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. 2023 Sep 25;51(5):360–371. doi: 10.1080/12298093.2023.2257999

Transcriptome Analysis Reveals the Putative Polyketide Synthase Gene Involved in Hispidin Biosynthesis in Sanghuangporus sanghuang

Jiansheng Wei a,b,†, Liangyan Liu c,†, Xiaolong Yuan b, Dong Wang b, Xinyue Wang b,d, Wei Bi b, Yan Yang e, Yi Wang b,✉
PMCID: PMC10621269  PMID: 37929012

Abstract

Hispidin is an important styrylpyrone produced by Sanghuangporus sanghuang. To analyze hispidin biosynthesis in S. sanghuang, the transcriptomes of hispidin-producing and non-producing S. sanghuang were determined by Illumina sequencing. Five PKSs were identified using genome annotation. Comparative analysis with the reference transcriptome showed that two PKSs (ShPKS3 and ShPKS4) had low expression levels in four types of media. The gene expression pattern of only ShPKS1 was consistent with the yield variation of hispidin. The combined analyses of gene expression with qPCR and hispidin detection by liquid chromatography-mass spectrometry coupled with ion-trap and time-of-flight technologies (LCMS-IT-TOF) showed that ShPKS1 was involved in hispidin biosynthesis in S. sanghuang. ShPKS1 is a partially reducing PKS gene with extra AMP and ACP domains before the KS domain. The domain architecture of ShPKS1 was AMP-ACP-KS-AT-DH-KR-ACP-ACP. Phylogenetic analysis shows that ShPKS1 and other PKS genes from Hymenochaetaceae form a unique monophyletic clade closely related to the clade containing Agaricales hispidin synthase. Taken together, our data indicate that ShPKS1 is a novel PKS of S. sanghuang involved in hispidin biosynthesis.

Keywords: Sanghuangporus sanghuang, transcriptome, hispidin biosynthesis, polyketide synthase, qRT-PCR

1. Introduction

Hispidin is a type of styrylpyrone, and it has antioxidant [1], antitumor [2,3], anti-inflammatory [4,5], antiviral [6], antiallergic [7], neuroprotective [8,9], antiangiogenic, and hypolipidemic properties [10,11]. Hispidin is a yellow pigment and was first isolated from Inonotus hispidus fungi [12]. Subsequently, hispidin has been identified in various fungal species of both the Hymenochaetaceae family, including species in the genera Inonotus (I. obliquus, I. radiatus, I. xeranticus), Hymenochaete, Phellinus [13], and Sanghuangporus [14], and the Hymenogastraceae family, including the genus Gymnopilus [15]. Hispidin and hispidin derivatives are also found in other fungal families such as the families Phaeolaceae [16], Omphalotaceae (Neonothopanus nambi) [17], Strophariaceae (Hypholoma sublateritium), and Cortinariaceae (Cortinarius glaucopus) [11]. Furthermore, hispidin derivatives were also identified in some plants, including Equisetum arvense [18], Pistacia atlantica [19], Leishmania amazonensis [20], and Goniothalamus umbrosus [21].

A previous tracer study showed that styrylpyrone was derived from phenylalanine via cinnamic acid, p-coumaric acid, and caffeoyl coenzyme-A and that the pyrone ring is derived from acetate in fungi [22]. Polyketide synthase (PKS) is the key enzyme involved in styrylpyrone biosynthesis, which condenses one cinnamic acid or its derivatives with two malonyl-CoA molecules. PKSs usually are classified into three types according to their architecture [23]. Additionally, most fungal PKSs are type I PKSs with basic domains consisting of ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and with additional catalytic domains, such as ketoreductase (KR), dehydratase (DH), enoylreductase (ER), methyltransferase (MeT), and thioesterase (TE). Fungal type I PKSs are also classified into aromatic or non-reducing PKSs, partially reducing PKSs, and highly reducing PKSs according to their domain architecture [24]. Several partially reducing PKSs were confirmed to be related to hispidin biosynthesis in bioluminescent and non-bioluminescent Agaricales [11]. In plants, a chalcone synthase (CHS) corresponding to a type III PKS has also been reported to be related to styrylpyrone biosynthesis in Piper nigrum [25]. Wu et al. [26] reported that a type III PKS (PmSPS1) from Piper methysticum catalyzed different substrates, such as hydrocinnamic acid, cinnamic acid, p-coumaric acid, and p-hydrocoumaric acid, into different styrylpyrones. However, it is still unclear that PKSs are involved in hispidin biosynthesis in Hymenochaetaceae family fungi.

