Faculty Profiles - TAKAMINE Kazunori

Enkang Zhu, Kentaro Hiramatsu, Taiga Inoue, Kazuki Mori, Kosuke Tashiro, Kiyotaka Fujita, Takefumi Karashima, Hideharu Takashita, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Hisanori Tamaki, Taiki Futagami . Deficiency of β-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308. . Bioscience, biotechnology, and biochemistry88 ( 7 ) 816 - 823 2024.4International journal

Language：English Publishing type：Research paper (scientific journal)

In this study, we investigated a deleterious mutation in the β-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial β-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose level during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

DOI： 10.1093/bbb/zbae046

Atsushi Nishitani, Kentaro Hiramatsu, Chihiro Kadooka, Kyoka Hiroshima, Kazutaka Sawada, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Masatoshi Goto, Hisanori Tamaki, Taiki Futagami . Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae. . Journal of bioscience and bioengineering137 ( 4 ) 281 - 289 2024.2

Language：English Publishing type：Research paper (scientific journal)

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

DOI： 10.1016/j.jbiosc.2024.01.010

Yuki Kobashi, Yumiko Yoshizaki, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . THI3 contributes to isoamyl alcohol biosynthesis through thiamine diphosphate homeostasis. . Journal of bioscience and bioengineering137 ( 2 ) 108 - 114 2023.12

Language：English Publishing type：Research paper (scientific journal)

Isoamyl alcohol is a precursor of isoamyl acetate, an aromatic compound that imparts the ginjo aroma to sake. The isoamyl alcohol biosynthesis pathway in yeasts involves the genes PDC1, PDC5, PDC6, ARO10, and THI3 encoding enzymes that decarboxylate α-ketoisocaproic acid to isovaleraldehyde. Among these genes, THI3 is the main gene involved in isoamyl alcohol biosynthesis. Decreased production of isoamyl alcohol has been reported in yeast strains with disrupted THI3 (Δthi3). However, it has also been reported that high THI3 expression did not enhance decarboxylase activity. Therefore, the involvement of THI3 in isoamyl alcohol biosynthesis remains unclear. In this study, we investigated the role of THI3 in isoamyl alcohol biosynthesis. While reproducing previous reports of reduced isoamyl alcohol production by the Δthi3 strain, we observed that the decrease in isoamyl alcohol production occurred only at low yeast nitrogen base concentrations in the medium. Upon investigating individual yeast nitrogen base components, we found that the isoamyl alcohol production by the Δthi3 strain reduced when thiamine concentrations in the medium were low. Under low-thiamine conditions, both thiamine and thiamine diphosphate (TPP) levels decreased in Δthi3 cells. We also found that the decarboxylase activity of cell-free extracts of the Δthi3 strain cultured in a low-thiamine medium was lower than that of the wild-type strain, but was restored to the level of the wild-type strain when TPP was added. These results indicate that the loss of THI3 lowers the supply of TPP, a cofactor for decarboxylases, resulting in decreased isoamyl alcohol production.

DOI： 10.1016/j.jbiosc.2023.11.006

Ryota Kobayashi, Yoshifuru Mitsui, Yumiko Yoshizaki, Kohki Takahashi, Kazunori Takamine, Keiichi Koyama . Controlling the growth of yeast by culturing in high magnetic fields . Journal of Magnetism and Magnetic Materials586 2023.11

Language：Japanese Publishing type：Research paper (scientific journal)

The effect of static magnetic fields up to a strength of 15 T on the growth of three types of yeast which were all classified as Saccharomyces cerevisiae was studied. The growth rate of the yeasts was suppressed by applying a magnetic field. The degree of suppression depended on the type of yeast. The reduction in the numbers of yeast cells was due to growth suppression, not sterilization. Among the three types of Saccharomyces cerevisiae, the degree of suppression on the growth of two yeasts was strongest in a magnetic field at 8 T with different suppression degree of 19% and 12% for a culture time of 24 h. Growth of the other yeast most suppressed by 10% at a magnetic field of around 4 T. Therefore, the yeasts have their characteristic magnetic field sensitivity on the growth. The results were discussed using a model based on the competition between yeast osmotic pressure changes by magnetic field and magnetic forces induced by magnetic field gradient. In low magnetic field regions, change of the osmotic pressure shrink the yeast cell and reduce the contact frequency between yeast cells and glucose-based ions. In higher magnetic field regions, effect of magnetic force become apparent, and recover the growth rate of the yeast. It suggested that the selective growth of yeast can be expected by in-magnetic-field culturing.

DOI： 10.1016/j.jmmm.2023.171193

Atsushi Nishitani, Kentaro Hiramatsu, Chihiro Kadooka, Kazuki Mori, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Kosuke Tashiro, Masatoshi Goto, Hisanori Tamaki, Taiki Futagami . Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii. . Journal of bioscience and bioengineering136 ( 6 ) 443 - 451 2023.9

Language：English Publishing type：Research paper (scientific journal)

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A. luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

DOI： 10.1016/j.jbiosc.2023.09.004

Wang Tiantian, Hanashiro Isao, Yoshizaki Yumiko, Kobashi Yuki, Noda Suzuka, Okutsu Kayu, Futagami Taiki, Tamaki Hisanori, Takamine Kazunori . Shochu koji microstructure and starch structure during preparation . Journal of Applied Glycoscienceadvpub ( 0 ) 109 - 117 2023.9Reviewed

Language：English Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Applied Glycoscience

In this study, we investigated the changes in composition-, microstructure-, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13%–20% of the total sugar content, with glucose representing over 70% of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization [DP] 4–73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6–30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

DOI： 10.5458/jag.jag.jag-2023_0006

Okutsu K., Yamamoto Y., Matsuo F., Yoshizaki Y., Futagami T., Tamaki H., Maeda G., Tsuchida E., Takamine K. . Characterization of aroma profiles of kokuto-shochu prepared from three different cultivars of sugarcane . Journal of Bioscience and Bioengineering135 ( 6 ) 458 - 465 2023.6

Language：Japanese Publisher：Journal of Bioscience and Bioengineering

Kokuto-shochu is a traditional Japanese spirit prepared from kokuto, obtained by evaporating water from sugarcane (Saccharum officinarum L.) juice. To clarify the effects of sugarcane cultivars on the sensory quality of kokuto-shochu, we investigated the flavor characteristics and composition of volatiles in kokuto-shochu prepared from kokuto using three different sugarcane cultivars, NiF8, Ni15, and RK97-14. Furthermore, experiments were conducted by using the cultivars collected between 2018 and 2020 to observe annual variations in their properties. The amino acid content of the three kokuto varieties did not differ significantly, but the amino acid content of NiF8 was two to five times higher than that of RK97-14, which was the same for all samples collected in the selected years. The browning degrees of kokuto were also higher in NiF8, and they were positively correlated to the amino acid contents of kokuto. The kokuto-like aroma of shochu made from Ni15 was stronger than that of shochu made from RK97-14. The concentration of ethyl lactate in shochu made from Ni15 was higher, however, the concentration of guaiacol was the lowest in the three cultivars’ products. Shochu made from NiF8 had the highest levels of Maillard reaction products (MRPs; pyrazines and furans), β-damascenone, and guaiacol amounts. In contrast, shochu made from RK97-14 tended to have a fruity flavor, and lower MRP levels than those made from NiF8. Thus, it was shown that sugarcane cultivars affect the sensory characteristics and volatiles in kokuto-shochu.

DOI： 10.1016/j.jbiosc.2023.03.010

Kentaro Hiramatsu, Atsushi Nishitani, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Hisanori Tamaki, Taiki Futagami . Efficient gene targeting in Aspergillus chevalieri used to produce katsuobushi. . Bioscience, biotechnology, and biochemistry87 ( 6 ) 672 - 682 2023.3International journal

Language：English Publishing type：Research paper (scientific journal)

In this study, we developed an efficient gene targeting system for the osmophilic fungus Aspergillus chevalieri, which is commonly used in the production of a dried bonito, katsuobushi. Specifically, we utilized the clustered regularly interspaced short palindromic repeats/Cas9 system to disrupt the ATP sulfurylase encoding sC gene. This results in methionine auxotroph and selenate-resistance. Additionally, we disrupted the DNA ligase IV encoding ligD gene, which is required for non-homologous end joining. Using the sC marker and selenate-resistance as a selection pressure, we were able to rescue the sC marker and generate a ΔligD ΔsC strain. We determined that the gene targeting efficiency of the ΔligD ΔsC strain was significantly higher than that of the parental ΔsC strain, which indicates that this strain provides efficient genetic recombination for the genetic analysis of A. chevalieri.

DOI： 10.1093/bbb/zbad033

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae . Seibutsu-kogaku Kaishi101 ( 2 ) 74 - 74 2023.2Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae

Yuki Kobashi, Eri Nakayama, Naoki Fukumori, Ayane Shimojima, Manami Tabira, Yuki Nishimura, Manami Mukae, Ai Muto, Naoto Nakashima, Kayu Okutsu, Yumiko Yoshizaki, Taiki Futagami, Kazunori Takamine, Hisanori Tamaki . Homozygous gene disruption in diploid yeast through a single transformation. . Journal of bioscience and bioengineering137 ( 1 ) 31 - 37 2023

Language：Japanese Publishing type：Research paper (scientific journal)

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

DOI： 10.1016/j.jbiosc.2023.11.003

Tiantian Wang, Isao Hanashiro, Yumiko Yoshizaki, Yuki Kobashi, Suzuka Noda, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . Shochu koji microstructure and starch structure during preparation . 70 ( 4 ) 109 - 117 2023Shochu koji microstructure and starch structure during preparationReviewed

曾伝濤, 劉夢迪, 吉﨑由美子，遠城道雄, 奥津果優，二神泰基，玉置尚徳，髙峯和則 . サトイモ焼酎の香味特性と焼酎粕の機能性 . 日本醸造協会誌117 ( 10 ) 701 - 711 2022.10サトイモ焼酎の香味特性と焼酎粕の機能性Reviewed

劉根僑, 吉﨑由美子，奥津果優, 二神泰基, 玉置尚徳, 髙峯和則 . サツマイモ固体糖化後仕込みによる芋焼酎製造の可能性 . 日本醸造協会誌117 ( 10 ) 712 - 723 2022.10サツマイモ固体糖化後仕込みによる芋焼酎製造の可能性Reviewed

CHRISTOPHER IMANSANTOSO Rimba, YOSHIZAKI Yumiko, OKUTSU Kayu, TAKAMINE Kazunori . Measuring the Alcohol Content in Shochu Moromi by Near-infrared Spectroscopy . BUNSEKI KAGAKU71 ( 7.8 ) 425 - 430 2022.8Reviewed

Language：Japanese Publisher：The Japan Society for Analytical Chemistry

We measured alcohol content in shochu moromi using near-infrared spectroscopy (NIRS) and verified the obtained values by comparing them with the distillation-oscillatory densimeter (D-OD) method. We prepared six types of shochu moromi made from three types of koji (white, black, and yellow koji) and two main ingredients (rice and sweet potato) and measured the alcohol content in shochu moromi using NIRS and D-OD. The gauze filtrate sample could not measure alcohol content using NIRS due to high turbidity. The NIRS sample needed paper or membrane filtration. The values of alcohol content measured using NIRS and D-OD were almost the same and had a high correlation. Furthermore, the measurement error of NIRS was smaller than that of D-OD. Thus, NIRS can measure the alcohol content in shochu moromi in a simple and reliable manner.

DOI： 10.2116/bunsekikagaku.71.425

Bohan Li, Yumiko Yoshizaki, Kayu Okutsu, Yuki Haruyama, Yuta Sato, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . Factors that cause replacement of shochu yeast by wild yeast upon Sashi-moto repetition . Journal of Bioscience and Bioengineering134 ( 4 ) 326 - 330 2022.8

Kazuki Mori, Chihiro Kadooka, Ken Oda, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Kosuke Tashiro, Masatoshi Goto, Hisanori Tamaki, Taiki Futagami . Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308 . Data in Brief41 107888 - 107888 2022.4Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308

Wang Zitai, Okutsu Kayu, Yoshizaki Yumiko, Futagami Taiki, Tamaki Hisanori, Maruyama Takuro, Toume Kazufumi, Komatsu Katsuko, Hashimoto Fumio, Takamine Kazunori . Effects of microbial fermentation on enzyme activity and volatile properties of Massa Medicata Fermentata(タイトル和訳中) . Traditional & Kampo Medicine9 ( 1 ) 10 - 17 2022.4Effects of microbial fermentation on enzyme activity and volatile properties of Massa Medicata Fermentata(タイトル和訳中)

Language：English Publisher：John Wiley & Sons Australia, Ltd

Massa Medicata Fermentata(MMF)はAspergillus属やRhizopus属による微生物発酵でコムギと数種のハーブから調製されるので、微生物発酵の影響について検討した。National Standard for Chinese Patent Drugsに従ってMMFの原材料を混合し、Aspergillus oryzae培養で調製(MMF-A)またはRhizopus oryzae培養で調製(MMF-R)した。MMFの調製により酵素活性は上昇した。MMF-AはMMF-Rよりもα-アミラーゼとリパーゼの活性が高かった。プロテアーゼ活性はMMF-Rがわずかに高かった。テルペノイドは発酵で低下したが、2,3-ブタンジオール、アセトイン、グアヤコールはMMFだけで検出された。1-オクテン-3-オールのC8化合物はMMF-RよりもMMF-Aで高く、4-ビニルグアヤコールとピラジンの芳香族化合物はMMF-Rで高かった。

Zitai Wang, Kayu Okutsu, Yumiko Yoshizaki, Taiki Futagami, Hisanori Tamaki, Takuro Maruyama, Kazufumi Toume, Katsuko Komatsu, Fumio Hashimoto, Kazunori Takamine . Effects of microbial fermentation on enzyme activity and volatile properties of Massa Medicata Fermentata . Traditional & Kampo Medicine9 ( 1 ) 10 - 17 2022.4

Zhe Zhao, Mina Sugimachi, Yumiko Yoshizaki, Xuan Yin, Xing-Lin Han, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . Correlation between key aroma and manufacturing processes of rice-flavor baijiu and awamori, Chinese and Japanese traditional liquors . FOOD BIOSCIENCE44 2021.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：ELSEVIER

In this study, we compared the flavor profiles Chinese rice-flavor baijiu and Japanese awamori-traditional liquors with similar manufacturing processes. Citric acid and lactic acid were the major acid constituents in the mash of awamori and rice-flavor baijiu, respectively. The amino acid content of awamori mash was greater than that of rice-flavor baijiu owing to the higher proteolytic activities in the former. The high amino acid content also induced yeast growth at the early stage of fermentation in awamori mash, thus increasing the fermentation rate. Sensory evaluation revealed that rice-flavor baijiu had a significantly stronger alcoholic, floral, and fruity aroma, whereas the awamori had a strong sour, koji-like, oily, and cereal-like aromas. We determined the aroma threshold of (+)-ethyl D-lactate (15,400 mu g/L) and (-)-ethyl L-lactate (12,500 mu g/L). Both compounds have a common sweet, fruity, and green leaves-like aroma and a different nuance; (+)-ethyl D-lactate has a peach- and citrus-like aromas, and (-)-ethyl L-lactate has an apple-like and milky aromas. In rice-flavor baijiu, (-)-ethyl Llactate constitutes the major isoform, as evident from the higher concentration of L (+)-lactic acid in the mash. Higher alcohols, (-)-ethyl L-lactate, and isoamyl acetate contents were greater in rice-flavor baijiu, while 1-octen-3-ol and acetic acid were greater in awamori. These results showed that solid-state saccharification contributed to the production of ethyl lactate by supplying in the mash a large amount of lactic acid produced by Rhizopus oryzae and koji contributed to the production of 1-octen-3-ol via the high fatty acid oxygenase activity produced by Aspergillus luchuensis.

DOI： 10.1016/j.fbio.2021.101375

Web of Science

Zhe Zhao, Mina Sugimachi, Yumiko Yoshizaki, Xuan Yin, Xing-Lin Han, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . Impact of solid-state saccharification on the flavor of rice-flavor baijiu . JOURNAL OF FOOD SCIENCE86 ( 11 ) 4958 - 4968 2021.11International journal

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：WILEY

Rice-flavor baijiu is a traditional Chinese liquor. The flavor profile and volatiles presented with or without the solid-state saccharification (SSS) were investigated to reveal the effects of SSS process on the quality of rice-flavor baijiu. The liquor prepared with SSS had a sweet flavor. It contained significantly higher contents of beta-phenylethyl alcohol, beta-phenylethyl acetate, and ethyl lactate with odor active value of >1. The liquor prepared without SSS had a cheese-like flavor. It was confirmed that the cheese-like flavor derived from butanoic acid was only detected in the liquor prepared without SSS. SSS facilitated the biosynthesis of beta-phenylethyl alcohol and ethyl lactate by supplying a large amount of phenylalanine and lactic acid at the initial stage of fermentation, and it prevented contamination. These results indicated that the SSS process contributed to produce the characteristic flavor compounds of rice-flavor baijiu. Practical Application Solid-state saccharification (SSS) process of rice-flavor baijiu contributes not only in brewing, but also in the production of the characteristic flavor compounds and the repression of the off-flavor derived from the contamination. Therefore, SSS is a critical process to control the flavor of rice-flavor baijiu.

DOI： 10.1111/1750-3841.15935

Chihiro Kadooka, Kazuki Mori, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Kosuke Tashiro, Hisanori Tamaki, Taiki Futagami . Chromosome-Level Genome Sequence of Aspergillus chevalieri M1, Isolated from Katsuobushi. . Microbiology resource announcements10 ( 37 September ) e0038521 2021.9International journal

Language：Japanese Publishing type：Research paper (scientific journal)

In this study, we report the chromosome-level genome sequence of the osmophilic filamentous fungus Aspergillus chevalieri M1, which was isolated from a dried bonito, katsuobushi. This fungus plays a significant role in the fermentation and ripening process. Thus, elucidating the sequence data for this fungus will aid in subsequent genomic research on the fungi involved in katsuobushi production.

DOI： 10.1128/MRA.00385-21

中光はるの, 王子泰, 奥津果優, 吉崎由美子, 二神泰基, 玉置尚徳, 丸山卓郎, 河野徳昭, 當銘一文, 小松かつ子, 高峯和則 . 漢方用薬「神麹」の製造時における植物添加の影響 . 和漢医薬学会学術大会要旨集38回 70 - 70 2021.9漢方用薬「神麹」の製造時における植物添加の影響

Kazuki Mori, Chihiro Kadooka, Atsushi Nishitani, Kayu Okutsu, Yumiko Yoshizaki, Kazunori Takamine, Kosuke Tashiro, Masatoshi Goto, Hisanori Tamaki, Taiki Futagami . Chromosome-Level Genome Sequence of the Black Koji Fungus Aspergillus luchuensis RIB2601. . Microbiology resource announcements10 ( 29 ) e0038421 2021.7International journal

蔵薗秀伍，吉﨑由美子，印璇，奥津果優，髙峯和則 . 青ヶ島で製造される焼酎 (青酎) の特徴について . 日本醸造協会誌116 ( 3 ) 182 - 194 2021.3青ヶ島で製造される焼酎 (青酎) の特徴について Reviewed

Zeng C. . Additional moisture during koji preparation contributes to the pigment production of red koji (Monascus-fermented rice) by influencing gene expression . Journal of Food Science86 ( 3 ) 969 - 976 2021.3

Zeng C. . The expression profiles of acid-stable α-amylase and acid-labile α-amylase of Aspergillus luchuensis mut. Kawachii effect on the microstructure of koji and alcohol fermentation . LWT139 2021.3

白石洋平, 竹浦　澪, 奥津果優, 吉﨑由美子, 二神泰基, 玉置尚徳, 和久豊, 髙峯和則 . 麴菌菌種の違いが芋焼酎の香味形成に及ぼす影響 (第1報) 酵素活性と芋焼酎醪の差異 . 日本醸造協会誌116 ( 1 ) 39 - 48 2021.1麴菌菌種の違いが芋焼酎の香味形成に及ぼす影響 (第1報) 酵素活性と芋焼酎醪の差異 Reviewed

白石洋平, 竹浦　澪, 奥津果優, 吉﨑由美子, 二神泰基, 玉置尚徳, 和久豊, 髙峯和則 . 麴菌菌種の違いが芋焼酎の香味形成に及ぼす影響 (第2報) 香気成分と官能評価での差異 . 日本醸造協会誌116 ( 1 ) 49 - 58 2021.1麴菌菌種の違いが芋焼酎の香味形成に及ぼす影響 (第2報) 香気成分と官能評価での差異 Reviewed

