Yan-Liang Lin

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Significance Antrodia cinnamomea, a mushroom, has long been used as a remedy for cancer, hypertension, and hangover. However, the molecular basis of its medicinal effects is unclear and its genome has not been studied. We obtained a genome draft and conducted gene annotation. Genome ontology enrichment and pathway analyses shed light on sexual development and metabolite biosynthesis. We identified genes differentially expressed between mycelium and fruiting body and also proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to production of medicinal metabolites. Genes of metabolite biosynthesis pathways showed expression enrichment for tissue-specific compounds in mycelium and in fruiting body. Our data will be useful for developing a strategy to increase the production of valuable metabolites. Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over

MicroRNA-like small RNAs prediction in the development of Antrodia cinnamomea.

100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.

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

Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.

Differential Gene Expression Network in Terpenoid Synthesis of Antrodia cinnamomea in Mycelia and Fruiting Bodies

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 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 A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-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 AcOSC 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 A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Characterization of S-(+)-linalool synthase from several provenances of Cinnamomum osmophloeum

Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea 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.

A genetic marker of 4-coumarate: coenzyme A ligase gene in the cinnamaldehyde-chemotype Cinnamomum osmophloeum

Cinnamomum osmophloeum is a commercially important tree species in Taiwan, which has numerous chemotypes with various secondary metabolite profiles. The linalool chemotype is important because it generates pure S-(+)-linalool with high yield. In the present study, the linalool synthase (LIS) genes were isolated from different provenances of C. osmophloeum. The recombinant proteins of LIS were able to generate the S-(+)-linalool from GPP and (E)-nerolidol from FPP. Kinetic analysis showed differing enzyme activities of similar proteins from different provenances. Two point mutations enhanced enzyme activity up to sixfold. Phylogenetic analysis and leaf volatiles composition analysis clustered the genes into designated groups according to their chemotypes. Because the CoLIS-LL gene has a particularly abundant transcript, we suggest that the W-box, recognized by the WRKY transcription factor, might be responsible for high differential gene expression. A marker based on the W-box sequence can be used to distinguish linalool chemotype.

Identification, Functional Characterization, and Seasonal Expression Patterns of Five Sesquiterpene Synthases in Liquidambar formosana

Abstract There are numerous chemotypes of Cinnamomum osmophloeum in Taiwan, each of which generates an identical profile of volatile secondary metabolites. Cinnamaldehyde is the major constituent of C. osmophloeum and its quantity varies between strains. The cinnamaldehyde-type C. osmophloeum contains abundance of cinnamaldehyde, which is an economically important product, which can be gained from the leaf essential oil. Here, the genes involved in cinnamaldehyde biosynthesis have been investigated and four candidate genes, phenylalanine ammonia-lyase (CoPAL), 4-coumarate: coenzyme A ligase 1 and 4 (Co4CL1 and Co4CL4), and cinnamoyl-CoA reductase (CoCCR), were selected as potential molecular marker typical for the cinnamaldehyde chemotype. Cinnamaldehyde was increased in Co4CL1, Co4CL4, and CoCCR transgenic plants. The results showed that the cinnamaldehyde and non-cinnamaldehyde chemotypes can be distinguished by a single nucleotide polymorphism in the substrate binding pocket region of Co4CL4, at residue 378 of Co4CL4. This polymorphism could be used as a potential molecular marker for identification of strains of C. osmophloeum, which belong to high-yield cinnamaldehyde producer type. In addition, this finding might provide a suitable strategy for biosynthesis of bioactive metabolites in the future.

Kirkendall voids formation in the reaction between Ni-doped SnAg lead-free solders and different Cu substrates

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 ( Liquidambar formosana 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., LfTPS01, LfTPS02, LfTPS03, and LfTPS04) and a bifunctional mono/sesquiterpene synthase ( LfTPS05) were identified from Formosan sweet gum. The gene expression of LfTPS01, LfTPS02, and LfTPS03 showed seasonal diversification, and, in addition, expression of LfTPS04 and LfTPS05 was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, trans-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.