RNA-Seq de Novo Assembly and Differential Transcriptome Analysis of Chaga (Inonotus obliquus) Cultured with Different Betulin Sources and the Regulation of Genes Involved in Terpenoid Biosynthesis

Abstract

Chaga (Inonotus obliquus) is a medicinal fungus used in traditional medicine of Native American and North Eurasian cultures. Several studies have demonstrated the medicinal properties of chaga’s bioactive molecules. For example, several terpenoids (e.g., betulin, betulinic acid and inotodiol) isolated from I. obliquus cells have proven effectiveness in treating different types of tumor cells. However, the molecular mechanisms and regulation underlying the biosynthesis of chaga terpenoids remain unknown. In this study, we report on the optimization of growing conditions for cultured I. obliquus in presence of different betulin sources (e.g., betulin or white birch bark). It was found that better results were obtained for a liquid culture pH 6.2 at 28 °C. In addition, a de novo assembly and characterization of I. obliquus transcriptome in these growth conditions using Illumina technology was performed. A total of 219,288,500 clean reads were generated, allowing for the identification of 20,072 transcripts of I. obliquus including transcripts involved in terpenoid biosynthesis. The differential expression of these genes was confirmed by quantitative-PCR. This study provides new insights on the molecular mechanisms and regulation of I. obliquus terpenoid production. It also contributes useful molecular resources for gene prediction or the development of biotechnologies for the alternative production of terpenoids.

1. Introduction

Inonotus obliquus, a member of the Hymenochaetaceae family of Basidiomycetes, is a medicinal fungus used in the traditional medicine of Native American and North Eurasian cultures. I. obliquus, commonly known as chaga or black diamond, is used by some Canadian First Nations to treat infections and tumors. Chaga is a black parasitic fungus that grows on the living trunks of the northern latitudes of America, Europe and Asia, and it is the primary pathogen of birch and other Betulaceae trees [1]. In nature, instead of a fruiting body, it usually forms an irregular shape of a sclerotial conk with the appearance of burnt charcoal, and it has been used for centuries as a folk medicine to treat and prevent multiple diseases. Over the last decade, studies have revealed that the extract of I. obliquus contains biologically active molecules derived from the specialized metabolism, supporting the effectiveness of chaga in traditional medicine. These specialized metabolites (e.g., polysaccharides, polyphenols and terpenoids) are responsible for I. obliquus’s medicinal effects including those of the antioxidant [1,2,3,4,5,6,7,8,9,10,11], antibacterial [1,7,12,13], anti-diabetic [14,15,16,17,18,19], and anticancer [16,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37] types.

Terpenoids consist of a large and diverse class of chemicals among the multitude of metabolites produced by fungi. Fungi employ terpenoids for an array of primary functions (growth and development) and for specialized activities, chemical interactions, and protection against abiotic and biotic factors. I. obliquus produces a diverse range of bioactive terpenoids exhibiting compelling therapeutic activity (e.g., anti-tumor, anti-inflammatory). Among the bioactive terpenoids found in chaga, sesquiterpenoids (e.g., bergamotene, selinene, and santalene) and triterpenoids (e.g., betulin, betulinic acid, lanosterol, inotodiol, and trametenolic acid) have been identified [1,35,38,39,40,41,42,43].

In addition, I. obliquus is able to catabolize metabolites from its birch host to grow and develop. White birch bark contains large amount of triterpenoids including betulin and betulinic acid, which represent up to 75% and 2% of the extractives, respectively [44,45]. It is not clear if betulin and betulinic acid detected in I. obliquus sclerotia result only from fungal de novo synthesis, from the transformation of birch terpenoid metabolites, or from both. It has been shown that I. obliquus cells are able to produce these triterpenoids in a submerged culture, suggesting the presence of genes encoding biosynthetic enzymes involved in their de novo production [46]. However, the identification and regulation of these genes remain unknown.

In fungi, terpenoid biosynthesis occurs via the mevalonate (MVA) metabolic pathway which can be divided into three parts (Appendix A—Figure A1). The first part (precursor pathway) involves a series of enzymatic reactions converting acetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the precursors of all terpenoids. For the second part (the sesquiterpenoid pathway), the cytosolic farnesyl diphosphate synthase (FPS) condenses two molecules of IPP with one molecule DMAPP head-to-tail sequentially to produce farnesyl diphosphate (FPP). FFP serves as a precursor for sesquiterpenoids, which are synthesized by terpene synthases and can be processed by other assorted enzymes. Lastly, the third part of the pathway involves reactions that catalyze the formation of three groups of triterpenoids: The amyrin-type, the lanosterol-type and the lupeol-type.

In recent years, high throughput sequencing platforms such as the Illumina RNA-Seq have been developed to study the transcriptome of model and non-model species, including plants, animals and microorganisms. This advanced sequencing technology has proven to be a cost-effective, rapid and powerful tool for investigating new functional genes and for detecting differentially expressed genes in diverse species. Currently, there is no genome information available for the Inonotus species; hence, the molecular basis of terpenoid production has not yet been elucidated. The aim of this study was to use de novo transcriptomic to generate a comprehensive profile of the genes related to terpenoid production in an I. obliquus cell culture supplemented or not with terpenoid substrates. Moreover, a better understanding of the metabolic pathway for the synthesis of I. obliquus pharmaceutically-relevant metabolites such as betulinic acid, betulin and inotodiol will enable tools to increase the production of these valued metabolites. Chaga’s host, i.e., white birch (Betula papyrifera), also produces betulin in high concentrations [47,48]. It has been reported that the use of microorganisms isolated from the same sources as the host substrate would increase the probability of obtaining derivatives of this substrate using the bioconversion approach [49]. Related to that, the co-culture of chaga with white birch bark residues could increase the conversion of betulin trapped in birch bark into betulinic acid to valorize forestry residues on one side, and, on the other side, it could elucidate the relationships between chaga and white birch bark.

In this study, the optimal growth conditions of I. obliquus cultured with or without betulin or white birch bark were investigated, and RNA was isolated. A cDNA library was then generated, and a whole transcriptomic analysis was performed using the Illumina HiSeq 4000. Whole-transcriptome was assembled de novo and analyzed, and differential expression studies confirmed by qRT-PCR unveiled the genes responsible for the terpenoid pathways in chaga.

2. Results and Discussion

2.1. Optimal Growth Conditions

The sclerotia of I. obliquus grow very slowly in nature, and artificial culture is difficult. To produce large amounts of biomass, I. obliquus was cultured using different growth conditions. Mycelial growth tests were carried out on a culture medium to determine optimal conditions. The growth of the I. obliquus mycelium was conducted under nine conditions (three different pH (5, 6.2 and 7.5) and three different temperatures (22, 28, and 37 °C) over 16 days in a solid medium. Figure 1 shows the results for two temperatures because at 37 °C, no growth of I. obliquus mycelium was observed (data not shown). At 28 °C, I. obliquus growth was significantly higher for all tested pH; however, pHs 5 and 6.2 showed the best results (Figure 1). The effect of temperature on chaga’s growth was only observed at pH 5. For example, on day 16 at pH 5 in the solid culture, fungal growth at 28 °C was twice the growth observed at 22 °C (Figure 1; from 12 mm at 22 °C to more than 24 mm at 28 °C). However, the effect of pHs 5 or 6.2 on fungal growth at 28 °C was not significantly different (Figure 1; circa 24 mm for both pH).

