上地 敬子 (ウエチ ケイコ)

UECHI Keiko

写真a

職名

助教

科研費研究者番号

70733426

現在の所属組織 【 表示 / 非表示

  • 専任   琉球大学   農学部   亜熱帯生物資源科学科   助教  

出身大学院 【 表示 / 非表示

  • 2009年04月
    -
    2011年03月

    香川大学  農学研究科  希少糖科学専攻  修士課程  修了

  • 2011年04月
    -
    2014年03月

    愛媛大学  連合農学研究科  生物資源利用学専攻  博士課程  修了

取得学位 【 表示 / 非表示

  • 愛媛大学 -  博士(農学)  農学

  • 香川大学 -  修士(農学)  農学

研究分野 【 表示 / 非表示

  • ライフサイエンス / 応用微生物学

論文 【 表示 / 非表示

  • Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

    Kozome D, Uechi K, Taira T, Fukada H, Kubota T, Ishikawa K

    Applied and environmental microbiology ( Applied and Environmental Microbiology )  88 ( 12 ) e0065222   2022年06月 [ 査読有り ]

    掲載種別: 研究論文(学術雑誌)

  • Preparation and Crystal Structure Analysis of High-Strength Film Derived from Nigeran Ester Derivatives

    Togo A.

    Journal of Fiber Science and Technology ( 一般社団法人 繊維学会 )  78 ( 8 ) 126 - 132   2022年 [ 査読有り ]

    掲載種別: 研究論文(学術雑誌)

     概要を見る

    <p>Nigeran (<i>α</i>-1,3-<i>alt</i>-<i>α</i>-1,4-glucan) is a linear glucan with alternating <i>α</i>-1,3- and <i>α</i>-1,4-glycosidic linkages. It is extracted from the cell walls of certain species in the genera <i>Aspergillus</i> and <i>Penicillium</i>. Nigeran can be esterified and used as a film, but its strength is only approximately 4–25 MPa. However, in the present study, high-strength nigeran ester films with tensile strengths of 100 MPa were successfully prepared by thermally stretching and annealing the melt-quenched films. Two-dimensional wide-angle X-ray diffraction (2D-WAXD) analysis revealed that the highly oriented films of nigeran butyrate (NGBu), nigeran valerate (NGVa), and nigeran hexanoate (NGHx) studied herein had two-fold helix symmetry along the molecular axis. Assuming the crystal systems to be orthorhombic, the unit lattice of each ester was calculated as NGBu (<i>a</i>=28.6Å, <i>b</i>=9.07Å, <i>c</i>=16.5Å), NGVa (<i>a</i>=31.5Å, <i>b</i>=9.07Å, <i>c</i>=16.2Å), and NGHx (<i>a</i>=36.7Å, <i>b</i>=9.07Å, <i>c</i>=16.2Å). In the calculated unit lattice parameters, only the <i>a</i>-axis was extended as the number of ester carbons increased.</p>

  • Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis

    Uechi K, Yaguchi H, Tokashiki J, Taira T, Mizutani O

    Applied and environmental microbiology   87 ( 21 ) e0114421   2021年10月 [ 査読有り ]

    掲載種別: 研究論文(学術雑誌)

     概要を見る

    Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched D-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA-seq to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB as nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, that in ΔgnsA and ΔagtCgnsA decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. Importance The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3-/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.

  • cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

    Takashima T, Henna H, Kozome D, Kitajima S, Uechi K, Taira T

    Planta ( Planta )  253 ( 6 ) 120 - 120   2021年05月 [ 査読有り ]

    掲載種別: 研究論文(学術雑誌)

     概要を見る

    MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

  • Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

    Yano S, Kanno H, Tsuhako H, Ogasawara S, Suyotha W, Konno H, Makabe K, Uechi K, Taira T

    Journal of bioscience and bioengineering ( Journal of Bioscience and Bioengineering )  131 ( 4 ) 348 - 355   2021年04月 [ 査読有り ]

    掲載種別: 研究論文(学術雑誌)

     概要を見る

    The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 μM.

全件表示 >>

科研費獲得情報 【 表示 / 非表示

  • 黒色Aspergillus属特異的な細胞壁多糖ニゲランの機能と生合成機構の解明

    若手研究

    課題番号: 19K15737

    研究期間: 2019年04月  -  2022年03月 

    代表者: 上地 敬子 

    直接経費: 3,200,000(円)  間接経費: 4,160,000(円)  金額合計: 960,000(円)

     概要を見る

    Aspergillus luchuensisなどの一部のAspergillus属糸状菌は窒素飢餓時にニゲランという細胞壁多糖を生産することが知られている。しかしながらニゲラン合成のメカニズムや生理学的な意義については不明な点が多く知見が少なかった。A. luchuensisを供試菌として窒素源の存在/非存在下でのRNA-Seq解析を実施し,窒素源非存在時に特異的に発現量が増加するα-1,3-グルカン合成酵素遺伝子を見出した。そこで本研究では,ニゲラン合成酵素遺伝子の同定とニゲラン合成のメカニズムを明らかにすることを目的とした。RNA-Seq解析で見出したニゲラン合成酵素候補遺伝子を破壊したA. luchuensisはニゲラン合成能を完全に失い,またニゲラン生産能を持たないA. oryzaeに同遺伝子を導入した結果,ニゲラン生産能を付与することができた。つまり本遺伝子はα-1,3-グルカン合成酵素ではなくニゲラン合成酵素をコードする遺伝子であることが世界で初めて明らかとなった。ニゲラン合成酵素遺伝子破壊株について,液体培養とプレート培養の比較,各種細胞壁合成阻害剤を含む培養条件下での表現型解析を行ったが,いずれの条件下でも野生株との顕著な変化は確認されなかった。また,RNA-Seq解析結果より,ニゲラン合成酵素遺伝子に隣接し,窒素飢餓時に転写量が増加するα-グルカノシルトランスフェラーゼ様遺伝子と機能未知遺伝子が見出され,両遺伝子の翻訳産物がニゲランの生産量や分子量に影響を及ぼすことが示唆された。

  • 微生物の代謝機能と酵素を利用した新しい希少糖およびヘテロ希少二糖生産技術の確立

    基盤研究(C)

    課題番号: 26450097

    研究期間: 2014年04月  -  2017年03月 

    代表者: 高田 悟郎, 櫻庭 春彦, 森本 兼司, 上地 敬子 

    直接経費: 3,900,000(円)  間接経費: 5,070,000(円)  金額合計: 1,170,000(円)

     概要を見る

    本課題は、D-アロース、D-アルトロース及びこれらの希少糖を含む二糖を新規の微生物の代謝機能と酵素を用いた新たな反応経路で生産する技術の確立のための基盤研究である。 本研究により、D-グルコシド3-デヒドロゲナーゼの大量生産に成功した。また、結晶化にも成功した。本酵素は様々な二糖類に作用し、化学的還元を組み合わせることで、α-1,4結合型、β-1,4結合型のアロシルグルコース、α-1,1結合型のアロシルアロース、β-1,4結合型のグロシルグルコースなど様々な希少糖含有二糖の生産が可能となった。