• 제목/요약/키워드: wood-rotting fungus

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Genome Sequencing and Genome-Wide Identification of Carbohydrate-Active Enzymes (CAZymes) in the White Rot Fungus Flammulina fennae

  • Lee, Chang-Soo;Kong, Won-Sik;Park, Young-Jin
    • 한국미생물·생명공학회지
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    • 제46권3호
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    • pp.300-312
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    • 2018
  • Whole-genome sequencing of the wood-rotting fungus, Flammulina fennae, was carried out to identify carbohydrate-active enzymes (CAZymes). De novo genome assembly (31 kmer) of short reads by next-generation sequencing revealed a total genome length of 32,423,623 base pairs (39% GC). A total of 11,591 gene models in the assembled genome sequence of F. fennae were predicted by ab initio gene prediction using the AUGUSTUS tool. In a genome-wide comparison, 6,715 orthologous groups shared at least one gene with F. fennae and 10,667 (92%) of 11,591 genes for F. fennae proteins had orthologs among the Dikarya. Additionally, F. fennae contained 23 species-specific genes, of which 16 were paralogous. CAZyme identification and annotation revealed 513 CAZymes, including 82 auxiliary activities, 220 glycoside hydrolases, 85 glycosyltransferases, 20 polysaccharide lyases, 57 carbohydrate esterases, and 45 carbohydrate binding-modules in the F. fennae genome. The genome information of F. fennae increases the understanding of this basidiomycete fungus. CAZyme gene information will be useful for detailed studies of lignocellulosic biomass degradation for biotechnological and industrial applications.

Genome-Wide Comparison of Carbohydrate-Active Enzymes (CAZymes) Repertoire of Flammulina ononidis

  • Park, Young-Jin;Kong, Won-Sik
    • Mycobiology
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    • 제46권4호
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    • pp.349-360
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    • 2018
  • Whole-genome sequencing of Flammulina ononidis, a wood-rotting basidiomycete, was performed to identify genes associated with carbohydrate-active enzymes (CAZymes). A total of 12,586 gene structures with an average length of 2009 bp were predicted by the AUGUSTUS tool from a total 35,524,258 bp length of de novo genome assembly (49.76% GC). Orthologous analysis with other fungal species revealed that 7051 groups contained at least one F. ononidis gene. In addition, 11,252 (89.5%) of 12,586 genes for F. ononidis proteins had orthologs among the Dikarya, and F. ononidis contained 8 species-specific genes, of which 5 genes were paralogous. CAZyme prediction revealed 524 CAZyme genes, including 228 for glycoside hydrolases, 21 for polysaccharide lyases, 87 for glycosyltransferases, 61 for carbohydrate esterases, 87 with auxiliary activities, and 40 for carbohydrate-binding modules in the F. ononidis genome. This genome information including CAZyme repertoire will be useful to understand lignocellulolytic machinery of this white rot fungus F. ononidis.

리그닌분해(分解)와 리그닌분해효소(分解酵素) 생산(生産)을 위한 목재부후균(木材腐朽菌)의 선발(選拔)과 평가(評價) (III) -리그닌분해균(分解菌) LSK-27에 의한 Manganese peroxidase 생산조건(生産條件)- (Screening and Evaluating of Wood-Rotting Fungi for Lignin Degradation and Ligninolytic Enzyme Production (III) - Conditions of Manganese Peroxidase Production by Lignin-Degrading Fungus LSK-27 -)

  • 정현채;박서기;김병수;박종열
    • Journal of the Korean Wood Science and Technology
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    • 제27권2호
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    • pp.53-61
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    • 1999
  • 리그닌분해균 LSK-27 균주로부터 Manganese peroxidase (MnP) 생산을 위한 배지조건과 Mn(II) 첨가효과를 검토하였다. LSK-27균주에 의한 균체외 MnP 생산에는 탄소원보다 질소원의 영향이 크게 나타났다. 질소원으로는 peptone 이나 yeast extract와 같은 복합 유기질소원이 효과적이었으며 특히 질소원의 농도가 높은 조건에서 MnP activity가 우수하였다. 질소원으로서 peptone 농도 1.0%까지는 뚜렷한 activity 증가를 가져왔으나 1.5%이상의 농도에서는 MnP activity 증가효과가 크게 나타나지 않았다. 반면에 탄소원의 농도에 의한 MnP activity 증가효과는 glucose농도 1.0% 에서 3.0%까지 거의 비슷한 수준이었다. Mn(II)는 높은 MnP 유도효과를 나타냈으며 첨가농도 100ppm 까지 MnP activity의 증가효과를 보였고 그 이상의 농도에서는 MnP 생산이 억제되었다. Mn(II)는 배양 2일 후에 첨가하였을 때 MnP activitv 증가가 가장 높게 나타났다.

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Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 한국균학회 2016년도 춘계학술대회 및 임시총회
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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