Discovery of New Secondary Metabolites by Epigenetic Regulation and NMR Comparison from the Plant Endophytic Fungus Monosporascus eutypoides

Abstract

Overexpression of the histone acetyltransferase and the ¹H NMR spectroscopic experiments of the endophytic fungus Monosporascus eutypoides resulted in the isolation of two new compounds, monosporasols A (1) and B (2), and two known compounds, pestaloficin C (3) and arthrinone (4). Their planar structures and absolute configurations were determined by spectroscopic analysis including high resolution electrospray ionization mass spectroscopy (HRESIMS), one-dimensional (1D) and two-dimensional (2D) NMR, and calculated electronic circular dichroism data. Compounds 1-2 were screened in cytotoxic bioassays against HeLa, HCT-8, A549 and MCF-7 cells. Our work highlights the enormous potential of epigenetic manipulation along with the NMR comparison as an effective strategy for unlocking the chemical diversity encoded by fungal genomes.

Keywords: Monosporascus eutypoides; NMR comparison; cyclopropane derivatives; cytotoxic activities; epigenetic regulation.

Figure 1

High-performance liquid chromatography (HPLC) chromatograms of the EtOAc extracts from the culture of WT (black) and MeHatOE (red), respectively (Chromatographic conditions: 0–2 min 60% MeOH in water, 2–25 min 60–100% MeOH/H₂O, 25–30 min 100% MeOH, t = 30 min, 1.0 mL/min, 254 nm).

Figure 2

¹H NMR spectra of the WT (brown) and MeHatOE (dark green), respectively (600 MHz, Acetone-d₆).

Figure 3

The parameters on the Agrobacterium tumefaciens-mediated transformation (ATMT) of M. eutypoides. (A) OD₆₀₀ of A. tumefaciens; (B) co-cultivation ratio of spores and A. tumefaciens; (C) temperature for co-cultivation; (D) time for co-cultivation. Bars denote standard error. Different letters in the same column in the same cultural condition indicate significant difference at p ≤ 0.05 level by the Tukey–Kramer multiple comparison test.

Figure 4

(A) Schematic diagram of the hat gene overexpression strategy. The hat promoter and hat gene were cloned in the pAg1-H3 vector to generate overexpression vector pAg1-H3-Hat. (B) PCR was performed to select MeHatOE. Hyg-F and Hyg-R were PCR primers. PC: positive control; NC: negative control; M: 100 bp ladder. (C) RT-PCR was used to detect the transcription of hat in MeHatOE relative to wild-type (H-9 and H-15 were randomly selected for RT-PCR experiments).

Figure 5

Structures of 1−4 from MeHatOE.

Figure 6

Key ¹H-¹H COSY (blue lines) and HMBC (red arrows) correlations of compounds 1 and 2.

Figure 7

Key NOESY correlations of compounds 1–2.

Figure 8

Experimental electronic circular dichroism (ECD) spectrum of 1–2 in MeOH and the calculated circular dichroism (CD) spectra of 1a–2a and 1b–2b.