Presentation Information
[3SBT-12]Solid-state cultivation-specific agmatine production mediated by a novel arginine decarboxylase in Aspergillus oryzae
○Naoki Akasaka1, Adina Amy Reikanisuji1, Jenna Hamaring Pinkan Kairupan1, Daisuke Watanabe1, Jun-ichi Maruyama2, Shinsuke Fujiwara3 (1. NAIST (Japan), 2. The Univ. of Tokyo (Japan), 3. Kwansei Gakuin Univ. (Japan))
Keywords:
Polyamine,Agmatine,Arginine decarboxylase,Aspergillus oryzae,Solid-state cultivation
Polyamines, such as agmatine, putrescine, spermidine, and spermine, are basic compounds comprising several amino groups and carbon chains, and are prerequisite for a wide spectrum of cellular processes including nucleic acid stabilization, translation regulation, and cell differentiation in all living organisms. Agmatine, a decarboxylated form of arginine generated by arginine decarboxylase (ADC), has gained attention as a promising agent for promoting a healthy lifespan because of its beneficial effects in alleviating lifestyle-related diseases and several neuropathological states. Fermented foods are richer in polyamines than in ordinary foodstuffs. Sake, a traditional Japanese alcoholic beverage, contains greater amounts of agmatine compared to other fermented foods, yet its ingredient rice contains no agmatine. Sake is produced via the multiple parallel fermentation (MPF), in which saccharification of steamed rice by Aspergillus oryzae and alcoholic fermentation by Saccharomyces cerevisiae proceed simultaneously, suggesting that agmatine in sake is derived from microbial metabolism. However, genome analyses suggest that ascomycetes including A. oryzae and S. cerevisiae lack ADC, and microorganisms responsible for agmatine production have remained unexplored.
To examine this, we first conducted MPF, simple alcoholic fermentation (SF) by cultivating S. cerevisiae in enzymatically saccharified rice syrup, and saccharification of steamed rice using koji, a solid starter culture of A. oryzae obtained by cultivating the fungus on steamed rice. Results showed that sake from MPF and rice syrup from saccharification contained equal amounts of agmatine whereas it was barely detected in rice alcohol from SF, indicating that A. oryzae synthesizes agmatine. The fungus produced agmatine during solid-state cultivation, but not under submerged conditions. Mass spectrometry imaging also revealed that arginine and agmatine were accumulated in steamed rice concomitant with mycelial elongation into rice grains during koji making, implying that an unidentified ADC is expressed in the invading (substrate) mycelia exclusively under solid-state cultivation. Peptide mass fingerprinting (LC-MS/MS) of the active fraction extracted from the substrate mycelia of A. oryzae RIB40 showed that the protein exhibiting ADC activity corresponded to the gene AO090102000327, annotated as a phosphatidylserine decarboxylase. The gene was designated Ao-adc1, and the recombinant Ao-ADC1 exhibited the low pH-dependent ADC activity. Crystal structure analysis revealed that Ao-ADC1 is a pyruvoyl-dependent ADC that matures through autocleavage of the inactive proenzyme generating active α and β subunits. The Ao-adc1 disruptant (Δadc1) produced no agmatine, highlighting that the agmatine production is solely attributable to Ao-ADC1. Furthermore, morphological observation indicated that aerial mycelia and the onset of conidiation were less fluffy and earlier, respectively, in Δadc1 than in RIB40, suggesting that agmatine may be involved in aerial hyphal growth and asexual development. To identify the signaling pathway inducing Ao-ADC1 expression, we focused on and disrupted laeA, a master regulator coordinating various cellular processes with environmental stimuli in Aspergillus species, leading to the almost complete loss of agmatine production in the laeA disruptant. This clearly indicates that the expression of Ao-ADC1 is controlled by LaeA.
To examine this, we first conducted MPF, simple alcoholic fermentation (SF) by cultivating S. cerevisiae in enzymatically saccharified rice syrup, and saccharification of steamed rice using koji, a solid starter culture of A. oryzae obtained by cultivating the fungus on steamed rice. Results showed that sake from MPF and rice syrup from saccharification contained equal amounts of agmatine whereas it was barely detected in rice alcohol from SF, indicating that A. oryzae synthesizes agmatine. The fungus produced agmatine during solid-state cultivation, but not under submerged conditions. Mass spectrometry imaging also revealed that arginine and agmatine were accumulated in steamed rice concomitant with mycelial elongation into rice grains during koji making, implying that an unidentified ADC is expressed in the invading (substrate) mycelia exclusively under solid-state cultivation. Peptide mass fingerprinting (LC-MS/MS) of the active fraction extracted from the substrate mycelia of A. oryzae RIB40 showed that the protein exhibiting ADC activity corresponded to the gene AO090102000327, annotated as a phosphatidylserine decarboxylase. The gene was designated Ao-adc1, and the recombinant Ao-ADC1 exhibited the low pH-dependent ADC activity. Crystal structure analysis revealed that Ao-ADC1 is a pyruvoyl-dependent ADC that matures through autocleavage of the inactive proenzyme generating active α and β subunits. The Ao-adc1 disruptant (Δadc1) produced no agmatine, highlighting that the agmatine production is solely attributable to Ao-ADC1. Furthermore, morphological observation indicated that aerial mycelia and the onset of conidiation were less fluffy and earlier, respectively, in Δadc1 than in RIB40, suggesting that agmatine may be involved in aerial hyphal growth and asexual development. To identify the signaling pathway inducing Ao-ADC1 expression, we focused on and disrupted laeA, a master regulator coordinating various cellular processes with environmental stimuli in Aspergillus species, leading to the almost complete loss of agmatine production in the laeA disruptant. This clearly indicates that the expression of Ao-ADC1 is controlled by LaeA.
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