Effect of epigenetic modulation on metabolites from endophytes isolated from Embelia ribes

Fungal endophytes share a symbiotic relationship with the host plants. Endophytes from medicinal plants produce metabolites similar to plants as well as some new metabolites, which serve as a promising medicinal source with, significant potential in the field of biomedicine. Epigenetic modifiers, such as DNA methyltransferase and histone deacetylase inhibitors, activate cryptic biosynthesis gene clusters, resulting in a significant increase in cryptic metabolite production. This study elucidated the alteration in the metabolite profiles of two endophytes isolated from the medicinal plant Embelia ribes after treatment with two epigenetic modulators.

This study assessed the effect of epigenetic modifiers-Azacitidine (AZ) and Sodium butyrate (SB)-on the metabolite profiles of Phomopsis azadirachtae and Diaporthe phaseolorum. Different concentrations of AZ and SB (1, 10, 50, 100, and 500 mM) were employed to assess their impact on the fungal endophyte cultures. Metabolome analysis was performed to observe the alteration of metabolites.

LC-MS analysis revealed 47 targeted metabolites in the AZ-treated P. azadirachtae culture. Treatment with AZ significantly affected the production of metabolites compared with the control. AZ treatment also altered the production of nine silent metabolites; namely dicerandrol B, phomosine A, epiepoxydon, taxol, cladosporine, phomonaphthalenone A, phomophyllin A, 3-indolepropionic acid (3-IPA) and ergosterol in P. azadirachtae culture. Two metabolites enhanced their production compared to the control. A total of 47 metabolites were identified in P. azadirachtae culture treated with SB, which also altered 11 silent metabolites and enhanced production of six metabolites; cytosporone B, phomophyllin A, phomosine A, phomosin B, laiolactol A, and ergosterol P by logarithmic analysis. Similarly, 41 metabolites were identified in D. phaseolorum culture treated with various concentrations of AZ. In D. phaseolorum culture treated with AZ, an epigenetic modification activated 11 silent metabolites-Cytochalasin N, bostrycoidin, phomonaphthalenone, phomopsterone, dicerandrol A, pinselin, indole-3-acetic acid, betulinic acid, phomophyllin A, dalienxanthone B and phomopoxide A. Two metabolites, phomosine A and zeatin riboside, were enhanced in majority of the AZ treatments compared to control by logarithmic analysis. SB treatment significantly modulated the metabolite profile of D. phaseolorum, with LC-MS analysis detecting 46 targeted compounds across different concentrations. The treatment activated 11 previously silent bioactive metabolites, including Ganodermaside D, lithocarpinol A, dalienxanthone B, cladospirone, dicerandrol B, libertellenone, phomonaphthalenone A, phomopoxide A, phomopsichin B, phomopsterone B, and cladospirone. Three metabolites, pinselin, dicerandrol A, and phmosine A was significantly enhanced in most of the SB treatments compared to control.

AZ treatment induced significant, concentration, dependent alterations in the metabolite profile of P. azadirachtae, with the most pronounced effects observed at the P1AZ concentration. Multivariate and clustering analyses revealed clear metabolic differentiation between treated and control cultures. A total of 47 targeted metabolites were detected under AZ treatment, including nine previously silent metabolites consistently induced across all concentrations. Notably, AZ exposure enhanced the production of phomophyllin A and phaseolorine, indicating the selective activation of cryptic biosynthetic pathways in P. azadirachtae. SB treatment significantly altered the secondary metabolite profile of P. azadirachtae in a dose-dependent manner. Metabolomic analysis detected 47 compounds in SB-treated cultures, with the most pronounced metabolic changes observed at the P50SB and P500SB concentrations. SB exposure activated a previously silent biosynthetic gene cluster responsible for the production of 11 metabolites. Furthermore, log fold-change analysis demonstrated significant and consistent upregulation of six metabolites across most SB treatments, highlighting SB's effectiveness in activating cryptic secondary metabolism in P. azadirachtae. In AZ-treated D. phaseolorum cultures, epigenetic alteration triggered 11 metabolites. Log fold change analysis reported significant upregulation of two metabolites. In D. phaseolorum, SB treatments detected 46 targeted compounds across different concentrations. The treatment activated 11 previously silent bioactive metabolites. and significantly increased the levels of three metabolites compared with controls. These findings demonstrate that the epigenetic modulators AZ and SB altered secondary metabolite profiles in fungal endophytes, indicating their potential to activate silent biosynthetic pathways. These findings support their use as exploratory tools for metabolite discovery, while highlighting the need for multi-omics and structural validation in future work.