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Article

Comparative Genomic Analysis and Metabolic Potential Profiling of a Novel Culinary-Medicinal Mushroom, Hericium rajendrae (Basidiomycota)

by
Jing Wei
1,2,3,
Min Cheng
1,
Jian-fang Zhu
2,
Yilin Zhang
1,
Kun Cui
2,
Xuejun Wang
1,* and
Jianzhao Qi
1,2,*
1
Shangluo Key Research Laboratory of Standardized Planting & Quality Improvement of Bulk Chinese Medicinal Materials, College of Biology Pharmacy & Food Engineering, Shangluo University, Shangluo 726000, China
2
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, 3 Taicheng Road, Xianyang 712100, China
3
Qinba Mountains of Bio-Resource Collaborative Innovation Center of Southern Shaanxi Province, Hanzhong 723001, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2023, 9(10), 1018; https://doi.org/10.3390/jof9101018
Submission received: 18 September 2023 / Revised: 10 October 2023 / Accepted: 11 October 2023 / Published: 15 October 2023
(This article belongs to the Special Issue Molecular Genetics and Genomics of Mushroom-Forming Fungi, 2nd Edition)
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Abstract

:
Hericium rajendrae is an emerging species in the genus Hericium with few members. Despite being highly regarded due to its rarity, knowledge about H. rajendrae remains limited. In this study, we sequenced, de novo assembled, and annotated the complete genome of H. rajendrae NPCB A08, isolated from the Qinling Mountains in Shaanxi, China, using the Illumina NovaSeq and Nanopore PromethION technologies. Comparative genomic analysis revealed similarities and differences among the genomes of H. rajendrae, H. erinaceus, and H. coralloides. Phylogenomic analysis revealed the divergence time of the Hericium genus, while transposon analysis revealed evolutionary characteristics of the genus. Gene family variation reflected the expansion and contraction of orthologous genes among Hericium species. Based on genomic bioinformation, we identified the candidate genes associated with the mating system, carbohydrate-active enzymes, and secondary metabolite biosynthesis. Furthermore, metabolite profiling and comparative gene clusters analysis provided strong evidence for the biosynthetic pathway of erinacines in H. rajendrae. This work provides the genome of H. rajendrae for the first time, and enriches the genomic content of the genus Hericium. These findings also facilitate the application of H. rajendrae in complementary drug research and functional food manufacturing, advancing the field of pharmaceutical and functional food production involving H. rajendrae.

1. Introduction

Mushrooms are a collective term for the fruiting bodies of specific physical forms of fungi, which come in a wide variety of species. For centuries, people have been consuming mushrooms for their nutritional value and health benefits. Mushrooms play a significant role in agriculture, industry, and medicine, and their impact on economic development and environmental protection is significant and likely to increase in the future. Hericium, a genus of the Russulales order and Hericiaceae family, is a globally renowned medicinal and edible mushroom [1]. Hericium erinaceus is its most common and famous member, and it is not only a rare edible mushroom but also a well-known traditional Chinese medicine fungus with a long history of use. Currently, H. erinaceus has been proven to be a functional edible mushroom that could help prevent and delay various age-related neurological disorders [2]. Its chemical components exhibit extraordinary pharmacological effects, including neurotrophic [3], anti-aging, antioxidant, and anti-neuroinflammatory activities [4].
Hericium rajendrae is a relatively little known but valuable and rare species of the genus Hericium, with distinct morphological differences from Hericium erinaceus. Compared to H. erinaceus, which has been extensively studied for its chemical composition, little research has been conducted on the chemical composition of H. rajendrae. Hericium alpestre, another rare species within the genus Hericium, was reported to contain cyathane diterpenoids [5] and phenolic compounds [6,7], with the former found to stimulate the production of nerve growth factor and brain-derived neurotrophic factor [5], and the latter shown to have anticancer activity [6,7].
In recent years, the emergence of third-generation sequencing technology and the maturity of T2T assembly technology have made the gene sequencing and assembly of medicinal and edible fungi more accurate and efficient. These technologies promote research on various aspects such as fungal life cycles, mating types, nutritional modes, and biosynthesis of bioactive metabolites. With the support of efficient sequencing technology and mature assembly technology, the genome of some valuable medicinal fungi such as Ganoderma lucidum [8], mulberry Sanghuang [9], H. erinaceus [10], Laetiporus sulphureus [11] and Inonotus hispidus [12] have been successfully decoded. This research will further contribute to their medical use and industrial development. Furthermore, the genome sequencing of some precious wild edible fungi, including Pleurotus giganteus [13], Agaricus sinodeliciosus [14] and A. bitorquis [15], will also promote the strain breeding and artificial cultivation of these species.
The genus Hericium includes several species of medicinal and culinary value, such as H. erinaceus, H. coralloides, and H. rajendrae. While the genomes of H. erinaceus [10,16] and H. coralloides [17,18] have been extensively studied, the genome of H. rajendrae remains unexplored. Despite the availability of a published H. rajendrae genome, its poor quality did not meet the requirements of relevant studies. To fill this gap, we sequenced and analyzed the complete genome of a wild H. rajendrae strain. High-quality assembly and annotation showed that H. rajendrae is dikaryon, and contains fifteen pseudochromosomes and four contigs. This is the first time that the genome of H. rajendrae has been sequenced and analyzed. Comparative analysis with the genomes of H. erinaceus and H. coralloides provided new insights into the genome, mating system and carbohydrate metabolism capabilities of H. rajendrae. Considering the promising activity of Hericium mushrooms against neurodegenerative diseases, we further evaluated their potential for secondary metabolite biosynthesis, identified cyathane diterpenoids, and analyzed the biosynthesis of these compounds. This study adds to the diversity of Hericium genome research and provides new insights into the genetics and physiology of these rare medicinal and culinary mushrooms.

2. Materials and Methods

2.1. Fungal Strain and Strain Culture

The fruiting body from a natural habitat was collected from the side veins of Qinling Mountain in Zhashui County, Shaanxi Province, China. By comparing the morphological characteristics of the specimens and performing ITS sequence alignment of the mycelium (Figure S1), the sample was identified as H. rajendrae and subsequently classified as H. rajendrae NPCB A08. Tissue isolation was carried out using fresh wild fruiting bodies of the strain NPCB A08. In order to obtain cultivable mycelium, small pieces of sterilized basidiospores were placed on potato dextrose agar (PDA, Difco, BD, USA) plates and cultured for one week. The identified mycelium was preserved in the Key Laboratory of Natural Products and Chemical Biology, College of Chemistry and Pharmacy, Northwest A&F University. This process ensures the availability of viable mycelium for further studies.

