The bHLH-zip transcription factor SREBP regulates triterpenoid and lipid metabolisms in the medicinal fungus Ganoderma lingzhi

Genome sequence analyses of G. lingzhi

We sequenced the genome of the G. lingzhi strain using a whole-genome shotgun sequencing strategy. As shown in Table 1, the sequences were assembled into 30 scaffolds with a total length of 49.15 Mb. The lengths of scaffolds ranged from 36,289 bp to 5,053,266 bp with an N50 scaffold size of 2.33 Mb, and the overall GC content is ~55.87%. By using K-mer analysis, these scaffolds covered 93.71% of the expected whole genome size (52.45 Mb) with a 0.023% read error rate, indicating the high quality of the genome sequence assembly (Supplementary Fig. 1). In total, 13,125 gene models were predicted, with an average mRNA and CDS length of 1,955.66 and 1,451.83 bp, respectively. On average, each predicted gene contained 6.04 exons, and in total, 79,238 exons were contained in all genes (Table 1).

Table 1 General characteristics of the G. lingzhi genome.

Nine public databases were used to annotate the function of predicted genes by the NCBI non-redundant, Pfam, NCBI clusters of orthologous groups of proteins, UniProt, Kyoto Encyclopedia of Genes and Genomes, Gene Ontology, Pathway, RefSeq, and InterProScan public databases. Overall, 12,802 genes were annotated to at least one function, accounting for 97.54% of all genes (Supplementary Table 1). The annotation results of nine databases were combined to facilitate subsequent research on gene search and exploration (details are shown in Supplementary Data 1).

The pathway of triterpenoid and ergosterol biosynthesis

Triterpenoids are one of the most important secondary metabolites with pharmacological activity. Ganoderic acid (GA), a type of triterpenoid, is an important medicinal component found in Ganoderma spp. To speculate on the GA biosynthetic pathway, the genes distributed in the mevalonate pathway (MVA) of the “terpenoid backbone biosynthesis (map00900)” pathway were evaluated, and 11 enzymes encoded by 13 genes existed in G. lingzhi, including two squalene monooxygenase genes and two farnesyl diphosphate synthase genes (Supplementary Table 2), which is not exactly consistent with previous reports of G. lucidum where the acetyl coenzyme A (CoA) acetyltransferase gene and farnesyl diphosphate synthase gene are each encoded by two genes22. We further summarized the potential triterpenoid biosynthesis pathway in G. lingzhi, as shown in Fig. 1. Compared with the well-studied upstream catalytic synthesis of lanosterol (MVA pathway), the steps following cyclization are largely unknown but most likely include a series of oxidation, reduction, and acylation reactions by the cytochrome P450 (CYP) superfamily. A total of 147 genes were annotated as CYPs by gene searching, as shown in Supplementary Data 2.

Fig. 1: Putative triterpenoid and ergosterol biosynthetic pathway in G. lingzhi.
figure 1

The representative triterpenoids of Ganoderma spp were marked in red, such as ganoderic acid A, D, and F. The dotted line with three arrows in ergosterol and GA biosynthetic pathway indicates the multiple enzymatic and speculative steps, respectively. Abbreviated word information is shown in Supplementary Table 2.

Lanosterol is also the common cyclic intermediate of ergosterol, which is one of the important components of the fungal cell membrane, and the various metabolic pathways of lanosterol are divergent27. We screened the G. lingzhi genes in the ergosterol biosynthesis pathway and found a complete pathway of ergosterol biosynthesis from lanosterol, which included 10 enzymes encoded by 17 genes (Supplementary Table 2). Enzyme-catalysed lanosterol to form zymosterol requires five enzymes (lanosterol 14-demethylase, delta14-sterol reductase, methyl sterol monooxygenase, sterol-4alpha-carboxylate 3-dehydrogenase, keto steroid reductase) to undergo eight steps. Then, zymosterol is further catalysed by five enzymes, sterol 24-C-methyltransferase, sterol isomerase, delta7-sterol 5-desaturase, sterol 22-desaturase, and delta 24-sterol reductase, to form ergosterol (Fig. 1, marked in blue; more detailed information can be found in Supplementary Table 2 and ko00100: steroid biosynthesis). In addition, we also analysed some other steroid biosynthetic enzymes starting with farnesyl diphosphate as a substrate, such as polycis-polyprenyl diphosphate synthase, farnesyltransferase type-1 subunit alpha and beta, endopeptidase, and farnesylcysteine lyase, in the G. lingzhi genome (Fig. 1, Supplementary Table 2).

