Presentation Information
[4Ferm-09]High-speed atomic force microscopy reveals rodlet film formation mechanism of Aspergillus oryzae hydrophobin RolA
○Nao Takahashi1, Tatsuya Kimura2, Yuki Terauchi3, Takumi Tanaka1, Akira Yoshimi4, Takahiro Watanabe-Nakayama2, Keietsu Abe1 (1. Tohoku University (Japan), 2. Kanazawa University (Japan), 3. Yamaguchi University (Japan), 4. Kyoto University (Japan))
Keywords:
Hydrophobin,Aspergillus oryzae,Self-assembly,High-speed AFM,Amyloid
Hydrophobins are biosurfactant proteins conserved in filamentous fungi. They coat the cell surface and function as a protective coat, contributing to the aerial dispersion of conidia, immune evasion, and the promotion of solid polymer degradation (1). Recently, we found that deletion of the hydrophobin rolA gene in Aspergillus oryzae reduces wall growth and culture viscosity during liquid culture, resulting in improved enzyme productivity. However, the mechanism by which RolA affects liquid culture remains completely unknown. To control liquid culture of A. oryzae through RolA, it is crucial to understand its fundamental properties, including its structure, in detail. Notably, RolA self-assembles into rod-shaped multimeric structures called rodlets, which adsorb strongly to interfaces and form a rigid amphiphilic rodlet film that coats conidial surface. Thus, in this study, we aimed to elucidate the mechanism by which RolA self-assembles to form the rodlet layer. To clarify how rodlets bundle together on an interface to form a dense coating, we performed direct in situ observation using high-speed atomic force microscopy (HS-AFM)(2).
RolA was expressed from a strain of A. oryzae overexpressing wild-type RolA and purified for the following experiments. Rodlet formation on a silicon substrate surface was monitored by HS-AFM where one AFM image was acquired every 12 s for approximately 30 min.
The observations showed that RolA first formed spherical structures on the substrate, then converted into rodlets, which elongated from both ends. Rodlet elongation was not continuous, but instead repeatedly alternated between growth and stop, suggesting that there were two distinct structural state at the rodlet ends: one in which monomers can bind and rodlets can elongate, and the other in which the monomers cannot bind. We also captured the process in which a rodlet elongated along a pre-existing rodlet (bundling process) and found that the rodlet elongated two times faster during the bundling. This reaction appears to lower the energy barrier between the growth and stop, suggesting that it can be regarded as surface-catalyzed elongation. This catalytic effect at the rodlet surface may promote rodlet alignment and thereby control the entire orientation of the rodlet film; our Monte Carlo simulations confirmed this. These findings provide important conceptual advances for understanding cell surface architectures of A. oryzae and may contribute to cell surface engineering.
(1) Takumi Tanaka et al., Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation. Microorganisms (2022)
(2) Nao Takahashi et al., High-speed atomic force microscopy reveals a surface-catalyzed elongation mechanism of the fungal functional amyloid hydrophobin RolA. PNAS (2026)
RolA was expressed from a strain of A. oryzae overexpressing wild-type RolA and purified for the following experiments. Rodlet formation on a silicon substrate surface was monitored by HS-AFM where one AFM image was acquired every 12 s for approximately 30 min.
The observations showed that RolA first formed spherical structures on the substrate, then converted into rodlets, which elongated from both ends. Rodlet elongation was not continuous, but instead repeatedly alternated between growth and stop, suggesting that there were two distinct structural state at the rodlet ends: one in which monomers can bind and rodlets can elongate, and the other in which the monomers cannot bind. We also captured the process in which a rodlet elongated along a pre-existing rodlet (bundling process) and found that the rodlet elongated two times faster during the bundling. This reaction appears to lower the energy barrier between the growth and stop, suggesting that it can be regarded as surface-catalyzed elongation. This catalytic effect at the rodlet surface may promote rodlet alignment and thereby control the entire orientation of the rodlet film; our Monte Carlo simulations confirmed this. These findings provide important conceptual advances for understanding cell surface architectures of A. oryzae and may contribute to cell surface engineering.
(1) Takumi Tanaka et al., Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation. Microorganisms (2022)
(2) Nao Takahashi et al., High-speed atomic force microscopy reveals a surface-catalyzed elongation mechanism of the fungal functional amyloid hydrophobin RolA. PNAS (2026)
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