Genomic and transcriptomic analyses of the medicinal PNAS PLUS fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development Mei-Yeh Jade Lua, Wen-Lang Fana, Woei-Fuh Wanga, Tingchun Chena, Yi-Ching Tanga, Fang-Hua Chub, Tun-Tschu Changc, Sheng-Yang Wangd,e,f, Meng-yun Lia, Yi-Hua Chena, Ze-Shiang Lina, Kai-Jung Yanga, Shih-May Chena, Yu-Chuan Tenga, Yan-Liang Linb, Jei-Fu Shawg, Ting-Fang Wangh, and Wen-Hsiung Lia,i,1 aBiodiversity Research Center, Academia Sinica, Taipei 11529, Taiwan; bSchool of Forestry and Resource Conservation, National Taiwan University, Taipei 10617, Taiwan; cTaiwan Forestry Research Institute, Taipei 10066, Taiwan; dDepartment of Forestry, National Chung-Hsing University, Taichung 402, Taiwan; eAgricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; fAgricultural Biotechnology Center, National Chung-Hsing University, Taichung 402, Taiwan; gDepartment of Biological Science and Technology, I-Shou University, Kaohsiung 84001, Taiwan; hInstitute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan; and iDepartment of Ecology and Evolution, University of Chicago, Chicago, IL 60637 Contributed by Wen-Hsiung Li, September 13, 2014 (sent for review June 10, 2014; reviewed by Robert Friedman, Kenneth Wollfe, and Takashi Gojobori) Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long basidium, whereas ergostanes are related to basidiomatal formation been used as a remedy for cancer, hypertension, and hangover, with and are produced only in basidium (6, 14). Significantly, the ergo- an annual market of over $100 million (US) in Taiwan. We obtained stane production of A. cinnamomea growing on C. kanehirai Hayata a 32.15-Mb genome draft containing 9,254 genes. Genome ontology is of higher efficiency and shows more compound variety than that enrichment and pathway analyses shed light on sexual development growing on other trees (14, 15). As to the metabolites of mycelium, and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, lanostane-type triterpenoids and the derivatives of maleic acid, antroquinonol, and antrocamphin. We identified genes differentially succinic acid and ubiquinone, have been reported (15). Despite its expressed between mycelium and fruiting body and 242 proteins in medicinal importance, however, there has been little research on the mevalonate pathway, terpenoid pathways, cytochrome P450s, its genetics. GENETICS and polyketide synthases, which may contribute to the production Currently, little is known about the mating system and the of medicinal secondary metabolites. Genes of secondary metabolite mechanism of meiotic control in A. cinnamomea. In Basidiomycota, biosynthetic pathways showed expression enrichment for tissue- the mating process is controlled by homeodomain-encoding genes specific compounds, including 14-α-demethylase (CYP51F1) in fruiting (HD) and G protein-coupled pheromone/receptor (P/R) genes. The body for converting lanostane to ergostane triterpenoids, coen- involvement of these genes in mating-type determination differs zymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting Significance body. Our data will be useful for developing a strategy to in- crease the production of useful metabolites. Antrodia cinnamomea, a mushroom, has long been used as a remedy for cancer, hypertension, and hangover. However, medicinal fungus | fruiting body | triterpenes | meiosis | P450 the molecular basis of its medicinal effects is unclear and its genome has not been studied. We obtained a genome draft n endemic mushroom (Polyporales, Basidiomycota) of and conducted gene annotation. Genome ontology enrichment ATaiwan, Antrodia cinnamomea (syn. Antrodia camphorate and pathway analyses shed light on sexual development and and Taiwanofungus camphorates) is restricted to the endemic metabolite biosynthesis. We identified genes differentially aromatic tree Cinnamomum kanehirai Hayata (1–3). A phyloge- expressed between mycelium and fruiting body and also pro- netic analysis using the nuclear large subunit (LSU) and internal teins in the mevalonate pathway, terpenoid pathways, cyto- transcribed spacer (ITS) sequences showed that the genus chrome P450s, and polyketide synthases, which may contribute Antrodia is not monophyletic (3). A. cinnamomea (T. camphorates) to production of medicinal metabolites. Genes of metabolite is in a clade that includes Taiwanofungus, Auriporia, Sarcoporia, biosynthesis pathways showed expression enrichment for tis- Dacryobolus,andAmylocystis. This clade is more closely related to sue-specific compounds in mycelium and in fruiting body. Our other genera in the order Polyporales such as Postia than to the data will be useful for developing a strategy to increase the rest of the genus Antrodia. No genomes from this clade have yet production of valuable metabolites. been sequenced. A. cinnamomea is rich in polysaccharides (4) and produces spe- Author contributions: M.