• Title/Summary/Keyword: polyporus brumalis

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Transformation of Terpene Synthase from Polyporus brumalis in Pichia pastoris for Recombinant Enzyme Production

  • An, Ji-Eun;Lee, Su-Yeon;Ryu, Sun-Hwa;Kim, Myungkil
    • Journal of the Korean Wood Science and Technology
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    • v.46 no.4
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    • pp.415-422
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    • 2018
  • Terpenoids have a wide range of biological functions and have extensive applications in the pharmaceutical, cosmetic, and flavoring industry. The white-rot fungus, Polyporus brumalis, is able to synthesize terpenoids via terpene synthase, which catalyzes an important step that forms a large variety of sesquiterpene products from farnesyl pyrophosphate (FPP). To improve the production of sesquiterpenes, the terpene synthase gene was isolated from Polyporus brumalis and was heterologously transformed into a Pichia pastoris strain. The open reading frame of the isolated gene (approximately 1.2 kb) was inserted into Pichia pastoris to obtain a recombinant enzyme. Five transformants were obtained and the expression of terpene synthase was analyzed at the transcript level by reverse transcription PCR (polymerase chain reaction) and at the protein level by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Expression of the terpene synthase gene product was elevated in the transformants and as expected the molecular weight of the protein was approximately 45 kDa. These recombinant enzymes have potential practical applications and future studies should focus on their functional characterization.

Biosynthesis of Eudesmane-type Sesquiterpenoids by The Wood-rotting Fungus, Polyporus brumalis, on Specific Medium, including Inorganic Magnesium Source

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • Journal of the Korean Wood Science and Technology
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    • v.44 no.2
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    • pp.253-263
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    • 2016
  • 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. Ten additional species of white rot fungi were inoculated in medium containing nutrients such as $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ at $28^{\circ}C$ for 25 days. After 10 days of incubation, eudesmane-type sesquiterpenes, ${\beta}$-eudesmane and ${\beta}$-eudesmol, were only synthesized during the growth phase of P. brumalis. Experiments excluding one nutrient at a time were conducted to determine the effects of inorganic nutrients on sesquiterpene biosynthesis. In conclusion, GC-MS analysis showed that biosynthesis of sesquiterpenes was differentially regulated by inorganic nutrients such as $MgSO_4$, $C_4H_{12}N_2O_6$, and $KH_2PO_4$. We found $MgSO_4$ supplementation to be vital for eudesmane-type sesquiterpene biosynthesis in P. brumalis; nitrogen ($C_4H_{12}N_2O_6$) and phosphate ($KH_2PO_4$) inhibited the synthesis of P. brumalis metabolites. Magnesium is a known cofactor of sesquiterpene synthase, which promotes ${\beta}$-eudesmol synthesis. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

Changes in Activities of Lignin Degrading Enzymes and Lignin Content During Degradation of Wood Chips by Polyporus brumalis (겨울우산버섯에 의한 목재칩의 리그닌 분해 효소 활성 및 리그닌 함량 변화)

  • Cho, Myung-Kil;Ryu, Sun-Hwa;Kim, Myungkil
    • Journal of the Korean Wood Science and Technology
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    • v.40 no.6
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    • pp.424-430
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    • 2012
  • In this study, laccase activity, rate of weight loss and degree of lignin degradation of pine wood chips were determined during the liquid and solid state incubation with Polyporus brumalis. The results showed that laccase enzyme activity at untreated wood chip was gradually decreased after 20 days, but enzyme activity with wood chip treatment showed 10 times higher than untreated ones at 60 incubation days. Rate of weight losses of pine chip and rate of lignin loss were 23.4% and 6.3% by P. brumalis during 80 incubation days. Gene expression of pblac1 from P. brumalis was 3 times increased under pine chip treatment at 40 incubation days. Consequently, laccase activity of white rot fungi, P. brumalis, was increased at incubation with wood chip and pblac1 gene was important factor of lignin degradation. Therefore, to regulate lignin degrading enzyme gene expression by using the tools of biotechnology will be able to develop superior strains and it will be useful for pretreatment of lignocellulosic biomass at bioethanol production.

Protoplast Isolation and Genetic Transformation of Polyporus brumalis (겨울우산버섯의 원형질체 분리와 유전자 형질전환)

  • Ryu, Sun-Hwa;Kim, Myung-Kil
    • Korean Journal of Microbiology
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    • v.50 no.4
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    • pp.372-375
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    • 2014
  • This experiment was undertaken to investigate proper conditions for protoplast isolation and genetic transformation of the white rot fungi, Polyporus brumalis. The protoplasts were formed from mycelia at a frequency of $1{\times}10^7/ml$ with 0.5% Usukizyme. The transformation vector (pHYgpt) was constructed using hygromycin resistance gene (hph) for the selectable maker. The yield was 100-160 transformants/${\mu}g$ DNA in a transformation mediated by 40% polyethylene glycol solution with aurintricarboxylic acid, heparin and supermidine. The genomic integration of the pHYgpt was confirmed by hph-specific PCR and the expected amplified band appeared only in the transformants. These results could be an efficient tool in gene engineering of the genus polyporus.

