Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Abstract

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.

Both galactosaminogalactan and α-1,3-glucan contribute to aggregation ofAspergillus oryzaehyphae in liquid culture

“…We disrupted the three α-1,3-glucan synthase genes in the industrial fungus Aspergillus oryzae (Δ agsA Δ agsB Δ agsC ; AGΔ) and confirmed the loss of α-1,3-glucan in the cell wall of the AGΔ strain, but the strain still formed small hyphal pellets in liquid culture (13). Although the AGΔ hyphae were not fully dispersed, the strain produced more recombinant polyesterase (cutinase) CutL1 than did a wild-type strain (WT-cutL1) because of the smaller pellets of the AGΔ strain (13). We predicted that another cell wall or cell surface component is responsible for hyphal aggregation in the AGΔ strain.…”

“…Aspergillus oryzae NS4 ( sC - , niaD - ) with Δ ligD (Δ ligD :: sC , Δ adeA :: ptrA ) was used for all genetic manipulations (20). All A. oryzae strains were cultured in standard Czapek–Dox (CD) medium as described previously (11, 13). The niaD - strains were cultured in CDE medium (CD medium containing 70 mM sodium hydrogen L(+)-glutamate monohydrate as the nitrogen source instead of sodium nitrate).…”

“…Conidia of A . oryzae used to inoculate flask cultures were isolated from cultures grown on malt medium, as described previously (13). YPD medium containing 2% peptone (Becton Dickinson and Company, Sparks, Nevada, USA), 1% yeast extract (Becton Dickinson and Company), and 2% glucose was used for flask culture to analyze growth characteristics.…”

“…Fragments containing the 3′ non-coding regions of ugeZ (amplicon 1) and sphZ (amplicon 2) derived from A. oryzae genomic DNA, and the adeA gene (amplicon 3) from the TOPO-2.1-adeA plasmid (13), were amplified by PCR. Amplicon 1 was amplified with the primers sphZ+ugeZ-LU and sphZ+ugeZ-LL+ade, amplicon 2 with the primers sphZ+ugeZ-RU+ade and sphZ+ugeZ-RL, and amplicon 3 with the primers sphZ+ugeZ-AU and sphZ+ugeZ-AL.…”

“…Conidia (final concentration, 1 × 10 5 /mL) of the wild-type, AGΔ, GAGΔ, and AG-GAGΔ strains were inoculated into 50 mL of YPD medium in 200-mL Erlenmeyer flasks and rotated at 120 rpm at 30°C for 24 h. The mean diameter of the hyphal pellets was determined as described previously (13).…”