Next Article in Journal
RNA Viruses as Tools in Gene Therapy and Vaccine Development
Next Article in Special Issue
Single-Molecule Real-Time (SMRT) Full-Length RNA-Sequencing Reveals Novel and Distinct mRNA Isoforms in Human Bone Marrow Cell Subpopulations
Previous Article in Journal
Population Genomics and Structure of the Critically Endangered Mariana Crow (Corvus kubaryi)
Previous Article in Special Issue
Genome Sequencing of Cladobotryum protrusum Provides Insights into the Evolution and Pathogenic Mechanisms of the Cobweb Disease Pathogen on Cultivated Mushroom
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Genes
Volume 10
Issue 3
10.3390/genes10030188
genes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of Gloeostereum incarnatum

by
Xinxin Wang
1,2,3,†,
Jingyu Peng
3,†,
Lei Sun
1,
Gregory Bonito
3,
Jie Wang
4,
Weijie Cui
1,
Yongping Fu
1,* and
Yu Li
1,*
1
Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun 130118, China
2
Department of Plant Protection, Shenyang Agricultural University, Shenyang 110866, China
3
Department of Plant, Soil, and Microbial Sciences, Michigan State University, East Lansing, MI, USA
4
Department of Plant Biology and Center for Genomics Enabled Plant Science, Michigan State University, East Lansing, MI, USA
*
Authors to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Genes 2019, 10(3), 188; https://doi.org/10.3390/genes10030188
Submission received: 17 December 2018 / Revised: 20 February 2019 / Accepted: 22 February 2019 / Published: 1 March 2019
(This article belongs to the Special Issue Advances in Single Molecule, Real-Time (SMRT) Sequencing)
Browse Figures
Versions Notes

Abstract

:
Gloeostereum incarnatum is a precious edible mushroom that is widely grown in Asia and known for its useful medicinal properties. Here, we present a high-quality genome of G. incarnatum using the single-molecule real-time (SMRT) sequencing platform. The G. incarnatum genome, which is the first complete genome to be sequenced in the family Cyphellaceae, was 38.67 Mbp, with an N50 of 3.5 Mbp, encoding 15,251 proteins. Based on our phylogenetic analysis, the Cyphellaceae diverged ~174 million years ago. Several genes and gene clusters associated with lignocellulose degradation, secondary metabolites, and polysaccharide biosynthesis were identified in G. incarnatum, and compared with other medicinal mushrooms. In particular, we identified two terpenoid-associated gene clusters, each containing a gene encoding a sesterterpenoid synthase adjacent to a gene encoding a cytochrome P450 enzyme. These clusters might participate in the biosynthesis of incarnal, a known bioactive sesterterpenoid produced by G. incarnatum. Through a transcriptomic analysis comparing the G. incarnatum mycelium and fruiting body, we also demonstrated that the genes associated with terpenoid biosynthesis were generally upregulated in the mycelium, while those associated with polysaccharide biosynthesis were generally upregulated in the fruiting body. This study provides insights into the genetic basis of the medicinal properties of G. incarnatum, laying a framework for future characterization of bioactive proteins and pharmaceutical uses of this fungus.

1. Introduction

Mushrooms are an important source of nutrition, and a growing body of evidence has indicated that mushrooms may have medicinal properties and human health benefits [1,2,3]. Gloeostereum incarnatum (family Cyphellaceae) is an edible mushroom, which grows as a saprophyte on broad-leaved trees [4]. G. incarnatum is native to China, but is popular in other regions in Asia too, such as Japan and Siberia [4]. Besides its savory taste, G. incarnatum is well-known for its medicinal properties. Antioxidant, immunomodulatory, anti-inflammatory, anti-proliferative, and antibacterial properties have been attributed to this mushroom [5,6,7]. Recent studies have shown that sesquiterpenes and polysaccharides are the main bioactive compounds underlying the beneficial effects of G. incarnatum [5,6,8].
With the rapid advancement of sequencing technologies, the number of available fungal genomes has increased [9,10]. However, genomes of medicinal mushrooms remain scarce. Recently, the genomes of a few medicinal mushrooms (e.g., Ganoderma lucidum, Antrodia cinnamomea, and Hericium erinaceus) were released, and proteins putatively associated with the pharmacological properties of these mushrooms were investigated [11,12,13]. Gene clusters associated with the synthesis of various bioactive secondary metabolites, such as terpenoids and polypeptides, have been identified in many medical mushroom genomes [11,13]. For instance, nine gene clusters associated with the cytochrome P450 (CYP) and triterpenoid pathways were identified in A. cinnamomea [11], while four gene clusters associated with terpene and polyketide biosynthesis were identified in H. erinaceus [13]. In G. lucidum, 24 physical CYP gene clusters, possibly involved in triterpenoid biosynthesis, were identified [12]. Although several bioactive compounds have been identified in G. incarnatum [5,6,8,14], the genetic basis of the medicinal benefits of this mushroom are largely unknown.
In this study, we used the Pacific Biosciences (PacBio) long-read sequencing platform [15] to perform the de novo assembly of the G. incarnatum genome. This is the first genome to be sequenced in the Cyphellaceae family. We also compared the transcriptome profiles of the mycelium and the fruiting body, the two major developmental stages of G. incarnatum. The sequenced genome of G. incarnatum presented herein is, to our knowledge, one of the most comprehensive assembled genomes of an edible mushroom. In this study, we aimed to (1) present a high-quality reference genome for G. incarnatum, which can be used for future analyses of genome function and genetic variation and (2) identify relevant functional genes, gene clusters, and signaling pathways associated with the saprophytic lifestyle and pharmaceutical properties of G. incarnatum. We specifically focused on terpene biosynthesis, cytochrome P450 enzyme biosynthesis, and polysaccharide production. Our study provides a valuable genomic and transcriptomic resource for future studies of the genetic basis of the medicinal properties of G. incarnatum. Such studies would represent a first step towards realizing the full potential of G. incarnatum as a source of pharmacologically active compounds on an industrial scale.

2. Materials and Methods

2.1. Fungal Material, Sequencing, and Genome Assembly

We isolated protoplast-derived monokaryons from the dikaryotic strain of the G. incarnatum commercial strain CCMJ2665. The monokaryons were obtained as described previously [16], except that the dikaryotic mycelia were incubated for 240 min at 30 °C in lywallzyme lysing enzyme. The single-nucleated genomic DNA of the G. incarnatum monokaryon strain was then used for genome sequencing and annotation. Genomic DNA was extracted using NuClean Plant Genomic DNA Kits (CWBIO, Beijing, China). The genome of G. incarnatum was sequenced on a PacBio Sequel long-read sequencing platform with a library insert size of 20 kb, at the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University (Changchun, China). Raw data were assembled with SMARTdenovo (https://github.com/ruanjue/smartdenovo). The completeness of the genome assembly was evaluated using Core Eukaryotic Genes Mapping Approach (CEGMA) [17] and Benchmarking Universal Single-Copy Orthologs (BUSCO; [18]). The whole-genome sequence of G. incarnatum has been deposited in GenBank (in submission). The genome reported in this study has been deposited in GenBank under the accession RZIO00000000.