Identification of a potential bHLH-zip transcription factor that regulates triterpene synthesis

At present, many upstream signalling molecules and functional proteins have been found to regulate GA biosynthesis in Ganoderma spp. However, the transcription factors that directly regulate the expression of triterpene biosynthesis genes are still unknown. The basic helix-loop-helix leucine zipper (bHLH-zip) transcription factor sterol regulatory element-binding protein (SREBP) is conserved in mammalian, worms, flies, and yeast, and functions in the regulation of sterol homoeostasis and lipid metabolism28. Overexpression studies indicate that SREBP positively regulates the expression of many sterol synthesis genes in the MVA pathway, such as 3-hydroxy-3-methylglutaryl CoA reductase (the rate-limiting enzyme of sterol biosynthesis), mevalonate kinase, squalene synthase, and fatty acid synthesis genes, such as fatty acid synthase and long-chain fatty acyl elongase, in mammals29,30. Because both sterol and triterpenoid synthesis occurs through the MVA pathway (Fig. 1), we speculated that SREBP may be a transcription factor directly regulating triterpene synthesis in G. lingzhi.

We used the SREBP of Trametes pubescens (GenBank accession number: OJT12715.1) to perform a homology BLAST against the G. lingzhi genome database. One gene (g2373) was identified as being a potential ortholog to SREBP. The SREBP gene of G. lingzhi had an ORF of 2532 bp located on scaffold 3 at positions 379,572–382,613 (Supplementary Fig. 2a). The deduced protein contains a bHLH DNA binding motif in its N-terminal domain (residues 197–294 aa, Supplementary Fig. 2b). We conducted a conservative analysis of the bHLH domain based on multiple sequence alignment and showed that bHLHs of G. lingzhi were highly conserved in comparison with other known bHLHs in several fungi, Trametes pubescens, Neurospora crassa and Stemphylium lycopersici, and less conserved in comparison with Mus musculus, Homo sapiens, and Drosophila willistoni (Supplementary Fig. 2b). The seven sequences contain 18 conserved residues, especially tyrosine residue (indicated in red) specific to the SREBP family of bHLH transcription factors31. In summary, the predicted SREBP protein from G. lingzhi has the characteristic features of SREBP proteins described in other organisms.

Utilizing DAP-Seq to identify direct target genes of SREBP in G. lingzhi

To elucidate the potential regulatory mechanism of SREBP on triterpenoid synthesis in G. lingzhi, we used DNA affinity purification sequencing (DAP-seq) for genome-wide identification of SREBP binding sites32. We identified a total of 2271 putative SREBP binding sites (details are shown in Supplementary Data 3). The binding sites were distributed mostly within promoter and transcription terminate site (TTS) regions, accounting for 50.37% (1144) and 26.42% (600), respectively, with very few sites located in intergenic regions, exons, and introns, accounting for 9.25%, 7.84%, and 6.12%, respectively (Fig. 2a). A stretch 2 kb up- and 500 bp downstream of the transcription start site was defined as promoter33. A total of 75.9% of putative SREBP binding sites were located within 2 kb of a known transcription start site (Fig. 2b). The sequences around the peaks were then analysed by MEME to search for enriched sequence motifs34. The best-fit core motif was 5′-GRVGRVGRVGR-3′ (E = 9.0 × 10−182), which was present in 2694 peaks with a p-value < 0.0001 (Fig. 2c), followed by 5′-GCAGAA-3′, 5′-GGCDAC-3′, 5′-GAGATGGGAGAR-3′, and 5′-AAAASAARAMAA-3′, which were the DNA motifs preferred by the G. lingzhi SREBPs (Supplementary Fig. 3, details are shown in Supplementary Data 4).

Fig. 2: Genome-wide identification of SREBP binding sites and motifs.
figure 2

a Relative binding-peak distribution across genomic regions. b Distance to the transcription start site (TSS) from SREBP binding sites. c The 2271 peak regions were analyzed for overrepresented motifs using MEME. The top-scoring motif is shown. The other preferred motifs are shown in Supplementary Fig. 3.