Sanghuang is well-known medical fungus in China, Japan, and Korea. Sanghuang was once incorrectly called Phellinus linteus, but recently, after detailed morphological and molecular phylogenetic studies, Sanghuang has been given the correct binomial name Sanghuangporus sanghuang [27]. Sanghuang possesses antitumor [28], antioxidant [29], anti-inflammatory [30], hypoglycemic, hypolipidemic [31], and immunomodulatory activities [32]. The bioactive compounds of Sanghuang mainly are comprised of triterpenoids [33], polysaccharides [34], and flavonoids [35]. Hispidin is an important bioactive flavonoid of Sanghuang [14]. Although more than three genomes of Sanghuang have been assembled and the putative pathway of flavonoid biosynthesis has also been deduced, the specific PKS involved in hispidin biosynthesis in Sanghuang is still unclear.

In this study, the transcriptome data from S. sanghuang producing and non-producing hispidin were obtained by RNA-Seq. The putative PKS involved in hispidin production was identified by bioinformatics analysis, and then it was confirmed by combined analyses utilizing qRT-PCR and liquid chromatography mass spectrometry (LCMS) coupled with ion-trap and time-of-flight technologies (LCMS-IT-TOF).

2. Materials and methods

2.1. Fungal culture and growth conditions

The S. sanghuang strain SH1 was obtained from Yan Yang from the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences. Genotypic identification of the S. sanghuang SH1 strain was authenticated by the fungal ITS sequences according to a previous study [36].

Sanghuangporus sanghuang SH1 was inoculated into malt-yeast (MY) solid medium (Difco, Lawrence, KS, USA). Then, a mycelia agar block (1 cm3) was transferred to 100 mL of new media. The carbon source of the media consisted of MY basal medium containing 2% (W/V) lactose (RT), mannitol (GLC), maltose (MYT), inositol (JC), glucose (PTT), and sorbitol (SLC), while the nitrogen sources of the media were prepared by the addition of 0.4% (W/V) of beef peptone (NRF), soy peptone (DDD), yeast extract (JMF), and malt extract (MYF) in the basal medium (maltose 1.8 g/L and glucose 6 g/L). The mycelia were harvested after cultivation at 28 °C for 2 weeks on a rotary shaker at 150 rpm.

2.2. LCMS analysis of hispidin

From the different culture media, S. sanghuang was collected by filtration and extracted using 100 mL of ethyl acetate. The extract was then evaporated with a Buchi Rotavapor (Buchi, Flawil, Switzerland). LCMS analyses were performed on the LCMS-IT-TOF system (Shimadzu, Kyoto, Japan) with an Agilent Eclipse Plus C18 column (100 × 2.1 mm i.d., 1.8 μm; Agilent Technologies, Santa Clara, CA, USA) at 30 °C. The injection volume was 2 μL for each LCMS analysis. MS experiments were conducted using an automatic pattern in positive ion mode. The Shimadzu Composition Formula Predictor was used to infer the molecular formula. For all S. sanghuang samples, hispidin production was confirmed through LCMS-IT-TOF analyses.