2.2. Genome Annotation

Three different strategies were used to predict genes in the G. incarnatum genome: Sequence homologies with four representative mushrooms; ab initio with Augustus [19], Genescan [20], GlimmerHMM [21], and SNAP [22]; and combining extrinsic and ab initio approaches with GLEAN (http://sourceforge.net/projects/glean-gene). GLEAN gene prediction results were used for subsequent analyses. Protein-coding genes were annotated by GLEAN using both ab initio and evidence-based methods [23]. Predicted genes were functionally annotated against several databases—National Center for Biotechnology Information (NCBI) non-redundant (nr), Swiss-Prot, and InterPro—using BLASTP searches (e-value ≤ 1 × 10−5). Gene annotations were refined using the following databases: Gene Ontology (GO) [24], Clusters of Orthologous Groups (KOG) [25], and Kyoto Encyclopedia of Genes and Genomes (KEGG) [26]. Transposon sequences were identified by aligning the assembled genome to the Repbase database [27] with RepeatMasker (version 3.3.0; http://www.repeatmasker.org/; [28]) and RepeatProteinMasker [22]. Tandem repeat sequences (TRF) were predicted with Tandem Repeat Finder [29]. Ribosomal RNA (rRNA) sequences were identified, based on sequence homology and also through use of de novo prediction strategies with rRNAmmer [30]. Transfer RNA (tRNA) genes were identified using tRNAscan-SE [31]. Non-coding RNAs, such as small nuclear RNA (snRNAs) and microRNAs (miRNAs), were predicted with Rfam [32].

2.3. Evolutionary Analysis and Phylogeny

The phylogenetic analysis was performed using single-copy genes shared across G. incarnatum and another nine fungal species (Omphalotus olearius, Gymnopus luxurians, Laccaria bicolor, Coprinopsis cinerea, Armillaria ostoyae, Lentinula edodes, Schizophyllum commune, Serpula lacrymans and Coniophora puteana). The “all against all” BLASTP searches were performed with a cutoff e-value of 1 × 10−7 for proteins from all species. The alignments of gene pairs were conjoined by solar. Only gene pairs with an alignment ratio (aligned region by total length) of more than 30% in both homologous genes were kept for the following gene family construction. Gene families were clustered using a sparse graph of gene relationships using the hierarchical clustering algorithm hcluster_sg 0.5.1 package. Finally, we identified single-copy genes which had only one homolog per taxon, and those genes were used to construct the phylogenetic tree. The protein sequences of these single-copy genes were aligned using MUSCLE [33] and the protein alignments were transformed into codon alignments with PAL2NAL. Gblocks was used to refine each codon alignment, and all refined alignments were concatenated to a super codon alignment. RAxML software (version 7.2.3) [34] was used to construct the phylogenetic tree using the maximum likelihood (ML) algorithm. The best-scoring ML tree was inferred using the rapid bootstrap analysis after 1000 runs. The divergence times among species were estimated using the mcmctree module in PAML [35] with the calibration time of Serpula lacrymans and Coniophora puteana according to Floudas et al. (2012).

2.4. Carbohydrate-Active Enzyme (CAZyme) Family Classification

The CAZymes in the G. incarnatum genome were identified by mapping the annotated protein sequences to the CAZy database (http://www.cazy.org/) [36] using BLASTP (cut-off e-value ≤ 1 × 10−5, identity ≥ 40% and coverage ≥ 40%). The recovered CAZymes were classified as glycoside hydrolases (GHs), auxiliary activities (AAs), carbohydrate-binding modules (CBMs), glycosyl transferases (GTs), polysaccharide lyases (PLs), and carbohydrate esterases (CEs).

2.5. Cytochrome P450 (CYP) Predictions

CYP proteins were predicted by aligning the gene models to the fungal P450 database (http://p450.riceblast.snu.ac.kr/index.php?a=view;) with BLASTP (e-value ≤ 1 × 10−5, matrix = BLOSUM62). CYP proteins were assigned to protein families based on Nelson’s nomenclature [37]. For protein sequences that aligned with multiple families, the top hit was chosen.

2.6. Secondary Metabolite Annotations

Secondary metabolite gene clusters were predicted with fungal AntiSMASH 3.0 (https://fungismash.secondarymetabolites.org/) [38], with the default parameter values.

2.7. RNA Sequencing of the Two Major Developmental Stages

Samples of the two major fungal developmental stages (mycelium and fruiting body) from the G. incarnatum strain CCMJ2665 were provided by the mushroom section of the Engineering Research Center of the Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University (Changchun, China). RNA extraction and quality control were performed following the processes of Fu et al. [16]. cDNA libraries were constructed, and 150 paired-end sequencing was performed on an Illumina HiSeq 4000 platform at Novogene Co., LTD (Beijing, China). Sequencing data have been deposited in the NCBI SRA (accession no. PRJNA510218).
Raw data were filtered to remove adapter sequences and low-quality reads for downstream analyses. The trimmed reads were mapped to the G. incarnatum genome using TopHat v2.0.12 [39]. The number of reads mapped to each gene was counted using HTSeq v0.6.1 [40]. Fragments per kilobase of transcript per million mapped reads (FPKM) values were used to calculate gene expression. Genes differentially expressed between developmental stages were identified using the DESeq package (1.18.0) [41] in R with adjusted p-value set to <0.05.

3. Results and Discussion

3.1. Genome Sequencing and Assembly

A high-quality reference genome for G. incarnatum was generated from a protoplast monokaryon isolated from the dikaryotic strain of a commercial G. incarnatum cultivar (CCMJ2665; see Table 1). The genomic DNA of G. incarnatum was sequenced on PacBio SMRT Sequel platform generating ~94× coverage of 3,642 Mbp of clean data, as shown in Table S1. Compared to other edible and medicinal mushrooms, the assembled genome of G. incarnatum (38.7 Mbp), as shown in Figure 1, was of an intermediate size; the Wolfiporia cocos genome was the largest (50.5 Mbp); and the Agaricus bisporus var. bisporus genome was the smallest (30.2 Mbp), as shown in Table 1 [11,12,13,42,43,44,45,46,47,48]. G. incarnatum had a guanine-cytosine (GC) content of 49%; GC content in the other mushroom genomes examined was 45.3–55.9% (Table 1). The genome of G. incarnatum was one of the most complete assembled genomes across all representative edible and medicinal mushrooms examined, consisting of 20 scaffolds with an N50 of 3.5 Mbp (Table 1; Figure 1). The completeness of the G. incarnatum genome assembly was analyzed with the CEGMA [17] and the single-copy orthologs test using Fungi BUSCOs [18]. The CEGMA analysis indicated that 96.8% of the core eukaryotic genes were mapped to the G. incarnatum genome. The BUSCO analysis suggested that the annotation set was well completed, with 93.1% complete BUSCOs and 4.5% missing BUSCOs. Thus, our results indicate that the G. incarnatum genome assembly is high quality.

