Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillus oryzae hyphae in liquid culture

Abstract

226 words 2 Text, 5,353 words 3 3Abstract Filamentous fungi generally form aggregated hyphal pellets in liquid culture. 1 We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did 2 not form hyphal pellets and their hyphae were fully dispersed, and we suggested that 3 α-1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae 4 α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we 5 hypothesized that another factor responsible for forming hyphal pellets remains in these 6 mutants. Here, we identified an extracellular matrix polysaccharide 7 galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae 8 (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. 9 Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that 10 GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG 11 fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. 12 Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, 13 suggesting that hydrogen bond formation by this group is important for aggregation. 14 Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many 15 filamentous fungi and thus may function in hyphal aggregation in these fungi. We also 16 demonstrated that production of a recombinant polyesterase, CutL1, was higher in the 17 AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal 18 aggregation factors of filamentous fungi may increase productivity in the fermentation 19 industry. 20 21 22Importance Production using filamentous fungi is an important part of the 1 fermentation industry, but hyphal aggregation in these fungi in liquid culture limits 2 productivity compared with that of yeast or bacterial cells. We found that 3 galactosaminogalactan and α-1,3-glucan both function in hyphal aggregation in 4 Aspergillus oryzae, and that the hyphae of a double mutant deficient in both 5 polysaccharides become fully dispersed in liquid culture. We also revealed the relative 6 contribution of α-1,3-glucan and galactosaminogalactan to hyphal aggregation. 7 Recombinant protein production was higher in the double mutant than in the wild-type 8 strain. Our research provides a potential technical innovation for the fermentation 9 industry that uses filamentous fungi, as regulation of the growth characteristics of A.10 oryzae in liquid culture may increase productivity. 11 12 13

The mechanisms of hyphal pellet formation mediated by polysaccharides, α-1,3-glucan and galactosaminogalactan, in Aspergillus species

“…Through functional analysis of α-1,3glucan synthase genes in Aspergillus nidulans, we found that the hyphae of an α-1,3-glucan-deficient strain are dispersed under liquid culture conditions with shaking, whereas wild-type mycelia form pellets [27]. We further revealed that, in addition to α-1,3-glucan, the extracellular secreted polysaccharide GAG contributes to the formation of hyphal pellets in the industrially used fungus A. oryzae [28]. This review describes the proposed mechanism of fungal pellet formation mediated by biochemical properties of cell wall polysaccharides, in particular α-1,3-glucan and GAG in Aspergillus species, and outlines the potential for controlling pellet formation by regulating the biosynthesis of the polysaccharides.…”

“…Understanding the cell wall structure is essential for the control of fungal pathogenesis, industrial productivity, and so on. We recently reported that α-1,3-glucan and GAG contribute to hyphal aggregation [28], and a study of the mechanism of hyphal aggregation mediated by the polysaccharides is ongoing. In this section, we review the biological functions of the polysaccharides related to hyphal aggregation and the mechanism of hyphal aggregation in liquid culture.…”

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

“…In N. crassa, deacetylation was correlated with the level of expression of a deacetylase [39][40][41] . Recently, it was shown that Aspergillus oryzae produces GAG with a degree of deacetylation near 50% 42 . Picomolar quantities of Agd3 are sufficient to cause soluble GAG adhesion in the GAG-ELLA assays suggesting that Agd3 is efficient, or that low levels of deacetylation are sufficient for adherence.…”

“…Fungal Agd3 orthologues have been identified previously within GAG gene clusters 5 . The production of GAG-like polymers has been confirmed in Aspergillus niger, Aspergillus parasiticus, A. oryzae, N. crassa, Penicillium frequentans, Paecilomyces sp., and Trichosporon asahii 5,40,42,[49][50][51][52][53] . Expression of poly-N-acetylgalactosamine deacetylases in both A. parasiticus and N. crassa has been confirmed and enzyme levels are linked to levels of polysaccharide produced suggesting co-regulation of polymer and deacetylase production 40,54 .…”

Fungal research in Japan: tradition and future

“…Filamentous fungi generally form aggregated hyphal pellets in liquid culture, which reduces cell density and fermentation productivity, however the α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets but fully dispersed. Besides α-1,3-glucan, an extracellular matrix polysaccharide galactosaminogalactan (GAG) is also involved in the aggregation in A. oryzae [ 10 ] (Fig. 3 , right).…”