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Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system

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Abstract

Hydrophobins are amphipathic secretory proteins with eight conserved cysteine residues and are ubiquitous among filamentous fungi. In the fungus Aspergillus oryzae, the hydrophobin RolA and the polyesterase CutL1 are co-expressed when the sole available carbon source is the biodegradable polyester polybutylene succinate-co-adipate (PBSA). RolA promotes the degradation of PBSA by attaching to the particle surface, changing its structure and interacting with CutL1 to concentrate CutL1 on the PBSA surface. We previously reported that positively charged residues in RolA and negatively charged residues in CutL1 are cooperatively involved in the ionic interaction between RolA and CutL1. We also reported that hydrophobin RodA of the model fungus Aspergillus nidulans, which was obtained via an A. oryzae expression system, interacted via ionic interactions with CutL1. In the present study, phylogenetic and alignment analyses revealed that the N-terminal regions of several RolA orthologs contained positively charged residues and that the corresponding negatively charged residues on the surface of CutL1 that were essential for the RolA–CutL1 interaction were highly conserved in several CutL1 orthologs. A PBSA microparticle degradation assay, a pull-down assay using a dispersion of Teflon particles, and a kinetic analysis using a quartz crystal microbalance revealed that recombinant A. nidulans RodA interacted via ionic interactions with two recombinant A. nidulans cutinases. Together, these results imply that ionic interactions between hydrophobins and cutinases may be common among aspergilli and other filamentous fungi.

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Acknowledgements

We thank Du Pont-Mitsui Fluorochemicals Co., Ltd. for the gift of the Teflon PFA350 suspension. We thank Katsuhiko Kitamoto of the University of Tokyo for providing the Aspergillus oryzae NSlD-tApEnBdIVdV2. We thank Hiroyuki Horiuchi of the University of Tokyo for providing the pUCpyroA2. We thank Daiki Sato, Megumi Nagayama, Yoonkyung Kim, Yuki Terauchi, Naoki Abe, and Natsumi Okada for helpful discussions and technical assistance.

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Authors and Affiliations

  1. Laboratory of Applied Microbiology, Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-0845, Japan

    Takumi Tanaka & Keietsu Abe

  2. Microbial Genomics Laboratory, New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, 980-8579, Japan

    Mayumi Nakayama, Toru Takahashi & Keietsu Abe

  3. Department of Microbial Resources, Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, 980-0845, Japan

    Kei Nanatani & Keietsu Abe

  4. Department of Applied Molecular Biology and Biochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan

    Youhei Yamagata

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  1. Takumi Tanaka
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Correspondence to Keietsu Abe.

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This work was supported, in part, by KAKENHI (Japan Society for the Promotion of Science), a Grant-in-Aid for Scientific Research (B) (grant no. 20380175 to Keietsu Abe), and a Challenging Exploratory Research grant (grant no. 24658281 to Keietsu Abe). This work was also supported, in part, by Japan Society for the Promotion of Science (JSPS) Research Fellowships for Young Scientists (grant no. 15J01183 to Takumi Tanaka).

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Tanaka, T., Nakayama, M., Takahashi, T. et al. Analysis of the ionic interaction between the hydrophobin RodA and two cutinases of Aspergillus nidulans obtained via an Aspergillus oryzae expression system. Appl Microbiol Biotechnol 101, 2343–2356 (2017). https://doi.org/10.1007/s00253-016-7979-5

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