2.3. RNA extraction and sequencing library construction

The LCMS analysis showed that S. sanghuang produced hispidin when it was cultured in MY medium containing 2% (W/V) lactose (RT) and sorbitol (SLC), and S. sanghuang did not produce hispidin when it was cultured in basal medium containing 0.4% (W/V) soy peptone (DDD) and sorbitol (SLC). Therefore, the mycelial samples of S. sanghuang cultured in different mediums (RT, SLC, DDD, and JMF) were collected, and samples from three replicates of each treatment were subjected to Illumina sequencing. Firstly, the mycelial samples of three replicates of each treatment were collected together for RNA extraction. RNA was extracted with the Transzol Up Plus RNA Kit (Transgen Biotech, Beijing, China). The sequencing library was constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), and the library was analyzed by the Agilent 2100 Bioanalyzer (Agilent Technologies). The constructed library was sequenced using the Illumina platform (Illumina, San Diego, CA, USA). The sequencing depth of the independent samples was 6G. Raw reads (FASTQ format) were quality filtered using FASTX-Toolkit61, and sequencing adaptors were removed with Trimmomatic. High-quality reads were mapped onto the reference genome of S. sanghuang (GCA_009806525.1) using HISAT2 [37].

2.4. Screening of differentially expressed genes

HTSeq statistics were used to compare the read count values of each gene with the original expression of the gene and to normalize gene expression using FPKM values (FPKM > 1). Analysis of differentially expressed genes (DEGs) was conducted using DESeq, with identification of DEGs according to the following criteria: |log2(expression fold change) | > 1, p < 0.05.

2.5. Functional analysis of DEGs

All DEGs were mapped to each term of the GO database (http://www.geneontology.org/), and the number of genes associated with each GO term was determined. Using the list of the names and numbers for each GO term, the hypergeometric test was used to determine significantly enriched GO terms among DEGs compared to the genomic background. All differentially expressed genes were also mapped onto the KEGG pathway, with Q-value ≤ 0.05 considered to be the threshold for significant enrichment by DEGs. Heatmaps were drawn using TBtools after normalizing the expression data.

2.6. Gene cloning and domain and phylogenetic analyses

Based on the transcriptome analysis, the genes with gene expression values of FPKM > 10 were ShPKS1, ShPKS2, and ShPKS5. Specific primers for gene cloning were designed based on the transcriptome sequences of these three PKS genes (Table S1). PCR was performed with super-fidelity DNA polymerase using cDNA as a template. The PCR product was cloned into the pUC19 vector with the pEASY-Uni Seamless Cloning and Assembly Kit (TransGen). Domain analysis was conducted using NCBI’s conserved domain database. Three PKSs from S. sanghuang were aligned with 23 fungal PKS sequences retrieved from GenBank (Table S2). The alignment was analyzed using Muscle as implemented in the MEGA 7 program. To obtain a confidence value for the aligned sequence dataset, bootstrap analysis with 1000 replicates was performed using MEGA 7. A phylogenetic tree was constructed using the maximum likelihood method.

2.7. Real time quantitative fluorescence PCR

The expression of three PKS genes in the four different culture media was detected by q-PCR with special primers (Table S1). RNA from the different samples was extracted with the EasyPure® Plant RNA Kit (TransGen). Then, cDNA synthesis was performed with a reverse transcriptase kit (TaKaRa Super RT Kit; TaKaRa, Beijing, China). The gene expression was detected by fluorescence quantitative PCR with a PCR thermal cycler (ABI 7300; Applied Biological System, Foster City, CA, USA). The tubulin gene was used as an internal control gene. At least two independent biological replicates and three technical replicates for each biological replicate were analyzed by q-PCR for each sample to ensure reproducibility and reliability.