-Y.J.L., J.-F.S., and W.-H.L. designed research; M.-Y.J.L., W.-L.F., cific ergostane triterpenoids, including antcins C and K (5), zhankuic W.-F.W., T.C., Y.-C. Tang, F.-H.C., T.-T.C., S.-Y.W., T.-F.W., and W.-H.L. performed research; acidsA,B,andC(6),andantrocamphin(7).A. cinnamomea has F.-H.C. and T.-T.C. contributed new reagents/analytic tools; M.-Y.J.L., W.-L.F., W.-F.W., T.C., long been the most valued medicinal fungus in Taiwan, as a remedy Y.-C. Tang, F.-H.C., S.-Y.W., M.-y.L., Y.-H.C., Z.-S.L., K.-J.Y., S.-M.C., Y.-C. Teng, Y.-L.L., T.-F.W., for various diseases, including hypertension, abdominal pains, hep- and W.-H.L. analyzed data; and M.-Y.J.L., W.-F.W., Y.-C. Tang, S.-Y.W., T.-F.W., and W.-H.L. atitis B, and cancers (8–11). Also, the extract of A. cinnamomea has wrote the paper. long been used to ameliorate the effects of alcohol intoxication. Reviewers: R.F., University of South Carolina; K.W., University College Dublin; and T.G., ThetotalmarketvalueofA. cinnamomea products in Taiwan King Abdullah University of Sciences and Technology. is estimated to be over 100 million US dollars per year (www. The authors declare no conflict of interest. businesstoday.com.tw/article-content-80392-93594). For these Data deposition: The sequences reported in this paper have been deposited at DDBJ/ reasons, numerous studies of its pharmacological properties have EMBL/GenBank database [accession nos. SRR1257388, SRR1258847, SRR1258848, SRR1258858, SRR1258102, SRR1258101, SRR1258110, SRR1258111, and SAMN02730102 been conducted (12). Also, others have investigated the composi- (PRJNA244959, Genome Project); SRR1258093, SRR1258113, SRR1257385, SRR1258114, tion of polysaccharides and lipopolysaccharides and their differ- SRR1258115, SRR1258116, SAMN02730102, SAMN02730103, SAMN02730107,and ences between basidium and mycelium and among strains (11, 13). SAMN02730108 (PRJNA244964, Transcriptome Project); JNBV00000000 (Whole Genome The main components isolated from the basidium of Shotgun Project)]. A. cinnamomea are classified into three categories: triterpenoids 1To whom correspondence should be addressed. Email: [email protected]. (including steroids), phenolic compounds, and polyacetylenes. The This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lanostane-type triterpenoids are produced in both mycelium and 1073/pnas.1417570111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1417570111 PNAS Early Edition | 1of10 Downloaded by guest on September 26, 2021 between the bipolar (one locus) and tetrapolar (two loci) mating isogroups from 11,622 and 19,478 isotig sequences, respectively (SI systems (16). For example, in the tetrapolar mushroom Coprinus Appendix,TablesS2andS3). An isotig represents an individual cinereus,theA genes in the HD locus promote the formation of transcript (exons and possible UTRs), whereas an isogroup repre- clamp connections and regulate conjugate division of the two nuclei sents a primary transcript (gene) containing a group of isotig from each monokaryotic mating partner, whereas the B genes in the sequences (isoforms of assembled transcripts). We examined P/R locus control the cellular fusions that complete clamp con- the cDNA assembly by searching the National Center for nections during growth and the nuclear migration required for Biotechnolgy Information (NCBI) database and found signif- dikaryosis. The clamp connection is a characteristic structure found icant hits to the protein-coding sequences of the polyporus only in fertile dikaryons with compatible A-andB-gene alleles (17). fungus Fibroporia radiculosa, Ceriporiopsis subvermispora, Although a report indicated that members in the Polyporale order Trametes versicolor, P. placenta,andDichomitus squalens. This display either bipolar or tetrapolar mating systems (16), the mating suggests a high quality of the transcript assembly, which then system of A. cinnamomea has not been characterized. enhanced the quality of our gene predictions. This study had three purposes. First,wecharacterizedthege- In parallel, we applied Illumina RNA-seq to analyze the quan- nome of A. cinnamomea to understand its gene content and genome titative transcriptome profiles of four A. cinnamomea tissues (stats structure. Second, we sequenced the transcriptomes of the mycelium of sequencing output, assembly, and read preprocessing are sum- and fruiting body to study the expression differences between my- marized in SI Appendix, Tables S2–S4). The highest mapping ratio celium