De Novo Sequencing and Transcriptome Analysis of Wolfiporia cocos to Reveal Genes Related to Biosynthesis of Triterpenoids

Abstract

Wolfiporia cocos Ryvarden et Gilbertson is a saprophytic fungus in the Basidiomycetes. Its dried sclerotium is widely used as a traditional crude drug in East Asia. Especially in China, the dried sclerotium is regarded as the silver of the Chinese traditional drugs, not only for its white color, but also its medicinal value. Furthermore, triterpenoids from W. cocos are the main active compounds with antitumor and anti-inflammatory activity. Biosynthesis of the triterpenoids has rarely been researched. In this study, the de novo sequencing of the mycelia and sclerotia of W. cocos were carried out by Illumina HiSeq 2000. A total of 3,484,996,740 bp from 38,722,186 sequence reads of mycelia, and 3,573,921,960 bp from 39,710,244 high quality sequence reads of sclerotium were obtained. These raw data were assembled into 60,354 contigs and 40,939 singletons, and 56,938 contigs and 37,220 singletons for mycelia and sclerotia, respectively. The transcriptomic data clearly showed that terpenoid biosynthesis was only via the MVA pathwayin W. cocos. The production of total triterpenoids and pachymic acid was examined in the dry mycelia and sclerotia. The content of total triterpenoids was 5.36% and 1.43% in mycelia and sclerotia, respectively, and the content of pachymic acid was 0.458% and 0.174%. Some genes involved in the triterpenoid biosynthetic pathway were chosen to be verified by qRT-PCR. The unigenes encoding diphosphomevalonate decarboxylase (Unigene 20430), farnesyl diphosphate synthase (Unigene 14106 and 21656), hydroxymethylglutaryl-CoA reductase (NADPH) (Unigene 6395_All) and lanosterol synthase (Unigene28001_All) were upregulated in the mycelia stage. It is likely that expression of these genes influences the biosynthesis of triterpenoids in the mycelia stage.

Traversing the fungal terpenome

“…24 Likewise, transcriptome analysis of the medicinally relevant Wolfiporia cocos identified several P450s that were upregulated simultaneously with its OSC. 205 Contrastingly, the genome of A. fumigatus shows that it contains multiple genes encoding OSC as opposed to a single copy. Furthermore, the putative enzymes related to helvolic acid 115 production are located in a gene cluster encoding protostadienol 118 synthase, multiple P450s, a reductase, acyltransferases and a dehydrogenase.…”

Biosynthesis of Terpenoid Natural Products in Fungi

“…Two genes, oxidosqualene synthase and squalene epoxidase, were identified in the antitumor clavaric acid producer Hypholoma sublaterium (a basidiomycete), and are expected to be required for the production of this secondary metabolite [225,226]. In an effort to elucidate the biosynthesis of lanosterane-type triterpenoids in Wolfiporia and Ganoderma, genomic and transcriptomic studies were published by several groups [227][228][229][230]. These studies suggested that Ganoderma has two oxidosqualene-lanosterol cyclases, with one homolog dedicated to either ergosterol or triterpenoid natural product biosynthesis.…”

De novo transcriptome sequencing of Flammulina velutipes uncover candidate genes associated with cold‐induced fruiting

“…Total RNA extraction was conducted from mycelium and primordia samples by using TRIzol reagent (Invitrogen, USA) followed by the integrity evaluation using Agilent Technology 2100 Bioanalyzer (Agilent Technologies, USA). Two cDNA libraries were generated as previous reported and paired‐end sequenced by Illumina Hiseq 2000 platform (Illumina, USA) at Beijing Genomics Institute (BGI) (Shenzhen, China). The raw data has been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession number SRP100485 and associated with Bioproject PRJNA369728.…”