Genomic characterization and identification of candidate genes for putative podophyllotoxin biosynthesis pathway in Penicillium herquei HGN12.1C

Abstract

Previous studies have reported the functional role, biochemical features and synthesis pathway of podophyllotoxin (PTOX) in plants. In this study, we employed combined morphological and molecular techniques to identify an endophytic fungus and extract PTOX derivatives. Based on the analysis of ITS sequences and the phylogenetic tree, the isolate was classified as Penicillium herquei HGN12.1C, with a sequence identity of 98.58%. Morphologically, the HGN12.1C strain exhibits white colonies, short-branched mycelia and densely packed hyphae. Using PacBio sequencing at an average read depth of 195×, we obtained a high-quality genome for the HGN12.1C strain, which is 34.9 Mb in size, containing eight chromosomes, one mitochondrial genome and a GC content of 46.5%. Genome analysis revealed 10 genes potentially involved in PTOX biosynthesis. These genes include VdtD, Pinoresinollariciresinol reductase (PLR), Secoisolariciresinol dehydrogenase (SDH), CYP719A23, CYP71BE54, O-methyltransferase 1 (OMT1), O-methyltransferase 3 (OMT3), 2-ODD, CYP71CU and CYP82D61. Notably, the VdtD gene in fungi shares functional similarities with the DIR gene found in plants. Additionally, we identified peltatin, a PTOX derivative, in the HGN12.1C extract. Docking analysis suggests a potential role for the 2-ODD enzyme in converting yatein to deoxypodophyllotoxin. These findings offer invaluable insights into the synthesis mechanism of PTOX in fungi, shedding light on the relationship between host plants and endophytes.

FIGURE 1

Bootstrap maximum‐likelihood phylogenetic trees of Penicillium herquei HGN12.C based on ITS region sequences in rRNA genes. Sequence alignment was performed using Clustal W in MEGA X. Bootstrap values obtained from 1000 replications of the dataset are displayed at the nodes.

FIGURE 2

Colony morphology of HGN12.1C strain on PDA medium after 5 days of culture: (A) Top; (B) Reverse; (C) The fungal mycelium of HGN12.1C at 40× objective; (D) The fungal spore of HGN12.1C at 40× objective).

FIGURE 3

Distribution of the top 15 gene ontology (GO) identified from the HGN12.1C strain genome according to Biological process (BP), Cellular component (CC), and Molecular function (MF).

FIGURE 4

Molecular docking of the g4336.t1 protein and substrate. (A) The model 3D structure of proteins shows the binding sites with active sites (red) and yatein (green). (B) The 2D interaction map of yatein with surrounding amino acids of the g4336.t1 protein. Ligand–protein interactions are marked by dashed lines.