We further searched the 1144 potential promoter binding sites and identified 1077 potential SREBP target genes of G. lingzhi (additional information on the peak locations and the nearest gene list is provided in Supplementary Data 3). These target genes were analysed by KEGG, and clusters for sesquiterpenoid and triterpenoid biosynthesis (p = 0.012), terpenoid backbone biosynthesis (p = 0.19), glycerolipid metabolism (p = 0.58) and glycerophospholipid metabolism (p = 0.40) were enriched as expected (Fig. 3a; details are shown in Supplementary Data 5). Several genes of those pathways were found in a KEGG enrichment analyses pursued a deeper analyses given the interest. Specifically, three genes, g4989, g3847, and g7601, encode the same germacrene-A synthase involved in sesquiterpenoid and triterpenoid biosynthesis. Two genes, g3941 and g1941, encoding mevalonate kinase and 3-hydroxy-3-methylglutaryl CoA synthetase, respectively, are involved in terpenoid backbone biosynthesis. Six genes, g4280, g4309, g1208, g5041, g6347, and g805, encoding cardiolipin-specific phospholipase, lysophospholipid acyltransferase (phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid biosynthesis), ethanolamine-phosphotransferase, diacylglycerol kinase, lysophospholipase I, and triacylglycerol/diacylglycerol lipase, respectively, are involved in glycerophospholipid/glycerolipid metabolism (Fig. 3a and b). On the other hand, in the identified SREBP target genes, 10 target genes belonged to CYPs (g5000, g4875, g4164, g11088, g10251, g3908, g11110, g6752, g87, and g7787), where g3908, g10251 and g7787 were involved in ergosterol biosynthesis (Supplementary Table 3).

Fig. 3: Screening of SREBP target genes for terpenoid and lipid metabolism.
figure 3

a KEGG analysis of SREBP target genes. Clusters for terpenoid and lipid metabolism were highlighted by the red line. b Schematic diagram showing the genomic locations of SREBP target genes that involved in terpenoid and lipid metabolism, and corresponding matched DNA sequence, promoter regions, and fitted motif. The conserved SREBP binding sequence was highlighted in blue shadow. c SREBP can bind to the DNA sequences in the targets promoter by EMSA. Line 1: the labelled DNA probe control. The labelled DNA probe was preincubated with 0.6 μg SREBP-bHLH protein (Line 2), and an unlabelled competitor DNA probe (200× labelled DNA probe) was added (Line 3). Arrows indicate positions of protein-bound (red) and free (black) DNA bands.

Further analysis of the SREBP-matched DNA sequence of the above target genes involved in terpenoid biosynthesis and lipid metabolism showed that only one and three SREBPs matched the DNA sequences of target genes g7601 and g6347/1208/g4309 fitted to the motifs 5′-GGCDAC-3′ and 5′-GAGATGGGAGAR-3′, respectively. The matched DNA sequences of the other target genes fitted to the motif 5′-GRVGRVGRVGR-3′. In addition, most SREBP-matched DNA sequences were located before the transcription start site, except for g5041, g4039 and g805 (Fig. 3b). Similar SREBP-matched DNA sequence characteristics also appeared in the 10 target genes belonging to the CYPs (Supplementary Table 3).

In order to verify the identified SREBP sites, we further purified the putative DNA binding domains (bHLHs) of the G. lingzhi SREBP proteins and DNA fragments corresponding to the promoter regions of the 10 target genes shown in Fig. 3b to carry out electrophoretic mobility gel shift assays (EMSAs). The purified bHLH protein could shift all 10 DNA fragments (Fig. 3c, line 2). The specificity of the SREBP–DNA complex was confirmed by adding an excess of unlabelled DNA. As expected, a weakened shift was detected when adding the unlabelled DNA (Fig. 3c, line 3).

SREBP overexpression increased triterpenoid, ergosterol and lipid biosynthesis

To further clarify the role of SREBP in triterpenoid and lipid metabolism, we characterized the metabolic changes of triterpenoids and lipids following overexpression of SREBP by RNA-seq, secondary metabolomics and lipid metabolomics analysis. We constructed a OE::SREBP vector named GLgpd-SREBP that carries the hygromycin B (Hyg) resistance gene as a selectable marker (Supplementary Fig. 4a–c). The transcription levels of SREBP were significantly increased in the transformants determined by RT-qPCR (Supplementary Fig. 4d). A Western blot analysis also showed a significant increase in the protein levels in the OE::SREBP strains. In particular, the nuclear version of SREBP in the OE::SREBP strains was significantly increased ~1.63–2.11-fold of the levels found in the WT strain (p < 0.01, Supplementary Fig. 4e, f).

