Differentially Expressed Genes (DEGs) in Umbelliferone (UMB) Producing Endophytic Fusarium oxysporum (ZzEF8) Following Epigenetic Modification.

Abstract

Despite several studies documenting secondary metabolite (SM) production by endophytes, their commercial use is often limited owing to the research lacunae in the underlying biosynthetic pathway and the corresponding metabolic flux. Combining epigenetic modulation with RNA-Seq analysis constitutes a promising approach for inducing regulatory gene(s) and thereby identifying their role in SM biosynthesis. Our earlier studies had identified the hypomethylating effects of prednisone in umbelliferone (UMB) (7-hydroxyl coumarin) producing endophytic Fusarium oxysporum isolate, ZzEF8 isolated from Zingiber zerumbet rhizomes. Hypomethylating effect of prednisone (300 μM) in ZzEF8 was validated in present experiments revealing decrease in 5-mC content (0.09 ± 0.01%) in prednisone treated ZzEF8 (PrZzEF8) compared to untreated control (UtZzEF8) (0.36 ± 0.01%). Subsequent RNA-Seq analysis detected transcriptional alterations in PrZzEF8 compared to UtZzEF8. Transcripts with significant differential expression (-2 ≥ fold change (FC) ≥ 2; q-value < 0.05) were detected for 64 transcripts, with 60 upregulated and four downregulated in PrZzEF8. Upregulated differentially expressed genes (DEGs) were annotated as transmembrane transporters, non-ribosomal peptide synthetase (NRPS), Type I and III polyketide synthase (PKS), phytoene dehydrogenase, bifunctional lycopene cyclase/phytoene synthase, geranylgeranyl pyrophosphate synthase and various genes involved in nutrient assimilation, transcription factors and transporters regulating metabolite export. Expression analysis of the selected DEGs were validated by qRT-PCR. Present study proposes UMB biosynthesis through acetate-malonate pathway from acetate units via a pentaketide intermediate in ZzEF8 instead of the phenylpropanoid pathway reported in plants. Study is of relevance as the insights gained into the UMB biosynthetic pathway in ZzEF8 will help in strategizing scale-up of UMB production.