3.2. Gene and Repeat Sequence Prediction and Annotation

To most accurately predict the protein-coding genes in the G. incarnatum genome, we used a homology-based prediction strategy (against four representative mushroom genomes) combined with de novo gene prediction approaches. We predicted 15,251 protein-coding genes, accounting for 57.46% of the assembled G. incarnatum genome (Table S2). The predicted protein-coding genes had an average length of 1456.86 bp and contained 4.38 exons (each with an average length of 264.46 bp). The protein-coding genes were functionally annotated against several databases: NCBI nr, Swiss-Prot, InterPro, GO, COG, and KEGG. Of the 15,251 protein-coding genes predicted, 72.62% had homologs in one or more of the databases searched (Table S2).
We identified ~5.9 Mbp of repeat sequences in the G. incarnatum genome. Of these repeat sequences, 0.49% were predicted to be tandem repeats and 14.46% to be transposons (TEs) (Figure 1; Table S3). Most of the predicted TEs were long terminal repeats (LTRs), representing 13.78% of the genome (Table S3). Of the non-coding RNA species we identified in the G. incarnatum genome, 161 were tRNAs and 44 were rRNAs (Table S4). Nine of the identified tRNAs were possible pseudogenes, and the remaining 152 anti-codon tRNAs corresponded to the 20 common amino acids (Table S4). We also predicted 18 miRNAs and 18 snRNAs; the snRNAs comprised 15 spliceosomal RNAs and three C/D box small nucleolar RNAs (Table S4).
KOG functionally classified 4243 (27.82%) of the predicted proteins. Of these, 499 genes were associated with “amino acid transport and metabolism”, 476 genes were associated with “carbohydrate transport and metabolism”, and 337 with “secondary metabolite biosynthesis, transport, and catabolism”. This suggests that several G. incarnatum proteins are involved in nutrient absorption, transformation, and the synthesis of secondary metabolites. Similarly, KEGG classification indicated that both “amino acid metabolism” and “carbohydrate metabolism” were enriched in G. incarnatum genes (603 and 654 genes, respectively). KEGG analysis indicated that another 161 proteins were assigned to “biosynthesis of other secondary metabolites”, and 59 proteins were associated with the “metabolism of terpenoids and polyketides”. As the medicinal properties of edible mushrooms are closely related to the biosynthesis of secondary metabolites [49], these compounds were the focus of the remainder of our study.

3.3. Comparative Genomics and Evolutionary Analysis

With the exception of the G. incarnatum genome assembled in this study, no complete genomes are available for other fungi in Cyphellaceae. Thus, our evolutionary analysis compared whole genome sequences of seven representative species of the Agaricales: O. olearius, G. luxurians, L. bicolor, C. cinerea, A. ostoyae, L. edodes, and S. commune. We also included genomes of two additional agaricomycetid species having fossil calibrations—S. lacrymans and C. puteana [50]. We found that the G. incarnatum genome includes 7384 gene families, with 10,075 (66.1%) genes having homologs in at least one of the other nine fungal species (Figure 1, Table S5). Interestingly, 5,176 (33.9%) unclustered genes and 469 unique gene families (containing 1369 genes) were G. incarnatum specific (Table S5). These G. incarnatum-specific genes were associated with diverse biological processes, including steroid biosynthesis, terpenoid backbone biosynthesis, and polysaccharide biosynthesis.
We then constructed an ML phylogeny for G. incarnatum and the nine additional fungal species, based on 1822 shared single-copy orthologous genes (Figure 2). These data indicate that G. incarnatum is phylogenetically closer to A. ostoyae, diverging ~174 million years ago (Figure 2). We also identified 325 significantly expanded gene families in the G. incarnatum genome (p ≤ 0.01) (Figure 2); these families were primarily associated with carbohydrate metabolism (starch/sucrose metabolism and glycolysis/gluconeogenesis), amino acid and lipid metabolism, and genetic and environmental information processing. However, caution is warranted when interpreting species’ evolutionary time estimates and gene family expansions and contractions based on genomes generated with different sequencing platforms, assembly methods, and selection of comparative analysis groups. Nevertheless, our analysis provides new insights into the phylogeny of G. incarnatum and other mushroom species based on whole-genome data.

3.4. The Decomposition of Wood by CAZymes

To further classify the proteins associated with lignin digestion during carbohydrate metabolism, we mapped the protein sequences of G. incarnatum to the CAZy database [36]. We identified 311 non-overlapping CAZymes in six families in G. incarnatum (Table S6); the CAZymes in G. incarnatum were more diverse and abundant than those of brown rot fungi [51]. The G. incarnatum CAZymes consisted of 164 GHs, 66 proteins with AAs, 42 CBMs, 41 GTs, 18 PLs, and 10 CEs (Figure 3).
As the GHs include many cellulase families (such as GH16, GH5, GH3, GH6, and GH7) [36], the remarkably higher number of GHs in G. incarnatum was not unexpected. As a saprotrophic mushroom, G. incarnatum is likely to require many GHs to decompose cellulose from its woody hosts. AAs were the next most abundant family of CAZymes in G. incarnatum; AAs identified in this species included 21 AA9, 18 AA1, and 14 AA3 enzyme families. These three AA families are also the most abundant AAs in other fungi [10,11,12,13]. However, only three AA2 family proteins, the lignin-modifying fungal peroxidases (PODs), were identified in G. incarnatum. PODs are the primary lignin decomposers in the model white rot fungus Phanerochaete chrysosporium and other fungal species [52]. As G. incarnatum is restricted to elm tree hosts, the few AA2s identified in this fungus may be sufficient to decompose lignins produced by elm. We also identified 21 genes encoding enzymes for pectin digestion in G. incarnatum. Thus, the wood-decaying mushroom, G. incarnatum, may utilize complex strategies to decompose plant cell walls.

3.5. Secondary Metabolites and Terpene Pathway

The pharmacological properties of medicinal mushrooms are largely conferred by secondary metabolites; these metabolites have received intense research attention [1,49,53]. Here, we used antiSMASH to search for gene clusters encoding secondary metabolites in G. incarnatum [38]. We identified 65 gene clusters: one saccharide, 15 terpene synthases (TSs), one fatty acid, three polyketide synthases (PKSs), two siderophores, one non-ribosomal peptide-synthetase (NRPS), and 37 putative gene clusters of unknown type (Figure 3).
Terpenoid biosynthesis is of particular interest as terpenoids are important pharmacologically active compounds in G. incarnatum [14,54]. G. incarnatum contains two unique sesquiterpene compounds, gloeosteretriol and incarnal [14,54]. Both compounds demonstrate antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, but not against any of the Gram-negative bacteria tested to date [14,54]. Incarnal extracted from G. incarnatum and another fungus in Cyphellaceae (Chondrostereum sp.) also shows potent cytotoxicity against several cancer cell lines [8,55]. To further investigate the biosynthesis of terpenoids in G. incarnatum, we mapped 35 proteins to 17 enzymes in the “terpenoid backbone biosynthesis” (KEGG: ko00900) pathway, and 10 proteins to four enzymes in the “sesquiterpenoid and triterpenoid biosynthesis” (KEGG: ko00909) pathway (Figure 4). The pathway mapping results suggested that the G. incarnatum terpenoids are likely to be synthesized through the mevalonate (MVA) pathway, not the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-d-xylulose 5-phosphate (MEP/DOXP) pathway. This is in line with the results for other mushrooms, such as G. lucidum and H. erinaceus [11,12].
Sesquiterpenoids are synthesized from farnesyl diphosphate (FPP) by various sesquiterpene synthases [56]. We located five genes encoding sesquiterpene synthases in G. incarnatum—three encoding trichodiene synthase (EC 4.2.3.6) and two encoding aristolochene synthase (EC 4.2.3.9; Figure 4). Interestingly, trichodiene is a precursor for the biosynthesis of the mycotoxin nivalenol, which is widely found in Fusarium species, and the biosynthesis of aristolochene, which is a precursor for the PR toxin found in Penicillium species [57,58]. To the best of our knowledge, neither of these compounds are produced by G. incarnatum. It would be interesting to know if trichodiene or aristolochene was a substrate for the synthesis of gloeosteretriol or incarnal in G. incarnatum. Based on structural similarity, incarnal might potentially be synthesized from trichodiene in conjunction with certain cyclization, bond-shift rearrangement, oxidation, and hydroxylation reactions. Further experiments are thus necessary to confirm the production of trichodiene and aristolochene, as well as their association with the biosynthesis of gloeosteretriol or incarnal, in G. incarnatum.
Regarding triterpenoid, two farnesyltransferases (EC 2.5.1.21), three squalene monooxygenases (EC 1.14.14.17), and one lanosterol synthase (EC 5.4.99.7) were encoded in the G. incarnatum genome. This suggested that G. incarnatum synthesizes squalene, (S)-2,3-epoxysqualene, and lanosterol, all of which are intermediates in the synthesis of triterpenoid and sterol [59]. Notably, lanosterane-type triterpenoids are produced by several medical mushrooms, including species of Ganoderma, Innonotus, and Antrodia (reviewed in [60]), although the relevant biosynthesis pathways are unknown. The existence of these triterpenoid-related proteins in G. incarnatum suggests that this species may produce previously uncharacterized triterpenoids.

