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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   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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• 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   2021.05 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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  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.

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• 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   2021.04 [ Peer Review Accepted ]

  Type of publication: Research paper (scientific journal)

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  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.

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• 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.