Dataset Information

Both Galactosaminogalactan and ?-1,3-Glucan Contribute to Aggregation of Aspergillus oryzae Hyphae in Liquid Culture.

ABSTRACT: Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that ?-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that ?-1,3-glucan functions in hyphal aggregation. However, Aspergillus oryzae ?-1,3-glucan-deficient (AG?) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAG?), we disrupted the genes required for GAG biosynthesis in an AG? mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAG? strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that ?-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAG? strain than in the wild-type and AG? strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.

SUBMITTER: Miyazawa K

PROVIDER: S-EPMC6753227 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Both Galactosaminogalactan and α-1,3-Glucan Contribute to Aggregation of Aspergillus oryzae Hyphae in Liquid Culture.

Miyazawa Ken K Yoshimi Akira A Sano Motoaki M Tabata Fuka F Sugahara Asumi A Kasahara Shin S Koizumi Ami A Yano Shigekazu S Nakajima Tasuku T Abe Keietsu K

Frontiers in microbiology 20190913

Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. However, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in th ...[more]

PMID: 31572319

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Project description:α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE ) or agsB (agsBOE ), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE , 41.6 ± 5.8 kDa; agsBOE , 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain-fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.

Dataset Information

Both Galactosaminogalactan and ?-1,3-Glucan Contribute to Aggregation of Aspergillus oryzae Hyphae in Liquid Culture.

Publications

Both Galactosaminogalactan and α-1,3-Glucan Contribute to Aggregation of <i>Aspergillus oryzae</i> Hyphae in Liquid Culture.

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