natural products from the EtOAc extracts. These products included structurally diverse aromatic polyketides fromPestalotiopsis sp. and C. indicum, and unique branchedfatty-acyl polyketides from C. cancroideum (panels 1–9,and 10–12 in Fig. 2, respectively).3,4)To determine whether the induction of fungal secondarymetabolite production by HDAC inhibitors is directly associated with chromatin-based epigenetic regulation throughhistone acetylation, we identified a non-reducing polyketidesynthase (NR-PKS) gene, pksCH-2, encoding the commonFig. 1. Reverse-phase HPLC profile of culture media extracts from Pestalotiopsis sp., Chaetomium indicum, and C. cancroideum, cultivated in the presence or absence of inhibitors.

50Fig. 2.

Examples of novel secondary metabolites isolated in this study.

intermediate of 1–5 in the draft genome of C. indicum. RTPCR analysis demonstrated that SBHA treatment resultedin an increase in pksCH-2 gene expression. Meanwhile,ChIP analysis indicated that this increase in expression wasaccompanied by a change in the acetylation state of theNR-PKS locus from an inactive hypo-acetylated, to a transcriptionally active hyper-acetylated state. These resultssuggest that a chromatin-based epigenetic process regulatedthe expression of pksCH-2 upon SBHA treatment, and thatthis regulation enhanced the production of a wide variety ofpolyketides in C. indicum.
ConclusionWe developed a chemical-based method for isolatingnovel secondary metabolites from fungi. Using the HDACinhibitors, SBHA and NA, we induced epigenetic changesin Pestalotiopsis sp., C. cancroideum and C. indicum, andsuccessfully isolated a variety of novel natural products.

This study demonstrates the practicality of this chemicalepigenetic method for identifying novel metabolites in awide variety of fungi through the stimulation of silent geneclusters.
References1)

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2) Cichewicz, R. H. (2010) Epigenome manipulation as a pathway tonew natural product scaffolds and their congeners. Nat. Prod. Rep.
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3) Asai, T., Yamamoto, T., Shirata, N., Taniguchi, T., Monde, K., Fujii,I., Gomi, K., Oshima, Y. (2013) Structurally diverse chaetophenolproductions induced by chemically mediated epigenetic manipulation of fungal gene expression. Org. Lett. 15: 3346–3349.
4) Asai, T., Taniguchi, T., Yamamoto, T., Monde, K., Oshima, Y. (2013)Structures of spiroindicumides A and B, unprecedented carbon skeletal spirolactones, and application of VCD exciton chirality methodin absolute configuration determination. Org. Lett. 15: 4320–4323.