Presentation Information
[P02-167]Elucidating a molecular mechanism underlying the production of agmatine by Aspergillus oryzae during solid-state cultivation
○Adina Amy Reikanisuji1, Jenna Hamaring Pinkan Kairupan1, Naoki Akasaka1, Jun-ichi Maruyama2, Daisuke Watanabe1 (1. NAIST (Japan), 2. The Univ. Tokyo (Japan))
Keywords:
LaeA,Starch,Aspergillus oryzae,Agmatine,Solid-state cultivation
Agmatine, a natural polyamine synthesized from arginine by arginine decarboxylase (ADC), has gained attention as a bioactive agent alleviating various neurological and lifestyle diseases. Our previous study revealed that Aspergillus oryzae, a filamentous fungus used in diverse traditional Japanese and Asian fermented foods, produces agmatine solely under solid-state cultivation using natural substrates such as steamed rice. Recently, we identified a novel ADC (Ao-ADC1) in A. oryzae, which is specifically expressed during solid-state cultivation. However, regulatory mechanisms, including signaling pathways that induce Ao-ADC1 expression, remain unexplored. This study aims to identify environmental stimuli to induce agmatine production and regulators governing Ao-ADC1 expression.
Steamed rice is characterized by high starch and low water contents. To examine their effects on agmatine production, A. oryzae RIB40 was cultivated on a minimal agar plate (1.5% agar) supplemented with 2 or 50% starch, and the ADC activity of cells was evaluated by in vitro assay. Cells grown on 50% starch exhibited ADC activity, while those grown on 2% starch showed no detectable activity, suggesting that high starch and/or low water content may contribute to Ao-ADC1 induction. To disentangle these factors, A. oryzae RIB40 was cultivated on a minimal agar plate (2% starch) solidified with 1.5 or 30% agar, and ADC activity was measured. Cells from both conditions showed no ADC activity, implying that low water content alone is insufficient for Ao-ADC1 induction. To verify the effect of starch on agmatine production, A. oryzae RIB40 was cultivated in a minimal liquid medium with various starch concentrations. Agmatine content in the culture supernatant increased in a starch concentration-dependent manner, and the strain accumulated 0.19, 0.28, and 0.53 mM agmatine in the presence of 10, 30, and 50% starch, respectively. The strain also produced a small amount of agmatine (0.08 mM) in the presence of 2% starch under submerged conditions, while no ADC activity was detected in cells grown on a minimal agar plate containing 2% starch and 1.5% agar. This may reflect slight differences in Ao-ADC1 expression between the two conditions. Collectively, these findings suggest that starch, but not low water content alone, is key for agmatine production in A. oryzae.
A previous study suggested that LaeA, a master regulator of secondary metabolism in Aspergillus spp., is involved in agmatine production (1). To investigate its role, the laeA disruptant (ΔlaeA) was constructed using A. oryzae RIB40 as a host via the CRISPR/Cas9 genome editing system. Sequencing analysis identified an insertion in the laeA gene, causing a frameshift mutation in the ΔlaeA strain. The parental and ΔlaeA strains were grown on steamed rice, and the solid cultures were subjected to in vitro assay to evaluate the ADC activity. The ADC activity was significantly lower in ΔlaeA (1.96 pmol/min/μgGlcNAc) than in the parental strain (238.02 pmol/min/μgGlcNAc). Consistently, the disruptant barely produced agmatine (0.02 mM) during solid-state fermentation of steamed rice, while RIB40 accumulated 2.53 mM agmatine. Considering that LaeA epigenetically controls the expression of target genes, these results suggest that Ao-ADC1 expression is transcriptionally upregulated via an unidentified signaling pathway mediated by LaeA in response to starch.
(1) Iwashita et al. 2017. Abstracts of the Annual Meeting of JSBBA p. 137.
Steamed rice is characterized by high starch and low water contents. To examine their effects on agmatine production, A. oryzae RIB40 was cultivated on a minimal agar plate (1.5% agar) supplemented with 2 or 50% starch, and the ADC activity of cells was evaluated by in vitro assay. Cells grown on 50% starch exhibited ADC activity, while those grown on 2% starch showed no detectable activity, suggesting that high starch and/or low water content may contribute to Ao-ADC1 induction. To disentangle these factors, A. oryzae RIB40 was cultivated on a minimal agar plate (2% starch) solidified with 1.5 or 30% agar, and ADC activity was measured. Cells from both conditions showed no ADC activity, implying that low water content alone is insufficient for Ao-ADC1 induction. To verify the effect of starch on agmatine production, A. oryzae RIB40 was cultivated in a minimal liquid medium with various starch concentrations. Agmatine content in the culture supernatant increased in a starch concentration-dependent manner, and the strain accumulated 0.19, 0.28, and 0.53 mM agmatine in the presence of 10, 30, and 50% starch, respectively. The strain also produced a small amount of agmatine (0.08 mM) in the presence of 2% starch under submerged conditions, while no ADC activity was detected in cells grown on a minimal agar plate containing 2% starch and 1.5% agar. This may reflect slight differences in Ao-ADC1 expression between the two conditions. Collectively, these findings suggest that starch, but not low water content alone, is key for agmatine production in A. oryzae.
A previous study suggested that LaeA, a master regulator of secondary metabolism in Aspergillus spp., is involved in agmatine production (1). To investigate its role, the laeA disruptant (ΔlaeA) was constructed using A. oryzae RIB40 as a host via the CRISPR/Cas9 genome editing system. Sequencing analysis identified an insertion in the laeA gene, causing a frameshift mutation in the ΔlaeA strain. The parental and ΔlaeA strains were grown on steamed rice, and the solid cultures were subjected to in vitro assay to evaluate the ADC activity. The ADC activity was significantly lower in ΔlaeA (1.96 pmol/min/μgGlcNAc) than in the parental strain (238.02 pmol/min/μgGlcNAc). Consistently, the disruptant barely produced agmatine (0.02 mM) during solid-state fermentation of steamed rice, while RIB40 accumulated 2.53 mM agmatine. Considering that LaeA epigenetically controls the expression of target genes, these results suggest that Ao-ADC1 expression is transcriptionally upregulated via an unidentified signaling pathway mediated by LaeA in response to starch.
(1) Iwashita et al. 2017. Abstracts of the Annual Meeting of JSBBA p. 137.
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