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• Characterization of an antifungal β-1,3-glucanase from <i>Ficus microcarpa</i> latex and comparison of plant and bacterial β-1,3-glucanases for fungal cell wall β-glucan degradation

  Takashima, T; Komori, N; Uechi, K; Taira, T

  PLANTA ( Planta )  258 ( 6 ) 116 - 116   2023.12 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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  Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall β-glucan, it was compared with β-1,3-glucanase with different substrate specificities. We obtained recombinant β-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I β-1,3-glucanase) and CcGluA (GH64 β-1,3-glucanase).

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• 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   2022.06 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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• 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 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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  <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>

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• 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 ( Applied and Environmental Microbiology )  87 ( 21 ) e0114421   2021.10 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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  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.

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• Synthesis and characterization of α-1,3-<i>alt</i>-α-1,4-glucan (nigeran) ester derivatives (vol 214, 123343, 2021)

  Togo, A; Uechi, K; Mizutani, O; Kimura, S; Iwata, T

  POLYMER   226   2021.06 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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• Grant-in-Aid for Scientific Research(C)

  Project Year: 2022.04  -   

• Grant-in-Aid for Scientific Research(C)

  Project Year: 2022.04  -  2025.03 

  Direct: 3,200,000 (YEN)  Overheads: 4,160,000 (YEN)  Total: 960,000 (YEN)

• Study on the production of rare disaccharides and rare sugars.

  Grant-in-Aid for Scientific Research(C)

  Project Year: 2014.04  -  2017.03 

  Investigator(s): Takata Goro, Sakuraba Haruhiko 

  Direct: 3,900,000 (YEN)  Overheads: 5,070,000 (YEN)  Total: 1,170,000 (YEN)

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  The objective of this study is the production of D-allose, D-altrose and their disaccharides using biotechnology method. From the result of this research, we succeeded to mass-production of recombinant D-glucoside 3-dehydrogenase and its crystalization. This enzyme showed the substrate specificity against various disaccharides. By combination of chemical hydrogenation, we achieved to produced various rare sugar containing disaccharide such as 1,4-a-allosylglucose, 1,4-b-allosylglucose, 1,1-a-allosylallose, and 1,4-b-gulosylglucose.

• Study on the production of rare disaccharides and rare sugars.

  Grant-in-Aid for Scientific Research(C)

  Project Year: 2014.04  -  2017.03 

  Investigator(s): Takata Goro, Sakuraba Haruhiko 

  Direct: 3,900,000 (YEN)  Overheads: 5,070,000 (YEN)  Total: 1,170,000 (YEN)

  　View Summary

  The objective of this study is the production of D-allose, D-altrose and their disaccharides using biotechnology method. From the result of this research, we succeeded to mass-production of recombinant D-glucoside 3-dehydrogenase and its crystalization. This enzyme showed the substrate specificity against various disaccharides. By combination of chemical hydrogenation, we achieved to produced various rare sugar containing disaccharide such as 1,4-a-allosylglucose, 1,4-b-allosylglucose, 1,1-a-allosylallose, and 1,4-b-gulosylglucose.