研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    堀 千明(ホリ チアキ), ホリ チアキ

所属(マスター)

  • 地球環境科学研究院 環境生物科学部門 環境分子生物学分野

所属(マスター)

  • 地球環境科学研究院 環境生物科学部門 環境分子生物学分野

独自項目

syllabus

  • 2021, 応用生物化学(生命システム工学), Applied Biochemistry (Biosystem Engineering), 修士課程, 総合化学院, 遺伝子、タンパク質、転写翻訳、酵素、生化学、進化分子工学、分子設計、タンパク質工学、生物の熱力学、バイオプラスチック、生分解、バイオテクノロジー、ゲノム、オミクス解析、細菌、糸状菌
  • 2021, 英語演習, English Seminar, 学士課程, 全学教育, 英語,化学,工学
  • 2021, 応用化学学生実験Ⅲ, Applied Chemistry Laboratory Ⅲ, 学士課程, 工学部, 微生物の培養,微生物の観察,遺伝子工学,形質転換,大量発現,タンパク質精製
  • 2021, 化学英語, English for Chemistry, 学士課程, 工学部, 化学,英語,単語,表記法
  • 2021, 環境と人間, Environment and People, 学士課程, 全学教育, バイオマス、農耕地、食料、エネルギー、有用物質、触媒、バイオエコノミー

PositionHistory

  • 研究戦略室室員, 2023年4月1日, 2024年3月31日

researchmap

プロフィール情報

学位

  • 博士(農学)(東京大学)

プロフィール情報

  • 堀, ホリ
  • 千明, チアキ
  • ID各種

    201601020460545298

対象リソース

業績リスト

研究キーワード

  • オミクス解析   糖質関連酵素   糸状菌   植物細胞壁   木材腐朽菌   

研究分野

  • ライフサイエンス / 応用生物化学
  • ライフサイエンス / ゲノム生物学
  • ライフサイエンス / 木質科学

経歴

  • 2022年04月 - 現在 北海道大学 大学院地球環境科学研究院 准教授
  • 2016年09月 - 2022年03月 北海道大学 工学研究院 応用化学部門 助教
  • 2018年08月 - 2018年08月 US department of Agriculture Forest Products Laboratory Madison Visiting researcher
  • 2017年01月 - 2017年03月 US department of Agriculture Forest Products Laboratory Madison Visiting researcher
  • 2015年04月 - 2016年08月 北海道大学 農学研究院 訪問研究員・博士研究員
  • 2013年04月 - 2016年03月 日本学術振興会 日本学術振興会(PD)
  • 2013年04月 - 2015年03月 理化学研究所 環境資源科学センター 訪問研究員
  • 2012年06月 - 2013年03月 US department of Agriculture Forest Products Laboratory Madison Visiting researcher
  • 2011年04月 - 2013年03月 日本学術振興会 特別研究員(DC2/PD)

学歴

  • 2009年04月 - 2012年03月   東京大学   大学院農学生命科学研究院   材料科学専修 博士課程
  • 2007年04月 - 2009年03月   東京大学   大学院農学生命科学研究院   材料科学専修 修士課程
  • 2003年04月 - 2007年03月   東京大学   農学部   植物資源プロセス学科

委員歴

  • 2021年04月 - 現在   日本木材学会・北日本支部会・研究会理事
  • 2019年04月 - 現在   糸状菌分子生物学研究会・若手の会運営委員
  • 2023年 - 2023年   細胞壁研究会   2023年度・実行委員
  • 2020年 - 2021年   7th International Conference on Plant Cell Wall Biology・実行委員
  • 2018年04月 - 2020年03月   応用化学部門・広報委員(部門内委員・コース紹介やホームページ運営)
  • 2019年   2019年度 糸状菌分子生物学カンファレンス・実行委員
  • 2019年   2019年度 日本生物工学会・北日本支部シンポジウム・実行委員
  • 2019年   2019年度 木材学会・年次大会・実行委員
  • 2018年   2018年度 日本生物工学会・若手の会・実行委員

受賞

  • 2022年04月 旭硝子財団 研究奨励賞
  • 2021年03月 日本農芸化学会 若手女性研究者賞
     バイオマス分解利用に関する基礎研究
  • 2020年06月 天野エンザイム科学技術振興財団 酵素応用シンポジウム研究奨励賞
     
    受賞者: 堀 千明
  • 2020年03月 日本木材学会 奨励賞
     
    受賞者: 堀 千明
  • 2018年11月 Novozymes Japan Research Fund 2018
     
    受賞者: 堀 千明
  • 2018年06月 秋山記念生命科学振興財団 奨励賞
     
    受賞者: 堀 千明
  • 2013年12月 理化学研究所 環境資源科学研究センター リトリート ポスター賞
     
    受賞者: 堀 千明
  • 2013年04月 日本学術振興会 特別研究員(PD)
     
    受賞者: 堀 千明
  • 2013年03月 US Department of Agriculture・Forest Products Laboratory Certificate of application
     
    受賞者: 堀 千明
  • 2011年04月 日本学術振興会 特別研究員(DC2)
     
    受賞者: 堀 千明
  • 2007年08月 日本木材学会 優秀ポスター賞
     
    受賞者: 堀 千明

論文

  • Hiroyuki Kato, Yasushi Takahashi, Hiromitsu Suzuki, Keisuke Ohashi, Ryunosuke Kawashima, Koki Nakamura, Kiyota Sakai, Chiaki Hori, Taichi E Takasuka, Masashi Kato, Motoyuki Shimizu
    Applied and environmental microbiology e0175323  2024年01月23日 
    White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.
  • 植物バイオマス分解利用に関する基礎研究〜多様な腐朽菌による木質成分の分解システムの解明と樹木による木質形成の改変について〜
    堀千明
    化学と生物 61 12 590 - 595 2023年12月 [査読有り][招待有り]
  • Ruy Matsumoto, Jakia Jerin Mehjabin, Hideki Noguchi, Toshizumi Miyamoto, Taichi E Takasuka, Chiaki Hori
    Applied and environmental microbiology e0027223  2023年04月26日 
    Perenniporia fraxinea can colonize living trees and cause severe damage to standing hardwoods by secreting a number of carbohydrate-activate enzymes (CAZymes), unlike other well-studied Polyporales. However, significant knowledge gaps exist in understanding the detailed mechanisms for this hardwood-pathogenic fungus. To address this issue, five monokaryotic P. fraxinea strains, SS1 to SS5, were isolated from the tree species Robinia pseudoacacia, and high polysaccharide-degrading activities and the fastest growth were found for P. fraxinea SS3 among the isolates. The whole genome of P. fraxinea SS3 was sequenced, and its unique CAZyme potential for tree pathogenicity was determined in comparison to the genomes of other nonpathogenic Polyporales. These CAZyme features are well conserved in a distantly related tree pathogen, Heterobasidion annosum. Furthermore, the carbon source-dependent CAZyme secretions of P. fraxinea SS3 and a nonpathogenic and strong white-rot Polyporales member, Phanerochaete chrysosporium RP78, were compared by activity measurements and proteomic analyses. As seen in the genome comparisons, P. fraxinea SS3 exhibited higher pectin-degrading activities and higher laccase activities than P. chrysosporium RP78, which were attributed to the secretion of abundant glycoside hydrolase family 28 (GH28) pectinases and auxiliary activity family 1_1 (AA1_1) laccases, respectively. These enzymes are possibly related to fungal invasion into the tree lumens and the detoxification of tree defense substances. Additionally, P. fraxinea SS3 showed secondary cell wall degradation capabilities at the same level as that of P. chrysosporium RP78. Overall, this study suggested mechanisms for how this fungus can attack the cell walls of living trees as a serious pathogen and differs from other nonpathogenic white-rot fungi. IMPORTANCE Many studies have been done to understand the mechanisms underlying the degradation of plant cell walls of dead trees by wood decay fungi. However, little is known about how some of these fungi weaken living trees as pathogens. P. fraxinea belongs to the Polyporales, a group of strong wood decayers, and is known to aggressively attack and fell standing hardwood trees all over the world. Here, we report CAZymes potentially related to plant cell wall degradation and pathogenesis factors in a newly isolated fungus, P. fraxinea SS3, by genome sequencing in conjunction with comparative genomic and secretomic analyses. The present study provides insights into the mechanisms of the degradation of standing hardwood trees by the tree pathogen, which will contribute to the prevention of this serious tree disease.
  • Yoshimi Nakano, Hitoshi Endo, Lorenz Gerber, Chiaki Hori, Ayumi Ihara, Masayo Sekimoto, Tomoko Matsumoto, Jun Kikuchi, Misato Ohtani, Taku Demura
    Frontiers in plant science 13 819360 - 819360 2022年 
    The secondary cell wall (SCW) in the xylem is one of the largest sink organs of carbon in woody plants, and is considered a promising sustainable bioresource for biofuels and biomaterials. To enhance SCW formation in poplar (Populus sp.) xylem, we developed a self-reinforced system of SCW-related transcription factors from Arabidopsis thaliana, involving VASCULAR-RELATED NAC-DOMAIN7 (VND7), SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN 1/NAC SECONDARY WALL THICKENING-PROMOTING FACTOR3 (SND1/NST3), and MYB46. In this system, these transcription factors were fused with the transactivation domain VP16 and expressed under the control of the Populus trichocarpa CesA18 (PtCesA18) gene promoter, creating the chimeric genes PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16. The PtCesA18 promoter is active in tissues generating SCWs, and can be regulated by AtVND7, AtSND1, and AtMYB46; thus, the expression levels of PtCesA18pro::AtVND7:VP16, PtCesA18pro::AtSND1:VP16, and PtCesA18pro::AtMYB46:VP16 are expected to be boosted in SCW-generating tissues. In the transgenic hybrid aspens (Populus tremula × tremuloides T89) expressing PtCesA18pro::AtSND1:VP16 or PtCesA18pro::AtMYB46:VP16 grown in sterile half-strength Murashige and Skoog growth medium, SCW thickening was significantly enhanced in the secondary xylem cells, while the PtCesA18pro::AtVND7:VP16 plants showed stunted xylem formation, possibly because of the enhanced programmed cell death (PCD) in the xylem regions. After acclimation, the transgenic plants were transferred from the sterile growth medium to pots of soil in the greenhouse, where only the PtCesA18pro::AtMYB46:VP16 aspens survived. A nuclear magnetic resonance footprinting cell wall analysis and enzymatic saccharification analysis demonstrated that PtCesA18pro::AtMYB46:VP16 influences cell wall properties such as the ratio of syringyl (S) and guaiacyl (G) units of lignin, the abundance of the lignin β-aryl ether and resinol bonds, and hemicellulose acetylation levels. Together, these data indicate that we have created a self-reinforced system using SCW-related transcription factors to enhance SCW accumulation.
  • Mana Iwata, Ana Gutiérrez, Gisela Marques, Grzegorz Sabat, Philip J Kersten, Daniel Cullen, Jennifer M Bhatnagar, Jagjit Yadav, Anna Lipzen, Yuko Yoshinaga, Aditi Sharma, Catherine Adam, Christopher Daum, Vivian Ng, Igor V Grigoriev, Chiaki Hori
    Scientific reports 11 1 12528 - 12528 2021年06月15日 
    Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.
  • Akira Yoshinari, Takuya Hosokawa, Marcel Pascal Beier, Keishi Oshima, Yuka Ogino, Chiaki Hori, Taichi E Takasuka, Yoichiro Fukao, Toru Fujiwara, Junpei Takano
    The Plant cell 33 2 420 - 438 2021年04月17日 
    Plants take up and translocate nutrients through transporters. In Arabidopsis thaliana, the borate exporter BOR1 acts as a key transporter under boron (B) limitation in the soil. Upon sufficient-B supply, BOR1 undergoes ubiquitination and is transported to the vacuole for degradation, to avoid overaccumulation of B. However, the mechanisms underlying B-sensing and ubiquitination of BOR1 are unknown. In this study, we confirmed the lysine-590 residue in the C-terminal cytosolic region of BOR1 as the direct ubiquitination site and showed that BOR1 undergoes K63-linked polyubiquitination. A forward genetic screen identified that amino acid residues located in vicinity of the substrate-binding pocket of BOR1 are essential for the vacuolar sorting. BOR1 variants that lack B-transport activity showed a significant reduction of polyubiquitination and subsequent vacuolar sorting. Coexpression of wild-type (WT) and a transport-defective variant of BOR1 in the same cells showed degradation of the WT but not the variant upon sufficient-B supply. These findings suggest that polyubiquitination of BOR1 relies on its conformational transition during the transport cycle. We propose a model in which BOR1, as a B transceptor, directly senses the B concentration and promotes its own polyubiquitination and vacuolar sorting for quick and precise maintenance of B homeostasis.
  • Chiaki Hori, Naoki Takata, Pui Ying Lam, Yuki Tobimatsu, Soichiro Nagano, Jenny C. Mortimer, Dan Cullen
    Scientific Reports 10 1 22043 - 22043 2020年12月16日 [査読有り]
     