In the wild, the growth and production of terpenoids by I. obliquus is likely influenced by substrates provided by its host. Thus, an investigation to determine the influence of white birch bark (WBB) on growth of I. obliquus under optimal pH and temperature conditions was carried out. For this purpose, I. obliquus cells were grown in presence or absence of WBB residues. Results showed that I. obliquus growth was significantly higher in presence of WBB (Figure 2). Specifically, after 16 days of co-culture, the I. obliquus control mycelia reached 23 mm diameter, whereas those that were WBB-treated amounted to 27 mm (Figure 2). This indicates that I. obliquus growth was stimulated by the presence of bark residues from its host.

The yield of fungal biomass is often higher in a liquid culture compared to a solid culture. Furthermore, a liquid culture is easier to manipulate in order to extract and obtain cell components such as metabolites, proteins or nucleic acids. Thus, the effect of pH on the kinetics of mycelial growth of I. obliquus in a liquid medium at 28 °C was investigated. A rapid increase in the biomass was detected within the first six days, concurrent with the cells being in the exponential growth phase with significant consumption of carbon. On day eight, the mycelial biomass reached the apex, and the optimal growth of I. obliquus was observed at pH 6.2 with 0.18 g/100 mL of mycelial dry weight compared to 0.10 g/100 mL at pH 5 and 0.14 g/100 mL at pH 7.5 (Figure 3).

By-products of metabolism are shuttled throughout the cell and utilized to grow. Thus, optimal growth may influence the production of terpenoids [46]. In this study, different culturing conditions were used to establish the best possible parameters for I. obliquus growth. According to the results, the best condition for I. obliquus growth is at pH 6.2 and at 28 °C in a liquid culture that continuously shaken at 150 rpm. These results are supported by other studies on I. obliquus reporting similar optimal growth conditions [46,50,51]. The results of growth in the liquid and solid culture medium support that pH 6.2 would be the optimal pH for optimal mycelium growth.

For subsequent analyses, an I. obliquus liquid culture grown at 28 °C and pH 6.2 were used, since they showed optimal growth and morphological properties. In addition, it provided an easier system for the collection of cells for RNA extraction.

2.2. Illumina Sequencing and de Novo Assembly

To better understand the molecular mechanisms underlying the differences in chaga’s transcriptome caused by the presence of terpenoid substrates, RNA-Seq was performed. RNA was extracted from three biological replicates of cultured I. obliquus in three different conditions: In the absence (control, n = 3) or presence of the terpenoid substrate betulin (BET, n = 3) or white birch bark (WBB, n = 4). Ten corresponding cDNA libraries were generated and sequenced using Illumina HiSeq 4000 PE100. The raw reads for each library were deposited to the NCBI Sequence Read Archive under the accession PRJNA526077 (Appendix A—

Table A1. Results of Illumina sequencing output and assembly for Inonotus obliquus.

Read Trimming and Clipping of Adapters
Raw paired reads a	229,970,255
Surviving paired reads b	219,288,500
Surviving paired reads (%) c	95.36
Trinity de novo Assembly
Nb. Transcripts d	196,273
Nb. Components d	42,787
Total Transcripts Length (bp)	494,954,509
Max. Transcript Length (bp)	2338
Min. Transcript Length (bp)	201
Median Transcript Length (bp)	1953
Mean Transcript Length (bp)	2521
N50 (bp) e	4052
GC percentage (%)	50.07
BLAST annotation and filtered annotated components
Nb. Transcripts	20,072
Nb. Components	2224
Total Transcripts Length (bp)	90,372,815
Max. Transcript Length (bp)	19,856
Min. Transcript Length (bp)	299
Median Transcript Length (bp)	4074
Mean Transcript Length (bp),	4502
N50 (bp) f	5512
GC percentage (%)	49.50

^a Number of paired reads obtained from the sequencer. ^b Number of remaining paired reads after the trimming step. ^c Percentage of surviving paired reads/Raw Paired Reads. ^d Trinity has created a list of transcripts (contigs). The transcripts are grouped in components loosely representing genes. Transcript names are prefixed by the component/gene name, e.g., transcripts c115_g5_i1 and c115_g5_i2 are derived from the same isolated de Bruijn graph and therefore share the same component/gene number c115_g5. ^e Corresponding contig length distribution N50 = 4052 bp (Figure 4). ^f Corresponding contig length distribution N50 = 5512 bp.

). A total of 229,970,255 raw paired reads were generated from all replicates of the three conditions of culturing. After filtering out low-quality sequences, 219,288,500 clean reads were obtained, corresponding to approximately 95% of the total raw reads (Appendix A—Table A1). Because of the lack of availability of information (i.e., genome, transcriptome) on I. obliquus or its related species, we combined all RNA-Seq libraries to build a deep transcriptome using de novo assembly. All clean reads obtained from the ten libraries were subsequently de novo assembled using the Trinity program (version 2.6.5), and a total of 196,273 transcripts with an average length of 2521 bp, and an N50 length of 4052 bp were obtained (Appendix A—Table A1). An evaluation of the size distribution showed that 96% of all transcripts of I. obliquus have lengths longer than 1 kb (Appendix A—Figure A2).

In a previous study, Zou et al. (2016) reported on the Illumina sequencing of Inonotus baumii [52] and obtained a total of 27,259,264 reads. After the Trinity de novo assembly, 30,051 unigenes with an average length of 561 bp and an N50 length of 831 bp were generated, which is two-to-three times less than our results [52]. In another research work on the saprophytic fungus Wolfiporia cocos, the Illumina sequencing of the transcriptome yielded a total of 38,722,186 reads, which were assembled into 60,354 contigs with an N50 of 765 bp [53]. In comparison to this, we obtained eight-to-ten times more reads, ensuring more coverage and allowing for a more contiguous assembly, as confirmed by the average transcript length of 2521 bp obtained here (Appendix A—Table A1).

The average transcript lengths of eukaryotic genes were greater than 1 kb. For example, the average transcript lengths of eukaryotic genes range from 1108 to 2667 bp in human and from 1135 to 1695 bp in yeast [54]. Additionally, eukaryotic proteins have an average size of 472 amino acid residues (i.e., 1419 bp), although the size of proteins from plant genomes are smaller than those of fungi and animals [55]. Based on our sequencing results, it could be concluded that the quality and the depth of this transcriptomic assembly is significantly improved compared to other fungal transcriptome studies.