2.2. Genome Sequencing, De Novo Assembly, and Annotation

2.2.1. Extraction of Genome DNA

The cultivation of H. rajendrae NPCB A08 mycelium was carried out in a controlled environment using potato dextrose broth (PDB, Difco, BD, USA) medium at a temperature of 25 °C with agitation at 200 rpm for a duration of one week. The purpose of this cultivation was to obtain a sufficient amount of fresh and viable mycelium. To ensure the purity and freshness of the mycelium, a series of washing steps were performed, including centrifugation, followed by rinsing with sterile water and then re-centrifugation to remove excess water. Genomic DNA extraction from the mycelium was performed using the sodium dodecyl sulfate technique. This involved grinding the mycelium with liquid nitrogen, and the DNA quality and concentration were assessed by agarose gel (0.75%) electrophoresis, Nanodrop One spectrophotometer (Thermo Fisher Scientific, CA, USA) and Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). Detailed information on the specific isolation and purification method can be found in a previous document [11].

2.2.2. Sequencing and De Novo Assembly

After assessing the quality and integrity of the DNA, it was randomly fragmented using a Covaris ultrasonic disruptor (Covaris, Woburn, MA, USA). Subsequently, Illumina sequencing pair-end libraries were constructed using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) with an insert size of 300 bp. The sequencing process was carried out on the Illumina NovaSeq6000 platform (Illumina, San Diego, CA, USA). To ensure high-quality data, the raw reads were subjected to a cleaning process to remove low-quality reads. This was accomplished using the SOAPnuke v2.1.8 (https://github.com/BGI-flexlab/SOAPnuke, accessed on 12 May 2023). Following the data filtering step, the clean data were utilized for subsequent analyses. For Oxford Nanopore sequencing, the libraries were prepared using the SQK-LSK109 ligation kit (Oxford Nanopore Technologies, Oxford, UK) following the standard protocol. Subsequently, the purified library was loaded onto primed R9.4 Spot-On Flow Cells and subjected to sequencing using a PromethION sequencer (Oxford Nanopore Technologies, Oxford, UK). To obtain the sequence data from the raw reads, base-calling analysis was conducted using the Oxford Nanopore GUPPY v6.4.6 (https://community.nanoporetech.com/downloads, accessed on 10 May 2023).
The NECAT software (https://github.com/xiaochuanle/NECAT, accessed on 10 July 2023) was used for genome correction and assembly, resulting in an initial assembly. This was followed by two rounds of error correction using the Racon V1.5 with default parameters based on the third-generation sequencing data. Subsequently, two rounds of error correction with second-generation reads were performed using the Pilon 1.2.4 (https://github.com/broadinstitute/pilon, accessed on 12 July 2023) with default parameters. Finally, the corrected genome was further processed using purge_haplotigs to remove heterozygous sequences, resulting in the final assembled genome.

2.2.3. Gene Prediction and Annotation

BRAKER v3.0.3 (https://github.com/Gaius-Augustus/BRAKER, accessed on 22 May 2023) was mainly used for gene sequence prediction. GeneMark—EX was then used to train the model, and AUGUSTUS v.3.5.0 (https://github.com/Gaius-Augustus/Augustus, accessed on 22 April 2023) was used for ORF prediction. The Rfam database was used to predict and classify non-coding RNAs using INFERNAL v1.1.4 (https://github.com/EddyRivasLab/infernal, viewed 22 April 2023). In addition, Repeat Modeller v2.0.4 (https://github.com/Dfam-consortium/RepeatModeler, accessed on 22 January 2023) was used to generate repetitive libraries by incorporating Rebase libraries, and Repeat-Masker v4.1.5 (https://github.com/rmhubley/RepeatMasker, accessed on 22 April 2023) was used for repetitive genomic sequence annotation. Finally, BLAST searches were performed against non-redundant protein sequences from National Center for Biotechnology Information (NCBI), Swiss-Prot, COG, and KEGG databases for gene product annotation. The above software runs on a dual-core server equipped with two Intel E5-2699V4 and 512 Gb RAM, running with default parameters.

2.3. Comparative Genomics Analysis

Genomic collinearity was analyzed and visualized using McscanX (https://github.com/wyp1125/MCScanX, accessed 4 July 2023). Clustering analysis of the comparative genomes of the genus Hericium species was performed using Orthofinder v2.5.5 (https://github.com/davidemms/OrthoFinder, accessed on 4 July 2023), which run using the following settings: -S diamond -M msa -T fasttree -t 88. The comparative genome results were visualized using jVenn (http://jvenn.toulouse.inra.fr/app/index.html, accessed 2 July 2023). To calculate the ratio of synonymous substitution rates (Ks) to nonsynonymous substitution rates (Ka) for each species of the genus Hericium, genome-wide replication analyses were performed using wgd v1.1.2 (https://github.com/arzwa/wgd, accessed 13 July 2023) and Para AT v2.0 (https://ngdc.cncb.ac.cn/tools/paraat, accessed 13 March 2023), and the results were visualized using Rstudio v4.20.

2.4. Transposon Element and the LTR-RT Analysis

Four types of transposon elements (TEs), including long terminal repeat (LTR), long interspersed element (LINE), short interspersed element (SINE) and DNA transposon element (DNA-TE), were predicted by RepeatModeler v2.0.2 (https://github.com/Dfam-consortium/RepeatModeler, accessed on 12 August 2023) and RepeatMasker v4.1.5 (https://www.repeatmasker.org/RepeatMasker/, accessed on 12 August 2023). First, the RepeatModeler software was used to construct a custom repeat library and then merge it with the repbase library. Repeat sequence annotation of the genome was then performed using RepeatMasker. LTR insertion times (LTR-RT) are identified and analyzed with the help of LTR_retriever v2.9.0 (https://github.com/oushujun/LTR_retriever, accessed on 12 August 2023).

2.5. Phylogenomic Analysis and Gene Family Variation Analysis

Phylogenetic analysis was performed to investigate the evolutionary relationships between Hericium strains and 37 other representative species of Basidiomycetes. Single-copy homologous genes were identified using OrthoFinder v2.5.5 with the parameters “-S diamond -M msa -T raxml-ng”. Divergence time prediction of 156 single-copy orthologous sequences from 40 strains was performed using the MCMCTree within PAML 4.9e (http://abacus.gene.ucl.ac.uk/software/paml.html, accessed on 12 March 2023). The calibrated points of several groups of recent ancestor divergence times were queried in TIMETREE 5 (http://www.timetree.org, accessed on 11 July 2023), including Ganoderma sinense vs. Grifola frondosa (84.2–135.2 MYA), and Laetiporus sulphureus vs. Gelatoporia subvermispora (137.2–164.3 MYA), Lyophyllum decastes vs. Tricholoma matsutake (90.6–118.1 MYA) and Paxillus involutus vs. Suillus brevipes (90.2–130.0 MYA). Gene family expansion and contraction were calculated using CAFÉ 4.2.1 (https://github.com/hahnlab/CAFE, accessed on 10 July 2023) with the identified orthologous gene families, which run as the following parameters: --cores 30 --fixed_lambda 0.0001.