    AbstractDeveloping an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.
  • Chiaki Hori, Xiang Yu, Jenny C Mortimer, Ryosuke Sano, Tomoko Matsumoto, Jun Kikuchi, Taku Demura, Misato Ohtani
    Plant biotechnology (Tokyo, Japan) 37 3 273 - 283 2020年09月25日 
    Growth of biomass for lignocellulosic biofuels and biomaterials may take place on land unsuitable for foods, meaning the biomass plants are exposed to increased abiotic stresses. Thus, the understanding how this affects biomass composition and quality is important for downstream bioprocessing. Here, we analyzed the effect of drought and salt stress on cell wall biosynthesis in young shoots and xylem tissues of Populus trichocarpa using transcriptomic and biochemical methods. Following exposure to abiotic stress, stem tissues reduced vessel sizes, and young shoots increased xylem formation. Compositional analyses revealed a reduction in the total amount of cell wall polysaccharides. In contrast, the total lignin amount was unchanged, while the ratio of S/G lignin was significantly decreased in young shoots. Consistent with these observations, transcriptome analyses show that the expression of a subset of cell wall-related genes is tightly regulated by drought and salt stresses. In particular, the expression of a part of genes encoding key enzymes for S-lignin biosynthesis, caffeic acid O-methyltransferase and ferulate 5-hydroxylase, was decreased, suggesting the lower S/G ratio could be partly attributed to the down-regulation of these genes. Together, our data identifies a transcriptional abiotic stress response strategy in poplar, which results in adaptive changes to the plant cell wall.
  • Chiaki Hori, Tomohiro Sugiyama, Kodai Watanabe, Jian Sun, Yuu Kamada, Toshihiko Ooi, Takuya Isono, Toshifumi Satoh, Shin-ichiro Sato, Seiichi Taguchi, Ken'ichiro Matsumoto
    POLYMER DEGRADATION AND STABILITY 179 109231 - 109231 2020年09月 [査読有り][通常論文]
     
    P[D-lactate (LA)-co-3-hydroxybutyrate (3HB)] is an artificial polyhydroxyalkanoate (PHA) containing unusual D-LA units. In this study, the P(D-LA-co-3HB)-degrading bacterial group in the soil was analyzed and the bacterial degradation of the D-LA clustering structure in the copolymer were evaluated by using chemically synthetic D-LA homo-oligomers. A total of 216 soil samples were screened on the basis of clear zone formation on agar plates containing emulsified P(64 mol% D-LA-co-3HB). The 16S rRNA analysis of the isolated bacteria resulted in the identification of eight Variovorax, three Acidovorax, and one Burkholderia strains, which are closely related to previously identified natural PHA-degrading bacteria. These bacteria nearly consumed the P(D-LA-co-3HB) emulsion in the liquid culture; however, a small amount of the D-LA fraction remained unconsumed, which should be attributable to the D-LA-clustering structure in the copolymer. Cultivation of the isolated bacteria with the D-LA homo-oligomers revealed that the oligomers with a degree of polymerization (DP) ranging from 10 to 30 were partly consumed by six Variovorax and one Acidovorax strains. In contrast, the oligomers with DP ranging from 20 to 60 were not consumed by the isolated bacteria. These results indicate that D-LA homo-oligomers with DP higher than approximately 20 are hardly degraded by the soil bacteria. Molecular dynamic simulation of the D-LA homo-oligomers indicated that the upper limit of DP is likely to be determined by the conformational structure of the oligomers in water. The information obtained in this study will be useful for the molecular design of biodegradable D-LA-containing polymers. (C) 2020 Elsevier Ltd. All rights reserved.
  • Chiaki Hori, Ruopu Song, Kazuki Matsumoto, Ruy Matsumoto, Benjamin B. Minkoff, Shuzo Oita, Hideho Hara, Taichi E. Takasuka
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 86 8 2020年04月 [査読有り][通常論文]
     
    Wood-devastating insects utilize their symbiotic microbes with lignocellulose-degrading abilities to extract energy from recalcitrant woods. It is well known that free-living lignocellulose-degrading fungi secrete various carbohydrate-active enzymes (CAZymes) to degrade plant cell wall components, mainly cellulose, hemicellulose, and lignin. However, CAZymes from insect-symbiotic fungi have not been well documented except for a few examples. In this study, an insect-associated fungus, Daldinia decipiens oita, was isolated as a potential symbiotic fungus of female Xiphydria albopicta captured from Hokkaido forest. This fungus was grown in seven different media containing a single carbon source, glucose, cellulose, xylan, mannan, pectin, poplar, or larch, and the secreted proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 128 CAZymes, including domains of 92 glycoside hydrolases, 15 carbohydrate esterases, 5 polysaccharide lyases, 17 auxiliary activities, and 11 carbohydrate-binding modules, were identified, and these are involved in degradation of cellulose and hemicellulose but not lignin. Together with the results of polysaccharide-degrading activity measurements, we concluded that D. decipiens oita tightly regulates the expression of these CAZymes in response to the tested plant cell wall materials. Overall, this study described the detailed proteomic approach of a woodwasp-associated fungus and revealed that the new isolate, D. decipiens oita, secretes diverse CAZymes to efficiently degrade lignocellulose in the symbiotic environment.IMPORTANCE Recent studies show the potential impacts of insect symbiont microbes on biofuel application with regard to their degradation capability of a recalcitrant plant cell wall. In this study, we describe a novel fungal isolate, D. decipiens oita, as a single symbiotic fungus from the Xiphydria woodwasp found in the northern forests of Japan. Our detailed secretome analyses of D. decipiens oita, together with activity measurements, reveal that this insect-associated fungus exhibits high and broad activities for plant cell wall material degradation, suggesting potential applications within the biomass conversion industry for plant mass degradation.
  • Maho Sudo, Chiaki Hori, Toshihiko Ooi, Shoji Mizuno, Takeharu Tsuge, Ken'ichiro Matsumoto
    Journal of bioscience and bioengineering 129 3 302 - 306 2019年10月18日 [査読有り][通常論文]
     