2.3. Functional Annotation of the RNA-Seq Data

After the Basic Local Alignment Serach Tool (BLAST) annotation of the de novo assembly against the uniprot_sprot.trinotate_v2.0.pep protein database, a total of 20,072 transcripts with an average length of 4502 bp and an N50 length of 5512 bp were obtained (Appendix A—Table A1). A large number of transcripts had a similarity with known genes, suggesting a large amount of sequences specific to I. obliquus.

Gene function was annotated based on the following databases: Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Protein family database (Pfam). A total of 86,246 transcripts (43.94% of assembled transcripts) had a match in the GO database with an E-value of 0 (

Table 1. Summary of functional annotation for Inonotus obliquus transcripts in public databases.

Description	Number of Transcripts	Percentage (%)
Blast-GO annotations	86,246	43.94
COG annotations	52,224	20.05
Pfam annotations	38,415	19.57
Total number of sequences not annotated	105,655	53.83
Total number of sequences annotated	90,618	46.17
Total of sequences transcripts	196,273	100.00

). Additionally, 52,224 transcripts (20.05% of assembled transcripts) and 38,415 transcripts (19.57%) showed similarity to sequences in the COG and Pfam databases, respectively (Table 1).

The functional GO classification of genes is considered to be an effective tool, offering controlled vocabulary and strictly defined biological process for annotating and performing functional analysis of a large number of annotated genes and their products in a selected organism [56]. The GO classification was performed on the 196,273 transcripts, and a total of 86,246 transcripts were classified into 41 functional groups, categorized into three main GO ontologies: Biological process, cellular component and molecular function. The results indicated that the most highly annotated GO category was the cellular component (49.63%) with cell, organelle and membrane being the most abundant, while only a few transcripts were attributed to symplast, cell junction and nucleoid (Figure 4). Under the category of molecular function (38.42%), catalytic activity and binding activity were, respectively, the largest categories. Regarding biological process (11.95%), the dominant subcategories included genes involved metabolic process and cellular process (Figure 4). In addition, important categories with low numbers of transcripts were also represented in the GO, such as transcription factor activity, protein binding and nucleic acid binding.

COG is a database where gene products from a common family ancestor are classified. From the I. obliquus transcriptome, COG-annotated putative proteins were functionally classified into, at least, 25 protein families involved in basic function, such as transcription, translation, signal transduction, cellular structure, biochemistry, metabolism, and molecular processing. The COG analysis of I. obliquus transcriptome led to the classification of 52,224 transcripts to COG classifications. The top COG categories included basic function prediction (16.63%), followed by amino acid transport and metabolism (11.65%), carbohydrate transport and metabolism (10.06%), translation, ribosomal structure and biogenesis (7.94%), inorganic ion transport and metabolism (7.03%), signal transduction, mechanisms (6.84%) and posttranslational modification, chaperones (5.13%) (Figure 5a). It is interesting that 4.45% of the I. obliquus annotated transcripts were ranked in the specialized metabolites biosynthesis, transport and catabolism, which suggests that specialized metabolism occurs in cultivated I. obliquus cells.

Protein domain analyses were performed using the Pfam database [57]. A total of 38,415 transcripts of I. obliquus were found to be associated with protein domains (Figure 5b). Compared to the I. baumi transcriptome, in which a total of 8276 transcripts contain at least one Pfam protein domain [52], our I. obliquus Pfam analysis was six times superior. This result provides another indication that our transcriptome was of good quality and had more depth.

Among the abundant protein families expressed, several were linked to primary metabolism enzymatic activities such as prenylprotease, S-adenosyl methyltransferase and phosphotransferase (PEP-utilizing enzyme). In addition, an important number of the expressed genes encoded predicted proteins from families implicated in ion transport, more specifically Nickel transport protein, a protein family proven to be essential for energy and nitrogen metabolism [58].

2.4. Differential Expression Analysis

Differential expression analyses were performed between databases of I. obliquus. Transcripts with adjusted p-values ≤0.05 and a fold change (log2FC) ≥1 were designated as significantly differentially expressed transcripts. For I. obliquus cells cultivated with betulin (control vs. BET), the results showed that 441 differentially expressed transcripts (139 up-regulated and 302 down-regulated) were identified. Twenty-two times more transcripts (9707) were differently expressed in cells cultivated with bark (control vs. WBB), with a total of 5070 annotated transcripts up-regulated and 4637 transcripts down-regulated (Figure 6). This suggest that the birch bark has very different molecular impacts and requires more transcriptome adjustment from I. obliquus than betulin.

The top twenty-five up- and down-regulated expressed transcripts in I. obliquus cells cultivated with betulin (Appendix A—

Table A2. Top 25 up-regulated transcripts of Inonotus obliquus library in library of cells cultivated in presence of betulin.

Rank	Log2 FC	Description	E-Value	Accession
1	1.66	Phosphatidylinositol 4-phosphate 3-kinase	1.0	O70173.1
2	1.65	Alpha-ketoglutarate-dependent xanthine dioxygenase	3 × 10−28	Q4QZZ9.1
3	1.59	Probable lysosomal cobalamin transporter	0.79	Q8K0B2.1
4	1.57	Major vault protein beta	0.53	P54659.1
5	1.48	Diphthine methyl ester synthase	9.8	Q7S949.1
6	1.46	ATP-dependent RNA helicase DEAH12	7 × 10−38	F4KGU4.1
7	1.44	No hit
8	1.44	No hit
9	1.43	No hit
10	1.42	No hit
11	1.40	Superoxide dismutase [Mn]	8.2	Q9UQX0.1
12	1.40	Orotate phosphoribosyltransferase	6.1	A6VLF2.1
13	1.33	No hit
14	1.32	Caffeic acid 3-O-methyltransferase	1.5	Q43239.1
15	1.32	No hit
16	1.31	30S ribosomal protein S7	0.23	B3EUF4.1
17	1.29	No hit
18	1.29	No hit
19	1.28	No hit
20	1.28	No hit
21	1.27	No hit
22	1.27	D-xylulose reductase	2 × 10−13	Q07993.1
23	1.27	No hit
24	1.26	No hit
25	1.26	No hit

and

Table A3. Top 25 down-regulated transcripts of Inonotus obliquus library in library of cells cultivated in presence of betulin.

