An Insight into the Secondary Metabolism of Muscodor yucatanensis: Small-Molecule Epigenetic Modifiers Induce Expression of Secondary Metabolism-Related Genes and Production of New Metabolites in the Endophyte

Abstract

Muscodor spp. are proficient producers of bioactive volatile organic compounds (VOCs) with many potential applications. However, all members of this genus produce varying amounts and types of VOCs which suggests the involvement of epigenetics as a possible explanation. The members of this genus are poorly explored for the production of soluble compounds (extrolites). In this study, the polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes from an endophyte, Muscodor yucatanensis Ni30, were cloned and sequenced. The PKS genes belonged to reduced, partially reduced, non-reduced, and highly reduced subtypes. Strains over-expressing PKS genes were developed through the use of small-molecule epigenetic modifiers (suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine). The putative epigenetic variants of this organism differed considerably from the wild type in morphological features and cultural characteristics as well as metabolites that were produced. Each variant produced a different set of VOCs distinct from the wild type, and several VOCs including methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)hexane-2,4-diol and 2-carboxymethyl-3-n-hexylmaleic appeared in the variant strains, the production of which could be attributed to the activity of otherwise silent PKS genes. The bioactive extrolite brefeldin A was isolated and characterized from the wild type. However, this metabolite was not detected in EV-1, but instead, two other products were isolated and characterized as ergosterol and xylaguaianol C. Hence, M. yucatanensis has the genetic potential to produce several previously undetectable VOCs and organic solvent soluble products. It is also the case that small-molecule epigenetic modifiers can be used to produce stable variant strains of fungi with the potential to produce new molecules. Finally, this work hints to the prospect that the epigenetics of an endophytic microorganism can be influenced by any number of environmental and chemical factors associated with its host plant which may help to explain the enormous chemical diversity of secondary metabolic products found in Muscodor spp.

The DNA‐methyltransferase inhibitor 5‐aza‐2‐deoxycytidine affects Humicola grisea enzyme activities and the glucose‐mediated gene repression

“…From the scientific literature, we believe that concentrations above this range would generate cytotoxic effects that could impair the enzyme production and gene regulation studies we aimed to address. The few studies that addressed the DNMT inhibitors effects on filamentous fungi growth employed the cytidine analogue 5‐AZ to evaluate the impact on secondary metabolites production . We have 5‐Aza, a more recently developed cytidine analogue which is incorporated only in the DNA, while 5‐AZ is incorporated both in DNA and RNA .…”

“…The 25 μM concentration is compatible to the studies which used the 5‐azacytidine (5‐AZ) analogue for the treatment of fungal cells . Due to the lack of genomic information and to biochemical techniques limitations, we could not evaluate if the Hg v t DNMTs were indeed inhibited by the drug concentration we have employed.…”

“…Chemical inhibitors of DMNTs, such as 5‐azacytidine (5‐AZ) and 5‐aza‐2‐deoxycytidine (5‐Aza), have been widely used to restore the correct DNA methylation profile in tumor cells, when associated with traditional chemotherapeutic agents (e.g., ). In filamentous fungi, growth in the presence of 5‐AZ led to an increase in secondary metabolites production . Nonetheless, the effect of these inhibitors on glycosyl hydrolase gene expressions has not yet been reported.…”

Insights in Fungal Bioprospecting in Mexico

“…According to GC-MS, the extracts and VOCs shared 12 compounds, including benzene derivatives, phenolic compounds, cyclopentadienes, esters, lactones, alkanes, aldehydes, and carboxylic acids [80]. Recently, Qadri and coworkers [81] isolated a new strain of this fungus (Ni30) from a leaf of Elleanthus sp. at the Reserva Natural Punta Gorda, Nicaragua.…”

“…The genus Acremonium Link contains more than 200 species, which could be saprophytic, pathogenic, or endophytic. This genus is known for producing many interesting bioactive secondary metabolites, like the β-lactam antibiotics cephalosporins, the tremorgenic indole-diterpene alkaloids lolitrems, sesquiterpenoids, diterpenoids and triterpenoids with different skeletons, ophiobiolin D, a few meroterpenoids, a variety of polyketides, and peptides [81]. Bioassay-guided fractionation of the mycelial extract from A. camptosporum MEXU 26354 led to the isolation of six heterodimeric polyketides, acremoxanthone E (93), acremoxanthone A-C (94, 95, and 71), and acremonidins A (72) and B (96) [82] (▶ Fig.…”