Genomic Features of Taiwanofungus gaoligongensis and the Transcriptional Regulation of Secondary Metabolite Biosynthesis

Abstract

Fungal secondary metabolites (SMs) have broad applications in biomedicine, biocontrol, and the food industry. In this study, whole-genome sequencing and annotation of Taiwanofungus gaoligongensis were conducted, followed by comparative genomic analysis with 11 other species of Polyporales to examine genomic variations and secondary metabolite biosynthesis pathways. Additionally, transcriptome data were used to analyze the differential expression of polyketide synthase (PKS), terpene synthase (TPS) genes, and transcription factors (TFs) under different culture conditions. The results show that T. gaoligongensis differs from other fungal species in genome size (34.58 Mb) and GC content (50.72%). The antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH) analysis reveals significant variation in the number of SM biosynthetic gene clusters (SMBGCs) across the 12 species (12-29), with T. gaoligongensis containing 25 SMBGCs: 4 PKS, 6 non-ribosomal peptide synthetase (NRPS), and 15 TPS clusters. The TgPKS1 gene is hypothesized to be involved in the biosynthesis of orsellinic acid or its derivatives, while TgPKS2 might catalyze the synthesis of 6-methylsalicylic acid (6MSA) and its derivatives. The TgTRI5 genes are suggested to synthesize tetracyclic sesquiterpene type B trichothecene compounds, while TgPentS may be involved in the synthesis of δ-cadinol, β-copaene, and α-murolene analogs or derivatives. Comparative genomic analysis shows that the genome size of T. gaoligongensis is similar to that of T. camphoratus, with comparable SMs. Both species share four types of PKS domains and five distinct types of TPS. Additionally, T. gaoligongensis exhibits a high degree of similarity to Laetiporus sulphureus, despite belonging to a different genus within the same family. Transcriptome analysis reveals significant variation in the expression levels of PKS and TPS genes across different cultivation conditions. The TgPKS1 and TgPKS4 genes, along with nine TgTFs, are significantly upregulated under three solid culture conditions. In contrast, under three different liquid culture conditions, the TgPKS3, TgTRI5-1, and TgTRI5-2 genes, along with twelve TgTFs, exhibit higher activity. Co-expression network analysis and TgTFs binding site prediction in the promoter regions of TgPKS and TgTPS genes suggest that TgMYB9 and TgFTD4 regulate TgPKS4 expression. TgHOX1, TgHSF2, TgHSF3, and TgZnF4 likely modulate TgPKS3 transcriptional activity. TgTRI5-1 and TgTRI5-5 expression is likely regulated by TgbZIP2 and TgZnF15, respectively. This study provides new insights into the regulatory mechanisms of SMs in T. gaoligongensis and offers potential strategies for enhancing the biosynthesis of target compounds through artificial intervention.

Keywords: T. gaoligongensis; biosynthesis gene cluster; secondary metabolite; transcriptional regulation; transcriptome sequencing; whole-genome sequence.

Figure 1

Functional annotation of T. gaoligongensis genes encoding the proteins: (a) eggNOG analysis; (b) KEGG analysis; (c) GO analysis.

Figure 1

Functional annotation of T. gaoligongensis genes encoding the proteins: (a) eggNOG analysis; (b) KEGG analysis; (c) GO analysis.

Figure 2

Distribution map of mutation types in the pathogen PHI phenotype of T. gaoligongensis.

Figure 3

CAZy functional classification chart of T. gaoligongensis.

Figure 4

TCDB Functional Classification Chart of T. gaoligongensis.

Figure 5

Comparison of biosynthesis of putative orsellinic acid biosynthetic gene clusters. The number after the region and the number before the decimal point represent the scaffold, and the number after the decimal point represents the gene cluster.

Figure 6

Comparison of biosynthesis of putative 6MSA biosynthetic gene clusters. The number after the region and the number before the decimal point represent the scaffold, and the number after the decimal point represents the gene cluster.

Figure 7

Comparative Analysis of Genes Surrounding TgPKS3 in T. gaoligongensis and Related Species. The number after the region and the number before the decimal point represent the scaffold, and the number after the decimal point represents the gene cluster.

Figure 8

Comparative Analysis of Genes Surrounding TgPKS4 and Related Species. The number after the region and the number before the decimal point represent the scaffold, and the number after the decimal point represents the gene cluster.

Figure 9

Scaffold containing SM biosynthesis gene cluster used for synteny analysis. From top to bottom: L. sulphureus, T. camphoratus2, T. gaoligongensis, T. camphoratus1.

Figure 10

Genomic inventory for terpenoid biosynthesis in T. gaoligongensis.

Figure 11

Phylogenetic Tree of TPS Proteins from 12 Fungal Strains. The TgTPS types are indicated in the figure: TC1 (T. camphoratus1), TC2 (T. camphoratus2), DQ (D. quercina), WC (W. cocos), LS (L. sulphureus), FR (F. radiculosa), FP (F. palustris), FS (F. schrenkii), FB (F. betulina), PP (P. placenta), NS (N. serialis).

Figure 12

Comparative Analysis of Genes Flanking Various Types of TPS in L. sulphureus and Fungi of the Taiwanofungus Genus.

Figure 13

Structural Characterization of the 10 TF Families in T. gaoligongensis. From left to right: Phylogenetic Tree of Proteins, Conserved Motif Analysis and Conserved Domain Analysis.

Figure 14

Interactive Heatmap of Gene Expression for (a) TgPKS, (b) TgTPS, and (c,d) TgTFs Under Different Cultivation Conditions. T: pea powder (5 g/L), KH₂PO₄ (1 g/L), MgSO₄ (0.5 g/L), yeast powder (5 g/L), and vita-min B1 (0.1 g/L).,NFT: T+Triton X-100 (100 μL) + C. kanehirae sawdust (5 g/L), YFT: T+ Triton X-100 (100 μL) + C. burmannii sawdust (5 g/L), YY: 15 mL MM medium +4 g Populus alba sawdust, YM: 15 mL MM medium +4 g Zea mays flour, YR: 15 mL MM medium +4 g Coix Coicis Semenurr.

Figure 15

Relative Expression of Differentially Expressed Genes by qRT–PCR. T: pea powder (5 g/L), KH₂PO₄ (1 g/L), MgSO₄ (0.5 g/L), yeast powder (5 g/L), and vita-min B1 (0.1 g/L). NFT: T+Triton X-100 (100 μL) + C. kanehirae sawdust (5 g/L), YFT: T+ Triton X-100 (100 μL) + C. burmannii sawdust (5 g/L), YY: 15 mL MM medium +4 g Populus alba sawdust, YM: 15 mL MM medium +4 g Zea mays flour, YR: 15 mL MM medium +4 g Coix Coicis Semenurr.

Figure 16

Interactive Heatmap of Gene Expression for (a) TgPKS, (b) TgTPS, and Co-expressed TgTFs Under Varying Cultivation Conditions. T: pea powder (5 g/L), KH₂PO₄ (1 g/L), MgSO₄ (0.5 g/L), yeast powder (5 g/L), and vitamin B1 (0.1 g/L). NFT: T+Triton X-100 (100 μL) + C. kanehirae sawdust (5 g/L), YFT: T+ Triton X-100 (100 μL) + C. burmannii sawdust (5 g/L), YY: 15 mL MM medium +4 g Populus alba sawdust, YM: 15 mL MM medium +4 g Zea mays flour, YR: 15 mL MM medium +4 g Coix Coicis Semenurr.

Figure 17

Derivatives of orsellinic acid in fungi.