3.6. The CYP Family

Although the pathway for terpenoid backbone biosynthesis in fungi is relatively well studied [61,62], the steps following terpenoid cyclization are largely unknown. The structural diversity of terpenoids depends on post-modification of many specific chemical groups. These modifications involve a series of hydroxylation, reduction, oxidation, and acylation reactions, largely mediated by CYPs (cytochrome P450s) [63,64,65]. In fungi, CYPs are especially important for xenobiotic degradation and the biosynthesis of several secondary metabolites, including terpenoids and polyketides [63]. Based on a comparative search of the Fungal Cytochrome P450 Database [66], 145 CYP proteins were identified in G. incarnatum. These proteins were classified into 57 families following Nelson’s nomenclature [37]. The family CYP5144 included the greatest number of G. incarnatum CYPs (16); CYP5144 also included the most CYPs in another medicinal mushroom, Lignosus rhinocerotis [46] (Table 2). It is thus likely that CYP5144 family proteins play key roles in the biosynthesis of terpenoids in G. incarnatum.
As previously noted, the G. incarnatum genome encoded two sesterterpenoid synthases—aristolochene synthase and trichodiene synthase. Interestingly, the trichodiene synthase genes (GI_10004653, GI_10004654, and GI_10004694), but not the aristolochene synthase gene, (GI_10003231) were identified in gene clusters containing several CYPs (i.e., CYP65X, CYP530A, and CYP617B; Figure 5). Based on the logic in the Fungal Cytochrome P450 Database (FDPD) pipeline [66], CYP530A and CYP617B were assigned to the families CYP512 and CYP5144, respectively. Both of these families may be involved in the biosynthesis of bioactive terpenoids in G. lucidum and L. rhinocerotis [12]. These results further support our hypothesis that incarnal, the bioactive sesterterpenoid produced by G. incarnatum, might be synthesized from trichodiene, mediated by CYPs. The second-largest family of CYPs identified in G. incarnatum was CYP620 (Table 2), which is a relatively rare family in other medicinal mushrooms (absent in G. lucidum, one in A. cinnamomea, and three in L. rhinocerotis) [11,12,46]. This indicates that CYP distributions and functions vary among medicinal mushrooms. The exact roles of the identified CYPs in terpenoid post-modification or other biological functions remain to be experimentally validated.

3.7. Polysaccharide Biosynthesis

Composition of G. incarnatum polysaccharides also had immunomodulatory and immuno-enhancing effects in a mice model [6]. Some of the most potent immunomodulatory polysaccharides produced by medical mushrooms are water soluble 1,3-β- and 1,6-β-glucans [67]. In G. incarnatum, we identified four 1,3-β-glucan synthases (K00706 and K01180), three UTP–glucose-1-phosphate uridylyltransferases (K00963), 12 GTPase-activating-associated proteins (K12492, K12493, K19838, K19844, K19845, K14319, K17265, K18470, K20315, and K19839), two hexokinases (K00844), and two phosphoglucomutases (K01835) (Table 3). We also identified 15 β-glucan biosynthesis-associated proteins (PF03935; Table 3); β-glucan biosynthesis-associated proteins were shown to be involved in the biosynthesis of 1,6-β-glucans in Saccharomyces cerevisiae [68]. The polysaccharide biosynthesis-related proteins identified in G. incarnatum are summarized in Table S5. Compared with five other species of medicinal mushrooms (Auricularia heimuer [69], A. cinnamomea [11], Sparassis latifolia [47], L. rhinocerotis [46], and G. lucidum [12]), G. incarnatum produced more 1,3-β-glucan synthases, GTPase-activating-associated proteins, and β-glucan biosynthesis-associated proteins, as well as similar numbers of UTP–glucose-1-phosphate uridylyltransferases, hexokinases, and phosphoglucomutases (Table S7). This suggests that G. incarnatum might produce more 1,3-β- and 1,6-β-glucans. In-parallel quantifications of 1,3-β- and 1,6-β-glucan production among these medicinal mushrooms during different growth phases should be performed and compared.

3.8. Transcriptomic Analysis

As the expression levels of target genes encoding pharmacologically relevant proteins in G. incarnatum might differ across developmental stages, we profiled the transcriptomes of two major developmental stages of G. incarnatum—the mycelium and the fruiting body. We generated 70,634,952 raw reads from the cDNA libraries of the two stages. After data filtering and trimming, 69,716,944 high-quality clean reads remained. Of these clean reads, 92% were successfully mapped to the G. incarnatum genome. Across both stages, 11,015 genes were expressed, with 944 genes expressed only in the mycelium, and 718 genes only expressed in the fruiting body (Figure 6). We identified 3524 differentially expressed genes (DEGs) in the fruiting body as compared to the mycelium (Figure 6). Of these 1822 were significantly upregulated in the fruiting body as compared to the mycelium, and 1702 were significantly downregulated (Figure 6).
The gene expression patterns in the mycelium and fruiting body of G. incarnatum varied depending on the type of secondary metabolite encoded. For example, 17 of 45 terpenoid biosynthesis-related genes (38%) were differentially expressed between the mycelium and the fruiting body (Figure 6, Table S8). Of these, 65% were upregulated in the mycelium as compared to the fruiting body (Figure 6, Table S8), indicating that the biosynthesis of terpenoid compounds might be greater in the mycelium of G. incarnatum. In contrast, 10 of the 23 genes associated with polysaccharide biosynthesis (43%) were differentially expressed between the mycelium and the fruiting body, with 70% of these being significantly upregulated in the fruiting body as compared to the mycelium (Figure 6). This indicates that the fruiting body of G. incarnatum might be a richer source of polysaccharides. These findings were consistent with those for terpenoid- and polysaccharide-related genes in H. erinaceus [13]. Therefore, different secondary metabolites might be more enriched at different fungal development stages. Due to the complexity of secondary metabolite biosynthesis, further studies should investigate the molecular mechanisms underlying the secondary metabolism of G. incarnatum.