2.6. CAZy Family, Microsatellite, and Cytochrome P450 Analyses

To annotate and classify genes encoding carbohydrate-active enzymes (CAZymes) from the genomes of three Hericium species and other edible fungi, the CAZy database (http://bcb.unl.edu/dbCAN2, accessed on 20 July 2023). A bubble plot of the CAZyme analysis was generated using the Complex Heatmap package in Rstudio v4.20.
Genome-wide microsatellites were identified using the Tandem Repeat Sequence Finder (TRF v4.09.1, https://github.com/Benson-Genomics-Lab/TRF, accessed on 20 July 2023) using default parameters. Simple sequence repeats (SSRs) with di- to hexa-nucleotide motifs were screened for marker development.
P450s were predicted and target protein sequences annotated using Diamond 2.1.8 (E-values < e−5) and the Hmmer v3.3.2 (filter parameter E-value < e−5; coverage > 0.35). The reference P450 sequences used for clustering analysis were obtained from the Fungal Cytochrome P450 Database (http://p450.riceblast.snu.ac.kr/index.php?a=view, accessed on 8 March 2022). For the phylogenetic tree analysis, 133 predicted P450 proteins from H. rajendrae NPCB A08 and several other Basidiomycota were selected from the fungal P450 database for clustering for accurate classification. Maximum likelihood trees were constructed using IQ-tree 2.2.2.6 (https://github.com/iqtree/iqtree2, accessed on 10 July 2023) with the options “-m MFP -bb 1000 -alrt 1000 -abayes -nt AUTO”.

2.7. Prediction and Cluster Analyses of Gene Clusters Involved in Secondary Metabolites

Prediction of biosynthetic gene clusters was accomplished with the fungal version of antiSMASH 7.0 (https://fungismash.secondarymetabolites.org/#!/start, accessed on 20 July 2023). Evolutionary tree-based cluster analysis is implemented via IQtree with the parameters described above. For a detailed analysis of multi-domain synthases such as NRPS and PKS, the package Synthaser [19] was used to analyze their domain characteristics. These structural domains include adenylation (A), acyl carrier protein (ACP), acyltransferase (AT), thiolation (T), thioesterase (TE), condensation (C), β-ketoacyl synthetase (KS), product template (PT), acyl carrier protein transacylase (SAT), thioesterase (TE), and thioester reductase (TR). The assessment of homology and similarity between two or more BGCs was performed using Clinker [20], which is based on the comparison of the sequence similarity of the encoded proteins. Visualization of the comparison results was achieved using clustermap.js [20], a tool embedded in Clinker to generate gene cluster comparison plots.

2.8. Metabolites Profiling of H. rajendrae NPCB A08

Five kg rice were divided into 100 1 L shake flasks and then 50 milliliters of sterile water was added. The mixture was soaked for two hours and then subjected to high pressure sterilization to prepare the culture medium (rice medium). After the strain was added to the culture medium, the culture was allowed to ferment at room temperature for 30 days. The culture was collected and subjected to three extractions with ten liters of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure, resulting in 76 g crude extract. Ten milligrams of the crude sample were subjected to high resolution mass spectrometry (HRMS) for GNPS (https://gnps.ucsd.edu) analysis, and the rest of the crude extract was separated by various chromatographic separation gears to obtain the monomer compounds. HRMS detection was performed using an AB Sciex TripleTOF 6600 mass spectrometer (AB Sciex, MA, USA) in positive ion modes. Molecular network analysis of HPLC-HRMS data of crude extracts was performed using GNPS (accessed on 17 July 2023) with default parameters. Finally, the molecular networks were visualized by Cytoscape 3.9.1. Details of the isolation of the monomeric compounds are described on the Supplementary Material. The structural characterization of the monomeric compounds was accomplished by high-resolution mass spectrometry and/or NMR spectroscopy, and the data were acquired via a Bruker Avance III 400 and 500 MHz NMR spectrometer, using TMS as an internal standard, with chemical shifts recorded in parts per million δ (ppm).

2.9. Data Availability

The ITS sequence of H. rajendrae NPCB A08 was registered in the NCBI GenBank under accession number OR646745 and the final genome assembly results and related data were submitted to NCBI under BioProject PRJNA1018320 and BioSample SAMN37432729, respectively. The network file based on positive-ion mode MS data can be found and accessed at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=7fbc2f60d1954567b8e592475c2ae3e2 (accessed on 31 July 2023). The NMR data of compounds 1, 4, 5, and 7 have been submitted to NP-MRD (Deposition ID: NPd000000335), and are available to download at this link: https://depositions.np-mrd,org/request-data/3424b45a-715d-4b7d-b57f-840e76057cf7 (accessed on 7 October 2023).

3. Results

3.1. Genome Sequence Assembly and Annotation of H. rajendrae NPCB A08

A pre-conducted genome survey based on Illumina sequencing indicated that the genome size of H. rajendrae NPCB A08 was less than 50 Mbp (Table S1). The presence of two peaks with a two-fold relationship in the K-mer curve indicated that the genome of H. rajendrae NPCB A08 was heterozygous, with a heterozygosity of 3.61% (Figure 1A, Table S1). This finding suggested that H. rajendrae was a dikaryon. The genome of H. rajendrae NPCB A08 was de novo assembled to 46.77 Mbp, which consists of fifteen pseudochromosomal molecules and four contigs, by combining Illumina sequencing data and nanopore sequencing data (Figure 1B, Table 1, and Tables S2–S4). The completeness of the genome assembly was assessed by a coverage of 99 95% (Table S5) and a BUSCO value of 91.6% (Table S5) based on the fungi_odb10 database.
The assembly quality of H. rajendrae NPCB A08 was superior to that of H. erinaceus CS-4 and H. coralloides FP101451 in terms of contig number and N50 (Table 1). There were 13,622 genes predicted by BRAKER, equipped with Augustus, which contain 13,418 protein-coding genes (Table S6), and 204 non-coding genes (Table S7). The BUSCO evaluation showed that 84.2% of the 13,418 coding genes were single-copy genes. These genes had an average length of 1715.42 bp, consisting of 93,499 exons and 80,081 introns, with average lengths of 182.74 bp and 74.07 bp, respectively (Table S8). In addition, 204 non-coding genes code 136 tRNAs, 38 rRNAs, 29 snRNAs, and one sRNA (Table S7). These findings provide insight into the genome structure of H. rajendrae.
To achieve comprehensive functional annotation of protein-coding genes, we conducted sequence similarity analysis and motif similarity search on a dataset consisting of 13,418 genes. Public databases such as NCBI nr (Figure S2), GO (Figure S3), COG (Figure S4), Uniprot, KEGG Pathway (Figure S5), Pfam (Figure S6), Refseq, and Interproscan were utilized for this purpose. Our analysis successfully annotated 11,716 genes (87.31%) at least once (Table S6). The annotation results obtained from the Nr library revealed that 9253 genes, accounting for 86.66% of all protein-coding genes, were annotated. Among these genes, 78.67% exhibited a significant match with H. alpestre, indicating a close relationship with Hericium species. Additionally, 10.77% showed similarity to Dentipellis fragilis, while 2.29% matched Bondarzewia mesentenica (Figure S2). This observation reflects the intergeneric variability of the H. rajendrae genome. These results emphasize the functional diversity of protein-coding genes in strain NPCB A08, as elucidated from various perspectives and levels of annotation.