    The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes β-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.
  • Satoko Yoshida, Seungill Kim, Eric K. Wafula, Jaakko Tanskanen, Yong-Min Kim, Loren Honaas, Zhenzhen Yang, Thomas Spallek, Caitlin E. Conn, Yasunori Ichihashi, Kyeongchae Cheong, Songkui Cui, Joshua P. Der, Heidrun Gundlach, Yuannian Jiao, Chiaki Hori, Juliane K. Ishida, Hiroyuki Kasahara, Takatoshi Kiba, Myung-Shin Kim, Namjin Koo, Anuphon Laohavisit, Yong-Hwan Lee, Shelley Lumba, Peter McCourt, Jenny C. Mortimer, J. Musembi Mutuku, Takahito Nomura, Yuko Sasaki-Sekimoto, Yoshiya Seto, Yu Wang, Takanori Wakatake, Hitoshi Sakakibara, Taku Demura, Shinjiro Yamaguchi, Koichi Yoneyama, Ri-ichiroh Manabe, David C. Nelson, Alan H. Schulman, Michael P. Timko, Claude W. DePamphilis, Doil Choi, Ken Shirasu
    CURRENT BIOLOGY 29 18 3041 - + 2019年09月 [査読有り][通常論文]
     
    Parasitic plants in the genus Striga, commonly known as witchweeds, cause major crop losses in sub-Saharan Africa and pose a threat to agriculture worldwide. An understanding of Striga parasite biology, which could lead to agricultural solutions, has been hampered by the lack of genome information. Here, we report the draft genome sequence of Striga asiatica with 34,577 predicted protein-coding genes, which reflects gene family contractions and expansions that are consistent with a three-phase model of parasitic plant genome evolution. Striga seeds germinate in response to host-derived strigolactones (SLs) and then develop a specialized penetration structure, the haustorium, to invade the host root. A family of SL receptors has undergone a striking expansion, suggesting a molecular basis for the evolution of broad host range among Striga spp. We found that genes involved in lateral root development in non-parasitic model species are coordinately induced during haustorium development in Striga, suggesting a pathway that was partly co-opted during the evolution of the haustorium. In addition, we found evidence for horizontal transfer of host genes as well as retrotransposons, indicating gene flow to S. asiatica from hosts. Our results provide valuable insights into the evolution of parasitism and a key resource for the future development of Striga control strategies.
  • Ken'ichiro Matsumoto, Jun Saito, Toshinori Yokoo, Chiaki Hori, Akihiro Nagata, Yuki Kudoh, Toshihiko Ooi, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 128 3 302 - 306 2019年09月 [査読有り][通常論文]
     
    Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1(Ps)STQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (Delta glcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1(Ps)STQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.
  • Chiaki Hori, Takashi Yamazaki, Greg Ribordy, Kenji Takisawa, Ken'ichiro Matsumoto, Toshihiko Ooi, Manfred Zinn, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 127 6 721 - 725 2019年06月 [査読有り][通常論文]
     
    Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is produced in engineered Escherichia coli harboring the genes encoding an LA-polymerizing enzyme (LPE) and monomer-supplying enzymes. In this study, high cell-density fed-batch jar fermentation was developed using xylose and/or glucose as the carbon source. Fed-batch fermentation was initially performed with 20 g/L sugar during the batch phase for 24 h, and subsequent sugar feeding from 24 to 86 h. The feeding rate was increased in a stepwise manner. When xylose alone was used for cultivation, the cells produced the polymer at 11.6 g/L, which was higher than the 43 g/L obtained using glucose as the sole carbon source. However, in the first 24 h the growth in the glucose culture was greater than in the xylose culture. Based on these results, glucose was used for cell growth (at the initial stage) and xylose was used for polymer production (at the feeding stage). As expected, in the glucose/xylose switching fermentation method, polymer production was significantly enhanced, eventually reaching 26.7 g/L. The enhanced polymer production obtained by using xylose was presumably due to overflow metabolism. In fact, during xylose feeding, acetic acid excretion was greater than that in case of the glucose grown culture, suggesting the channeling of the metabolic flux from acetyl-CoA towards polymer production over into the tricarboxylic acid cycle in the xylose-fed cultures. Therefore, this sequential glucose/xylose feed strategy is potentially useful for production of acetyl-CoA derived compounds in E. coli. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.
  • J. Musembi Mutuku, Songkui Cui, Chiaki Hori, Yuri Takeda, Yuki Tobimatsu, Ryo Nakabayashi, Tetsuya Mori, Kazuki Saito, Taku Demura, Toshiaki Umezawa, Satoko Yoshida, Ken Shirasu
    PLANT PHYSIOLOGY 179 4 1796 - 1809 2019年04月 [査読有り][通常論文]
     
    Striga species are parasitic weeds that seriously constrain the productivity of food staples, including cereals and legumes, in Sub-Saharan Africa and Asia. In eastern and central Africa, Striga spp. infest as much as 40 million hectares of smallholder farmland causing total crop failure during severe infestation. As the molecular mechanisms underlying resistance are yet to be elucidated, we undertook a comparative metabolome study using the Striga-resistant rice (Oryza sativa) cultivar 'Nipponbare' and the susceptible cultivar 'Koshihikari'. We found that a number of metabolites accumulated preferentially in the Striga-resistant cultivar upon Striga hermonthica infection. Most apparent was increased deposition of lignin, a phenylpropanoid polymer mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatic units, around the site of interaction in Nipponbare. The increased deposition of lignin was accompanied by induction of the expression of corresponding enzyme-encoding genes in the phenylpropanoid pathway. In addition, perturbing normal lignin composition by knocking down or overexpressing the genes that regulate lignin composition, i.e. p-COUMARATE 3-HYDROXYLASE or FERULATE 5-HYDROXYLASE, enhanced susceptibility of Nipponbare to S. hermonthica infection. These results demonstrate that enhanced lignin deposition and maintenance of the structural integrity of lignin polymers deposited at the infection site are crucial for postattachment resistance against S. hermonthica.
  • Tomohiro Watanabe, Hisaya Kojima, Kazuhiro Umezawa, Chiaki Hori, Taichi E. Takasuka, Yukako Kato, Manabu Fukui
    FRONTIERS IN MICROBIOLOGY 10 316  2019年02月 [査読有り][通常論文]
     
    Even in the current era of metagenomics, the interpretation of nucleotide sequence data is primarily dependent on knowledge obtained from a limited number of microbes isolated in pure culture. Thus, it is of fundamental importance to expand the variety of strains available in pure culture, to make reliable connections between physiological characteristics and genomic information. In this study, two sulfur oxidizers that potentially represent two novel species were isolated and characterized. They were subjected to whole-genome sequencing together with 7 neutrophilic and chemolithoautotrophic sulfur-oxidizing bacteria. The genes for sulfur oxidation in the obtained genomes were identified and compared with those of isolated sulfur oxidizers in the classes Betaproteobacteria and Gammaproteobacteria. Although the combinations of these genes in the respective genomes are diverse, typical combinations corresponding to three types of core sulfur oxidation pathways were identified. Each pathway involves one of three specific sets of proteins, SoxCD, DsrABEFHCMKJOP, and HdrCBAHypHdrCB. All three core pathways contain the SoxXYZAB proteins, and a cytoplasmic sulfite oxidase encoded by soeABC is a conserved component in the core pathways lacking SoxCD. Phylogenetically close organisms share same core sulfur oxidation pathway, but a notable exception was observed in the family 'Sulfuricellaceae'. In this family, some strains have either core pathway involving DsrABEFHCMKJOP or HdrCBAHypHdrCB, while others have both pathways. A proteomics analysis showed that proteins constituting the core pathways were produced at high levels. While hypothesized function of HdrCBAHypHdrCB is similar to that of Dsr system, both sets of proteins were detected with high relative abundances in the proteome of a strain possessing genes for these proteins. In addition to the genes for sulfur oxidation, those for arsenic metabolism were searched for in the sequenced genomes. As a result, two strains belonging to the families Thiobacillaceae and Sterohbacteriaceae were observed to harbor genes encoding ArxAB, a type of arsenite oxidase that has been identified in a limited number of bacteria. These findings were made with the newly obtained genomes, including those from 6 genera from which no genome sequence of an isolated organism was previously available. These genomes will serve as valuable references to interpret nucleotide sequences.
  • Kei Kumakura, Chiaki Hori, Hiroki Matsuoka, Kiyohiko Igarashi, Masahiro Samejima
    JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE 99 2 529 - 535 2019年01月 [査読有り][通常論文]
     