4. Conclusions

In this study, we presented the first whole-genome sequence of G. incarnatum, which is the first sequenced genome for a fungus belonging to Cyphellaceae. The G. incarnatum genome is one of the most completely assembled edible mushroom genomes available to date, consisting of 20 scaffolds with an N50 of 3.5 Mbp. The remarkably higher number of GHs and AAs in G. incarnatum may contribute to the active decomposition of lignin and cellulose of its woody hosts. We identified 65 gene clusters involved in the biosynthesis of secondary metabolites in the G. incarnatum genome. We also investigated the functions of the proteins involved in terpenoid biosynthesis; terpenoids are one of the main types of pharmacologically active compounds produced by G. incarnatum. We found two sesquiterpenoid synthase genes, one encoding aristolochene synthase and the other encoding trichodiene synthase, in gene clusters enriched with CYP genes. This suggested that CYPs play an active role in the post-modification of aristolochene and trichodiene sesquiterpenoids. We also predicted 38 proteins involved in polysaccharides biosynthesis, another main class of bioactive compounds in G. incarnatum. Genes involved in terpenoid biosynthesis were generally upregulated in mycelium, while the polysaccharide biosynthesis-related genes were upregulated in the fruiting body. These results provide a foundation for future studies of the genetic basis underlying the medicinal properties of G. incarnatum.

Supplementary Materials

Supplementary materials can be found at https://www.mdpi.com/2073-4425/10/3/188/s1.

Author Contributions

Conceptualization and supervision, Y.L. and Y.F.; sample preparation, W.C.; formal analysis and writing, J.P. and X.W.; software, J.P., L.S. and J.W.; critical review, G.B.

Funding

This research was funded by the Special Fund for Agro-Scientific Research in the Public Interest (No. 201503137); the Program of Creation and Utilization of Germplasm of Mushroom Crop of “111” Project (No. D17014); National-Level International Joint Research Center (2017B01011).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

SMRTSingle-Molecule, Real-Time
KEGGKyoto Encyclopedia of Genes and Genomes
CYPcytochrome P450
CAZymescarbohydrate-active enzymes
DEGdifferentially expressed genes
FPKMfragments per kilobase of transcript per million mapped reads

