Differential Gene Expression Network in Terpenoid Synthesis of <i>Antrodia cinnamomea</i> in Mycelia and Fruiting Bodies

<i>Antodia cinnamomea</i>, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in <i>A. cinnamomea</i> contribute to its most important bioactivities. We identified several terpenoid compounds in <i>A. cinnamomea</i> and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network

Differential Gene Expression Network in Terpenoid Synthesis of <i>Antrodia cinnamomea</i> in Mycelia and Fruiting Bodies

<i>Antodia cinnamomea</i>, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in <i>A. cinnamomea</i> contribute to its most important bioactivities. We identified several terpenoid compounds in <i>A. cinnamomea</i> and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network

Characterization of the 2,3-Oxidosqualene Cyclase Gene from <i>Antrodia cinnamomea</i> and Enhancement of Cytotoxic Triterpenoid Compound Production

<i>Antrodia cinnamomea</i> is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of <i>A. cinnamomea</i> are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of <i>A. cinnamomea</i>. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from <i>A. cinnamomea</i> with degenerate primers and designated it as <i>AcOSC</i>. The full length <i>AcOSC</i> cDNA was subcloned into a yeast expression vector, and <i>AcOSC</i> activity was confirmed. RT-PCR results showed that <i>AcOSC</i> expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. <i>Agrobacterium</i>-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the <i>AcOSC</i> transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in <i>A. cinnamomea</i> oxidosqualene cyclase overexpression strains compared to controls. In summary an <i>Agrobacterium</i>-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured <i>A. cinnamomea.</i

Identification, Functional Characterization, and Seasonal Expression Patterns of Five Sesquiterpene Synthases in <i>Liquidambar formosana</i>

Terpenoids are a large group of important secondary metabolites that are involved in a variety of physiological mechanisms, and many are used commercially in the cosmetics and pharmaceutical industries. During the past decade, the topic of seasonal variation in terpenoid biosynthesis has garnered increasing attention. Formosan sweet gum (<i>Liquidambar formosana</i> Hance) is a deciduous tree species. The expression of terpene synthase and accumulation of terpenoids in leaves may vary in different seasons. Here, four sesquiterpene synthases (i.e., <i>LfTPS01</i>, <i>LfTPS02</i>, <i>LfTPS03</i>, and <i>LfTPS04</i>) and a bifunctional mono/sesquiterpene synthase (<i>LfTPS05</i>) were identified from Formosan sweet gum. The gene expression of <i>LfTPS01</i>, <i>LfTPS02</i>, and <i>LfTPS03</i> showed seasonal diversification, and, in addition, expression of <i>LfTPS04</i> and <i>LfTPS05</i> was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, <i>trans</i>-β-caryophyllene, α-copaene/δ-cadinene, and nerolidol/linalool, respectively. The data indicated that the sesquiterpenoid content in the essential oil of Formosan sweet gum leaves shows seasonal differences that were correlated to the sesquiterpene synthase gene expression

Gene ontology classification and KEGG annotation of DEGs between wild-type fruiting body and liquid cultured mycelium.

<p>MY: unigenes upregulated in liquid cultured mycelium, FB: unigenes upregulated in wild-type fruiting bodies. (a) GO annotation. 2,282 unigenes from DEGs were analyzed with blast2GO to obtain the GO terms. And the GO term were classified with CateGOrizer and separated into three major categories. (b) KEGG annotation. 2,282 unigenes from DEGs were submitted to KAAS to get the KEGG metabolic pathway classification.</p

Differentially expressed gene analysis of wild-type fruiting bodies and liquid cultured mycelium of <i>A</i>. <i>cinnamomea</i>.

<p>F: wild-type fruiting bodies; M: liquid cultured mycelium. (a) Scatter plot of unigenes from <i>A</i>. <i>cinnamomea</i> RNA-seq. (b) Pie chart of the DEG distribution in wild-type fruiting bodies and liquid cultured mycelium. FDR <0.05 and fold change ≥2 or ≤0.5 were defined as differential expression.</p

RT-PCR of <i>DCL-2</i> (Contig_799), <i>QDE-2</i> (argonaute protein, Contig_2130), <i>DCL-1</i> (Contig_5359) and 18s rRNA genes in <i>A</i>. <i>cinnamomea</i>.

<p>RT-PCR of <i>DCL-2</i> (Contig_799), <i>QDE-2</i> (argonaute protein, Contig_2130), <i>DCL-1</i> (Contig_5359) and 18s rRNA genes in <i>A</i>. <i>cinnamomea</i>.</p

Predicted novel milRNA candidates in <i>A</i>. <i>cinnamomea</i>.

<p>M: <i>A</i>. <i>cinnamomea</i> WSY-01 mycelium (MY) library, F: wild-type fruiting body library (FB), B: in both libraries.</p><p>Predicted novel milRNA candidates in <i>A</i>. <i>cinnamomea</i>.</p

milRNAs identified in this study with Northern blot.

<p>milRNAs identified in this study with Northern blot.</p

General features of the sRNAs and predicted miRNA in <i>A</i>. <i>cinnamomea</i>.

<p>(a) length distribution of total clean reads of sRNA library from MY and FB, (b) 5′ end nucleotide frequency of sRNAs from MY and FB, (c) length distribution of predicted novel milRNAs, (d) 5′ end nucleotide frequency of predicted novel milRNAs.</p