3.2. Comparative Genomic Analysis within Hericium Species

Comparison of genome sizes indicates that the genome of H. rajendrae NPCB A08 is larger than that of H. erinaceus CS-4, but smaller than that of H. coralloides tvtc0002. However, the number of proteins encoded by H. rajendrae NPCB A08 is greater than that of both H. erinaceus CS-4, and H. coralloides tvtc0002 (Table 1). Collinearity analysis showed that the fifteen pseudochromosomes of H. rajendrae NPCB A08 have high homology with the fifteen chromosomes of H. erinaceus CS-4, but homologous relationships hardly exist on the same numbered chromosomes (Figure 1A). Further analysis showed that most genomic regions of these two Hericium species showed highly syntenic to that of H. rajendrae (Figure S7). Comparative analysis of orthologous genes among the three Hericium species identified a total of 7410 groups, meanwhile H. rajendrae NPCB A08 contains more unique orthologous groups (125) than that of H. erinaceus CS-4 (238) and H. coralloides tvtc0002 (86). The number of homologous genes shared between H. rajendrae NPCB A08 and H. erinaceus CS- 4 (1162) is much greater than that of H. coralloides tvtc0002 shared with H. rajendrae NPCB A08 (593) and H. erinaceus CS-4 (444), respectively (Figure 1C).
To gain further insight into these differences, a genome-wide duplication analysis based on nonsynonymous substitution rate (Ka) and synonymous substitution rates (Ks) were performed. The Ka/Ks curves of the three Hericium species were different. The trends of the Ka/Ks curves of H. coralloides and H. erinaceus were relatively close to each other, while the Ka/Ks curves of H. rajendrae were significantly different. This finding reflects that the three species have experienced different degrees of evolutionary selection pressure, while H. rajendrae has suffered more pure selection (Figure 1D).
Given the important role of transposon elements (TEs) in genomic constitute, TEs in the genus Hericium were used for further analysis. The content of four types of TE, LTR (Gypsy and Copia), LINE, SINE and DNA TEs, was investigated. The TE content in H. rajendrae (7.66% of the 46.77 Mb genome) was slightly higher than that identified in H. coralloides (6.05% of the 35.33 Mb genome) and H. erinaceus (5.76% of the 41.88 Mb genome), whereas it was much lower than that of Lactarius deliciosus 48 (23.70% of the 96.04 Mb genome), the control species (Figure 1E). Cross-genome comparisons showed that LTRs contributed the most to the TE expansion in the four taxa. H. rajendrae had 1462 Gypsy-LTRs, which were the dominant TEs and occupied 5.13% of the genome (Figure 1E). The continuous insertion of intact Gypsy- and Copia-LTRs in H. rajendrae since nearly one MYA was observed, and it had a distinct polymorphic distribution (Figure 1F). In the genera Hericium and L. deliciosus, the maximal bursts of LTRs were close to one MYA. However, a second burst of apparent LTR occurred in H. erinaceus, which occurred at approximately 16–17 MYA (Figure 1G).

3.3. Phylogenetic and Gene Family Variation Analysis

To shed light on the evolutionary history of H. rajendrae, a phylogenetic tree reconstruction and species divergence time estimation of 40 edible and medicinal fungi (Table S9), with Ustilago maydis as an outgroup, were conducted from 156 conserved single-copy orthologous proteins (Figure 2). The inferred phylogenetic tree was strongly supported by bootstrap values. The mean divergence time of Russulales containing the genus Hericium and the group containing Ployporales, Atheliales, and Gloeophyllales was 172.115 MYA with 95% highest posterior density (HPD) of 112.559–231.349 MYA. The emergence of the genus Hericium is estimated to have occurred at a crown age of 27.503 MYA (95% HPD of 15.258–42.530 MYA). Among the three species of the genus Hericium, H. rajendrae has the closest evolutionary affinity with H. erinaceus, with a divergence time of 16.115 MYA (95% HPD of 8.737–25.586 MYA), whereas that between H. rajendrae and H. coralloides was estimated to be 204.64 Mya (95% HPD of 125.66–348.17 MYA).
Further analysis based on reconstructed evolutionary trees revealed complex gene contraction and expansion events in 28,144 gene families in the genomes of these 40 species (Table S9). In the genus of Hericium, 108/42, 108/601, and 464/173 gene families were found to have undergone expansion/contraction in H. coralloides, H. erinaceus, and H. rajendrae, respectively. Among them, H. rajendrae had the most significant expansion events, affecting a total of 313 gene families (Figure 2).

3.4. CAZymes Analysis

CAZymes, as members of gene families, play a vital role in the genome. Specifically, CAZymes found in mushrooms have the ability to utilize cellulose- and lignin-rich substrates like wood chips and straw to obtain nutrients for their own growth and development [21,22]. Considering the nutritional value of H. rajendrae and its demand for large-scale cultivation, we conducted an investigation on its CAZymes. Our findings revealed the presence of 360 genes encoding 380 CAZymes in H. rajendrae. These included 186 glycoside hydrolases (GHs), 162 auxiliary activities (AAs), 60 glycosyltransferases (GTs), 35 glycoesterases (CEs), fourteen carbohydrate-binding modules (CBMs), and twelve polysaccharide lyases (PLs) (Figure 3A, Table S10). Notably, twenty genes, such as g1089, encoded enzymes with dual CAZyme domains. Comparison of the CAZyme profiles of H. rajendrae with those of H. coralloides and H. erinaceus did not reveal any significant differences (Figure 3A, Table S10). Furthermore, our analysis of 33 edible fungi’s CAZyme repertoires showed that the number and types of CAZymes were not species-specific. Among these fungi, the strains Lactarius deliciosus EDB83 exhibited the highest similarity to the genus Hericium in terms of their CAZyme profile (Figure 3A, Table S10, Dataset S1). Additionally, H. rajendrae, H. erinaceus, and H. coralloides possessed twenty, ten, and fifteen CAZymes with dual domains, respectively. Cluster analysis indicated a high level of relatedness among most of the CAZymes with dual domains (Figure 3A,B).

3.5. Cytochrome P450 Family Analysis

Cytochrome P450s (CYPs) are another important family of genes in organisms that play important roles in various biological processes. In fungi, CYPs are extensively involved in their primary and stimulated metabolic processes. To gain a deeper understanding of the number and types of CYPs in H. rajendrae, we used Pfam prediction based on structural domain features to screen them. In total, we identified 121 genes encoding 133 different CYPs. Through clustering analysis, we determined a clear classification of the P450s in H. rajendrae NPCB A08 by comparing the protein sequences of these 133 proteins with representative fungal P450 sequences from the Cytochrome P450 Database (Figure S8). These clustering results clearly illustrate the classification of P450s in H. rajendrae NPCB A08. The cluster analysis revealed the presence of nineteen CYP subfamilies and eight uncertain groups (Figure 4). Among the identified CYP families, the CYP5144 family has the largest number of members with 30, followed by CYP5037 (20) and CYP5139 (10), while the remaining subfamily members are all less than 10. In addition, the 30 P450s are scattered in eight ambiguous groups, accounting for 22.56% (Figure 5). These unidentified P450s indicate the presence of potential novel P450 types that require further analysis and identification.