References

  1. Wasser, S.P. Medicinal mushroom science: Current perspectives, advances, evidences, and challenges. Biomed. J. 2014, 37, 345–356. [Google Scholar] [CrossRef] [PubMed]
  2. Song, C.; Liu, Y.; Song, A.; Dong, G.; Zhao, H.; Sun, W.; Ramakrishnan, S.; Wang, Y.; Wang, S.; Li, T.; et al. The Chrysanthemum nankingense genome provides insights into the evolution and diversification of chrysanthemum flowers and medicinal traits. Mol. Plant 2018, 11, 1482–1491. [Google Scholar] [CrossRef] [PubMed]
  3. Guggenheim, A.G.; Wright, K.M.; Zwickey, H.L. Immune modulation from five major mushrooms: Application to integrative oncology. Integr. Med. 2014, 13, 32–44. [Google Scholar]
  4. Petersen, R.H.; Parmasto, E. A redescription of Gloeostereum incarnatum. Mycol. Res. 1993, 97, 1213–1216. [Google Scholar] [CrossRef]
  5. Zhang, Z.-F.; Lv, G.-Y.; Jiang, X.; Cheng, J.-H.; Fan, L.-F. Extraction optimization and biological properties of a polysaccharide isolated from Gleoestereum incarnatum. Carbohydr. Polym. 2015, 117, 185–191. [Google Scholar] [CrossRef] [PubMed]
  6. Wang, D.; Li, Q.; Qu, Y.; Wang, M.; Li, L.; Liu, Y.; Li, Y. The investigation of immunomodulatory activities of Gloeostereum incaratum polysaccharides in cyclophosphamide-induced immunosuppression mice. Exp. Ther. Med. 2018, 15, 3633–3638. [Google Scholar] [CrossRef] [PubMed]
  7. Lull, C.; Wichers, H.J.; Savelkoul, H.F. Antiinflammatory and immunomodulating properties of fungal metabolites. Mediat. Inflamm. 2005, 2005, 63–80. [Google Scholar] [CrossRef] [PubMed]
  8. Asai, R.; Mitsuhashi, S.; Shigetomi, K.; Miyamoto, T.; Ubukata, M. Absolute configurations of (−)-hirsutanol A and (−)-hirsutanol C produced by Gloeostereum incarnatum. J. Antibiot. 2011, 64, 693–696. [Google Scholar] [CrossRef] [PubMed]
  9. Liu, W.; Chen, L.; Cai, Y.; Zhang, Q.; Bian, Y. Opposite polarity monospore genome de novo sequencing and comparative analysis rreveal the possible heterothallic life cycle of Morchella importuna. Int. J. Mol. Sci. 2018, 19, 2525. [Google Scholar] [CrossRef]
  10. Dai, Y.; Su, W.; Yang, C.; Song, B.; Li, Y.; Fu, Y. Development of novel polymorphic EST-SSR markers in Bailinggu (Pleurotus tuoliensis) for crossbreeding. Genes 2017, 8, 325. [Google Scholar] [CrossRef] [PubMed]
  11. Lu, M.Y.; Fan, W.L.; Wang, W.F.; Chen, T.; Tang, Y.C.; Chu, F.H.; Chang, T.T.; Wang, S.Y.; Li, M.Y.; Chen, Y.H.; et al. Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development. Proc. Natl. Acad. Sci. USA 2014, 111, E4743–E4752. [Google Scholar] [CrossRef] [PubMed]
  12. Chen, S.; Xu, J.; Liu, C.; Zhu, Y.; Nelson, D.R.; Zhou, S.; Li, C.; Wang, L.; Guo, X.; Sun, Y.; et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat. Commun. 2012, 3, 913. [Google Scholar] [CrossRef] [PubMed]
  13. Chen, J.; Zeng, X.; Yang, Y.L.; Xing, Y.M.; Zhang, Q.; Li, J.M.; Ma, K.; Liu, H.W.; Guo, S.X. Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus. Sci. Rep. 2017, 7, 10151. [Google Scholar] [CrossRef] [PubMed]
  14. Takazawa, H.; Kashino, S. Incarnal. A new antibacterial sesquiterpene from Basidiomycetes. Chem. Pharm. Bull. 1991, 39, 555–557. [Google Scholar] [CrossRef] [PubMed]
  15. Li, C.; Lin, F.; An, D.; Wang, W.; Huang, R. Genome sequencing and assembly by long reads in plants. Genes 2017, 9, 6. [Google Scholar] [CrossRef] [PubMed]
  16. Fu, Y.; Dai, Y.; Yang, C.; Wei, P.; Song, B.; Yang, Y.; Sun, L.; Zhang, Z.W.; Li, Y. Comparative transcriptome analysis identified candidate genes related to Bailinggu mushroom formation and genetic markers for genetic analyses and breeding. Sci. Rep. 2017, 7, 9266. [Google Scholar] [CrossRef] [PubMed]
  17. Parra, G.; Bradnam, K.; Korf, I. CEGMA: A pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 2007, 23, 1061–1067. [Google Scholar] [CrossRef] [PubMed]
  18. Simao, F.A.; Waterhouse, R.M.; Ioannidis, P.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO: Assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 2015, 31, 3210–3212. [Google Scholar] [CrossRef] [PubMed]
  19. Stanke, M.; Keller, O.; Gunduz, I.; Hayes, A.; Waack, S.; Morgenstern, B. AUGUSTUS: Ab initio prediction of alternative transcripts. Nucleic Acids Res. 2006, 34, W435–W439. [Google Scholar] [CrossRef] [PubMed]
  20. Burge, C.; Karlin, S. Prediction of complete gene structures in human genomic DNA. J. Mol. Biol. 1997, 268, 78–94. [Google Scholar] [CrossRef] [PubMed]
  21. Allen, J.E.; Majoros, W.H.; Pertea, M.; Salzberg, S.L. JIGSAW, GeneZilla, and GlimmerHMM: Puzzling out the features of human genes in the ENCODE regions. Genome Biol. 2006, 7 (Suppl. 1), S9. [Google Scholar] [CrossRef]
  22. Korf, I. Gene finding in novel genomes. BMC Bioinform. 2004, 5, 59. [Google Scholar] [CrossRef] [PubMed]
  23. Elsik, C.G.; Mackey, A.J.; Reese, J.T.; Milshina, N.V.; Roos, D.S.; Weinstock, G.M. Creating a honey bee consensus gene set. Genome Biol. 2007, 8, R13. [Google Scholar] [CrossRef] [PubMed]
  24. Ashburner, M.; Ball, C.A.; Blake, J.A.; Botstein, D.; Butler, H.; Cherry, J.M.; Davis, A.P.; Dolinski, K.; Dwight, S.S.; Eppig, J.T.; et al. Gene ontology: Tool for the unification of biology. The gene ontology consortium. Nat. Genet. 2000, 25, 25–29. [Google Scholar] [CrossRef] [PubMed]
  25. Tatusov, R.L.; Galperin, M.Y.; Natale, D.A.; Koonin, E.V. The COG database: A tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000, 28, 33–36. [Google Scholar] [CrossRef] [PubMed]
  26. Kanehisa, M.; Goto, S.; Kawashima, S.; Okuno, Y.; Hattori, M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 2004, 32, D277–D280. [Google Scholar] [CrossRef] [PubMed]
  27. Jurka, J.; Kapitonov, V.V.; Pavlicek, A.; Klonowski, P.; Kohany, O.; Walichiewicz, J. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 2005, 110, 462–467. [Google Scholar] [CrossRef] [PubMed]
  28. Tarailo-Graovac, M.; Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinform. 2009. [Google Scholar] [CrossRef]
  29. Benson, G. Tandem repeats finder: A program to analyze DNA sequences. Nucleic Acids Res. 1999, 27, 573–580. [Google Scholar] [CrossRef] [PubMed]
  30. Lagesen, K.; Hallin, P.; Rodland, E.A.; Staerfeldt, H.H.; Rognes, T.; Ussery, D.W. RNAmmer: Consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007, 35, 3100–3108. [Google Scholar] [CrossRef] [PubMed]
  31. Lowe, T.M.; Eddy, S.R. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997, 25, 955–964. [Google Scholar] [CrossRef] [PubMed]
  32. Gardner, P.P.; Daub, J.; Tate, J.G.; Nawrocki, E.P.; Kolbe, D.L.; Lindgreen, S.; Wilkinson, A.C.; Finn, R.D.; Griffiths-Jones, S.; Eddy, S.R.; et al. Rfam: Updates to the RNA families database. Nucleic Acids Res. 2009, 37, D136–D140. [Google Scholar] [CrossRef] [PubMed]
  33. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef] [PubMed]
  34. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef] [PubMed]
  35. Yang, Z. PAML 4: Phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 2007, 24, 1586–1591. [Google Scholar] [CrossRef] [PubMed]
  36. Cantarel, B.L.; Coutinho, P.M.; Rancurel, C.; Bernard, T.; Lombard, V.; Henrissat, B. The Carbohydrate-Active EnZymes database (CAZy): An expert resource for Glycogenomics. Nucleic Acids Res. 2009, 37, D233–D238. [Google Scholar] [CrossRef] [PubMed]
  37. Nelson, D.R. The cytochrome p450 homepage. Hum. Genom. 2009, 4, 59–65. [Google Scholar]
  38. Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H.U.; Bruccoleri, R.; Lee, S.Y.; Fischbach, M.A.; Muller, R.; Wohlleben, W.; et al. antiSMASH 3.0—A comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 2015, 43, W237–W243. [Google Scholar] [CrossRef] [PubMed]