3. Results

3.1. Effects of different media on hispidin yield of S. sanghuang

The mycelia of S. sanghuang were inoculated into different media and cultivated with shaking for 2 weeks. Then, each culture medium was harvested and extracted with ethyl acetate. The crude extracts were detected by LCMS-IT-TOF. The detection of hispidin was confirmed by comparison to a reference sample (Sigma-Aldrich, St. Louis, MO, USA) with the same retention time (tR = 6.0 min) and mass spectra (positive, m/z 247.0593, [M + H]+, −2.5 mD; Figure S1) and UV spectrum (Figure S2). The detection results showed that the extracts of RT, SLC, and GLC cultures contained hispidin, while hispidin was not detected in MYT, JC, PTT, NRF, DDD, JMF, and MYF cultures (Figure 1).

Figure 1.

Figure 1.

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Detection of hispidin in extracts of different Sanghuang cultures. SLC, RT, GLC, MYF, DDD, JMF, NRF, MYT, JC, and PTT indicate different extracts from each different medium, respectively.

3.2. RNA sequencing and read mapping onto S. sanghuang reference genome

Libraries constructed from S. sanghuang mycelia in media (RT, SLC, DDD, and JMF) were sequenced using the Illumina platform. From each of the four samples, we obtained at least 45,288,766 bp, and after filtering we obtained at least 41,019,250 bp. The total sequence similarity with the reference genome was higher than 94% and for Q20 and Q30 sequences, the similarity was >97 and >94%, respectively (Table 1). The raw data were deposited in NCBI with accession number SRX20095548.

Table 1.

Transcriptome sequencing data statistics.

Sample Raw reads Clean reads Total mapped Q20 (%) Q30 (%)
SLC 45288766 41882100 39529649 (95.19%) 97.85 94.03
RT 48614202 43937560 40898845 (95.76%) 97.96 94.48
JMF 45501220 41019250 46162738 (94.90%) 97.83 94.38
DDD 53615258 49314890 37522071 (95.97%) 98.25 95.01
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In total, 12,307 protein-coding genes were predicted according to the reference genome. The predicted genes were annotated with known databases, and 6632 genes were annotated in the GO database, 3622 in the KEGG database, 8957 in the eggNOG database, and 7105 in the Swiss-Prot database. The greatest number of genes was annotated in the NR database, with 12,016 (Figure 2).

Figure 2.

Figure 2.

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Gene function annotation.

3.3. Differentially expressed gene analysis

DEG analysis using DESeq identified a total of 2,077 DEGs (Figure 3), of which 308 were identified in the JMF_vs_DDD comparison (95 upregulated, 213 downregulated), 472 in the RT_vs_DDD comparison (226 upregulated, 246 downregulated), 342 in the RT_vs_JMF comparison (249 upregulated, 93 downregulated), 386 in the SLC_vs_DDD comparison (143 upregulated, 243 downregulated), 238 in the SLC_vs_JMF comparison (133 upregulated, 105 downregulated), and 331 in the SLC_vs_RT comparison (110 upregulated, 221 downregulated) (Figure 3, Table S3).

Figure 3.

Figure 3.

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Statistical summary of the differentially expressed gene analysis.

3.4. GO functional enrichment analysis of DEGs

To explore the functions of DEGs, we performed GO enrichment analysis on DEGs. The DEGs in the RT_vs_JMF comparison were classified into cellular component, molecular function, and biological process categories (Figure 4). The most significantly enriched category in both RT_vs_JMF and SLC_vs_JMF comparisons was oxidoreductase activity in the molecular function domain (Figure 4, Figure S4). The second most significant enrichment of the RT_vs_JMF and SLC_vs_JMF comparisons was iron ion binding and cofactor binding, respectively. The most significantly enriched term in the RT_vs_DDD and JMF_vs_DDD comparisons was transmembrane transport in the biological process domain (Figures S3 and S6). The most enriched term in the SLC_vs_DDD comparison was protein unfolding in the biological process domain (Figure S5). The most enriched term in the SLC_vs_RT category was the integral component of the membrane and intrinsic component of the membrane in the cellular component domain (Figure S7).

Figure 4.

Figure 4.