3.6. Identification of the Mating Genes and SSR Marker Development

The development of mushroom-shaped fruiting bodies in basidiomycetes is a complex process, in which mating is a key step regulated by specific mating loci [23]. Mating loci (MAT) are located in different chromosomal regions on the genome. Heterozygous cooperation dominates the mating types of fungi, which can be divided into a bipolar mating type and tetrapolar mating type. Among them, the tetrapolar mating system is the most extensive and complex sexual reproduction control system discovered so far in Basidiomycetes [24]. Considering that the reasons for the formation of the fruiting bodies of Hericium mushrooms are still unknown, and the demand for cultivation of the H. rajendrae due to the huge market vacancies, it is necessary to analyze and characterize its mating system.
The matA locus of H. rajendrae was found to be located on Chr1 by a homologous sequence search with the mitochondrial intermediate peptidase (mip, HE02G000648) codon gene and a homeodomain transcription factor 2-codon gene (HD2, HE02G000668) of H. erinaceus CS-4 [10]. Genes on the matA locus encode a glycosyltransferase family 8 protein (glgen, g5593), an unknown conserved fungal protein (βfg, g5596) two HD transcription factors (HD1, g5598, and HD2, g5621), an mip (g5599) (Figure 5A), whereas the matB locus contains at least five unclustered ste3, including g3950, g3952, g3953, g3965, and g3966, located on Chr2 (Figure 5B). The corresponding coding products of the matA and matB loci between H. rajendrae NPCB A08 and H. erinaceus CS-4 showed high similarity (Figure 5A,B). Current analyses indicate that the matA and matB loci are not located on the same chromosome, suggesting the existence of a quadrupolar mating system in H. rajendrae. However, this finding only reveals the intricate genomic structure of H. rajendrae mating loci. Further investigations are needed to fully understand sexual reproduction and fruiting body formation in H. rajendrae.
Microsatellite markers are an important molecular breeding tool that will have potential application in screening for elite varieties of H. erinaceus [10]. Therefore, microsatellite sequences in the genome of H. rajendrae were characterized and developed as genetic markers. Typical SSRs with repeat sequence lengths between one and six bp were searched on the genome of H. rajendrae, and a total of 1448 typical SSRs were found. The total length of SSRs was about 25.18 kb, accounting for 0.054% of the total genomic length. Among them, tri-nucleotide SSRs were the most abundant repeat type, accounting for 49.59% of the total repeats, followed by di-nucleotide (23.90%) and mono-nucleotide (17.75%) SSRs. Further comparisons showed that the SSR traits of H. rajendrae were highly similar to those reported for H. erinaceus [10] and predicted for H. coralloides (Figure 5C, Table S11).

3.7. The BGCs for Secondary Metabolite Analysis

In view of the long edible history and significant medicinal value of H. rajendrae, we analyzed its biosynthetic potential of secondary metabolites. The genome of the strain NPCB A08 was predicted using the webtool antiSMASH, and 24 secondary metabolite biosynthetic gene clusters (BGCs) containing 31 core genes were discovered (Table 2). The core genes consisting of eleven terpenoid synthase, a non-ribosomal peptide synthase (NRPS) and 16 NRPS-likes enzymes, a polyketide synthase (PKS), and two ribosomally synthesized and post-translationally modified peptide like enzymes (RiPP-likes) are distributed on nine chromosomes (Chr2–6, 9–12, 14, and 15) (Figure 6A, Table 2).
Among the eleven terpenoid synthesis-related enzymes (Table S12), there are eight sesquiterpene synthases (STSs). Cluster analysis of STSs from the strain NPCB A08 and identified STSs from the basidiomycete revealed eight STSs belonging to Clade I, II, and IV, and no members belonging to Clade III (1,11-cyclization of (2E, 6E)-FPP) (Figure 6B, Figure S9). Similarly, the FPPs of H. erinaceus and H. coralloides do not have members of Clade III. In the case of H. rajendrae, Clade II contained four FPPs, while Clade I and III each contain two FPPs (Figure 6B). Three enzymes associated with triterpene synthesis, squalene oxidase (g11649.t1), squalene synthase (g12602.t1), and lanosterol synthase (g12602.t1) were identified sequence similarity comparison (Table S12).
Cluster analysis of the 16 NRPS-like enzymes revealed that g9771.t1 and g7538.t1 were in relative outgroup (Figure 6C). Further structural compositional analysis revealed that these two enzymes predicted by antiSMASH to be NRPS-like were labeled NRPS_PKS and NRPS, respectively, because the former contains a KS domain at the C-terminus and the latter contains a C domain at the N-terminus. The other 14 NRPS-like enzymes were uniformly characterized by A-T-TR domains (Figure 6D).
The PKS g84366.t1 shows almost 100% identify with HE04T003470.1, an identified orsellinic acid synthase (OAS) from H. erinaceus [25]. Hercor1_45461 from H. coralloides is another predicted OAS, which displays over 80.00% identify with g84366.t1 and HE04T003470.1. The identifies of these three OASs with other identified OASs of basidiomycetes ranged from 40.00% to 80.00% (Figure 6E). Domain analysis reflects that these OASs share almost the same domain composition (SAT-KS-AT-PT-ACP-ACP-TE), except for an additional ACP domain in PKS2 (Figure 6F). These OASs are speculated to synthesize OA using acetyl-coenzyme A and malonyl-coenzyme A as substrates (Figure 6G). Cluster 5 contains not only g8436.t1 and 2 NRPS-likes (g8434.t1 and g8441.t1), but also multiple postmodifying enzymes, which suggests that this BGC may encode structurally complex products (Figure 6G). Cluster 17 (g7538.t1) and cluster 20 (g9771.t1) each contain an unusual NRPS-like and several postmodifying enzymes, and it is worthwhile to investigate their products (Figure 6G).