    BACKGROUND Mushrooms have been widely considered as health foods as their extracts have anti-hypertensive and anti-tumor activities. After a thorough literature survey, we hypothesized that enzymes in mushroom extracts play an important role in synthesizing functional molecules. Therefore, in this study, proteins extracted from reishi mushroom (Ganoderma lucidum), which is used in oriental medicine, were identified by the proteomic approach, and appropriate extraction methods for improving angiotensin-converting enzyme (ACE) inhibitory activities were investigated. RESULTS Various glycoside hydrolases (GHs), such as beta-N-acetylhexosaminidase (GH family 20), alpha-1,2-mannosidase (GH family 47), endo-beta-1,3-glucanase (GH family 128), and beta-1,3-glucanase (GH152), that degrade glycans in the fruiting body were identified. The residual glucanase activities generated beta-oligosaccharides. Additionally, the glutamic acid protease of the peptidase G1 family was determined as the major protein in the extract, and the residual peptidase activity of the extracts was found to improve ACE inhibitory activities. Finally, it was observed that extraction at 50 degrees C is suitable for yielding functional molecules with high ACE inhibitory activities. CONCLUSION Water extraction is generally believed to extract only functional macromolecules that exist in mushroom fruiting bodies. This study proposed a new concept that describes how functional molecules are produced by enzymes, including proteases and GHs, during extraction. (c) 2018 Society of Chemical Industry
  • Chiaki Hori, Makoto Yoshida, Kiyohiko Igarashi, Masahiro Samejima
    J. Jpn. Wood Sci. Soc. 65 4 173 - 188 2019年 [査読有り]
     
    Wood decay fungi can efficiently decompose and metabolize recalcitrant plant cell walls, which consist of cellulose, hemicellulose and lignin. Their unique degradation processes and related enzymes have been extensively studied toward various industrial and environmental applications. The recent advances in DNA sequencing technologies and informatics have made more than 250 fungal genomes publicly available, and enabled comparative omics analyses to provide new insights regarding diversity of fungal wood degradation processes, their origins and evolutions. In this review, at first, the background of wood decay types and related enzymes was summarized, and then new insights on the molecular mechanism of the diversity of wood degradation by both white-rot and brown-rot fungi as disclosed from the current comparative genome analyses were presented. Moreover, the origin and evolutions of wood decay fungi based on molecular clock analyses were discussed.
  • Kiyoshi Sakuragi, Chiaki Hori, Kiyohiko Igarashi, Masahiro Samejima
    JOURNAL OF WOOD SCIENCE 64 6 845 - 853 2018年12月 [査読有り][通常論文]
     
    Ammonia pretreatment is a promising technique for enhancing enzymatic saccharification of lignocellulosic biomass. However, an enzymatic cocktail suitable for the breakdown of pretreated biomass samples is still being developed. The basidiomycete Phanerochaete chrysosporium is a well-studied fungus with regard to bioconversion of lignocellulosic biomass. In the present work, we analyzed proteins secreted by P. chrysosporium grown on untreated and ammonia-treated birch wood meal. Fungal growth, xylanase activity, and extracellular protease activity increased in the media containing the ammonia-treated biomass; however, cellulase production decreased compared to that observed in the untreated biomass. Secreted extracellular proteins were separated by two-dimensional electrophoresis and identified by liquid chromatography ion-trap mass spectrometry. Fifty-five spots corresponding to secreted proteins were chosen for further analysis. In the culture with ammonia-treated biomass, the relative concentration of a xylanase belonging to glycoside hydrolase (GH) family 11 increased, while acetyl xylan esterases belonging to carbohydrate esterase family 1 decreased. Moreover, GH family 10 xylanases were promoted proteolysis in the culture of ammonia-treated biomass, leading to the loss of family 1 carbohydrate-binding modules. These results indicated that P. chrysosporium produced enzymes related to the recognition of structural changes on xylan with de-acetylation and introduction of nitrogen by ammonia pretreatment of birch wood meal.
  • Chiaki Hori, Jill Gaskell, Dan Cullen, Grzegorz Sabat, Philip E. Stewart, Kathleen Lail, Yi Peng, Kerrie Barry, Igor V. Grigoriev, Annegret Kohler, Laure Fauchery, Francis Martin, Carolyn A. Zeiner, Jennifer M. Bhatnagar
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 84 20 2018年10月 [査読有り][通常論文]
     
    Fungi play a key role cycling nutrients in forest ecosystems, but the mechanisms remain uncertain. To clarify the enzymatic processes involved in wood decomposition, the metatranscriptomics and metaproteomics of extensively decayed lodgepole pine were examined by RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), respectively. Following de novo metatranscriptome assembly, 52,011 contigs were searched for functional domains and homology to database entries. Contigs similar to basidiomycete transcripts dominated, and many of these were most closely related to ligninolytic white rot fungi or cellulolytic brown rot fungi. A diverse array of carbohydrate-active enzymes (CAZymes) representing a total of 132 families or subfamilies were identified. Among these were 672 glycoside hydrolases, including highly expressed cellulases or hemicellulases. The CAZymes also included 162 predicted redox enzymes classified within auxiliary activity (AA) families. Eighteen of these were manganese peroxidases, which are key components of ligninolytic white rot fungi. The expression of other redox enzymes supported the working of hydroquinone reduction cycles capable of generating reactive hydroxyl radicals. These have been implicated as diffusible oxidants responsible for cellulose depolymerization by brown rot fungi. Thus, enzyme diversity and the coexistence of brown and white rot fungi suggest complex interactions of fungal species and degradative strategies during the decay of lodgepole pine.IMPORTANCE The deconstruction of recalcitrant woody substrates is a central component of carbon cycling and forest health. Laboratory investigations have contributed substantially toward understanding the mechanisms employed by model wood decay fungi, but few studies have examined the physiological processes in natural environments. Herein, we identify the functional genes present in field samples of extensively decayed lodgepole pine (Pinus contorta), a major species distributed throughout the North American Rocky Mountains. The classified transcripts and proteins revealed a diverse array of oxidative and hydrolytic enzymes involved in the degradation of lignocellulose. The evidence also strongly supports simultaneous attack by fungal species employing different enzymatic strategies.
  • Ken'ichiro Matsumoto, Midori Iijima, Chiaki Hori, Camila Utsunomia, Toshihiko Ooi, Seiichi Taguchi
    BIOMACROMOLECULES 19 7 2889 - 2895 2018年07月 [査読有り][通常論文]
     
    Engineered D-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaCl(ps)STQK) efficiently produces poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaCl(ps)STQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaCl(ps)STQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of 3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA-co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA-co-3HB) biosynthetic systems.
  • Chiaki Hori, Kenta Oishi, Ken'ichiro Matsumoto, Seiichi Taguchi, Toshihiko Ooi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 125 6 632 - 636 2018年06月 [査読有り][通常論文]
     
    In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1(ps)(S325T/ Q481IC). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaCl(ps) towards enhanced P(2HB) accumulation in E. coil by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaCl(ps)(S325T/Q481K). Among them, the cells expressing PhaCl(ps)(S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaCl(ps)(ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaClps(ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaCl(ps) mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB). (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Ken'ichiro Matsumoto, Chiaki Hori, Ryunosuke Fujii, Masahiro Takaya, Takashi Ooba, Toshihiko Ooi, Takuya Isono, Toshifumi Satoh, Seiichi Taguchi
    BIOMACROMOLECULES 19 2 662 - 671 2018年02月 [査読有り][通常論文]
     
    Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.
  • Mikio Ueta, Chiaki Hori, Yutaka Tamai, Yusuke Yamagishi, Toshizumi Miyamoto, Yuzou Sano
    J Jpn Wood Sci Soc 64 1 1 - 9 2018年 [査読有り][通常論文]
     
    This study aimed to clarify the factors that influence tree susceptibility to infection by the white-rot fungus Perenniporia fraxinea, by inoculating it into stems of four tree species at butt and breast height, and comparing the alterations in the xylem three to five months after inoculation. The four tree species include Robinia pseudoacacia, Cerasus sargentii, Ulmus davidiana var. japonica and Picea glehnii; the former two species are easily infected by this fungus, whereas the latter two species are not. Polymerase chain reaction (PCR) analysis detected the inoculated fungus only in the butt xylem of R. pseudoacacia and C. sargentii and the cell walls in the butt xylem tissues of these species were eroded by hyphae. Other xylem alterations (e.g., water accumulation and cell occlusion) differed among tree species, whereas no apparent difference was observed between butt and breast height. For example, water accumulation occurred in the discolored wood tissues around the inoculated holes in both R. pseudoacacia and U davidiana var. japonica, while dehydration occurred in the light-colored wood tissues around the inoculated hole in P. glehnii. In the vicinity of the inoculation holes, resin deposits formed in P. glehnii, whereas cell occlusions by tyloses/gums occurred in the other three species. These results suggest that susceptibility to P. fraxinea infection differs not only among tree species but also between heights above ground. It is likely that no particular response to fungal inoculation in the xylem tissues of these four tree species is closely associated with their susceptibility to the infection by P. fraxinea.
  • Camila Utsunomia, Chiaki Hori, Ken'ichiro Matsumoto, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 124 6 635 - 640 2017年12月 [査読有り][通常論文]
     