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Gene ontology (GO) enrichment of the differentially expressed genes in the RT_vs_JMF comparison. The x-axis represents the GO terms, and the ordinate is − log10 (p-value) of the enriched GO terms. CC, cellular component; MF, molecular function; BP, biological process.

3.5. KEGG enrichment analysis of DEGs

To better understand the biological functions of DEGs, their enrichment in KEGG database pathways was analyzed. Among the DEGs in the RT_vs_JMF comparison, they were enriched in five categories including metabolism, genetic information processing, cellular processes, environmental information processing, and human diseases. Most DEGs in the RT_vs_JMF comparison were enriched in metabolism, and their most significant enrichment was observed for pentose and glucuronate interconversions (Figure 5). Indeed, the main DEGs (from JMF_vs_DDD, RT_vs_DDD, SLC_vs_DDD, SLC_vs_JMF, and SLC_vs_RT comparisons) were annotated in the metabolism category (Figures S8–12). The most significant enrichment of the RT_vs_DDD comparison was pyruvate metabolism. The most and second most significant enrichments of the SLC_vs_DDD comparison were tyrosine metabolism and pyruvate metabolism, respectively. Additionally, the most significant enrichment of the SLC_vs_JMF comparison was ascorbate and aldarate metabolism. The most significant enrichment of the SLC_vs_RT comparison and JMF_vs_DDD was histidine metabolism and glycerolipid metabolism, respectively.

Figure 5.

Figure 5.

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Kyoto encyclopedia of genes and genomes (KEGG) enrichment of differentially expressed genes in the RT_vs_JMF comparison. The x-axis represents the KEGG pathway, and the ordinate is the − log10 (p-value) of the enriched KEGG terms.

3.6. Transcriptome profiling of five PKS genes in different media

According to the genome annotation, there were five PKS genes in S. sanghuang. Transcriptome analysis showed that the genes ShPKS3 and ShPKS4 had a low expression level in all four medium cultures of S. sanghuang. The gene expression of ShPKS5 was not significantly different among the four media. The expression of ShPKS2 in DDD medium was higher than in the other three media (JMF, RT, and SLC). Additionally, the expression of ShPKS1 cultured in both RT and SLC media was higher than in both DDD and JMF media. Only the expression pattern of ShPKS1 was positively correlated with the yield of hispidin across the four media (Figure 6).

Figure 6.

Figure 6.

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Heat map of five differentially expressed PKS genes of Sanghuangporous sanghuang in four media. The size and color indicate the level of gene expression. Large spot size and red color represent high expression, while small spot size and blue color represent low expression.

3.7. Gene cloning and domain architecture analysis

To confirm the transcriptome analysis results, the full cDNAs of ShPKS1, ShPKS2, and ShPKS5 were obtained by PCR with cDNA (Figure S13). The full cDNAs of ShPKS3 and ShPKS4 were not amplified by PCR (result not shown). Then, the obtained PKS was cloned into a vector. Three PKS genes were then confirmed by sequencing. The ORF of ShPKS5 was a 6780 bp gene encoding a 2259 amino acid protein (accession number, OQ865374). Domain analysis showed that ShPKS5 encodes a non-reducing PKS. Seven catalytic domains were identified in ShPKS5 based on the presence of conserved motifs. It had starter unit acyltransferase (SAT), beta-ketoacyl-ACP synthase (KS), acyltransferase (AT), product template (PT), two acyl carrier protein (ACP), and thioesterase (TE) domains. The domain architecture of ShPKS5 was SAT-KS-AT-PT-ACP-ACP-TE.

The ORF of ShPKS2 was a 5472 bp gene encoding an 1823 amino acid protein (accession number, OQ865373). Compared with ShPKS5, ShPKS2 had one more dehydratase (DH) and β-ketoacyl reductase (KR) domain but no TE domain. The domain architecture of ShPKS2 was KS-AT-DH-KR-ACP. Domain analysis showed that ShPKS2 encodes a partially reducing PKS. A partially reducing PKS with this domain architecture (KS-AT-DH-KR-ACP) is usually involved in 6-methylsalicylic acid [38]. However, ShPKS2 had more DH domains than common partially reducing PKSs, and it is thus a new type of partially reducing PKS.