Six RNA libraries from WT and OE::SREBP strains of G. lingzhi were sequenced using Illumina paired-end sequencing technology. The average number of clean reads for each sample was 23.20 million (Supplementary Fig. 5a). The mapping ratio of each sample against the reference genome ranged from 91.59% to 92.84% (Supplementary Fig. 5a, b). The mapped reads were assembled and compared with original annotations of the genome. The transcript regions without annotation obtained by the above processes are defined as novel transcripts. Excluding short transcripts (coding peptides with <50 amino acids) or those containing only one exon, 3996 novel transcripts were discovered in this project (detailed information of the novel transcripts shown in Supplementary Data 6, and has been deposited at NCBI: PRJNA738334, SRR17081370).

A systematic analysis of expression profiles through RNA-seq identified 4601 differentially expressed genes (DEGs, fold change ≥ 2 and false discovery rate < 0.01), including 2152 upregulated and 2449 downregulated genes, in the OE::SREBP strain compared to the WT strain (Fig. 4a, Supplementary Data 7). Venn analysis showed that 177 upregulated DEGs belonged to SREBP target genes by integrating RNA-seq and DAP-seq data (Fig. 4b). The 2152 upregulated DEGs were enriched for functional categories involved in metabolic activities by KEGG. In particular, steroid biosynthesis (p-value = 6.18E−5), sesquiterpenoid and triterpenoid biosynthesis (p-value = 8.70E−3), glycerophospholipid metabolism (p-value = 7.35E−1), glycerolipid metabolism (p-value = 8.04E−1), fatty acid biosynthesis (p-value = 2.78E−1), and sphingolipid metabolism (p-value = 4.53E−1) were enriched as uppathway in OE::SREBP strain compared to WT strain (Fig. 4c and Supplementary Data 8). Specifically, 22 upregulated DEGs were involved in steroid and triterpenoid biosynthesis, in which 11 DEGs involved in the MVA pathway, such as squalene synthase (g1847), lanosterol synthase (g1881), and SREBP targets 3-hydroxy-3-methylglutaryl CoA synthetase (g1941) and mevalonate kinase (g3941), and the other eleven ERGs, such as delta 24-sterol reductase (ERG4, g1634) and sterol 22-desaturase (ERG5, g288), and SREBP targets sterol isomerase (ERG2, g7787) and lanosterol 14-demethylase (ERG11, g3908). In addition, twenty-two upregulated DEGs were involved in glycerophospholipid/glycerolipid metabolism and contained three SREBP targets: ethanolamine-phosphotransferase (g1208), lysophospholipid acyltransferase (g4309), and cardiolipin-specific phospholipase (g4280). We also found that the SREBP targets ceramide synthase (g4387) and CYPs (g4875 and g4164) were significantly upregulated in the OE::SREBP strain compared to the WT strain (Fig. 4d).

Fig. 4: Transcriptional profiling revealed that the transcription factor SREBP regulates triterpenoid, ergosterol and lipid biosynthesis genes expression in G. lingzhi.
figure 4

a Volcano plot on differential expression genes between WT and OE::SREBP strains. Significant DEGs were defined with the adjusted fold change (FC) ≥ 2 and false discovery rate (FDR) < 0.01. Green-, red- and black-dots are down-regulated, up- regulated and without significant difference, respectively. b Venn analysis the SREBP target gene expression levels by integrating RNA-seq and DAP-seq data. c KEGG analysis of the up-regulation DEGs in OE::SREBP strain. d Differential expression of triterpenoid, sterol and lipid biosynthesis genes ranked by degree of log2 fold change from a and b. The SREBP direct target genes are shown in red line. The abbreviation information is shown in Supplementary Table 2. e qRT-PCR analyses of ten selected SREBP target genes in WT and OE::SREBP strains. 1 μM and 10 μM fatostatin (abbreviated as Fato, CAS 298197-04-3, Simga) were added at shaking for 5 days and then maintained until the 7th days in liquid cultures of OE::SREBP strains. CK indicated the OE::SREBP strains not treated with fatostatin. The expression level of each gene from the WT strains was arbitrarily designated a value of 1. The values are the means ± SD of three independent experiments. Different letters indicate significant differences between the lines (n = 3 independent experiments, P < 0.05, according to Duncan’s multiple range test).

