麹菌の転写因子KpeAは平板培養でのコウジ酸生産の制御因子として見出されたが，その後，広く二次代謝と分生子形成の制御に関わることが明らかとなった。種麹製造及び製麹は分生子形成の制御を伴う醸造工程であり，また，米麹中の二次代謝物は醸造物の品質や安全性に影響する。そこで本研究では，実際の醸造における培養工程である種麹製造と製麹において，KpeAの遺伝子破壊株と高発現株の形質を調べることでKpeAの醸造における役割を明らかにすることを目的とした。 製造した種麹の状貌と遺伝子解析から，KpeAは，分生子形成の主要因子であるBrlAの遺伝子発現制御を介して，分生子形成を促進する役割を果たしていることが示された。製麹においても米麹に着生する分生子に差が生じたことから，KpeAは醸造工程においても分生子形成に重要な転写因子であることが分かった。一方，米麹中のコウジ酸量が破壊株で顕著に増加した。コウジ酸の推定生合成酵素KojAの遺伝子発現解析から，KpeAは平板培養と同様に，製麹においてもコウジ酸生産を転写レベルで制御することが確認された。さらに破壊株の麹抽出液は，コウジ酸が多く含まれるために褐変しにくいことが明らかとなったことから，KpeAの遺伝子破壊によるコウジ酸生産の増加が，米麹の褐変抑制に効果があることが示された。 以上よりKpeAは種麹製造と製麹において，分生子形成やコウジ酸生産を制御しており，実際の醸造においても重要な役割を果たす転写因子であることが確認された。KpeAの遺伝子破壊株は，米麹の酵素活性が若干低下するものの，製麹において分生子が形成しにくく，かつ米麹の褐変性が低いといった清酒醸造に使用する麹菌として好ましい特性をもつため，KpeAは醸造における麹菌の育種ターゲットの新しい候補として期待できる。

We previously identified a novel transcription factor, KpeA, in Aspergillus oryzae. KpeA is involved in secondary metabolism and conidiation when A. oryzae is cultured in liquid or agar medium. To clarify the role of KpeA in sake brewing, we analyzed the functionality of KpeA in rice koji making and seed koji making. In seed koji making, the kpeA disruption (ΔkpeA) strain showed longer aerial hyphae and the number of conidia was one-third that of the control strain. In accordance with conidiation, expression of brlA, a pivotal gene for conidiation, was also decreased in the ΔkpeA strain, whereas it was increased in the kpeA overexpression (OE) strain. These results suggested that, similar to the phenomenon that occurs in agar culture, KpeA induced conidiation via positive regulation of brlA expression in seed koji making. Rice koji samples made with ΔkpeA strain, OE strain, and control strain were analyzed for conidiation, enzyme activity, and secondary metabolites. The OE strain and the control strain formed conidia after 46 and 72 h of inoculation, respectively, whereas the ΔkpeA strain did not. In contrast, activities of representative hydrolytic enzymes in rice koji did not differ among the strains. Regarding secondary metabolism, we confirmed that kpeA is responsible for the enhanced kojic acid production via inducing the expression of the putative biosynthesis gene, kojA. Notably, the browning of rice koji extract was not observed in the ΔkpeA strain sample. We confirmed that additional dosage of kojic acid at the level of the rice koji of the ΔkpeA strain prevented the browning of rice koji extract of the control sample. As a result, kpeA disruption led to increased kojic acid amount in rice koji, which consequently prevented the browning of rice koji. Taken together, our results showed that KpeA regulates secondary metabolism and conidiation of A. oryzae during rice koji making and seed koji making. Although enzyme activities of the ΔkpeA strain were reduced slightly, we consider that the observed phenotypes, such as suppressed conidiation or repression of the browning of rice koji, are beneficial for rice koji or rice koji making. Thus, we propose that kpeA could be a new target for the breeding of A. oryzae for traditional brewing.

E

4

P

論文

Article