    D-Lactate (LA)-based oligomers (D-LAOs) are unusual oligoesters consisting of D-LA and D-3-hydroxybutyrate that are produced and secreted by engineered Escherichia coli grown on glucose. The cells heterologously express LA-polymerizing polyhydroxyalkanoate synthase and monomer-supplying enzymes. In this study, we attempted to identify the D-LAO secretion route in E. con, which is thought to be mediated by intrinsic membrane proteins. To this end, a loss-of-function screening of D-LAO secretion was carried out using 209 single-gene membrane protein deletants, which are involved in the transport of organic compounds. Among the deletants of the outer membrane-associated proteins, Delta ompF and Delta ompG exhibited diminished D-LAOs secretion and elevated intracellular D-LAO accumulation. When the ompF and ompG expression levels were down- and up-regulated with plasmids harboring these genes, the secreted amounts of the D-LAOs were changed in correspondence with their expression levels. These results suggest that porins mediate D-LAOs transport through the outer membrane. In particular, OmpF is likely to be the major porin involved in the spontaneous secretion of D-LAOS due to the high basal expression of ompF in the parental strain. Among the deletants of the inner membrane-associated proteins, the Delta mngA, Delta argT, Delta macA, Delta citA and Delta cpxA strains were selected by the screening. These genes are also candidate transporters related to D-LAO secretion, suggesting the presence of multiple secretion routes across the inner membrane. To the best of our knowledge, this is the first report on the mechanism of the microbial secretion of oligoesters. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Camila Utsunomia, Tatsuya Saito, Ken'ichiro Matsumoto, Chiaki Hori, Takuya Isono, Toshifumi Satoh, Seiichi Taguchi
    JOURNAL OF POLYMER RESEARCH 24 10 2017年09月 [査読有り][通常論文]
     
    D-Lactate (LA)-based oligomers (D-LAOs), consisting of D-LA and D-3-hydroxybutyrate (D-3HB), are biobased compounds which are produced and spontaneously secreted by recombinant Escherichia coli. By supplementing the bacterial cultivation with diethylene glycol (DEG), the oligomers featuring hydroxyl groups at both ends of their structures, the D-LAOs-DEG, can be efficiently biosynthesized. In the present work, we attempted to verify the feasibility of D-LAOs-DEG as building blocks to be assembled into LA-based poly(ester-urethane) via polyaddition reactionwith diisocyanate. The polymeric products were demonstrated by SEC and the urethane bound formation in the polymer was determined by FT-IR analysis, indicating that the polymerization was successfully performed. These results suggested that the one-step biosynthesized D-LAOs-DEG are potential substrates for the synthesis of LA-based poly(ester-urethane) and can be further applied to the synthesis of other LA copolymers.
  • Camila Utsunomia, Ken'ichiro Matsumoto, Sakiko Date, Chiaki Hori, Seiichi Taguchi
    JOURNAL OF BIOSCIENCE AND BIOENGINEERING 124 2 204 - 208 2017年08月 [査読有り][通常論文]
     
    Recently, we have succeeded in establishing the microbial platform for the secretion of lactate (LA)-based oligomers (D-LAOs), which consist of D-LA and D-3-hydroxybutyrate (D-3HB). The secretory production of D-LAOs was substantially enhanced by the supplementation of diethylene glycol (DEG), which resulted in the generation of DEG-capped oligomers at the carboxyl terminal (referred as D-LAOS-DEG). The microbial D-LAOs should be key compounds for the synthesis of lactide, an important intermediate for polylactides (PLAs) production, eliminating the costly chemo-oligomerization step in the PLA production process. Therefore, in order to demonstrate a proof-of-concept, here, we attempted to convert the D-LAOS-DEG into lactide via metal-catalyzed thermal depolymerization. As a result, D-LAOS-DEG containing 68 mol% LA were successfully converted into lactide, revealing that the DEG bound to D-LAOs-DEG does not inhibit the conversion into lactide. However, the lactide yield (4%) was considerably lower than that of synthetic LA homooligomers (33%). We presumed that 3HB units in the polymer chain blocked the lactide formation, and therefore, we investigated the LA enrichment in the oligomers. As the results, the combination of an LA-overproducing Escherichia coli mutant (Mid and ApflA) with the use of xylose as a carbon source exhibited synergistic effect to increase LA fraction in the oligomers up to 89 mol%. The LA-enriched D-LAOS-DEG were converted into lactide with greater yield (18%). These results demonstrated that a greener shortcut route for PLA production can be created by using the microbial D-LAOS secretion system. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.
  • Yoshihisa Yoshimi, Yumi Sugawara, Chiaki Hori, Kiyohiko Igarashi, Satoshi Kaneko, Yoichi Tsumuraya, Toshihisa Kotake
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 3 475 - 481 2017年03月 [査読有り][通常論文]
     
    Arabinogalactan-proteins (AGPs) are highly diverse plant proteoglycans found on the plant cell surface. AGPs have large arabinogalactan (AG) moieties attached to a core-protein rich in hydroxyproline (Hyp). The AG undergoes hydrolysis by various glycoside hydrolases, most of which have been identified, whereas the core-proteins is presumably degraded by unknown proteases/peptidases secreted from fungi and bacteria in nature. Although several enzymes hydrolyzing other Hyp-rich proteins are known, the enzymes acting on the core-proteins of AGPs remain to be identified. The present study describes the detection of protease/peptidase activity toward AGP core-proteins in the culture medium of winter mushroom (Flammulina velutipes) and partial purification of the enzyme by several conventional chromatography steps. The enzyme showed higher activity toward Hyp residues than toward proline and alanine residues and acted on core-proteins prepared from gum arabic. Since the activity was inhibited in the presence of Pefabloc SC, the enzyme is probably a serine protease.
  • Keisuke Ohashi, Chiaki Hori, Taichi Takasuka
    J Jpn Biochem. Soc. 89 5 739 - 743 2017年 [査読有り]
  • Xueqing Song, Xiang Yu, Chiaki Hori, Taku Demura, Misato Ohtani, Qiang Zhuge
    FRONTIERS IN PLANT SCIENCE 7 612  2016年05月 [査読有り][通常論文]
     
    Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2. 7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2. 7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSHRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSHRK2.7 overexpression. Furthermore, PtSnRK2. 7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes.
  • 高須賀太一, 堀 千明, James Elinger, 飛松裕基
    化学と生物 54 3 156 - 158 2016年 [査読有り]
  • Xueqing Song, Misato Ohtani, Chiaki Hori, Arika Takebayasi, Ryoko Hiroyama, Nur Ardiyana Rejab, Takaomi Suzuki, Taku Demura, Tongming Yin, Xiang Yu, Qiang Zhuge
    PLANT BIOTECHNOLOGY 32 4 337 - 341 2015年12月 [査読有り][通常論文]
     
    Type 2C protein phosphatase (PP2C) is a central player in abscisic acid (ABA) signaling transduction, which is required for plant growth, development and stress responses. In Arabidopsis, group A PP2Cs inhibit activity of the SNF1-related protein kinases 2 (SnRK2) family via physical interaction. To clarify whether this scheme is conserved in woody plants, we experimentally isolated the genes homologous to three members of group A PP2Cs (PtABI1, PtAHG1 and PtAHG3) and 12 SnRK2s (PtSnRK2.1-2.12) from a model tree Populus trichocarpa, and examined their interaction using a yeast two-hybrid assay. Our results showed that only three PtSnRK2 proteins had a positive interaction with PtPP2Cs: PtSnRK2.10 possessed strong interaction activity with all three PtPP2Cs, while significant, but relatively weak, interactions were observed with PtSnRK2.6 and PtSnRK2.9. These three PtSnRK proteins are grouped into subclass 2 or 3, which are considered to be ABA-dependent kinases in Arabidopsis. These findings suggest that physical interaction between SnRK2 and PP2C is also conserved in poplars and may be involved in the ABA signaling pathway in tree plants.
  • 堀千明, 五十嵐圭日子, 鮫島正浩
    化学と生物 53 6 381 - 388 2015年
  • Chiaki Hori, Takuya Ishida, Kiyohiko Igarashi, Masahiro Samejima, Hitoshi Suzuki, Emma Master, Patricia Ferreira, Francisco J. Ruiz-Duenas, Benjamin Held, Paulo Canessa, Luis F. Larrondo, Monika Schmoll, Irina S. Druzhinina, Christian P. Kubicek, Jill A. Gaskell, Phil Kersten, Franz St John, Jeremy Glasner, Grzegorz Sabat, Sandra Splinter BonDurant, Khajamohiddin Syed, Jagjit Yadav, Anthony C. Mgbeahuruike, Andriy Kovalchuk, Fred O. Asiegbu, Gerald Lackner, Dirk Hoffmeister, Jorge Rencoret, Ana Gutierrez, Hui Sun, Erika Lindquist, Kerrie Barry, Robert Riley, Igor V. Grigoriev, Bernard Henrissat, Ursula Kuees, Randy M. Berka, Angel T. Martinez, Sarah F. Covert, Robert A. Blanchette, Daniel Cullen
    PLOS GENETICS 10 12 e1004759  2014年12月 [査読有り][通常論文]
     
    Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.
  • Chiaki Hori, Jill Gaskell, Kiyohiko Igarashi, Phil Kersten, Michael Mozuch, Masahiro Samejima, Dan Cullen
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 80 7 2062 - 2070 2014年04月 [査読有り][通常論文]
     
    The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.
  • Chiaki Hori, Jill Gaskell, Kiyohiko Igarashi, Masahiro Samejima, David Hibbett, Bernard Henrissat, Dan Cullen
    MYCOLOGIA 105 6 1412 - 1427 2013年11月 [査読有り][通常論文]
     
    To degrade the polysaccharides, wood-decay fungi secrete a variety of glycoside hydrolases (GHs) and carbohydrate esterases (CEs) classified into various sequence-based families of carbohydrate-active enzymes (CAZys) and their appended carbohydrate-binding modules (CBM). Oxidative enzymes, such as cellobiose dehydrogenase (CDH) and lytic polysaccharide monooxygenase (LPMO, formerly GH61), also have been implicated in cellulose degradation. To examine polysaccharide-degrading potential between white- and brown-rot fungi, we performed genomewide analysis of CAZys and these oxidative enzymes in 11 Polyporales, including recently sequenced monokaryotic strains of Bjerkandera adusta, Ganoderrna sp. and Phlebia brevispora. Furthermore, we conducted comparative secretome analysis of seven Polyporales grown on wood culture. As a result, it was found that genes encoding celluloses belonging to families GH6, GH7, GH9 and carbohydrate-binding module family CBM1 are lacking in genomes of brown-rot polyporales. In addition, the presence of CDH and the expansion of LPMO were observed only in white-rot genomes. Indeed, GH6, GH7, CDH and LPMO peptides were identified only in white-rot polypores. Genes encoding aldose 1-epimerase (ALE), previously detected with CDH and celluloses in the culture filtrates, also were identified in white-rot genomes, suggesting a physiological connection between ALE, CDH, cellulose and possibly LPMO. For hemicellulose degradation, genes and peptides corresponding to GH74 xyloglucanase, GH10 endo-xylanase, GH79 beta-glucuronidase, CE1 acetyl xylan esterase and CE15 glucuronoyl methylesterase were significantly increased in white-rot genomes compared to brown-rot genomes. Overall, relative to brown-rot Polyporales, white-rot Polyporales maintain greater enzymatic diversity supporting lignocellulose attack.
  • 堀千明, 五十嵐圭日子, 鮫島正浩
    Cellul Commun 19 4 189 - 194 セルロース学会 2012年12月01日 [査読有り][通常論文]
  • Hitoshi Suzuki, Jacqueline MacDonald, Khajamohiddin Syed, Asaf Salamov, Chiaki Hori, Andrea Aerts, Bernard Henrissat, Ad Wiebenga, Patricia A. vanKuyk, Kerrie Barry, Erika Lindquist, Kurt LaButti, Alla Lapidus, Susan Lucas, Pedro Coutinho, Yunchen Gong, Masahiro Samejima, Radhakrishnan Mahadevan, Mamdouh Abou-Zaid, Ronald P. de Vries, Kiyohiko Igarashi, Jagjit S. Yadav, Igor V. Grigoriev, Emma R. Master
    BMC GENOMICS 13 444  2012年09月 [査読有り][通常論文]
     
    Background: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome.Results: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood.Conclusions: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.
  • Kenta Higashijima, Chiaki Hori, Kiyohiko Igarashi, Toshiharu Enomae, Akira Isogai
    STUDIES IN CONSERVATION 57 3 164 - 171 2012年07月 [査読有り][通常論文]
     
    With the aim of developing a new easy-to-use method for rescuing flood-damaged paper, the effect of saltwater on the inhibition of fungal growth on paper was investigated. This procedure could be used instead of, or assisted by freeze drying. Cellulose-digesting Trichoderma reesei, three types of fungi representative of fungi growing on paper (T. reesei, Aspergillus terreus, and Aureobasidium pullulans) and various naturally airborne fungi were cultured on cellulosic materials in liquid media containing artificial seawater with different salt concentrations. The addition of salts successfully inhibited the growth of T. reesei on microcrystalline cellulose at the concentration of 3.2% (m/m) or higher. The critical salt concentration, 3.2%, is within the general range of salt content in seawater. Other solutions of salts similar to sodium chloride also inhibited fungal growth. Although the observed growth-inhibiting effect was attributed to the high osmotic pressure of the salt solution, physiological effects depending on ion species used were also considered to be possible. The growth of all three types of fungi on copy paper was inhibited effectively when the salt concentration was increased. The growth of various fungi on pure cellulose, with enough oxygen supplied to pores, was completely inhibited (as assessed by visual examination) for 24 days at salt concentrations of 3.5% (m/m) or greater. The fact that the effect of saltwater on cellulosic materials was observed even under optimum medium conditions implies that fungi would be considerably inhibited on flood-damaged paper immersed in saltwater. This method is a promising first aid measure when circumstances do not allow for flood-damaged paper to be dried immediately.
  • Dimitrios Floudas, Manfred Binder, Robert Riley, Kerrie Barry, Robert A. Blanchette, Bernard Henrissat, Angel T. Martinez, Robert Otillar, Joseph W. Spatafora, Jagjit S. Yadav, Andrea Aerts, Isabelle Benoit, Alex Boyd, Alexis Carlson, Alex Copeland, Pedro M. Coutinho, Ronald P. de Vries, Patricia Ferreira, Keisha Findley, Brian Foster, Jill Gaskell, Dylan Glotzer, Pawel Gorecki, Joseph Heitman, Cedar Hesse, Chiaki Hori, Kiyohiko Igarashi, Joel A. Jurgens, Nathan Kallen, Phil Kersten, Annegret Kohler, Ursula Kuees, T. K. Arun Kumar, Alan Kuo, Kurt LaButti, Luis F. Larrondo, Erika Lindquist, Albee Ling, Vincent Lombard, Susan Lucas, Taina Lundell, Rachael Martin, David J. McLaughlin, Ingo Morgenstern, Emanuelle Morin, Claude Murat, Laszlo G. Nagy, Matt Nolan, Robin A. Ohm, Aleksandrina Patyshakuliyeva, Antonis Rokas, Francisco J. Ruiz-Duenas, Grzegorz Sabat, Asaf Salamov, Masahiro Samejima, Jeremy Schmutz, Jason C. Slot, Franz St. John, Jan Stenlid, Hui Sun, Sheng Sun, Khajamohiddin Syed, Adrian Tsang, Ad Wiebenga, Darcy Young, Antonio Pisabarro, Daniel C. Eastwood, Francis Martin, Dan Cullen, Igor V. Grigoriev, David S. Hibbett
    SCIENCE 336 6089 1715 - 1719 2012年06月 [査読有り][通常論文]
     
    Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.
  • Chiaki Hori, Hitoshi Suzuki, Kiyohiko Igarashi, Masahiro Samejima
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 78 10 3770 - 3773 2012年05月 [査読有り][通常論文]
     
    Cellobiose dehydrogenase (CDH) gene transcripts were quantified by reverse transcription-PCR (RT-PCR) in cultures of Phanerochaete chrysosporium supplemented with various cello- and xylooligosaccharides in order to elucidate the mechanism of enhanced CDH production in xylan/cellulose culture. Cellotriose and cellotetraose induced cdh expression, while xylobiose and xylotriose induced expression of cellobiohydrolase genes, especially cel7C.
  • Elena Fernandez-Fueyo, Francisco J. Ruiz-Duenas, Patricia Ferreira, Dimitrios Floudas, David S. Hibbett, Paulo Canessa, Luis F. Larrondo, Tim Y. James, Daniela Seelenfreund, Sergio Lobos, Ruben Polanco, Mario Tello, Yoichi Honda, Takahito Watanabe, Takashi Watanabe, Ryu Jae San, Christian P. Kubicek, Monika Schmoll, Jill Gaskell, Kenneth E. Hammel, Franz J. St John, Amber Vanden Wymelenberg, Grzegorz Sabat, Sandra Splinter BonDurant, Khajamohiddin Syed, Jagjit S. Yadav, Harshavardhan Doddapaneni, Venkataramanan Subramanian, Jose L. Lavin, Jose A. Oguiza, Gumer Perez, Antonio G. Pisabarro, Lucia Ramirez, Francisco Santoyo, Emma Master, Pedro M. Coutinho, Bernard Henrissat, Vincent Lombard, Jon Karl Magnuson, Ursula Kuees, Chiaki Hori, Kiyohiko Igarashi, Masahiro Samejima, Benjamin W. Held, Kerrie W. Barry, Kurt M. LaButti, Alla Lapidus, Erika A. Lindquist, Susan M. Lucas, Robert Riley, Asaf A. Salamov, Dirk Hoffmeister, Daniel Schwenk, Yitzhak Hadar, Oded Yarden, Ronald P. de Vries, Ad Wiebenga, Jan Stenlid, Daniel Eastwood, Igor V. Grigoriev, Randy M. Berka, Robert A. Blanchette, Phil Kersten, Angel T. Martinez, Rafael Vicuna, Dan Cullen
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 14 5458 - 5463 2012年04月 [査読有り][通常論文]
     
    Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.
  • Chiaki Hori, Kiyohiko Igarashi, Akira Katayama, Masahiro Samejima
    FEMS MICROBIOLOGY LETTERS 321 1 14 - 23 2011年08月 [査読有り][通常論文]
     
    Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.
  • Maki Ishiguro, Chiaki Hori, Akira Katayama, Kiyohiko Igarashi, Koji Takabatake, Satoshi Kaneko, Masahiro Samejima
    J Jpn Wood Sci Soc 56 6 388 - 396 2010年 [査読有り][通常論文]
     
    To identify the extracellular enzymes produced by Enoki-take mushrooms (Flammulina velutipes), we performed secretome analysis using transcriptomic sequence database of the fungus. F. velutipes was cultivated using various types of biomass and related polysaccharides as carbon sources and total RNA was extracted from the mycelia obtained from each culture. The normalized cDNA library constructed from the total RNA was sequenced by 2nd generation DNA sequencer and subsequently a transcriptomic sequence database of F. velutipes was constructed. The extracellular proteins produced by the fungus in the cellulose-degrading culture were separated by 2-dimensional gel electrophoresis and the obtained spots were identified using the transcriptomic sequence database. A total of 41 protein spots were assigned to corresponding contig sequences, and the various enzymes related to carbohydrate degradation were identified, suggesting the validity of the database for the secretome analysis without total genomic sequence or genome-wide annotation.
  • Kiyohiko Igarashi, Takuya Ishida, Chiaki Hori, Masahiro Samejima
    APPLIED AND ENVIRONMENTAL MICROBIOLOGY 74 18 5628 - 5634 2008年09月 [査読有り][通常論文]
     
    The wood decay fungus Phanerochaete chrysosporium has served as a model system for the study of lignocellulose conversions, but aspects of its cellulolytic system remain uncertain. Here, we report identifying the gene that encodes the glycoside hydrolase (GH) family 45 endoglucanase (EG) from the fungus, cloning the cDNA, determining its heterologous expression in the methylotrophic yeast Pichia pastoris, and characterizing the recombinant protein. The cDNA consisted of 718 bp, including an open reading frame encoding a 19-amino-acid signal peptide, a 7-amino-acid presequence at the N-terminal region, and a 180-amino-acid mature protein, which has no cellulose binding domain. Analysis of the amino acid sequence revealed that the protein has a low similarity (< 22%) to known fungal EGs belonging to the GH family 45 (EGVs). No conserved domain of this family was found by a BLAST search, suggesting that the protein should be classified into a new subdivision of this GH family. The recombinant protein has hydrolytic activity toward amorphous cellulose, carboxylmethyl cellulose, lichenan, barley beta-glucan, and glucomannan but not xylan. Moreover, a synergistic effect was observed with the recombinant GH family 6 cellobiohydrolase from the same fungus toward amorphous cellulose as a substrate, indicating that the enzyme may act in concert with other cellulolytic enzymes to hydrolyze cellulosic biomass in nature.

MISC

書籍等出版物

  • Gene Expression Systems in Fungi: Advancements and Applications.
    堀 千明 (担当:共著範囲:Chapter 6. Prospects for Bioprocess Development Based on Recent Genome Advances in Lignocellulose Degrading Basidiomycetes.)
    Springer International Publishing 2016年

所属学協会

  • 日本農芸化学会   糸状菌分子生物学研究会   日本木材学会   

共同研究・競争的資金等の研究課題

  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    研究期間 : 2023年06月 -2026年03月
  • 日本学術振興会:科学研究費補助金・基盤(B)
    研究期間 : 2021年04月 -2025年03月 
    代表者 : 堀千明
     
    樹木は樹皮や木部に抗菌性物質となる二次代謝産物を生産することにより、菌の侵入を防いでおり、特に針葉樹は大量に蓄積しているため腐朽されにくい。しかし針葉樹分解性白色腐朽菌Phlebiopsis giganteaは一早くこれら樹木防御を突破しコロニーを形成する(Hori et al. 2014 PLoS Genet.)。これまでの研究で、P. giganteaは針葉樹の抽出成分に豊富に含まれる二次代謝産物を生物変換する高い能力を保持している可能性を示した(Hori et al. 2014 PLoS Genet.; Iwata et al. 2020)。そこで本研究では、針葉樹二次代謝産物の本菌による変換過程を網羅的に明らかにすることで、本菌が樹木防御を突破できる一因の解明へと繋げる。 昨年度、伐採したばかりのマツから取り出した抽出成分のメタボローム解析法をGCMSによって確立し、本プラットフォームを利用する事で、マツ抽出成分および成分変換物を網羅的に解析した。今年度は、マツ抽出成分の中でも主要な成分を対象として、腐朽菌への成長阻害機能があるかどうかを確認した。さらに、P. giganteaの代謝産物を明らかにするための液体培養条件を検討することで、本菌が主要成分を消費していることを確認した。今後、本培養系を解析することで、変換物の同定や変換に関わる代謝や酵素を明らかにすることが可能となる。
  • Elucidating the plant-microbe interactions of the wood decay fungus Perenniporia fraxinea, a serious pathogen of hardwood trees
    US Department of Energy Joint Genome Institute:JGI proposal 505700
    研究期間 : 2019年09月 -2024年08月 
    代表者 : 堀 千明
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2022年06月 -2024年03月 
    代表者 : 堀 千明
     
    本研究は、自然界で樹木分解として獲得された担子菌が保有する卓越した難分解性ポリマー分解能力を、合成ポリマーのポリエステル分解へと適応化させることを想定した探索的萌芽研究である。これまで申請者は、樹木の多様化に合わせて、長い進化の過程で、担子菌は多様なポリマー分解機構を獲得してきたことを明らかにした。その過程で、針葉樹分解へと進化した珍しい担子菌Phlebiopsis giganteaから針葉樹に多く含まれる脂質類を分解する新規高機能性リパーゼを同定・特徴解析した(Hori et al. PLoS Genet. 2014; Iwata et al. Sci. Rep. 2021*責任著者;堀 千明,特許出願2021-024557)。本研究では、本酵素が合成ポリマーのエステル結合を分解するかを検討し、さらに高機能化することを目標としている。 本年度は、種々のポリエステルと本酵素を反応させ、反応物解析を行うことで分解活性を測定した。さらに、商業的リパーゼについても分解活性を測定し、本酵素と比較した。その結果、本酵素が高いポリエステル分解活性を保持することを明らかにした。 また、本酵素については、国際特許 PCT/JP2022/6467 Chiaki Hori (2022/02/17)を申請しており、それを基に海外酵素会社との共同研究によって市販させることができており、応用化研究の基盤を整えつつある。
  • 糸状菌が生産する新規樹木分解酵素の探索とその応用
    一般財団法人杉山産業化学研究所:2023年度研究助成
    研究期間 : 2023年07月 -2024年03月
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 飛松 裕基, 堀 千明, 久住 亮介, 梅澤 俊明, 今井 友也
     
    複雑多様なリグニンの分子構造は、維管束植物の進化・環境適応と密接に関係する木質 細胞壁の本質的特徴であると同時に、脱炭素社会構築 を担う木質バイオマス利用の重要阻害要因でもある。しかしながら、リグニンの分子構造と木質細胞壁の生理機能・超分子構造 ・ 各種バイオマス利用特性の関係性の理解は未だ著しく欠落しているのが現状である。本研究では、木質の生理学的機能・超分子構造・利用特性に及ぼすリグニンの寄与を体系的に明らかにすることを目的とし、リグニンの量・構造を系統的に変化させたリグニン改変組換え植物を作出し、その細胞壁の化学構造・超分子構造・生理学的特性・化学変換利用特性を体系的に調べ、改変されたリグニンの量・構造との関 係性を明らかにする。同時に、代謝工学的に制御したリグニンの反応特性に合わせた木質の成分分離・触媒分解反応の最適化を通じ、木質化学変換利用効率の相乗的向上を図る。令和2年度は、リグニンを改変した幾つかの新たなゲノム編集イネの作出に成功し、その細胞壁の化学構造の詳細を各種化学分析や2D NMR解析により明らかにした。過去に作出したリグニン改変組換えイネと共に、一部のイネ株については、固体NMR法及び広角X線法(WAXD)による細胞壁超分子構造の解析も行い、リグニンの量的・質的改変が細胞壁多糖の集合状態に影響することを実験的に証明した。さらに詳細な細胞壁超分子構造の解析を目的に、狭角X線法(SAXS)を用いた細胞壁中における多糖およびリグニンの混合状態の解析のための予備実験も行った。また、種々の植物種におけるリグニン生合成機構の解明、代謝工学、生物分解等を目的とする共同研究において、リグニン構造解析やイメージング解析の実験も行った。これらの成果の一部を纏め、論文発表及び学会発表を行った。
  • 樹木が生産する抗菌成分を担子菌が克服する分子相互作用の解明
    旭硝子財団:化学・生命分野 研究奨励
    研究期間 : 2021年04月 -2023年03月 
    代表者 : 堀千明
  • 日本学術振興会:科学研究費助成事業
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 堀 千明
     