  39. Trapnell, C.; Pachter, L.; Salzberg, S.L. TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics 2009, 25, 1105–1111. [Google Scholar] [CrossRef] [PubMed]
  40. Anders, S.; Pyl, P.T.; Huber, W. HTSeq—A Python framework to work with high-throughput sequencing data. Bioinformatics 2015, 31, 166–169. [Google Scholar] [CrossRef] [PubMed]
  41. Anders, S.; Huber, W. Differential expression analysis for sequence count data. Genome Biol. 2010, 11, R106. [Google Scholar] [CrossRef] [PubMed]
  42. Shim, D.; Park, S.G.; Kim, K.; Bae, W.; Lee, G.W.; Ha, B.S.; Ro, H.S.; Kim, M.; Ryoo, R.; Rhee, S.K.; et al. Whole genome de novo sequencing and genome annotation of the world popular cultivated edible mushroom, Lentinula edodes. J. Biotechnol. 2016, 223, 24–25. [Google Scholar] [CrossRef] [PubMed]
  43. Gupta, D.K.; Ruhl, M.; Mishra, B.; Kleofas, V.; Hofrichter, M.; Herzog, R.; Pecyna, M.J.; Sharma, R.; Kellner, H.; Hennicke, F.; et al. The genome sequence of the commercially cultivated mushroom Agrocybe aegerita reveals a conserved repertoire of fruiting-related genes and a versatile suite of biopolymer-degrading enzymes. BMC Genom. 2018, 19, 48. [Google Scholar] [CrossRef] [PubMed]
  44. Shu, S.; Chen, B.; Zhou, M.; Zhao, X.; Xia, H.; Wang, M. De novo sequencing and transcriptome analysis of Wolfiporia cocos to reveal genes related to biosynthesis of triterpenoids. PLoS ONE 2013, 8, e71350. [Google Scholar] [CrossRef] [PubMed]
  45. Morin, E.; Kohler, A.; Baker, A.R.; Foulongne-Oriol, M.; Lombard, V.; Nagye, L.G.; Ohm, R.A.; Patyshakuliyeva, A.; Brun, A.; Aerts, A.L.; et al. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche. Proc. Natl. Acad. Sci. USA 2012, 109, 17501–17506. [Google Scholar] [CrossRef] [PubMed]
  46. Yap, H.Y.; Chooi, Y.H.; Firdaus-Raih, M.; Fung, S.Y.; Ng, S.T.; Tan, C.S.; Tan, N.H. The genome of the Tiger Milk mushroom, Lignosus rhinocerotis, provides insights into the genetic basis of its medicinal properties. BMC Genom. 2014, 15, 635. [Google Scholar] [CrossRef] [PubMed]
  47. Xiao, D.; Ma, L.; Yang, C.; Ying, Z.; Jiang, X.; Lin, Y.Q. De novo sequencing of a Sparassis latifolia genome and its associated comparative analyses. Can. J. Infect. Dis. Med. Microbiol. 2018, 2018, 1857170. [Google Scholar] [CrossRef] [PubMed]
  48. Kurata, A.; Fukuta, Y.; Mori, M.; Kishimoto, N.; Shirasaka, N. Draft genome sequence of the basidiomycetous fungus Flammulina velutipes TR19. Genome Announc. 2016, 4. [Google Scholar] [CrossRef] [PubMed]
  49. Zhong, J.J.; Xiao, J.H. Secondary metabolites from higher fungi: Discovery, bioactivity, and bioproduction. Adv. Biochem. Eng. Biotechnol. 2009, 113, 79–150. [Google Scholar] [CrossRef] [PubMed]
  50. Floudas, D.; Binder, M.; Riley, R.; Barry, K.; Blanchette, R.A.; Henrissat, B.; Martinez, A.T.; Otillar, R.; Spatafora, J.W.; Yadav, J.S.; et al. The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 2012, 336, 1715–1719. [Google Scholar] [CrossRef] [PubMed]
  51. Sista Kameshwar, A.K.; Qin, W. Comparative study of genome-wide plant biomass-degrading CAZymes in white rot, brown rot and soft rot fungi. Mycology 2018, 9, 93–105. [Google Scholar] [CrossRef] [PubMed]
  52. Martinez, A.T.; Ruiz-Duenas, F.J.; Martinez, M.J.; Del Rio, J.C.; Gutierrez, A. Enzymatic delignification of plant cell wall: From nature to mill. Curr. Opin. Biotechnol. 2009, 20, 348–357. [Google Scholar] [CrossRef] [PubMed]
  53. Lee, H.Y.; Moon, S.; Shim, D.; Hong, C.P.; Lee, Y.; Koo, C.D.; Chung, J.W.; Ryu, H. Development of 44 novel polymorphic SSR markers for determination of shiitake mushroom (Lentinula edodes) cultivars. Genes 2017, 8, 109. [Google Scholar] [CrossRef] [PubMed]
  54. Gao, J.; Yue, D.C.; Cheng, K.D.; Wang, S.C.; Yu, K.B.; Zheng, Q.T.; Yang, J.S. Gloeosteretriol, a new sesquiterpene from the fermentation products of Gloeostereum incarnatum S. Ito et Imai. Yao Xue Xue Bao = Acta Pharm. Sin. 1992, 27, 33–36. [Google Scholar]
  55. Li, H.J.; Chen, T.; Xie, Y.L.; Chen, W.D.; Zhu, X.F.; Lan, W.J. Isolation and structural elucidation of chondrosterins F-H from the marine fungus Chondrostereum sp. Mar. Drugs 2013, 11, 551–558. [Google Scholar] [CrossRef] [PubMed]
  56. Christianson, D.W. Unearthing the roots of the terpenome. Curr. Opin. Chem. Biol. 2008, 12, 141–150. [Google Scholar] [CrossRef] [PubMed]
  57. Hidalgo, P.I.; Ullan, R.V.; Albillos, S.M.; Montero, O.; Fernandez-Bodega, M.A.; Garcia-Estrada, C.; Fernandez-Aguado, M.; Martin, J.F. Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: Cross talk of secondary metabolite pathways. Fungal Genet. Biol. FG B 2014, 62, 11–24. [Google Scholar] [CrossRef] [PubMed]
  58. Schothorst, R.C.; van Egmond, H.P. Report from SCOOP task 3.2.10 “collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states”. Subtask: Trichothecenes. Toxicol. Lett. 2004, 153, 133–143. [Google Scholar] [CrossRef] [PubMed]
  59. Benveniste, P. Biosynthesis and accumulation of sterols. Annu. Rev. Plant Biol. 2004, 55, 429–457. [Google Scholar] [CrossRef] [PubMed]
  60. Rios, J.L.; Andujar, I.; Recio, M.C.; Giner, R.M. Lanostanoids from fungi: A group of potential anticancer compounds. J. Nat. Prod. 2012, 75, 2016–2044. [Google Scholar] [CrossRef] [PubMed]
  61. Schmidt-Dannert, C. Biosynthesis of terpenoid natural products in fungi. Adv. Biochem. Eng. Biotechnol. 2015, 148, 19–61. [Google Scholar] [CrossRef] [PubMed]
  62. Kanehisa, M.; Goto, S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000, 28, 27–30. [Google Scholar] [CrossRef] [PubMed]
  63. Cresnar, B.; Petric, S. Cytochrome P450 enzymes in the fungal kingdom. Biochim. Biophys. Acta 2011, 1814, 29–35. [Google Scholar] [CrossRef] [PubMed]
  64. Sanglard, D.; Loper, J.C. Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis: Identification of a new P450 gene family. Gene 1989, 76, 121–136. [Google Scholar] [CrossRef]
  65. Mansuy, D. The great diversity of reactions catalyzed by cytochromes P450. Comp. Biochem. Physiol. Part C Pharmacol. Toxicol. Endocrinol. 1998, 121, 5–14. [Google Scholar] [CrossRef]
  66. Park, J.; Lee, S.; Choi, J.; Ahn, K.; Park, B.; Park, J.; Kang, S.; Lee, Y.H. Fungal cytochrome P450 database. BMC Genom. 2008, 9, 402. [Google Scholar] [CrossRef] [PubMed]
  67. Xu, Z.; Chen, X.; Zhong, Z.; Chen, L.; Wang, Y. Ganoderma lucidum polysaccharides: Immunomodulation and potential anti-tumor activities. Am. J. Chin. Med. 2011, 39, 15–27. [Google Scholar] [CrossRef] [PubMed]
  68. Montijn, R.C.; Vink, E.; Muller, W.H.; Verkleij, A.J.; Van Den Ende, H.; Henrissat, B.; Klis, F.M. Localization of synthesis of beta1,6-glucan in Saccharomyces cerevisiae. J. Bacteriol. 1999, 181, 7414–7420. [Google Scholar] [PubMed]
  69. Yuan, Y.; Wu, F.; Si, J.; Zhao, Y.F.; Dai, Y.C. Whole genome sequence of Auricularia heimuer (Basidiomycota, Fungi), the third most important cultivated mushroom worldwide. Genomics 2017. [Google Scholar] [CrossRef] [PubMed]
Genes 10 00188 g001 550
Figure 1. The Gloeostereum incarnatum genome and comparative genomics analysis. (A) The G. incarnatum genome. Outside to inside of concentric circles show assembly scaffold number, gene density, non-coding RNA (ncRNA), GC count and GC skew. (B) Unique and homologous gene families. The number of unique and shared gene families is shown in each of the diagram components and the total number of gene families for each fungus is given in parentheses.