To validate our transcriptomics data, 10 up-DEGs belonging to the SREBP targets mentioned above were selected to determine the mRNA expression levels by qRT–PCR. The results show that the gene expression levels of 10 selected SREBP targets were significantly upregulated, ranging from 3.9- to 22.6-fold in the OE::SREBP strain (Fig. 4e), which is consistent with the RNA-seq results. Fatostatin, a specific inhibitor of SREBP, can impair the activation of SREBP35,36. To further validate the up-regulation role of SREBP in the 10 SREBP targets, fatostatin was added to the fermentation medium of OE::SREBP strain for detecting mRNA expression levels. As illustrated in Fig. 4e, an significantly decrease in gene expression levels of ten selected SREBP targets were observed when the exogenous fatostatin was added in OE::SREBP strain. A 10 μg/mL fatostatin treatment in OE::SREBP strain led to not significantly different in mRNA expression levels than that of WT strain. These results indicate that SREBP positively regulates the expression of triterpenoid, ergosterol and lipid biosynthesis genes.

To further elucidate the role of SREBP in regulating triterpenoid, ergosterol and lipid biosynthesis, we performed targeted secondary metabolic profiling and lipid metabolic profiling between the WT and OE::SREBP strains. We identified 35 differential secondary metabolites including 29 upregulated, and 251 differential lipid metabolites, including 244 upregulated (VIP ≥ 1, and fold change ≥1.5 or ≤0.67), in the OE::SREBP strain compared to the WT strain (Supplementary Data 9). In particular, we observed that the contents of mevalonic acid, lanosterol, and 13 different GAs were significantly upregulated in the OE::SREBP strain (Fig. 5a). Significant accumulation of 25 diacylglycerol (DG) species and 28 phosphatidylcholine (PC) species was observed in the OE::SREBP strain (Fig. 5b and c).

Fig. 5: The roles of SREBP in metabolic abundance of GAs, sterols, and lipids.
figure 5

a Heatmap showing the changes in the contents of detected GAs and sterols in WT and OE::SREBP strains. Up and Insig were indicated the significantly upregulated and no significantly differential metabolites, respectively, in the OE::SREBP strain compared to WT strain. Representative glycerolipids and glycerophospholipids, DG and PC, are shown in b and c, respectively. The significant differential metabolites had variable importance in projection (VIP) ≥ 1 and fold change ≥1.5 or ≤0.67. d The contents of cellular total GA, ergosterol, lanosterol (abbreviated as lano), GA-C2 and CYP in WT and OE::SREBP strains. 1 μM and 10μ M fatostatin were added at shaking for 5 days and then maintained until 7th days in liquid cultures of OE::SREBP strains. The values are the means ± SD of three independent experiments. Different letters indicate significant differences between the lines (n = 3 independent experiments, P < 0.05, according to Duncan’s multiple range test).

Furthermore, the cellular total GA, ergosterol, lanosterol, and GA-C2 contents were determined in WT and OE::SREBP strains. As shown in Fig. 5d, the total GA, ergosterol, lanosterol, and GA-C2 contents of the OE::SREBP strain were significantly increased by ~1.87-, 1.84-, 1.89-, and 2.75- fold, respectively, compared to the WT, which is consistent with the metabonomics data results. In addition, the effects of overexpression SREBP on GA, ergosterol, lanosterol, and GA-C2 contents were recovered when OE::SREBP strain were treated with the SREBP inhibitor fatostatin. A 10 μg/mL fatostatin treatment in OE::SREBP strain led to not significantly different in total GA, ergosterol, lanosterol, and GA-C2 contents than that of WT strain (Fig. 5d). These above metabolic results is consistent with the effects of overexpression SREBP on gene expression. Moreover, we analyzed cellular CYP content to confirm the role of SREBP on GA biosynthesis. Overexpression SREBP led to a significantly increase of ~1.63-fold in the CYP content compared with the WT strain. The effects of overexpression SREBP on CYP content were also recovered when OE::SREBP strain were treated with the 10 μg/mL fatostatin (Fig. 5d).

Taken together, these results showed that overexpression of SREBP led to significant increases in GA (triterpenoid), ergosterol and lipid metabolism by facilitating transcription of target genes related to the GA, ergosterol and lipid biosynthesis.

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