    これまでの研究では、植物成分の分解に関わる酵素(セルラーゼ等)について、単離された微生物から一つずつ生化学的解析を進めることで詳細な分子機能が明らかになってきた。しかし、環境下において様々な微生物が存在する場合の植物分解についてはほとんど知見が得られていなかった。そこで、本研究では実際に自然環境下で選抜された酵素について生化学的解析を行い、より効率的な植物分解メカニズムを明らかにできた。このような知見は植物バイオマス成分を分解する糖化技術を効率化することに繋がると考えている。
  • 日本学術振興会:科学研究費補助金・基盤(B)
    研究期間 : 2018年04月 -2022年03月 
    代表者 : 渥美正太
     
    本研究では褐藻成分アルギン酸分解産物であるDEHU分子を細胞内に輸送し、1,4-butanediolを大腸菌細胞内で合成出来る変異株の作出を目的としている。これまでの研究において、DEHU分子輸送タンパク質遺伝子3種(low copy plasmid)、3種のDEHU reductase (dehR)と3種の5-keto-4-deoxy-D-glucarate dehydratase (KdgD)および1種の2-ketoglutarate semialdehyde dehydrogenase(xylA)(midium copy plasmid)について遺伝子合成後、クローニングを行い、それらを大腸菌icd遺伝子欠損株に形質転換した。また、1,4-butanediol合成のための遺伝子2種2-keto acid decarboxylase(kdc)とalcohol dehydrogenase(adh)についても遺伝子合成およびクローニング後に、大腸菌icd遺伝子欠損株に形質転換済みである。後者のkdcとadh遺伝子については、2-ketoisovalarateを基質に、isobutanolが合成出来る事を確認済みであり、上記DEHU分子が調製でき次第、上記プラスミドの組み合わせ27種類について、試験予定である。DEHU分子については、精製アルギン酸を基質に、3種類のアルギン酸分解酵素を反応させ、DEHU分子を含むアルギン酸酵素加水分解産物を調製中である。
  • 樹木抽出成分の分解に関与する酵素の探索と利用
    天野エンザイム科学技術振興財団:研究奨励賞
    研究期間 : 2020年04月 -2021年03月 
    代表者 : 堀千明
  • 科学技術振興機構:戦略的な研究開発の推進 戦略的創造研究推進事業 ACT-X
    研究期間 : 2019年 -2021年 
    代表者 : 堀 千明
     
    地表上で最も多くの炭素を蓄積する樹木をいち早く分解できる寄生性の腐朽菌(キノコ)を、炭素循環における先駆的分解者として位置づけ、その樹木分解機構を解明します。本研究では、樹木と寄生性腐朽菌の相互作用させた時の機序をマルチオミックス解析によって分子レベルで解明し、寄生性を付与する能力とそれを担う仕組み(分子メカニズム)を世界に先駆けて明らかにします。
  • Gene expression in the unusual white rot fungus Phlebiopsis gigantea
    US Department of Energy:JGI
    研究期間 : 2017年09月 -2020年08月
  • ショウジョウバエを使って「つわり」を考える
    北海道大学部局横断シンポジウム:第1回若手共同研究助成
    研究期間 : 2019年12月 -2020年03月 
    代表者 : 田中 暢明
  • 科学研究費補助金・基盤(C):科学研究費助成事業
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 大井俊彦
     
    石油由来のプラスチックによる海洋汚染が世界的問題となっている。その中で自然環境中で分解し水と二酸化炭素まで分解できる生分解性プラスチックが注目されている。本研究課題では、我々が新規に開発したバイオプラスチックである乳酸コポリマー[P(LA-co-3HB)]の乳酸(LA)と3-ヒドロキシブタン酸(3-HB)のポリマー分子チュノ両モノマー組成と環境微生物による分解性を評価した。 初めに乳酸コポリマーの微生物生産法を検討し、乳酸分率を制御した培養方法を確立した。得られたコポリマーをフィルム化して環境中での分解を評価するとともに、分解に関与する環境微生物の系統解析を実施した。
  • 植物バイオマス分解に関わる新規酵素の探索とその利用
    秋山記念生命科学振興財団:2018年度研究助成(推奨)
    研究期間 : 2018年04月 -2019年03月 
    代表者 : 堀 千明
  • Exploration of the novel enzymes related to deconstruction of woody extractives by white-rot fungi
    Novozymes Japan:Research Fund 2018
    研究期間 : 2018年04月 -2019年03月 
    代表者 : 堀 千明
  • 立木加害性の木材腐朽菌による木材分解機構の解明
    科学研究費補助金:若手(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 堀 千明
  • 若手研究者学会参加支援事業
    北海道大学:平成30年度 フロンティア化学教育研究センター
    研究期間 : 2018年09月 -2018年09月 
    代表者 : 堀 千明
  • 木材腐朽菌による植物抽出物質の分解メカニズムの解明
    北海道大学FCC:H29若手若手研究者フィージビリティ・スタディ支援事業
    研究期間 : 2017年10月 -2018年03月 
    代表者 : 堀 千明
  • 科学研究費補助金:特別研究員奨励費
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 堀 千明
     
    本研究では、担子菌分解機構に関わる酵素群を植物改変のための遺伝子材料として利用することで、易分解性バイオマス生産植物の作出を目指す。植物細胞壁の分解性において重要なファクターになるキシランの構造を変化させるために、木粉培地で担子菌が多く生産したキシラン分解酵素を選択した。昨年度までに組換えポプラの細胞壁解析について終了していたため、本年度では得られた組換えポプラの細胞壁形成に与える影響について二次壁形成に関わる遺伝子の転写産物量の変化を測定することにより詳細な分子メカニズムを明らかにする予定であった。そこで、3種の変異株についてマイクロアレイ解析を行った。GH10の変異株においては2倍以上増加した遺伝子群についてGO解析をしたところ、細胞壁形成関連遺伝子群に変化がみられた。そこで、細胞壁形成関連遺伝子を抽出し、詳細な遺伝子発現変化を解析した。その結果、野生株と比較してリグニン合成に関わるいくつかのLaccase遺伝子の発現量が増加しており、これらが変異体の細胞壁形成の変化に関わっている可能性が考えられた。これら研究を通し、菌類由来のキシラン分解酵素を導入したポプラでは、単純にキシランが分解されたポプラが出来上がるのではなく、そのような変化をポプラは感知し細胞壁形成を分子レベルで変化させることを明らかにした。本研究の本来の目的である易分解性ポプラの作出には成功しなかったが、細胞壁構造を遺伝子工学的手法を用いて変化させる上で重要な知見が得られたと考えている。
  • 科学研究費補助金:特別研究員奨励費
    研究期間 : 2011年04月 -2013年03月 
    代表者 : 堀 千明
     
    本研究では、分解機構に新たな知見を与えるために、単一バイオマス成分を使用した新しいモデル系を取り入れ、セクレトーム解析およびRNAseqを利用したトランスクリプトーム解析にて観察することを目的としている。 昨年度に、担子菌Phanerochaete chrysosporiumがセルロース培地にキシランを添加すると、初期成長速度が早くなるとともに、菌体外酵素生産が増加することを明らかにした。それらはキシラン分解関連酵素だけでなく、セルロースの酸化的分解に関与すると考えられるセロビオース脱水素酵素(CDH)および糖質加水分解酵素(GH)ファミリー61タンパク質の生産も促進されることが明らかとなった。 そこで今年度では、キシラン添加時に観察された現象を引き起こす原因となるキシランの構造を明らかにすることを目的とした。キシランは、キシロースがβ-1,4結合した主鎖にアラビノースやグルクロン酸、アセチル基などの側鎖が付加されたヘテロ多糖である。側鎖構造の違うキシランを添加した培地で本菌を生育させ、菌体量や菌体外酵素を解析したところ、ほとんど同じ傾向の結果が得られたことから、主鎖構造が菌体に影響を与えたことが推測された。そこで、直鎖状のキシロオリゴ糖(重合度1-4)に対するCDHの遺伝子応答を定量PCR解析によって調べたところ、キシロースには反応しない一方でキシロオリゴ糖には顕著に反応した。しかしながら、それらの応答強度は対照として測定したセロオリゴ糖に対する遺伝子応答強度よりも著しく小さかった。そこで、セロオリゴ糖を生産する主要なセルラーゼ遺伝子についてもキシロオリゴ糖に対する遺伝子応答を観察したところ、一部のセルラーゼ遺伝子(Cel7C)が顕著に二量体以上のキシロオリゴ糖に顕著に反応する事が明らかとなった。これらの結果から、キシロオリゴ糖によって誘導生産された一部セルラーゼのセルロース分解によってセロオリゴ糖の生成が促され、このセロオリゴ糖によりセルロース分解関連酵素の遺伝子全体が連鎖的に強く発現誘導されるといった増強作用を持つカスケード的応答機構が存在する可能性を示した。 そこで、キシロースおよび重合度2-4のキシロオリゴ糖に対するゲノムワイドな遺伝子応答を観察するために、Illuminaシーケンサーを用いて全転写産物解析を行った。糖質関連酵素遺伝子における発現応答を解析したところ、キシラン分解酵素よりも一部のセルロース分解関連酵素遺伝子が、キシロオリゴ糖に対する高い発現応答能をもつことが明らかとなり、上述したような応答機構の存在が強く示唆された。

産業財産権

  • 特願2021-024557:担子菌由来の新規な高機能性リパーゼの利用  2021年02月
    堀千明  北海道大学工学研究院


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