Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae

Ami Koizumi; Ken Miyazawa; Makoto Ogata; Yuzuru Takahashi; Shigekazu Yano; Akira Yoshimi; Motoaki Sano; Masafumi Hidaka; Takanori Nihira; Hiroyuki Nakai; Satoshi Kimura; Tadahisa Iwata; Keietsu Abe

doi:10.3389/ffunb.2022.1061841

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae

Front Fungal Biol. 2023 Jan 10:3:1061841. doi: 10.3389/ffunb.2022.1061841. eCollection 2022.

Authors

Ami Koizumi¹, Ken Miyazawa^{1

2}, Makoto Ogata³, Yuzuru Takahashi⁴, Shigekazu Yano⁴, Akira Yoshimi^{5

6

7}, Motoaki Sano⁸, Masafumi Hidaka⁹, Takanori Nihira¹⁰, Hiroyuki Nakai¹⁰, Satoshi Kimura¹¹, Tadahisa Iwata¹¹, Keietsu Abe^{1

5}

Affiliations

¹ Laboratory of Applied Microbiology, Department of Biochemistry, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
² Laboratory of Filamentous Mycoses, Department of Fungal Infection, National Institute of Infectious Diseases, Tokyo, Japan.
³ Faculty of Food and Agricultural Sciences, Fukushima University, Fukushima, Japan.
⁴ Department of Biochemical Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa, Japan.
⁵ ABE-Project, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
⁶ Laboratory of Environmental Interface Technology of Filamentous Fungi, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
⁷ Laboratory of Terrestrial Microbial Ecology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
⁸ Genome Biotechnology Laboratory, Kanazawa Institute of Technology, Hakusan, Japan.
⁹ Laboratory of Enzymology, Department of Biochemistry, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
¹⁰ Faculty of Agriculture, Niigata University, Niigata, Japan.
¹¹ Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

Abstract

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

Keywords: Aspergillus oryzae; cell wall; filamentous fungi; glycosylphosphatidylinositol-anchored protein; oligosaccharide; α-1,3-glucan; α-amylase.

Grants and funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Nos. JP20H02895, JP26292037 (KA), JP18K05384 (AY), JP18J11870, and JP20K22773 (KM). This work was also supported by the Institute for Fermentation, Osaka (Grant No. L-2018–2–014) (KA), and New Energy and Industrial Technology Development Organization (NEDO), JPNP20011 (KA).