The ORF of ShPKS1 was a 7722 bp gene encoding a 2574 amino acid protein (accession number, OQ865372). ShPKS1 also encodes a partially reducing PKS. In contrast to ShPKS5, it had three ACP domains and an AMP domain. The domain architecture of ShPKS1 was AMP-ACP-KS-AT-DH-KR-ACP-ACP. It is similar to the hispidin synthase of H. sublateritium (AMP-ACP-KS-AT-DH-KR-ACP) [11]. Thus, ShPKS1 is a putative hispidin synthase in S. sanghuang.

3.8. Phylogenetic analysis of non-reducing PKSs

The amino acid sequence of the KS domain of three PKSs from S. sanghuang and several other fungal PKSs was used to generate multiple alignments and phylogenetic trees (Figure 3). The high-reducing PKSs of Aspergillus niger PKS1 and A. tubingensis PKS1 were the outgroup, and ShPKS5 was grouped with Sanghuangporus baumii PKS2 and I. obliquus PKS2 into a unique clade. Additionally, the clade including ShPKS5 was the sister clade of orsellinic acid synthase. Thus, ShPKS1 and ShPKS1 belonged to a partially reducing PKS clade. The clade of ShPKS1, S. baumii PKS1, and I. obliquus PKS1 was the sister clade of hispidin synthase (Figure 7). The phylogenetic analysis also indicated that ShPKS1 is a putative hispidin synthase.

Figure 7.

Figure 7.

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Phylogenetic relationship of Sanghuangporous sanghuang polyketide synthase (PKS) with other fungal PKSs. The deduced PKS protein was aligned with fungal PKS sequences retrieved from GenBank. The PKSs of the S. sanghuang clade obtained are marked in bold.

3.9. Expression patterns of PKS transcripts

To confirm the transcriptome analysis results, we assessed by q-PCR the expression of three PKS genes in different media (Figure 8). The q-PCR results showed that the expression of ShPKS5 in soy peptone (DDD) medium was slightly higher than in the other three mediums. Additionally, the expression of ShPKS5 in DDD medium was much higher than in the other three media. The gene expression of ShPKS1 in sorbitol (SLC) and lactose (RT) was much higher than in DDD and yeast extract (JMF). The qPCR results were thus consistent with transcriptome analysis.

Figure 8.

Figure 8.

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Gene expression of polyketide synthase (PKS) of Sanghuangporous sanghuang in different media.

4. Discussion

In fungi and plants, styrylpyrones and flavonoids are derived from phenylalanine. PKS plays an important role in the biosynthesis of styrylpyrones and flavonoids. In plants, the formation of the styrylpyrone scaffold is catalyzed by styrylpyrone synthase, a type III PKS (CHS-like) that condenses one hydroxycinnamoyl-CoA molecule with two malonyl-CoA molecules [25]. Chalcone formation, the most crucial step in flavonoid biosynthesis, is catalyzed by a type III PKS (CHS) that condenses p-coumaroyl-CoA with three malonyl-CoA molecules [39]. Although type III PKS (CHS-like) has also been found in fungi, the known CHS-like PKS in fungi is not involved in styrylpyrone and flavonoid biosynthesis [39]. For example, the first fungal type III PKS to be functionally characterized was resorcylic acid synthase from Neurospora crassa [39]. In fungi, the functional similarity with CHS of plants is through a hybrid NRPS-PKS involved in flavonoid biosynthesis, which has a A-T-KS-AT-DH-KR-ACP-TE domain architecture [40–42]. Additionally, a hybrid PR-PKS is involved in styrylpyrone biosynthesis in fungi. Hispidin synthase was first characterized in bioluminescent Agaricales [43]. The hispidin synthase (HispS) of bioluminescent fungi has a AMP-ACP-KS-AT-ACP domain architecture. Moreover, the HispS in non-bioluminescent fungi has more DH and KR domains, and its domain architecture is AMP-ACP-KS-AT-DH-KR-ACP [11]. In the present study, we found that ShPKS1 (AMP-ACP-KS-AT-DH-KR-ACP-ACP) is a putative HispS. It has one more ACP domain than known HispS proteins. Thus, it may be a new kind of HispS. The reported clustered genes are related to styrylpyrone and flavonoid biosynthesis in fungi [40,41,43]. However, we did not find this cluster of ShPKS1 genes in the genome analysis (result not shown). It is possible that there is a novel pathway of styrylpyrone biosynthesis utilized in S. sanghuang.

