Deletion of the Histone Deacetylase HdaA in Endophytic Fungus Penicillium chrysogenum Fes1701 Induces the Complex Response of Multiple Bioactive Secondary Metabolite Production and Relevant Gene Cluster Expression

Abstract

Epigenetic regulation plays a critical role in controlling fungal secondary metabolism. Here, we report the pleiotropic effects of the epigenetic regulator HdaA (histone deacetylase) on secondary metabolite production and the associated biosynthetic gene clusters (BGCs) expression in the plant endophytic fungus Penicillium chrysogenum Fes1701. Deletion of the hdaA gene in strain Fes1701 induced a significant change of the secondary metabolite profile with the emergence of the bioactive indole alkaloid meleagrin. Simultaneously, more meleagrin/roquefortine-related compounds and less chrysogine were synthesized in the ΔhdaA strain. Transcriptional analysis of relevant gene clusters in ΔhdaA and wild strains indicated that disruption of hdaA had different effects on the expression levels of two BGCs: the meleagrin/roquefortine BGC was upregulated, while the chrysogine BGC was downregulated. Interestingly, transcriptional analysis demonstrated that different functional genes in the same BGC had different responses to the disruption of hdaA. Thereinto, the roqO gene, which encodes a key catalyzing enzyme in meleagrin biosynthesis, showed the highest upregulation in the ΔhdaA strain (84.8-fold). To our knowledge, this is the first report of the upregulation of HdaA inactivation on meleagrin/roquefortine alkaloid production in the endophytic fungus P. chrysogenum. Our results suggest that genetic manipulation based on the epigenetic regulator HdaA is an important strategy for regulating the productions of secondary metabolites and expanding bioactive natural product resources in endophytic fungi.

Keywords: Penicillium chrysogenum; endophytic fungi; meleagrin; metabolic regulation; roquefortine.

Figure 1

Phylogenetic tree analyses of HdaA in the strain Fes170 and its homologs from different species. Branch lengths are in proportion to distance.

Figure 2

Generation and phenotype of the ΔhdaA strain. (A) Schematic illustration for hdaA disruption. The bleoR gene is amplified from the plasmid pZeo, and the bleomycin is used for the selection of transformants bearing the bleoR gene. Transformation was performed by homologous recombination using the protoplast transformation method. (B) The phenotype of the ΔhdaA and WT strains grown on PDA plates (25 °C for 5 days).

Figure 3

Secondary metabolite profiles of ΔhdaA and WT strains. (A) HPLC analysis of secondary metabolite profiles. (B) Relative amounts of four main products 1–4 in ΔhdaA compared with the WT. Mean values with asterisks are significant. (C) The chemical structure of the four main products detected in this study: 1, chrysogine; 2, meleagrin; 3, roquefortine C; 4, roquefortine F. The analysis for each strain was performed in triplicate. Mean values with asterisk are significant at p < 0.01.

Figure 4

Transcriptional analysis of the chrysogine biosynthetic gene cluster. (A) Organization of the chrysogine biosynthetic gene cluster (BGC). (B) Quantitative RT-PCR analysis of the chrysogine BGC. The analysis for each strain was performed in triplicate. Data are shown as fold change relative to the first trial of the WT. Mean values with asterisk are significant at p < 0.01.

Figure 5

Transcriptional analysis of the meleagrin/roquefortine biosynthetic gene cluster. (A) Organization of the meleagrin/roquefortine BGC. (B) Quantitative RT-PCR analysis of the meleagrin/roquefortine BGC. The analysis for each strain was performed in triplicate. Data are shown as fold change relative to the first trial of the WT. Mean values with asterisk are significant at p < 0.01.