Biodegradation of Phthalic acid by White rot Fungus, Polyporus brumalis (백색부후균 Polyporus brumalis에 의한 프탈산의 분해)

  • Lee, Soo-Min;Park, Ki-Ryung;Lee, Sung-Suk;Kim, Myung-Kil;Choi, In-Gyu
    • Journal of the Korean Wood Science and Technology
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    • v.33 no.1 s.129
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    • pp.48-57
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    • 2005
  • Phthalate esters are known as plasticizers and some of them suspected as endocrine disrupting chemicals. In this study, in order to identify the mechanism of phthalate esters degradation by white rot fungus, phthalic acid, which is major metabolite in the biodegradation of phthalate esters, was used. Phthalic acid 50 ppm was treated in culture medium with Polyporus brumalis. The availability of ABTS oxidation was different from control and phthalic acid treated group after 4 days of incubation. The activity was gradually increased in control group, but not in phthalic acid treated group. Especially, esterase activity of control group was maximized at 10 days of incubation, and then decreased while the activity of phthalic acid treated group was increased. Glucose was used as a carbon source, and the difference of glucose consumption by control and phthalic acid treated group was not significant. However, after 6 days of incubation the residual glucose in culture medium was rapidly decreased. The consumption rate of phthalic acid treated group was lower than control. These results might indicate that the absorption of phthalic acid in culture medium was occurred by mycelium and metabolized through some pathways as that of glucose was. To clearify the chemical modification of phthalic acid in culture medium, phthalic acid was reacted under in vitro condition which mycelium was excluded. The metabolites were analyzed by GC/MS. The results showed that phthalic acid was converted to phthalic acid anhydride by the extracellular enzymes of P. brumalis.

Phylogenetic relationships of genera Polyporus on the basis of ITS region sequences (rDNA의 ITS 부위 염기서열 분석에 의한 겨울우산버섯(Polyporus)속 균주의 유전적인 유연관계 분석)

  • Lee, Chan-Jung;Jhune, Chang-Sung;Cheong, Jong-Chun;Kong, Won S.
    • Journal of Mushroom
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    • v.10 no.1
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    • pp.37-43
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    • 2012
  • This study was carried to identify a correct species and asses genetic diversity within the same species of Polyporus spp. preserved in Division of applied Microbiology. Contaminated isolates showed different growth rates, morphology and color of hyphae. We have reconstructed the phylogenetic tree of a select group of Polyporus spp. using nucleotide sequences of the internal transcribed spacer region(ITS) region. The phylogenetic tree was constructed by using the neighbor-joining method. PELF primers of 20-mer were used to assess genetic diversity of preserved isolates. Sequence analysis showed that three strains were different species and four strains were identified completely different nomenclature. According to the analysis of ITS sequences, the genus Polyporus clustered into five distinct group, most of which correlated with species-groups identified by RAPD method. Four isolates included strain PM02 showed high similarity with P. arcularius, four isolates included strain PM03 high similarity with P. alveolaris, three isolates included strain PM01 high similarity with P. tuberaster, and PM 06 and PM04 high similarity with P. brumalis and P. squamossus. Isolates were collected in the United States(PM10, PM11) was identified as P. alveolarius and P. arcularius. RAPD analysis of genetic polymorphisms of genus Polyporus showed a very different band patterns. As the result of RAPD and ITS region sequences analysis for preserved isolates, it seems likely that 6 isolates of Polyporus spp. may be need to reclassify or eliminate from preserved catalogue.

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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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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Comparison of Mycelial Growth Characteristics According to Culture Conditions of Ulleungdo Collection Strains (울릉도 수집 균주의 배양조건에 따른 균사 생장 특성 비교)