3.8. Secondary Metabolic Profiling and Putative Biosynthesis for Erinacines

H. rajendrae, a rare species of the genus Hericium, has been poorly studied in terms of its metabolites. Considering the high genomic similarity among H. rajendrae, H. erinaceus and H. coralloides, the secondary metabolites of H. rajendrae were investigated by HPLC-HRMS with the help of GNPS (Figure S10). The structures of partial monomeric compounds were confirmed by NMR analysis. A total of fifteen compounds were identified by comparison of their MS and MS2 characteristics with the previous literatures, including erinacine A (1), erinacine B (2), erinacine G (3), erinacine E (4), erinacine F (5), erinacine T (6), erinacine Z1 (7), 8, Hericinoid B (9), CJ-14,544 (10), CP-412,065 (11), and Hericinoid C (12) (Table 3, Figure 7 and S10). All of these compounds are cyathane-type diterpenoids. Among them, 1, 4, 5, and 7 were further confirmed by NMR (Table S13, Figures S11–S15). Almost all of these compounds identified in H. rajendrae were found in H. erinaceus [2]. Compounds 1, 2, 4 and 5 have been reported in H. flagellum, a synonym specimen of H. alpestre [5].
The biosynthetic pathway for erinacines derived from H. erinaceus has been elucidated [32,33], and in view of the high similarity of the genomes of the three Hericium species and the convergence of the metabolites of H. erinaceus and H. rajendrae, it is speculated that the BGCs for erinacines are also present in the genomes of H. rajendrae and H. coralloides. Using the BGC for erinacines from H. erinaceus (yamabushitake Y2) as a clue [32], the BGCs for erinacines were identified from H. rajendrae and H. coralloides. The sequence identity of the protein encoded by the corresponding gene on the BGC is not less than 67% (Figure 8A). The enzymes encoded by g9677 and g9585 are responsible for the synthesis of geranylgeranyl diphosphate (GGPP) and the cyclization of GGPP to the cyathane diterpene backbone cyatha-3,12-diene. The three CPYs then sequentially hydroxylate C14 (g9586.t1), the methyl group of C12 (g9580.t1), and C11 to form cyathatriol. The C11 of cyathatriol then undergoes acylation to form 11-O-acetyl cyathatriol. 11-O-acetylcyathatriol is catalyzed by the rare xylosyltransferase (g9587.t1) to the cyathane xyloside, erinacine Q, which is then catalyzed by postmodifier proteins (g1840.t1 and g9579.t1) and non-enzymatic catalysts to form 1, 2, and possibly erinacine C (Figure 8B).

4. Discussion

The genus Hericium is a well-known taxon in the order Russulales because of its specialized morphology, wide range of biological activities, and valuable nutritional value. In this study, we report the genome of a wild rare Hericium species, H. rajendrae, from the Qinling Mountains, which is the first time that the genome of H. rajendrae is reported at the chromosome level. Due to recent advances in sequencing technology, the assembly and annotation quality of H. rajendrae is superior to those of previously reported Hericium species (Table 1). Genomic synteny (Figure 1B), LTR insertion time (Figure 1G), and CAZymes (Figure 3A) analysis revealed interspecific convergence among Hericium species, whereas comparative genomic analysis based on orthologous groups revealed that H. rajendrae contains more unique genes (Figure 1C) and Gypsy-LTRs (Figure 1F). Analysis of contraction and expansion based on single-copy immediate homologous genes showed that H. rajendrae possessed a much higher number of expansions than those of H. erinaceus and H. coralloides (Figure 2), which may be related to the presence of a large number of Gypsy-LTRs present in H. rajendrae (Figure 1E). These results provide valuable insights into the complexity of the genome of the genus Hericium.
Considering the rarity of the source of H. rajendrae, artificial domestication and subsequent large-scale cultivation are inevitable. Therefore, it is meaningful to analyze and characterize the mating system of H. rajendrae. SSR-based development of molecular breeding markers in a variety of edible mushrooms including Agaricus bisporus [34], Lentinula edodes [35], Flammulina velutipes [36], and Pleurotus tuoliensis [37] has gained in-depth and extensive research, which is an important contributor to the large-scale cultivation of edible mushrooms. SSR analysis of the genome of H. rajendrae shows diversity of repetitive sequences. The investigation of these repetitive sequences contributes to the development of molecular markers for the H. rajendrae genome.
Mushrooms exert certain well-known pharmacological effects by producing specific types of biologically active secondary metabolites. For example, ganoderic acids demonstrate immunomodulatory activity [38], and Styrylpyrone compounds derived from the genera Inonotus [39] and Phellinus [40] show antioxidant properties. The anti-neurodegenerative activity of H. erinaceus is closely associated with the production of erinacines [3] and hericenones [2]. While the biosynthesis for erinacines has been identified in Hericium erinaceus [32,33], this study suggests that their BGCs also exist in H. rajendrae and H. coralloides (Figure 8). Given the promising medicinal value of erinacines, the identification of the BGCs for erinacines in H. rajendrae and H. coralloides is more significant. Hericenones are a class of OA-containing meroterpenoids, and the OAS from Hericium erinaceus have been uncovered [25]. Similarly, the OAS in H. rajendrae and H. coralloides were also identified in this study (Figure 6E,F). Considering the scarcity of sources of these compounds, the reconstruction of their biosynthetic pathways using Aspergillus oryzae to achieve high production [25,32] shows remarkable application value.

5. Conclusions

Hericium rajendrae is a rare medicinal edible mushroom with multiple biological activities and nutritional value. In this study, we present, for the first time, the complete genome of H. rajendrae at the chromosomal level. Comparative genomic analysis revealed the sequence similarities and compositional differences in the genome among Hericium species, and phylogenetic and evolutionary analysis revealed the divergence time of the Hericium genus and the variations in their gene families. The well-assembled genome and functional annotation provided important clues for the study of mating loci, CAZymes, and SSRs in this wild mushroom, contributing to its artificial cultivation and elite strain breeding. Cluster analysis reflected the diversity of CYPs in H. rajendrae and the convergence of FPPs in the Hericium genus. Core genes-based investigations reflected the biosynthetic diversity of secondary metabolites in Hericium species. Metabolite profiling based on molecular networks and gene cluster comparisons revealed the BGC for erinacines in H. rajendrae and inferred the biosynthetic pathway of erinacines in H. rajendrae. This study not only provides a high-quality H. rajendrae genome for the first time, but also delves into genomic characteristics and functional elements, including mating genes and biosynthesis core genes. These findings enrich the genomic content of the Hericium genus and will contribute to the development and utilization of H. rajendrae as alternative medicine agents and functional foods.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof9101018/s1. Table S1. Estimation of genome size of H. rajendrae NPCB A08; Table S2. Statistics of Illumina NovaSeq sequencing data mapping of H. rajendrae NPCB A08 genome; Table S3. Statistics of Oxford Nanopore PromethION sequencing data volume of H. rajendrae NPCB A08 genome; Table S4. Statistics on assembly results; Table S5. Statistics of BUSCO evaluation of H. rajendrae NPCB-A08 genome; Table S6. Statistics of H. rajendrae NPCB A08 protein-coding gene annotation; Table S7. Statistics of non-coding RNA annotation results in H. rajendrae NPCB-A08 genome; Table S8. Annotation Statistics of coding gene from the H. rajendrae NPCB-A08 genome; Table S9. Species genomes for evolutionary inference and gene family variation. Table S10. The composition of CAZymes of edible mushrooms; Table S11. Number and relative abundance of SSRs identified in the genus Hericium; Table S12. Terpenoid biosynthesis related enzymes in H. rajendrae NPCB A08 genome; Table S13. 1H and 13C NMR Spectroscopic data of compounds 1, 4, 5, and 7; Figure S1. ITS alignment of the strain NPCB A08; Figure S2. Species distribution of the top 10 homologous sequences using the annotation results from the Nr database; Figure S3. Statistical map of functional annotation classification based on GO database; Figure S4. Statistical Chart of COG Functional Annotated Classification; Figure S5. KEGG Pathway Functional Classification Chart; Figure S6. Domain annotation based on the Pfam database; Figure S7. Comparative genome and genomic collinearity analysis among the genus Hericium; Figure S8. P450s Cluster analysis of the strain NPCB-A08 and other Basidiomycetes; Figure S9. FPPs Cluster analysis of the strain NPCB-A08 and other Basidiomycetes; Figure S10. Molecular network analysis of metabolites from the mycelium and fruiting bodies of the strain NPCB-A08; Figure S11. The LC-ESI-HRMS and LC-ESI-HRMS/MS spectrums of isolates from the strain NPCB A08; Figure S12. The NMR spectrums of compound 1 (MeOD) from the strain NPCB A08; Figure S13. The NMR spectrums of compound 4 (MeOD) from the strain NPCB A08; Figure S14. The NMR spectrums of compound 5 (MeOD) from the strain NPCB A08; Figure S15. The NMR spectrums of compound 7 (MeOD) from the strain NPCB A08; Dataset 1. CAZymes of 33 mushrooms; Dataset 2. SSR.