Although genome annotation results showed that five PKSs exist in the S. sanghuang genome, we only cloned three PKSs (ShPKS1, ShPKS2, and ShPKS5). It is possible that ShPKS3 and ShPKS4 had low expression levels in the cultured medium. The transcriptome analysis also showed low expression levels of ShPKS3 and ShPKS4. Commonly, many genes and gene clusters are silenced under standard fermentation conditions [44]. The strategy of one-strain many compounds (OSMAC) has been shown as a simple and powerful tool that can activate many silent biogenetic gene clusters in microorganisms to make more natural products [45]. In the future, we aim to utilize the OSMAC strategy to discover the function of silenced PKSs in S. sanghuang.

The culture conditions, such as media composition, temperature, osmolarity, and pH, influence the secondary metabolite production of fungi [46]. Li et al. [47] reported that different carbon sources influence the yield of hispidin in P. linteus, which produced a high yield of hispidin in glucose and yeast extract medium. However, we found S. sanghuang did not produce hispidin in a medium with glucose and yeast extract. This implies that there is a different regulatory mechanism in S. sanghuang and P. linteus.

Although several genomic studies deduced the pathway of hispidin biosynthesis in Hymenochaetaceae fungi, they did not deduce the specific PKS involved in hispidin biosynthesis [3,48–51]. In the present study, based on genome and transcriptome comparative analysis and combined analyses of gene expression and hispidin production, it was determined that ShPKS1 is involved in hispidin biosynthesis. This result identifies a specific PKS for subsequent confirmation of the function of hispidin synthase in S. sanghuang through gene silencing and heterologous expression.

5. Conclusions

We first confirmed ShPKS1 was related to hispidin biosynthesis in S. sanghuang. Three different media were screened for the production of hispidin by S. sanghuang SH1. The function of ShPKS1 was identified by combined analyses of gene expression with qPCR and hispidin detection by LCMS-IT-TOF. Domain analysis showed that ShPKS1 is a partially reducing polyketide synthesis gene with extra AMP and ACP domains before its KS domain. The domain architecture of ShPKS1 was determined to be AMP-ACP-KS-AT-DH-KR-ACP-ACP. Phylogenetic analysis also showed that ShPKS1 and other PKS genes from Hymenochaetaceae form a unique clade that is closely related to the clade containing Agaricales hispidin synthase genes.

Supplementary Material

Supplemental Material
Click here for additional data file. (1.7MB, docx)

Funding Statement

The research was funded by General Project of Basic Research Program in Yunnan Province (project no. 202101AT070218), the National Natural Science Foundation of China (Project No. 31860177), and the Reserve Talents for Young and Middle-aged Academic and Technical Leaders of the Yunnan Province (Project No. 202205AC160044).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Publicly available datasets were analyzed in this study. All newly generated sequences were deposited in GenBank with accession number SRX20095548.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
Click here for additional data file. (1.7MB, docx)

Data Availability Statement

Publicly available datasets were analyzed in this study. All newly generated sequences were deposited in GenBank with accession number SRX20095548.

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