  • Kim, Minkyeong;Ahn, Chorong;Kim, Changmu
    • The Korean Journal of Mycology
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    • v.48 no.2
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    • pp.75-85
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    • 2020
  • The collection of biological data of indigenous species must comply with the Nagoya Protocol. Fungi contain various bioactive substances making them an attractive source of several products, including food and medicines. In this study, we investigated the growth characteristics of five indigenous fungal strains, Fomitiporia punctata, Polyporus ulleungus, P. brumalis, Gymnopus subnudus, and Tyromyces kmetii, isolated from samples collected in the Ulleungdo Island. The growth rates for each strain were assessed across various temperatures (20 ℃ to 35 ℃), culture media (Potato dextrose agar, Malt extract & Yeast extract agar, Malt extract agar, Malt extract & peptone agar, Sabouraud dextrose agar, and Modified Melin-Norkrans agar), and pH conditions (4.0 to 8.0). Additionally, we assessed the mycelial growth characteristics in liquid culture. The mycelial growth in different media varied across species; specifically, F. punctata (in MMNA), G subnudus (in MMNA), and P. brumalis (in MEPA) showed rapid growth. Optimal growth temperatures ranged between 25 ℃ and 30 ℃ for most species, with the exception of T. kmetii and P. brumalis, which were able to grow across all the temperatures tested. P. brumalis showed the best growth rate, whereas P. ulleungus showed the lowest growth potential. The optimal pH conditions for mycelial growth ranged between 4.0 and 5.0. In experiments using culture flasks, the dry weight of the culture filtrates decreased with the increasing incubation time and showed a significant decrease between 1 and 6 months of incubation, indicating that the five strains take longer than a month to fully use the culture media. Our findings highlight and establish the optimal growth conditions for five different fungal species that can be used in future application studies.

Screening of Outstanding White Rot Fungi for Biodegradation of Organosolv Lignin by Decolorization of Remazol Brilliant Blue R and Ligninolytic Enzymes Systems (Remazol Brilliant Blue R 탈색능과 리그닌 분해 효소시스템을 이용한 유기용매 리그닌 생분해 우수 균주 선별)

  • Hong, Chang-Young;Kim, Ho-Yong;Jang, Soo-Kyeong;Choi, In-Gyu
    • Journal of the Korean Wood Science and Technology
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    • v.41 no.1
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    • pp.19-32
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    • 2013
  • In this study, outstanding white rot fungi for biodegradation of organosolv lignin were selected on the basis of their ligninolytic enzyme system. Fifteen white rot fungi were evaluated for their ability to decolorize Remazol Brilliant Blue R (RBBR) in SSC and MEB medium, respectively. Six white rot fungi (Ceriporiopsis subvermispora, Ceriporia lacerate, Fomitopsis insularis, Phanerochaete chrysosporium, Polyporus brumalis, and Stereum hirsutum) decolorized RBBR rapidly in SSC medium within 3 days. The protein contents as well as the activities of manganese peroxidase (MnP) and laccase for 6 selected fungi were determined on the SSC medium with and without organosolv lignin. Interestingly, extracellular protein concentrations were determined to relative higher for S. hirsutum and P. chrysosporium in the presence of organosolv lignin than others. On the other hands, each fungus showed a different ligninolytic enzyme pattern. Among them, F. insularis resulted the highest ligninolytic enzyme activities on incubation day 6, indicating of 1,545 U/mg of MnP activity and 1,259 U/mg of laccase activity. In conclusion, $STH^*$ and FOI were considered as outstanding fungi for biodegradation of organosolv lignin, because $STH^*$ showed high extracellular protein contents and ligninolytic enzyme activities over all, and ligninolytic enzyme activities of FOI were the highest among white rot fungi used in this study.

Biological Pretreatment of Softwood Pinus densiflora by Three White Rot Fungi

  • Lee, Jae-Won;Gwak, Ki-Seob;Park, Jun-Yeong;Park, Mi-Jin;Choi, Don-Ha;Kwon, Mi;Choi, In-Gyu
    • Journal of Microbiology
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    • v.45 no.6
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    • pp.485-491
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    • 2007
  • The effects of biological pretreatment on the Japanese red pine Pinus densiflora, was evaluated after exposure to three white rot fungi Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. Change in chemical composition, structural modification, and their susceptibility to enzymatic saccharification in the degraded wood were analyzed. Of the three white rot fungi tested, S. hirsutum selectively degraded the lignin of this sortwood rather than the holocellulose component. After eight weeks of pretreatment with S. hirsutum, total weight loss was 10.7%, while lignin loss was the highest at 14.52% among the tested samples. However, holocellulose loss was lower at 7.81 % compared to those of C. lacerata and P. brumalis. Extracelluar enzymes from S. hirsutum showed higher activity of ligninase and lower activity of cellulase than those from other white rot fungi. Thus, total weight loss and changes in chemical composition of the Japanese red pine was well correlated with the enzyme activities related with lignin- and cellulose degradation in these fungi. Based on the data obtained from analysis of physical characterization of degraded wood by X-ray Diffractometry (XRD) and pore size distribution, S. hirsutum was considered as an effective potential fungus for biological pretreatment. In particular, the increase of available pore size of over 120 nm in pretreated wood powder with S. hirsutum made enzymes accessible for further enzymatic saccharification. When Japanese red pine chips treated with S. hirsutum were enzymatically saccharified using commercial enzymes (Cellulclast 1.5 L and Novozyme 188), sugar yield was greatly increased (21.01 %) compared to non-pre treated control samples, indicating that white rot fungus S. hirsutum provides an effective process in increasing sugar yield from woody biomass.