Author Contributions

Conceptualization and funding acquisition, J.W. and J.Q.; methodology, software, validation, and investigation, J.W., M.C., K.C., X.W. and J.Q.; data curation and writing—original draft preparation, J.W., M.C. and X.W.; writing—review and editing, M.C., Y.Z. and J.Q.; visualization, J.-f.Z. and J.Q. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Key R&D Projects in Shaanxi Province of China (No. 2023-YBSF-164), the Educational Commission of Shaanxi Province (20JY009), the Specialized Scientific Research Fund Projects of Academician Shengyong Zhang (18YSZX004), the doctoral research funding (17SKY013) from Shangluo University, the Youth Talent Promotion Program of Shaanxi University Association for Science and Technology (20200208), and Shaanxi Science and Technology Innovation Team (2022TD-56).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Wenbing Gong from the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, for providing the genomic annotation files of H. erinaceus CS-4.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Genomic characterization and comparative genomic analysis. (A) Genomic collinearity analysis between H. rajendrae NPCB A08 and H. erinaceus CS-4. From the outside to the inside are I. Chromosome and Contigs; II–IV. GC-density, GC-skew, AT-skew (window size 10 kb), V. Gene-density (window size 100 kb), VI. Whole-genome collinearity analysis based on protein-coding genes between H. rajendrae NPCB A08 and H. erinaceus CS-4. (B) K-mer assessment curves for genome size and heterozygosity. (C) Venn schematic of comparative genomes within the genus Hericium. (D) Ka/Ks comparison within the genus Hericium. (E) Comparison of TE families in the genus Hericium and L. deliciosus. MYA indicates million years ago. (F) Insertion bursts of Gypsy and Copia elements in H. rajendrae NPCB A08. (G) Comparison of temporal patterns of intact LTR insertion bursts in the four taxa.
Figure 1. Genomic characterization and comparative genomic analysis. (A) Genomic collinearity analysis between H. rajendrae NPCB A08 and H. erinaceus CS-4. From the outside to the inside are I. Chromosome and Contigs; II–IV. GC-density, GC-skew, AT-skew (window size 10 kb), V. Gene-density (window size 100 kb), VI. Whole-genome collinearity analysis based on protein-coding genes between H. rajendrae NPCB A08 and H. erinaceus CS-4. (B) K-mer assessment curves for genome size and heterozygosity. (C) Venn schematic of comparative genomes within the genus Hericium. (D) Ka/Ks comparison within the genus Hericium. (E) Comparison of TE families in the genus Hericium and L. deliciosus. MYA indicates million years ago. (F) Insertion bursts of Gypsy and Copia elements in H. rajendrae NPCB A08. (G) Comparison of temporal patterns of intact LTR insertion bursts in the four taxa.
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Figure 2. Phylogeny and gene family variation. The evolutionary relationship and expanded and contracted gene families among Hericium species and 37 representative medicinal Basidiomycetes. The maximum likelihood method credibility tree was inferred from 40 single-copy orthologous genes. All nodes received full bootstrap support. The divergence time is labeled as the mean crown age for each node, while the 95% highest posterior density is also given within the Hericium clade. The black numbers at the branches indicate the corresponding divergence times in MYA. The proportion of expansion and contraction in the genome of each species was displayed before its species name.
Figure 2. Phylogeny and gene family variation. The evolutionary relationship and expanded and contracted gene families among Hericium species and 37 representative medicinal Basidiomycetes. The maximum likelihood method credibility tree was inferred from 40 single-copy orthologous genes. All nodes received full bootstrap support. The divergence time is labeled as the mean crown age for each node, while the 95% highest posterior density is also given within the Hericium clade. The black numbers at the branches indicate the corresponding divergence times in MYA. The proportion of expansion and contraction in the genome of each species was displayed before its species name.
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Figure 3. CAZymes analysis of Hericium and related edible mushrooms. (A) Composition comparison of CAZymes among 33 edible fungi including H. rajendrae NPCB A08. (B) Clustering analysis of CAZymes with bi-domains of Hericium mushrooms.
Figure 3. CAZymes analysis of Hericium and related edible mushrooms. (A) Composition comparison of CAZymes among 33 edible fungi including H. rajendrae NPCB A08. (B) Clustering analysis of CAZymes with bi-domains of Hericium mushrooms.
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Figure 4. Maximum likelihood tree of 133 cytochrome P450s from H. rajendrae NPCB A08. Each cytochrome P450 family is shown in a separate color, and the branch reliability value of over 50 is marked on the corresponding branch node.
Figure 4. Maximum likelihood tree of 133 cytochrome P450s from H. rajendrae NPCB A08. Each cytochrome P450 family is shown in a separate color, and the branch reliability value of over 50 is marked on the corresponding branch node.
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Figure 5. Identification of the mating genes and comparison of SSR abundance of Hericium species. Structural diagram of the genes on the matA locus (A) and matB locus (B) of H. rajendrae, the numbers on the similarity diagrams indicate the identity between corresponding genes. (C) Relative abundance of six type SSRs in the genus Hericium.
Figure 5. Identification of the mating genes and comparison of SSR abundance of Hericium species. Structural diagram of the genes on the matA locus (A) and matB locus (B) of H. rajendrae, the numbers on the similarity diagrams indicate the identity between corresponding genes. (C) Relative abundance of six type SSRs in the genus Hericium.
