Host metabolite producing endophytic fungi isolated from Hypericum perforatum.

Aruna Vigneshwari,Noémi Kiss,Csaba Vágvölgyi,Tamás Papp,András Szekeres,Anikó Németh,Biljana Škrbić,Dezső Csupor,Dávid Rakk,Sándor Kocsubé,Vijai Gupta

doi:10.1371/journal.pone.0217060

Abstract

In the present study, endophytic fungi have been isolated from various parts of the medicinal herb Hypericum perforatum (St. John’s Wort), which is known as a source of medically important metabolites. The isolated strains were cultured in liquid media and their ability to synthesize hypericin, the secondary metabolite of the host and its suspected precursor, emodin was tested analyzing the extracts of the fermentation broth and the mycelia. The HPLC-UV analysis of the chloroform/methanol extracts of the mycelia revealed that three isolates were able to produce emodin (SZMC 23771, 19.9 ng/mg; SZMC 23772, 20.8 ng/mg; SZMC 23769, 427.9 ng/mg) and one of them also could synthesize hypericin (SZMC 23769, 320.4 ng/mg). These results were also confirmed via UHPLC-HRMS technique both in full scan and MS/MS mode. The strains producing only emodin belong to the section Alternata of the genus Alternaria, while the isolate producing both metabolites was identified as Epicoccum nigrum. The mycelial extracts of E. nigrum and the Alternaria sp. SZMC 23772 showed higher inhibitory activities in the antimicrobial tests against the six selected bacteria compared to the hypericin and emodin standards in the applied concentration (100 μg/mL), while in case of the Alternaria sp. SZMC 23771 lower inhibition activities were observed on Staphylococcus aureus and Streptomyces albus than the pure compounds.

Highlights

The present study was based on the observation that certain endophytes are able to produce the same metabolites as their plant hosts and they can serve as novel microbial sources of bioactive plant metabolites [37,38]
Only a single case has been reported until today when the plant metabolite, hypericin could be produced by an endophyte [6,22]
Based on the recent classification of the Alternaria genus, the support values (Bayesian posterior probabilities, RAxML bootstrap) of the section Alternata were sufficiently high for the discrimination [28]

Summary

Introduction

The resulted octaketide chain undergoes both cyclization and decarboxylation reactions to form emodin anthrone, which is oxidized to emodin probably by the enzyme emodinanthrone-oxygenase and a condensation reaction yields a dianthrone leading to the formation of protohypericin and of hypericin [7] This biosynthetic pathway is generally accepted and some genes encoding enzymes potentially involved in the biosynthesis have been already analyzed by generation sequencing technology [8]. The spatial distribution of the chemical components of the biosynthetic pathway in planta were determined with desorption electrospray ionization mass spectrometry imaging (DESI-MSI) [9] and matrix free UV-laser desorption/ionization mass spectrometric imaging (LDI-IMS) [10] as well as by matrix-assisted laser desorption/ionization high-resolution mass spectrometry (HRMS) techniques [11] In these studies, hypericin was found to be localized in the dark glands on leaves of H. perforatum, but the proposed precursor, emodin anthrone, could not be visualized. The present study focused on the isolation and identification of endophytic fungi, which can produce hypericin and emodin, the host metabolites of H. perforatum

Materials and methods

Result

Findings

Discussion