Rank	Log2 FC	Description	E-Value	Accession
1	−1.61	Isoleucine-tRNA ligase	1 × 10−42	Q21926.1
2	−1.55	N-acetyltransferase eso1	2 × 10−20	O42917.1
3	−1.52	Probable inactive purple acid phosphatase 16	0.47	Q9SR79.1
4	−1.48	No hit
5	−1.45	ATPase family AAA domain-containing protein 1	2 × 10−52	F6QV99.2
6	−1.44	E3 SUMO-protein ligase NSE2	1 × 10−8	Q7ZXH2.1
7	−1.42	No hit
8	−1.42	Cyclin-dependent kinase 1	0.21	P04551.1
9	−1.42	H/ACA ribonucleoprotein complex subunit NHP2	3 × 10−48	P32495.2
10	−1.40	Structural maintenance of chromosomes protein 1	2 × 10−31	O94383.2
11	−1.39	No hit
12	−1.38	No hit
13	−1.38	Tyrosine-protein kinase receptor ver-4	1.8	Q21041.2
14	−1.37	No hit
15	−1.37	Proteasome-activating nucleotidase 2	2.4	Q5UT56.1
16	−1.37	Structural maintenance of chromosomes protein 2	0.0	P41003.2
17	−1.36	Structural maintenance of chromosomes protein 6	3 × 10−159	P53692.1
18	−1.36	Condensin complex subunit 3	4 × 10−110	Q10429.1
19	−1.36	No hit
20	−1.36	Deoxynucleoside triphosphate triphosphohydrolase	1 × 10−102	Q0VCA5.1
21	−1.35	NFX1-type zinc finger-containing protein 1	5 × 10−42	Q8R151.3
22	−1.35	Cysteine-rich receptor-like protein kinase 41	0.001	O23081.2
23	−1.34	U3 small nucleolar RNA-associated protein 3	0.40	Q12136.1
24	−1.34	No hit
25	−1.33	H/ACA ribonucleoprotein complex subunit cbf5	9 × 10−102	O43102.1

) or white birch bark (Appendix A—

Table A4. Top 25 up-regulated transcripts of Inonotus obliquus library of cells cultivated in presence of white birch bark.

Rank	Log2 FC	Description	E-Value	Accession
1	6.09	Phosphatidylinositol 4-phosphate 3-kinase	1.0	O70173.1
2	5.89	Major vault protein beta	0.53	P54659.1
3	5.89	No hit
4	5.75	DNA-directed RNA polymerase subunit beta	1.1	Q0ANP4.1
5	5.75	No hit
6	5.71	Acetyl-CoA acetyltransferase IB	2.0	Q04677.3
7	5.70	No hit
8	5.70	Orotate phosphoribosyltransferase	6.1	A6VLF2.1
9	5.67	Methyl-CpG-binding domain-containing protein 11	2.5	Q9LW00.1
10	5.64	F-box DNA helicase 1	2.6	F1ND48.2
11	5.62	No hit
12	5.59	No hit
13	5.58	No hit
14	5.56	Glutamyl-tRNA reductase	1.5	A4SV44.1
15	5.54	No hit
16	5.53	Segregation and condensation protein A	4.5	P47455.1
17	5.49	Nascent polypeptide-associated complex subunit beta	1.2	A5DF06.2
18	5.48	No hit
19	5.48	Glucose-6-phosphate isomerase	0.39	Q83XM3.1
20	5.47	Caffeic acid 3-O-methyltransferase	1.5	Q43239.1
21	5.44	No hit
22	5.43	No hit
23	5.43	Protein AegA	2.7	P37127.2
24	5.43	No hit
25	5.43	No hit

and

Table A5. Top 25 down-regulated transcripts of Inonotus obliquus library of cells cultivated in presence of white birch bark.

Rank	Log2 FC	Description	E-value	Accession
1	−5.03	No hit
2	−4.91	Tryptophan synthase alpha chain	4.6	P50382.2
3	−4.30	No hit
4	−4.15	No hit
5	−3.98	No hit
6	−3.76	No hit
7	−3.75	No hit
8	−3.74	No hit
9	−3.71	No hit
10	−3.69	GTP-binding protein EngA	7.9	Q3AAU6.1
11	−3.67	No hit
12	−3.65	Uncharacterized protein C12orf29 homolog	6.0	Q8BHN7.1
13	−3.60	Protein numb homolog	0.33	P49757.2
14	−3.56	No hit
15	−3.56	No hit
16	−3.53	No hit
17	−3.53	Leucine-rich repeat transmembrane protein FLRT1	5.7	Q6RKD8.2
18	−3.51	Manganese peroxidase H4	1 x 10−132	P19136.1
19	−3.51	No hit
20	−3.50	L-ascorbate peroxidase, cytosolic	3.0	P48534.2
21	−3.45	No hit
22	−3.44	No hit
23	−3.43	DNA polymerase epsilon catalytic subunit A	1.7	Q9WVF7.3
24	−3.41	Putative lipase C4A8.10	0.001	O14162.2
25	−3.39	Glutamate racemase	3.4	P63638.1

) were identified. After blasting the top twenty-five up- and down-regulated transcripts against the NCBI database, 48% could not be annotated (no hit). Among top-regulated transcripts with predicted function, 16% have an E-value of 10⁻⁵ or less (Appendix A—Table A2, Table A3, Table A4 and Table A5). Interestingly, the transcript annotated as phosphatidylinositol 4-phosphate 3-kinase ranked first up-regulated for both treatments, suggesting the importance of the signaling pathways involved in cell proliferation, cell survival, and intracellular protein trafficking (Appendix A—Table A2 and Table A4). Phosphatidylinositol 4-phosphate 3-kinase is a conserved enzyme involved in the regulation of phosphatidylinositol 4-phosphate, which is crucial for maintaining morphology, regulating lipid storage, Golgi function and actin cytoskeleton organization [59]. The signaling cascade activated downstream of this enzyme implicates the mitogen-activated protein kinase (MAPK) pathway, which regulates the filamentous growth of yeast [60]. This suggests a putative role for phosphatidylinositol 4-phosphate 3-kinase in the regulation of the morphology of the mycelium and filamentous growth in I. obliquus cells in presence of WBB or betulin.

In addition, caffeic acid 3-O methyltransferase, which catalyzes the conversion of caffeic acid to ferulic acid, was up-regulated in both treatments (Appendix A—Table A2 and Table A4). Phenolic acids, such as caffeic acid and ferulic acid, possess antioxidant properties that play key roles in the synthesis of polyphenols and protection against UV and oxidative stress [6,61,62,63]. The up-regulated condition of caffeic acid 3-O-methyltransferase in our transcriptomic results suggests an important role for ferulic acid in cultivated I. obliquus cells for protection or polyphenol synthesis.

The most down-regulated transcripts found in the betulin-cultivated cells were annotated as transcripts involved in cellular processes such as DNA replication, transcription, cell division and proliferation, i.e., t-RNA ligase, cyclin-dependent kinase, ribonucleoprotein complex subunits, chromosome proteins, and condensin complex subunits. This suggests betulin-induced cellular processes in I. obliquus cells (Appendix A—Table A3).

For WBB-cultivated cells (Appendix A—Table A5), most down-regulated transcripts found annotated for protein involved in metabolic processes such as several peroxidases, lipases, and racemases. This suggests that cultivation with WBB reduced the expression of genes encoding proteins involved in ligninolysis, i.e., to metabolize substrates/degrade bark molecules [64]. Effective lignin and plant cell wall degradation is possible through the action of enzymes from filamentous fungi, and it has been reported that fungi of the basidiomycetes family express several enzymes of the peroxidase family in their transcriptomes, such as lignin- and manganese-peroxidases known for their ability to degrade plant cell walls [65]. The down-regulated peroxidase transcripts in I. obliquus cells cultivated with WBB suggests that WBB may leak repressors for peroxidases to prevent its degradation from the mycelium.

2.5. Genes Involved in the Biosynthesis of Terpenoids in Cultured I. Obliquus Cells

The presence and identification of terpenoids in I. obliquus cells have been already studied and are well documented [1,22,28,35,40,41,43,46,66,67]. In this study, narrow BLAST searches were achieved to identify distinct transcripts encoding enzymes presumably taking part in terpenoid biosynthesis (Appendix A—Figure A1). Eighteen transcript sequences from the mevalonate pathway involved in terpenoid biosynthesis were found in the transcriptomic data of I. obliquus (

Table 2. Summary of the coding DNA sequence (CDS) of biosynthetic gene transcripts identified from the I. obliquus transcriptome known to be involved in terpenoid metabolism. All transcripts were deposited in the publicly available Genbank sequence database with the corresponding accession numbers listed in the material and methods section. * Accession number to UniprotKB/Swiss-Prot.

Name	Number of Reads (Control)	Length (nt)	CDS (nt)	Top Annotation	Species	E Value	* Accession Number
AACT1	8769	1655	816	Acetyl-CoA acetyltransferase	Danio rerio	1 × 10−64	Q6AZA0.1
AACT2	13	1247	1167	Acetyl-CoA acetyltransferase	Xenopus laevis	4 × 10−149	Q6GN02.1
HMGS	15,322	2277	1434	3-hydroxy-3-methylglutaryl CoA synthase	Ustilago maydis	0	Q4P3F1.1
HMGR	12,463	6041	2814	3-hydroxy-3-methylglutaryl CoA reductase	Phycomyces blakesleeanus	0	Q12649.2
MVK	323	1872	927	Mevalonate kinase	Homo sapiens	3 × 10−39	Q03426.1
PMK	4024	5489	1440	Phosphomevalonate kinase	Arabidopsis thaliana	2 × 10−61	Q9C6T1.1
PMD	10,182	4189	1209	Diphosphomevalonate decarboxylase	Ganoderma lucidum	0	G9BIY1.1
IPI	1668	1830	969	Isopentenyl diphosphate isomerase	Xanthophyllomyces dendrorhous	2 × 10−106	O42641.1
FPS	5469	4639	771	Farnesyl diphosphate synthase	Schizosaccharomyces pombe	7 × 10−113	O14230.1
MUS1	5999	4901	1251	Alpha-muurolene synthase	Coprinopsis cinerea	1 × 10−104	A8NE23.1
MUS2	2651	5374	1281	Alpha-muurolene synthase	Coprinopsis cinerea	7 × 10−169	A8NE23.1
PRS	55	2054	258	Delta (6)-protoilludene synthase	Armillaria gallica	1 × 10−20	P0DL13.1
SQS1	2007	3770	1476	Squalene synthase	Ustilago maydis	2 × 10−154	Q92459.2
SQS2	4675	2525	1332	Squalene synthase	Ustilago maydis	4 × 10−147	Q92459.2
SQE	9978	6694	1164	Squalene epoxidase	Homo sapiens	3 × 10−88	Q14534.3
LAS1	3491	2500	1695	Lanosterol synthase	Pneumocystis carinii	0	Q96WJ0.1
LAS2	5	2447	2020	Lanosterol synthase	Pneumocystis carinii	0	Q96WJ0.1
AO	4214	6834	1596	11-oxo-beta-amyrin 30-oxidase	Glycyrrhiza uralensis	2 × 10−39	H1A988.1
CEN3	1727	2261	1254	Centromere protein 3	Schizosaccharomyces pombe	9 × 10−22	Q9USR9.2
GluRL	21	1656	831	Glutamate tRNA ligase	Pelodictyon luteolum	2 × 10−35	Q3B256.1

). As expected, no genes involved in the non-mevalonate pathways were identified, suggesting the absence of this pathway in chaga. Similarly, the transcriptome from I. baumii reported no genes from the non-mevalonate pathway [52].

From the precursor pathway leading to IPP/DMAPP (Appendix A—Figure A1), several transcript variants of orthologous genes were identified (Table 2). For example, AACT genes have been cloned and characterized from various species including zebra fish, frog and human. Two human (Homo sapiens (Hs)) isoforms (HsAACT1 and HsAACT2) were identified to be ubiquitous and important enzymes found in different intracellular locations. The cytosolic HsAACT1 catalyzes the formation of the acetoacetyl-CoA required for sterol, including cholesterol biosynthesis. The exact function of HsAACT2 is not known, but patients with HsAACT2 deficiency have shown severe mental retardation and hypotonus [68]. Similarly, the model plant Arabidopsis thaliana (At) has two isoforms of AACT, where AtAACT1 is primarily expressed in the vascular system and AtAACT2 is deeply present in root tips, top stems, young leaves, and anthers. The characterization of T-DNA insertion of mutated alleles for each AtAACT locus established that AtAACT2 function is necessary for normal male gamete transmission and embryogenesis, whereas plants lacking AtAACT1 are viable with no apparent growth phenotype [69]. In yeast, AACT (also called erg10) encodes a multimeric enzyme, but the exact subunit structure has not been defined [68].

Transcripts encoding each enzyme involved in the formation of terpenoid precursors IPP and DMAPP (Appendix A—Figure A1) were identified in the transcriptome of I. obliquus (Table 2). According to the number of reads, the HMGS, HMGR, and PDM transcripts were the most abundant with E values of 0, suggesting that these gene sequences are well conserved.

Sesquiterpene synthases play an essential role in diversifying the skeletal structure of sesquiterpenoids by catalyzing the very complex cyclisation of the common precursor, FPP [38,70,71,72]. Sesquiterpenoids have been reported in Inonotus [43]; however, no biosynthetic genes have been identified. BLASTx searches of I. obliquus transcriptome led to the identification of three sesquiterpene synthases: Two transcript variants of the muurolene synthase (MUS1 and MUS2) and one of the protoilludene synthase (PRS) (Table 2). MUS1 and MUS2 are only 36% identical at the nucleotide level, suggesting different functions, i.e., substrates for enzymatic activities. Similarly, the E values of MUS1, MUS2 and particularly PRS suggest that their function may be different than orthologous sequences, suggesting that I. obliquus possess the machinery to produce structurally different sesquiterpenoids. Muurolene, protoilludene, and derivatives, also considered volatile organic compounds (VOCs), are produced by several fungal species. The rich bouquet of several VOCs contribute to the aroma and flavor of mushrooms. Though the ecological function of fungal volatiles is unknown, studies showed their biological activities as antibiotics, antioxidants or inhibitor of germination [72,73,74].

Lee et al. (2016) performed a transcriptomic analysis of the white rot fungus Polyporus brumalis which led to the identification of two transcripts, germacrene A synthase and trichodiene synthase, involved in sesquiterpenoid biosynthesis [75]. However, the final products of these enzymes were not detected in the cultivated fungus [75]. To our knowledge, this study is the first to report the presence of transcripts encoding sesquiterpenoid-forming enzymes in Inonotus.

Triterpenoids from Inonotus species include high pharmaceutical valued metabolites. For example, the lanosterol-derived triterpenoid inotodiol and the lupeol derivative betulinic acid are well known bioactive triterpenoids from I. obliquus [17,19,24,25,27,28,33,36,76,77]. Though several studies have shown the presence of triterpenoids in I. obliquus, only one gene, SQS, has been identified [78]. In order to increase knowledge on genes involved in triterpenoids production, a search for these genes was carried out in the transcriptome of I. obliquus. Two transcript variants of squalene synthase (SQS1 and SQS2) were identified with a 95% sequence identity to each other (SQS2 has an insertion at the 3′ end) (Table 2). Our SQS1 transcript sequence is identical to previously characterized SQS from I. obliquus [78].

A single transcript for AO was identified in the transcriptome of I. obliquus, whereas two transcript variants, LAS1 and LAS2, were found (Table 2). LAS1 and LAS2 possess a 98% sequence identity, but LAS2 is longer and far less expressed than LAS1, suggesting the importance of LAS1 in the production of lanosterol and its derivatives in I. obliquus cell cultures (Table 2).

According to the literature, only one terpenoid gene, SQS, has been characterized from I. obliquus, whereas nine genes related to terpenoids biosynthesis where identified in I. baumii [52,78]. The transcriptome generated in the current study allowed for the identification of all (17) orthologous genes involved in terpenoid metabolism. Furthermore, variants of the AACT, MUS, SQS, and LAS genes were identified, indicating that these steps may be regulated differently in I. obliquus. For example, the higher expression of AACT1, MUS1, and LAS1 suggests the importance of theses isoforms in terpenoid formation of I. obliquus-cultivated cells.

The digital expression of each terpenoid-associated transcript was studied for each growth condition (Figure 7). Upstream precursor genes, such as AACT1 and PMD, had a higher expression in I. obliquus cells compared to specific downstream (sesqui and triterpenoid) genes such as PRS and LAS2 (Figure 8). From the precursor pathway, the most strongly expressed transcript, AACT1, remained highly expressed across the different treatments, suggesting no transcriptional regulation under those conditions, whereas PMD, SQS2 and SQE were up-regulated in presence of BET and WBB (Figure 7). In contrast, HMGR, MUS1, and AO were down-regulated in presence of the terpenoid substrate, particularly in I. obliquus cells cultivated with WBB. HMGR is a rate-limiting enzyme in the terpenoid biosynthesis. It is possible that the addition of terpenoid substrates promotes negative feedback regulation mechanisms. For example, HMGR, which generates mevalonate, a critical intermediate in the biosynthesis of terpenoids, is exposed to large amount of feedback regulation through various mechanisms that are influenced by end-products, light, and hormones [79,80,81,82].

2.6. qRT-PCR Validation of RNA-Seq Gene Expression Data

To validate the RNA-Seq digital expression data, eight transcripts encoding terpenoid biosynthetic enzymes were selected for the qRT-PCR analysis. For control reference genes, transcripts with no/low variation were extracted from the database using a custom method developed by dos Santos et al. (2019), which has shown to outperform pre-defined references genes [83]. Indeed, centromere protein 3 (CEN3) and glutamine RNA ligase (GluRL) show no/low difference of expression among the conditions tested indicating that they are good reference genes for qRT-PCR study (Figure 7).

All eight transcripts qRT-PCR analyses are consistent with RNA-Seq results (Figure 8). This indicates that our transcriptome was reliable and that we could make reasonable inferences from the differentially expressed transcripts. For example, as observed for the digital expression, SQS2 was up-regulated and HMGR was down-regulated in presence of terpenoid substrates ( Figure 7 and Figure 8).

Interestingly, qRT-PCR analysis showed a downregulation of FPS in the presence of terpenoid substrates, suggesting a negative feedback regulation on this important step in the sesqui and triterpenoid biosynthesis. In contrast, SQS2 expression increased in the presence of WBB, suggesting a positive regulation. This opposite regulation, negative on FPS and positive on SQS2, may be due to the presence of terpenoids such as FPP in bark residues. Altogether, the results suggest that few key reactions in the terpenoid pathway are regulated at the transcriptional level in presence of terpenoid substrates in I. obliquus cell cultures.

The amounts of valuable terpenoids accumulated remained low under exogenous induction in a fungal culture. The metabolic engineering of microorganisms is an interesting alternate route for the production of these important compounds. To do this, a better understanding of the terpenoid biosynthetic pathway and the genes involved in this pathway is required. However, the lack of genomic information about I. obliquus hinders the development of alternative production methods. In this work, the best conditions for growth of I. obliquus cells cultivated in presence of terpenoid substrates, betulin or white birch bark were examined, and the corresponding transcriptomes obtained after next-generation sequencing and de novo assembly. Transcriptome analysis identified eighteen transcripts encoding enzymes implicated in the terpenoid metabolism. Comparative analyses of the transcriptomes yielded valuable information with respect to the wide variety of genes implicated in terpenoid metabolism in I. obliquus cells under the described conditions. It would be interesting to compare the laboratory-adapted isolate of I. obliquus to the wild type fungus to monitor gene expression implicated in the host-pathogen interaction between chaga and its host, birch trees.

3. Materials and Methods

3.1. Chemicals and Reagents

Standard betulinic acid (97%) was purchased from Adipogen Corp. (San Diego, CA, USA). Standard betulin (98%) was purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Methanol, acetonitrile and dimethylsulphoxide (DMSO), ethyl acetate, and acetic acid were HPLC grade from Fisher Chemical. The other cited chemicals were of analytical grade. Media components (yeast extract, malt extract, agar, etc.) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA).

3.2. Fungal Culture and Growth Conditions

I. obliquus was provided and cultured by the Biopterre laboratory (La Pocatière, Qc, Canada). The stock of I. obliquus was maintained on a potato dextrose agar (PDA) medium, and the stock culture was stored at 4 °C. The identity of the isolate was confirmed by PCR amplification followed by Sanger sequencing of the ribosomal internal transcribed spacer (ITS) DNA region. The sequence data were deposited to the NCBI database under the GenBank accession number MN239482. To optimize the mycelial growth of I. obliquus in laboratory, yeast malt broth (YMB) and yeast malt agar (YMA) media were selected based on previous reported studies [51,67,85]. The YMB and YMA contained 3 g/L of yeast extract and 3 g/L of malt extract. Different growth condition parameters of the liquid and solid culture of I. obliquus, were tested: Three pH (5, 6.2, and 7.5) and three temperatures (22 °C, 28 °C, and 37 °C) in presence or not of white birch bark (50 µg/mL) or betulin (15 µg/mL).

3.2.1. Optimization of Growth Conditions for Solid Medium

To optimize the mycelial culture on solid media, the effect of pH, temperature, and the presence of white birch bark (50 µg/mL) were investigated. I. obliquus was initially grown for 14 days on a YMA medium in petri dish in absence of light. A disc of 1 cm² was cut after 14 days of culture, and then it was transferred into other plate under different conditions for 14 days in absence of light. The growth diameter was measured, in triplicate, every two days on the Petri dish [86,87]. In order to sustain the culture of I. obliquus, mycelium was maintained on YMA Petri dishes for three months in an incubator at 28 °C and then stored at 4 °C. Based on preliminary results obtained from the optimization of growth in a solid culture, and in order to reduce the parameters in liquid cultures, the optimization of the liquid cultures of I. obliquus was carried out at 28 °C, which yielded the best growth of the isolate.

3.2.2. Optimization of Growth Conditions for Liquid Medium

To initiate a liquid culture of I. obliquus, a one-centimeter² disc was collected from a fresh solid culture of mycelium in a YMA medium. The mycelium pre-culture was grown in flask of 250 mL with 30 mL of yeast malt broth (YMB) at 28 °C on a rotary shaker incubator at 150 rpm for 8 days. Then, to investigate the effect of pH (5, 6.2 and 7.5), an aliquot of 10 mL of the mycelium was transferred to 250 mL flask containing 100 mL of YMB at different pH, incubated at 28 °C on a rotary shaker (Thermofisher Scientific) at 150 rpm in absence of light for 8 days. The mycelium was sampled every 48 h by sequential filtration, washed several times with distilled water, and then was dried overnight at 70 °C.

3.3. Isolation of RNA

Ten mL aliquote of 8 days I. obliquus liquid pre-culture were aseptically added to 250 mL flask containing 100 mL of a YMB medium supplemented or not with 0.2% DMSO (Control), 15 µg/mL of betulin (BET), or 50 µg/mL of white birch bark (WBB), and grown for 8 days on a rotary incubator at 150 rpm and 28 °C in absence of light. The I. obliquus mycelium was collected after 8 days of growth. The mycelium was filtered, frozen with liquid N₂, and then placed in a 2 mL tube with 2 mL of TRIzol reagent (Fisher Scientific, Toronto, Canada). The mycelia were crushed using the TissueLyser II (QIAGEN, Qiagen Retsch GmbH, Hannover, Germany) using the stainless steel beads with 5 mm diameter (QIAGEN, Qiagen Retsch GmbH, Hannover, Germany) at speed of 30 strokes per second for 3 min until a homogeneous sample was obtained. The tubes were incubated on ice for 5 min, and then 400 µL of chloroform were added. The tubes were vigorously vortexed for 15 s and incubated in an ice bath for 3 min. After centrifugation at 13,000× g for 15 min at 4 °C, two phases were obtained, which were separated by cellular debris—the clear phase of the supernatant was recovered and transferred into another clean 1.5 mL Eppendorf tube. RNA was precipitated with the addition of 700 μL isopropanol, followed by incubation at −20 °C for 3 h. Finally, precipitated RNA was collected by centrifugation at 13,000× g for 15 min at 4 °C, and the pellets were washed once with 1 mL of ice cold 75% ethanol and air dried briefly at room temperature. Then, they were centrifuged again at 13,000× g for 5 min at 4 °C. The RNA pellets were resuspended using 20 μL of nuclease free water. The quality and quantity of RNA extracted from the samples under different conditions were verified using the Nanodrop and the Bioanalyzer. Only samples with an RNA Integrity number higher than 8 were selected for Illumina sequencing.

3.4. Transcriptome Sequencing, de Novo Assembly and Functional Annotation

Sequencing was performed using Illumina HiSeq 4000, PE 100 paired ends, at McGill University and Genome Québec Innovation Centre (Montreal, QC, Canada) on cDNA Libraries converted from isolated high-quality mRNA. Among the raw reads obtained from Illumina sequencing, low quality reads, reads with unknown nucleotides, and reads with adapters were found (

Table 3. List of Inonotus obliquus RNA-Seq library sequenced using Illumina HiSeq4000 deposited to the NCBI Sequence Read Archive under the accession PRJNA526077.

Library	Nb of Reads	Nb of Bases
RNA-Seq of Inonotus obliquus cell culture: Control 1	41,874,533	8,374,906,600
RNA-Seq of Inonotus obliquus cell culture: Control 2	19,534,645	3,906,929,000
RNA-Seq of Inonotus obliquus cell culture: Control 3	22,008,711	4,401,742,200
RNA-Seq of Inonotus obliquus cell culture: Betulin1	25,264,465	5,052,893,000
RNA-Seq of Inonotus obliquus cell culture: Betulin2	20,205,852	4,041,170,400
RNA-Seq of Inonotus obliquus cell culture: Betulin3	22,305,852	4,461,046,200
RNA-Seq of Inonotus obliquus cell culture: White birch bark 1	17,766,031	3,553,206,200
RNA-Seq of Inonotus obliquus cell culture: White birch bark 2	16,318,641	3,263,728,200
RNA-Seq of Inonotus obliquus cell culture: White birch bark 3	28,913,696	5,872,739,200
RNA-Seq of Inonotus obliquus cell culture: White birch bark 4	15,777,829	3,155,565,800
Total	229,970,255	46,083,926,800

). Trimmomatic was used to remove the adapters from sequencing and trimmed reads from the 3′ end and further filtered all reads below 50 bp in order to obtain clean reads [88]. However, once the data from the surviving pairs were generated, Trinity normalization was performed to remove redundant reads in datasets without affecting its k-mer content [89]. De novo assembly of cleaned and standardized reads was performed using Trinity (v2.6.5) assembler [90]. Trinity method assembles the RNA-Seq reads into full-length transcripts, which are called contigs or unigenes. All the unigenes were aligned to the public protein database uniprot_sprot.trinotate_v2.0.pep protein database using the BLASTX program against the NCBI BLAST families using an E-value of 10⁻⁵. Top BLAST hits were used for annotation of component/gene for each transcript. To quantify the gene transcript abundance, the raw RNA-Seq reads were mapped to assembled transcripts with Bowtie [91] using default parameters. The gene transcript abundance was calculated as transcripts per kilobase million (TPM) using the RSEM package [92].

Functional annotations of unigenes were performed using Trinotate (http://trinotate.github.io/), by aligning our transcripts to Swiss Institute of Bioinformatics databases (Swiss-Prot) with BLASTX and by identifying protein domains (HMMER/PFAM), protein signal peptides, transmembrane domains prediction (signalP/tmHMM), and Clusters of Orthologous Groups (COG). Then, the functional annotation of unigenes was compared to currently curated annotation databases (EMBL Uniprot eggNOG/GO Pathways databases). The Trinity assembly and functional annotation of unigenes were integrated as an annotation report into an SQLite database.

Gene Ontology (GO) annotations for our unigenes were obtained using the Swiss-Prot. The WEGO 2.0 software was used to obtain the GO functional classification for all unigenes and to better understand the distribution of gene functions in I. obliquus at the macro level. All annotated sequences were linked to the GO terms in the database, and a quantification of the number of sequences for each of the different terms was subsequently calculated. The raw reads under the accession number PRJNA526077 were deposited to the Sequence Read Archive [93].

3.5. qRT-PCR Analysis

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on I. obliquus in different culture conditions (control, with white birch bark (WBB) or with betulin (BET). The qRT-PCR assay was performed using a CFX connect Real-Time System by BioRad and analyzed using BioRad CFX Maestro 1.1. (BioRad laboratories) including statistics analysis using an ANOVA.

Two micrograms of total RNA extracted from different culture conditions were reverse transcribed into single-stranded complementary DNA using the High-Capacity RNA-to-cDNA™ Kit supplied by Thermo Fisher Scientific, following their protocol. qRT-PCR was performed in triplicates in a 10 µL total volume reaction containing 6 µL of SYBR Lo-ROX mix from Bioline SensiFAST SYBR Lo-ROX mix kit (FrogaBio, Toronto, ON, Canada), 2 µL cDNA and 2 µL of 200 µM of specific primers. The primers were designed using the conserved sequences of the variant of the transcripts selected using Clustal tool on Galaxy platform. The primers were aligned to the target genes sequences using the Integrated DNA Technology (www.idtdna.com), Primer3 and Tm Calculator (New England Biolabs, tmcalculator.neb.com) to select suitable annealing temperatures for each primer. The qRT-PCR was performed for the following gene transcripts: Acetoacetyl-CoA synthetase (AACS), hydroxymethylglutaryl-CoA synthase A (HMGS); 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl pyrophosphate synthase (FPS), squalene synthase (SQS), squalene monooxygenase (SQE), β-amyrin synthase (βAS) and lanosterol synthase (LAS). The primers used for the qRT-PCR assay are listed in

Table 4. Primers used for qRT-PCR validation of gene expression.

Gene	Forward Primer (5′-3′)	Reverse primer (5′-3′)	Product Size (bp)
AACT2	CCGATCACTGTGAAGGGTAAG	TGGTGCGATAGGGAAATCTATG	273
HMGS	CAGTGTCGACTACCCCGTTT	GTGTACATGTTTCCGCAACG	280
HMGR	GTCGTTCCTGGTGTGAGGTT	GCGTCTTAGTGGCCAGAGTC	243
FPS	TCATGCACGAAACGACTCTC	CCGAAGCAGTCGAGGTAGTC	276
SQS2	CTTCGAGGGTTGGACACAAT	GACGTCGCGGTAAGAAGAAG	282
SQE	GTCCTGTCCTACTCTACCAAATC	GTCATTCCACCTCCAGTCAA	289
LAS	GCCTGAAGGCTGTGCTTTAC	TCTGCAAGGAAAGCATTGTG	246
AO	GCTTACTCCTGCGTTCAGTAA	TCCGAAACCTCCTCCATAGT	375
CEN3	AGGTCGACCGAGAAGTCG	CTTGAACTTTCTTACGTTTG	300

. qRT-PCR data were standardized using reference genes. From the transcriptomic data of I. obliquus, we selected as reference genes, the two genes with lowest variability in their expression under the different culture conditions. The Ct values for the seven genes of interests were normalized to the Ct value of the reference gene centromere protein 3 (CEN3) and glutamine RNA ligase (GluRL).

The amplification was carried out under the following conditions: 2 min of polymerase activation at 95 °C, followed by 39 cycles of 5 sec of denaturation at 95 °C, 10 sec of annealing, and 30 s of extension and fluorescence data acquisition at 72 °C were measured. After verifications of unique and clear melt curve and unique band obtained on agarose gel electrophoresis, RT-qPCR efficiency and linearity were taken into account for next step of qRT-PCR. The percentage of efficiency qRT-PCR was chosen between 90% and 110% and a standard curve correlation coefficient (R2) ≥96 [94].

3.6. Accession Numbers

The sequences described in this paper have been deposited in the National Center for Biotechnology Information Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/) under the accession number PRJNA526077. Gene transcript sequences were deposited in Genbank with the following accession numbers for nucleotide sequences: Acetoacetyl-CoA transferase 1 (MK825552); acetoacetyl-CoA transferase 2 (MK825553); HMG-CoA synthase (MK825554); HMGR, HMG-CoA reductase; (MK825555); MVA kinase (MK825556); phosphoMVA kinase (MK825557); diphosphoMVA decarboxylase (MK82558); IPP isomerase (MK825559); FPP synthase (MK825560); Muurolene synthase 1 (MK825561); Muurolene synthase 2 (MK825562); Protoilludene synthase (MK825563); Squalene synthase 1 (MK825564); Squalene synthase 2 (MK825565); Squalene epoxidase (MK825566); Lanosterol synthase 1 (MK825567); Lanosterol synthase 2 (MK825568); 11-oxo-β-amyrin 30-oxidase (MK825569); Centromere protein 3 (MK825570); Glutamine RNA ligase (MK825571) and Glyceraldehyde 3-phosphate deshydrogenase (MK825572).

4. Conclusions

This research allowed us to define the best growth conditions to conduct qRT-PCR and RNA-Seq de novo assembly of Québec Inonotus obliquus. The best growing conditions in liquid and solid cultures were determined for optimal production yield of Inonotus obliquus mycelia. It was found that optimal conditions were obtained at pH 6.2, 28 °C, and using YMB as a culture medium. In this study, we provide the first transcriptomic report generated by next-generation sequencing (Illumina HiSeq4000) of the medicinal fungus chaga, I. obliquus, grown under different conditions. The assembled transcriptomes generated 196,273 transcripts. High-quality transcriptomes were obtained, and based on genes annotations, we were allowed to identify of transcripts encoding 18 enzymes playing a key role in the terpenoid pathway. This led us to identify novel gene sequences encoding biosynthetic enzymes involved in the terpenoid pathway in chaga. The transcriptomic analysis of this medicinal fungus has led to the discovery of new genes that can be used as a reference for future genetic and genomic studies on chaga and other related medicinal species. Moreover, this transcriptome assembly may also be used as a reference platform for studies on fungus Hymenochaetaceae family. Beyond the biological impact, the data can be used as a reference to provide gene sequences for metabolic engineering and should pave the way for advanced fungal biology and biotechnology to produce valuable chaga terpenoids.