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Figure 6. Analysis of genes involved in secondary-metabolite biosynthesis. (A) Distribution of biosynthetic core genes for natural products on the chromosomes, (B,D) phylogenetic tree analysis for STSs (B) and NRPS-likes (D), (C,F) domain analysis of NRPS-likes (C) and OASs (F), (E) percent identity matrix of seven OASs of Basidiomycota, an asterisk indicates that the function of the corresponding gene is predicted, (G) schematic representation of OA biosynthesis, and (H) schematic diagram of the composition of postulated clusters 5, 17, and 20.
Figure 6. Analysis of genes involved in secondary-metabolite biosynthesis. (A) Distribution of biosynthetic core genes for natural products on the chromosomes, (B,D) phylogenetic tree analysis for STSs (B) and NRPS-likes (D), (C,F) domain analysis of NRPS-likes (C) and OASs (F), (E) percent identity matrix of seven OASs of Basidiomycota, an asterisk indicates that the function of the corresponding gene is predicted, (G) schematic representation of OA biosynthesis, and (H) schematic diagram of the composition of postulated clusters 5, 17, and 20.
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Figure 7. GNPS-based molecular network identification of metabolites from the fruiting bodies of H. rajendrae.
Figure 7. GNPS-based molecular network identification of metabolites from the fruiting bodies of H. rajendrae.
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Figure 8. Biosynthesis of erinacines from the genus Hericium. (A) Comparison of BGCs for erinacines from the three Hericium species. (B) Proposed biosynthetic pathway of erinacines from H. rajendrae. GGPP synthase indicates geranylgeranyl diphosphate synthase, GGPP cyclase indicates geranylgeranyl diphosphate cyclase. UDP-GT indicates glycosyltransferase.
Figure 8. Biosynthesis of erinacines from the genus Hericium. (A) Comparison of BGCs for erinacines from the three Hericium species. (B) Proposed biosynthetic pathway of erinacines from H. rajendrae. GGPP synthase indicates geranylgeranyl diphosphate synthase, GGPP cyclase indicates geranylgeranyl diphosphate cyclase. UDP-GT indicates glycosyltransferase.
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Table 1. Genomic comparison within Hericium species.
Table 1. Genomic comparison within Hericium species.
EntryH. rajendrae
NPCB A08		H. erinaceus
CS-4		H. coralloides
FP101451
Sequencing technologyIllumina NovaSeq 6000
Nanopore PromethION		PacBio RSII,
Illumina Hiseq X-Ten		PacBio
Sequencing depth118×750×122×
No. of contig1952125
No. of chromosome1515NA
Total length (bp)46,767,96541,880,34055,905,675
Largest length (bp)5,307,7526,077,0301,366,710
Contig N50 (bp)3,238,8773,208,415711,881
BUSCO (%)91.696.497.5
Heterozygosity (%)3.610.02260.847
GC content (%)52.5752.3053.64
No. of protein-coding genes13,41810,62012,369
GenBank accession No.PRJNA1018320GCA_006506795.2JGI_Hercor1
ReferencesThis study[10][18]
NA indicates not available.
Table 2. Putative BGCs responsible for secondary metabolites in the strain NPCB A08.
Table 2. Putative BGCs responsible for secondary metabolites in the strain NPCB A08.
Cluster No.LocationStart (bp)End (bp)Core Gene IDCore Gene Type
1Chr2860,561963,214g3046.t1NRPS-like
g3050.t1NRPS-like
g3052.t1NRPS-like
2Chr22,123,6062,187,764g3442.t1NRPS-like
3Chr3228,685291,856g7981.t1NRPS-like
4Chr3318,507352,028g8004.t1terpene
g8005.t1terpene
5Chr31,992,9542,074,528g8434.t1NRPS-like
g8436.t1T1PKS
g8441.t1NRPS-like
6Chr426,21552,908g11649.t1terpene
7Chr41,486,1211,513,187g12043.t1terpene
8Chr43,476,8343,542,535g12602.t1terpene
g12610.t1NRPS-like
9Chr5493,923554,595g177.t1NRPS-like
10Chr5674,234733,369g226.t1NRPS-like
11Chr5868,813933,044g285.t1NRPS-like
12Chr51,747,2641,775,280g362.t1terpene
13Chr52,071,0282,102,333g468.t1terpene
14Chr52,804,2022,824,688g741.t1NRPS
15Chr6198,427229,557g10218.t1terpene
16Chr9354,078414,897g7354.t1NRPS-like
17Chr91,012,1501,076,675g7538.t1NRPS-like
18Chr101,369,6431,433,801g1354.t1NRPS-like
19Chr11197,542257,730g9668.t1fungal-RiPP
g9673.t1fungal-RiPP
20Chr11492,774559,259g9771.t1NRPS-like
21Chr12460,518492,887g11367.t1terpene
22Chr141,503,0131,567,208g7114.t1NRPS-like
23Chr15211,721g2467.t1terpene
24Chr15115,836146,976g2512.t1terpene
Table 3. The identified secondary metabolites from H. rajendrae NPCB A08.
Table 3. The identified secondary metabolites from H. rajendrae NPCB A08.
No.Putative MetaboliteMolecular FormulaAdductm/zReference
1Erinacine AC25H36O6[M + H]+433.258[26]
2Erinacine BC25H36O6[M + H]+433.258[26]
3Erinacine GC25H36O8[M + H]+465.285[27]
4Erinacine EC25H36O6[M + Na]+455.243[27]
5Erinacine FC25H36O6[M + Na]+455.243[27]
6Erinacines TC25H38O7[M + Na]+473.269[28]
7Erinacine Z1C25H38O7[M + Na]+473.269[5]
88C26H38O5[M + H]+431.242[29]
9Hericinoid BC26H36O6[M + H]+445.197[30]
10CJ-14,544C25H34O6[M + H]+431.182[31]
11CP-412,065C25H34O6[M + H]+431.182[30]
12Hericinoid CC25H34O7[M + K]+485.577[30]
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MDPI and ACS Style

Wei, J.; Cheng, M.; Zhu, J.-f.; Zhang, Y.; Cui, K.; Wang, X.; Qi, J. Comparative Genomic Analysis and Metabolic Potential Profiling of a Novel Culinary-Medicinal Mushroom, Hericium rajendrae (Basidiomycota). J. Fungi 2023, 9, 1018. https://doi.org/10.3390/jof9101018

AMA Style

Wei J, Cheng M, Zhu J-f, Zhang Y, Cui K, Wang X, Qi J. Comparative Genomic Analysis and Metabolic Potential Profiling of a Novel Culinary-Medicinal Mushroom, Hericium rajendrae (Basidiomycota). Journal of Fungi. 2023; 9(10):1018. https://doi.org/10.3390/jof9101018

Chicago/Turabian Style

Wei, Jing, Min Cheng, Jian-fang Zhu, Yilin Zhang, Kun Cui, Xuejun Wang, and Jianzhao Qi. 2023. "Comparative Genomic Analysis and Metabolic Potential Profiling of a Novel Culinary-Medicinal Mushroom, Hericium rajendrae (Basidiomycota)" Journal of Fungi 9, no. 10: 1018. https://doi.org/10.3390/jof9101018

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