Figure 1. The Gloeostereum incarnatum genome and comparative genomics analysis. (A) The G. incarnatum genome. Outside to inside of concentric circles show assembly scaffold number, gene density, non-coding RNA (ncRNA), GC count and GC skew. (B) Unique and homologous gene families. The number of unique and shared gene families is shown in each of the diagram components and the total number of gene families for each fungus is given in parentheses.
Genes 10 00188 g001
Genes 10 00188 g002 550
Figure 2. The Gloeostereum incarnatum genome evolutionary analysis. The number of expanded (green) and contracted (red) gene families is shown at each branch. The estimated divergence time (MYA: million years ago) is shown at the bottom. MRCA: most recent common ancestor.
Figure 2. The Gloeostereum incarnatum genome evolutionary analysis. The number of expanded (green) and contracted (red) gene families is shown at each branch. The estimated divergence time (MYA: million years ago) is shown at the bottom. MRCA: most recent common ancestor.
Genes 10 00188 g002
Genes 10 00188 g003 550
Figure 3. (A) Annotation of carbohydrate-related genes in the G. incarnatum genome; (B) secondary metabolite-related gene clusters in the G. incarnatum genome. T1PK: Type I polyketide synthases; NRPS: nonribosomal peptide synthetase.
Figure 3. (A) Annotation of carbohydrate-related genes in the G. incarnatum genome; (B) secondary metabolite-related gene clusters in the G. incarnatum genome. T1PK: Type I polyketide synthases; NRPS: nonribosomal peptide synthetase.
Genes 10 00188 g003
Genes 10 00188 g004 550
Figure 4. “Terpenoid backbone biosynthesis” (KEGG: ko00900) and “sesquiterpenoid and triterpenoid biosynthesis” (KEGG: ko00909) pathways of G. incarnatum. Red boxes indicate the presence of the enzymes, whereas white boxes indicate enzyme is not present.
Figure 4. “Terpenoid backbone biosynthesis” (KEGG: ko00900) and “sesquiterpenoid and triterpenoid biosynthesis” (KEGG: ko00909) pathways of G. incarnatum. Red boxes indicate the presence of the enzymes, whereas white boxes indicate enzyme is not present.
Genes 10 00188 g004
Genes 10 00188 g005 550
Figure 5. Genetic structures of sesterterpenoid synthase genes and their neighboring genes. Each gene is represented by an arrow. The aristolochene synthase gene (GI_10003231) is indicated by green color; the trichodiene synthase genes (GI_10004653, GI_10004654 and GI_10004694) are indicated by light blue color; the cytochrome P450 (CYP) genes are indicated by red color; choline dehydrogenase genes are indicated by yellow color; The Sec1-like protein genes are indicated by purple color.
Figure 5. Genetic structures of sesterterpenoid synthase genes and their neighboring genes. Each gene is represented by an arrow. The aristolochene synthase gene (GI_10003231) is indicated by green color; the trichodiene synthase genes (GI_10004653, GI_10004654 and GI_10004694) are indicated by light blue color; the cytochrome P450 (CYP) genes are indicated by red color; choline dehydrogenase genes are indicated by yellow color; The Sec1-like protein genes are indicated by purple color.
Genes 10 00188 g005
Genes 10 00188 g006 550
Figure 6. Comparative transcriptome profiling of the mycelium and the fruiting body of G. incarnatum: (A) Fruiting bodies of G. incarnatum; (B) Venn diagram of the genes expressed in the mycelium and/or the fruiting body; (C) number of genes being significantly downregulated or upregulated in the fruiting body compared with the mycelium; (D) heatmap of the genes associated with the biosynthesis of polysaccharides and terpenes.
Figure 6. Comparative transcriptome profiling of the mycelium and the fruiting body of G. incarnatum: (A) Fruiting bodies of G. incarnatum; (B) Venn diagram of the genes expressed in the mycelium and/or the fruiting body; (C) number of genes being significantly downregulated or upregulated in the fruiting body compared with the mycelium; (D) heatmap of the genes associated with the biosynthesis of polysaccharides and terpenes.
Genes 10 00188 g006
Table
Table 1. Comparison of genome assembly among representative edible mushrooms.
Table 1. Comparison of genome assembly among representative edible mushrooms.
OrganismAccessionGenome Size (Mbp)GenomeScaffoldN50 (Kbp)GC Content (%)Protein-Coding GenesSequencing Method
Gloeostereum incarnatum 38.794×20350049.015,251PacBio Sequel
Lentinula edodesLSDU0000000046.160×31366345.313,426PacBio RSII; Illumina HiSeq 2500
Agrocybe aegeritaPRJEB2191744.8253×12276849.214,113PacBio RSII; Illumina HiSeq 2500
Hericium erinaceusPRJN36133839.4200×51953853.19895Illumina MiSeq; Hiseq 2500
Antrodia cinnamomeaJNBV0000000032.2878×360103550.69254Roche 454; Illumina GAIIx
Ganoderma lucidumAGAX0000000043.3440×82138855.916,113Roche 454; Illumina GAII
Wolfiporia cocosAEHD0000000050.540×348253952.212,212Sanger; Roche 454
Inonotus baumiiLNZH0000000031.6186×21726747.68455Illumina HiSeq
Agaricus bisporus var. bisporusAEOK0000000030.28.3×29230046.610,438Sanger
Lignosus rhinocerotisAXZM0000000034.3180×13389053.710,742Illumina Hiseq 2000
Sparassis latifoliaLWKX0000000048.1601×47264151.412,471Illumina HiSeq 2500
Flammulina velutipesBDAN0000000035.3132×513015049.613,843Illumina HiSeq 2500
Table
Table 2. Summary of the CYP genes in the G. incarnatum genome.
Table 2. Summary of the CYP genes in the G. incarnatum genome.
FamilySubfamilyCorresponding Gene NumberTotal Gene NumberFamilySubfamilyCorresponding Gene NumberTotal Gene Number
CYP5144C,F15,116CYP675A33
CYP620A,B,E,H1,1,4,28CYP682B33
CYP5015C66CYP504A33
CYP5014F,H2,35CYP51F33
CYP5068B55CYP55A33
CYP5080B,D3,25CYP65J,X1,12
CYP5093A55CYP5070A22
CYP505C,D3,14CYP5074A22
CYP535A44CYP5078A22
CYP536A44CYP5081A22
CYP617A,B1,23CYP5125A22
CYP5037B33CYP540B22
CYP5110A33CYP630B22
CYP530A33Others--30
Table
Table 3. Summary of the polysaccharide biosynthesis-related proteins in G. incarnatum.
Table 3. Summary of the polysaccharide biosynthesis-related proteins in G. incarnatum.
Enzyme FamilyKO TermEC NumberGene NumberGene Name
1,3-β-glucan synthaseK01180EC:3.2.1.61GI_10004256
K00706EC:2.4.1.343GI_10014134, GI_10014600, GI_10010064
UTP–glucose-1-phosphate uridylyltransferaseK00963EC:2.7.7.93GI_10009949, GI_10009950, GI_10009951
HexokinaseK00844EC:2.7.1.12GI_10010509, GI_10009252
PhosphoglucomutaseK01835EC:5.4.2.22GI_10003989, GI_10014463
GTPase-activating-associated proteinK12492-1GI_10009154
K19838-1GI_10009280
K12493-1GI_10004440
K14319-1GI_10004658
K19845-2GI_10004984, GI_10007354
K19839-3GI_10003462, GI_10010380, GI_10012746
K19844-2GI_10014590, GI_10000357
K18470-1GI_10014667

Share and Cite

MDPI and ACS Style

Wang, X.; Peng, J.; Sun, L.; Bonito, G.; Wang, J.; Cui, W.; Fu, Y.; Li, Y. Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of Gloeostereum incarnatum. Genes 2019, 10, 188. https://doi.org/10.3390/genes10030188

AMA Style

Wang X, Peng J, Sun L, Bonito G, Wang J, Cui W, Fu Y, Li Y. Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of Gloeostereum incarnatum. Genes. 2019; 10(3):188. https://doi.org/10.3390/genes10030188

Chicago/Turabian Style

Wang, Xinxin, Jingyu Peng, Lei Sun, Gregory Bonito, Jie Wang, Weijie Cui, Yongping Fu, and Yu Li. 2019. "Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of Gloeostereum incarnatum" Genes 10, no. 3: 188. https://doi.org/10.3390/genes10030188

APA Style

Wang, X., Peng, J., Sun, L., Bonito, G., Wang, J., Cui, W., Fu, Y., & Li, Y. (2019). Genome Sequencing Illustrates the Genetic Basis of the Pharmacological Properties of Gloeostereum incarnatum. Genes, 10(3), 188. https://doi.org/10.3390/genes10030188

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop