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.

Keywords: Inonotus obliquus; RNA-Seq; betulinic acid; biosynthesis; chaga; de novo transcriptome; specialized metabolism; terpenoid.

Figure A1

Proposed biosynthetic pathway leading to multiple terpenoids in I. obliquus. Enzymes for which corresponding genes have been isolated from Inonotus are shown in bold black whereas the ones from other species are shown in bold grey. Broken arrow represents more than one biochemical reaction. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonic acid; MVAP, MVA phosphate; MVAPP, MVA diphosphate; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; AACT, Acetoacetyl-CoA transferase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MVK, MVA kinase; PMK, phosphoMVA kinase; PMD, diphosphoMVA decarboxylase; IPI, IPP isomerase; FPS, FPP synthase; MUS, Muurolene synthase; PRS, Protoilludene synthase; SQS, Squalene synthase; SQE, Squalene epoxidase; AS, Amyrin synthase; βAO, 11-oxo-β-amyrin 30-oxidase; LAS, Lanosterol synthase; LUS, Lupeol synthase.

Figure A2

Sequence length distribution of transcripts of I. obliquus after Trinity de novo assembly.

Figure 1

Effect of pH and temperature on Inonotus obliquus growth. I. obliquus was grown on a yeast malt agar medium (YMA) in Petri dishes for 16 days at 22 or 28 °C. YMA pH was adjusted with addition of an HCl or NaOH solution to obtain final pHs of 5, 6.2 and 7.5. Diameter (in mm) corresponding to fungal growth was measured every two days starting at day seven. Results represent the average (± error bars) growth of three biological repetitions per condition. Statistical significance is annotated with an asterix (*), according to ANOVA test results with p ˂ 0.05.

Figure 2

Inonotus obliquus cell culture growth in presence of white birch bark. I. obliquus was grown on a solid medium pH 6.2 at 28 °C for 16 days on agar plates containing a yeast malt agar (Control) medium (white bars) or YMA supplemented with white birch bark (WBB) fragments (black bars). The measurements of the diameter (mm) of growth were taken every two days starting at day seven. Results show the average (± error bars) growth of three biological repetitions per condition. Statistical significance is annotated with an asterix (*) according to ANOVA test results with p ˂ 0.05.

Figure 3

Effect of pH on Inonotus obliquus biomass. I. obliquus was grown in a liquid medium of a yeast malt broth medium (YMB) at 28 °C for eight days. The pH of the medium was adjusted by adding HCl or NaOH to obtain three final pH values of 5, 6.2 and 7.5. At day eight, I. obliquus cells were collected, filtered and dried at 70 °C overnight. Results represent the average (± error bars) dry weight (mg) of three biological repetitions per condition.

Figure 4

Gene Ontology (GO) terms of 41 functional groups of expressed transcripts from Inonotus obliquus.

Figure 5

Annotation of the Inonotus obliquus transcriptome. (a) Cluster of orthologous groups (COG). (b) Protein family database (Pfam) classification of transcripts from Inonotus obliquus.

Figure 6

Venn diagram summarization of the differential expression comparisons. The number of differentially expressed genes (DEGs) in each circle represents the amount of DEGs between the different comparisons betulin (BET) versus control and white birch bark (WBB) versus control. Only the annotated genes were included. The overlapping number specifies the mutual DEGs between the distinctive comparisons and the non-overlapping numbers define the genes exclusive to each condition. Indicated in the diagram are the numbers of up-regulated ($\uparrow$) and down-regulated DEGs (↓).

Figure 7

Heatmap of the digital expression levels of transcripts encoding terpenoid biosynthetic enzymes in the Inonotus obliquus transcriptome. Results are in transcripts per million (TPM) with the legend from low (blue) to high (red) expressed transcript.

Figure 8

Comparison of expression profiles of eight representative transcripts from I. obliquus cells supplemented without (control) or with betulin (BET) or white birch bark (WBB), as measured by RNA-Seq and quantitative reverse transcription PCR (qRT-PCR). The eight transcripts are assigned to the terpenoid pathway in Appendix A—Figure A1. Columns represent expression determined by qRT-PCR (left y-axis), while lines represent digital expression by RNA-Seq in TPM values (right y-axis). The x-axis indicates different growth conditions (control, BET, and WBB). Graphs are plotted using normalized ddCt values scaled to control. Centromere protein 3 (CEN3) was used for internal reference. Expression fold change and error bars were calculated using the comparative 2^−ΔΔCt method [84]. Bars represent the mean standard deviation of three independent replicates. Abbreviations are defined in Appendix A—Figure A1.