Nakamura E. . Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae . Journal of Bioscience and Bioengineering131 ( 1 ) 68 - 76 2021.1

Genqiao Liu, Juna Serikawa, Kayu Okutsu, Yumiko Yoshizaki, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . Impact of fermentation temperature on the quality and sensory characteristics of imo-shochu . Journal of the Institute of Brewing127 ( 4 ) 417 - 423 2021

KURAZONO Shugo, YOSHIZAKI Yumiko, YIN Xuan, OKUTSU Kayu, TAKAMINE Kazunori . Characteristics of shochu（aochu）made on Aogashima Island . JOURNAL OF THE BREWING SOCIETY OF JAPAN116 ( 3 ) 182 - 194 2021

Language：Japanese Publisher：Brewing Society of Japan

In this study, we examined the current production methods and the aroma characteristics of aochu, which is a kind of shochu made on Aogashima Island. It was confirmed from a field survey that there were eight aochu manufacturers. Five of them made koji by individually, while the rest jointly made koji. The preparation of moromi, distillation and bottling were done in one place. Some manufacturers inoculated the steamed materials with tane-koji and tomo-koji while others used the leaves of Asplenium antiquum Makino and utilized wild microorganisms. One manufacturer added cultured yeast to the moromi. The aroma characteristics of twelve samples of aochu and six samples of control-shochu produced in Kyushu were examined. The acidity, ultraviolet absorption, and thiobarbitur acid value in aochu were higher than those of the control-shochu. Consequently, 69 aroma components were determined in the GC-MS analysis. When compared with the control, ethyl isobutyrate, ethyl isovalerate, ethyl benzoate, isovaleraldehyde, 2-methylbutylaldehyde, isovaleraldehyde, pentanal, hexanal, heptanal, 2-octenal, benzaldehyde, 2-nonenal, hexanol, 1-octen-3-ol, caproic acid, caprylic acid, capric acid, furfural and DMTS were higher in aochu. Finally, the principal component analysis performed on the 69 quantified aroma components found that clusters were formed for each koji making method. Sensory evaluation revealed that the characteristic aromas of aochu were “grassy” and “a sour smell.”

DOI： 10.6013/jbrewsocjapan.116.182

SHIRAISHI Yohei, OKUTSU Kayu, YOSHIZAKI Yumiko, FUTAGAMI Taiki, TAMAKI Hisanori, WAGU Yutaka, TAKAMINE Kazunori . Effect of the koji variety of Aspergillus species on the flavor formation of imo-shochu（Part 2） Difference in volatile compounds and sensory evaluations of imo-shochu . JOURNAL OF THE BREWING SOCIETY OF JAPAN116 ( 1 ) 49 - 58 2021

Language：Japanese Publisher：Brewing Society of Japan

In order to clarify t he effects of differences in koji type on the variety of the aroma and taste of imo-shochu, we prepared imo-shochu with yellow, black, and white koji and investigated their aroma components and sensory characteristics. GC-MS analyses revealed that the concentrations of higher alcohols, acetic acid ethyl esters, and sulfur compounds were higher in imo-shochu prepared with yellow koji than those with black or white koji. The aldehyde and terpene contents in imo-shochu prepared with white or black koji were higher than that with yellow koji. Meanwhile, imo-shochu prepared with white koji contained more DL-2-methylbutyrate, and imo-shochu prepared with black koji contained more methyl salicylate and 1-octen-3-ol compared to the other shochu. Results of sensory evaluations, showed that imo-shochu prepared with yellow koji had stronger koji-like, baked confectionery-like, and herb-like flavors. Imo-shochu prepared with white koji was evaluated as roasted and sharp, and that with black koji evaluated as roasted, oily, and mild. Furthermore, it was first demonstrated that the shochu prepared with different types of koji could be distinguished by their sensory characteristics in blind tests. From the above results, the empirical fact that differences in the flavor of imo-shochu by the variety of koji was confirmed at the chemical level.

DOI： 10.6013/jbrewsocjapan.116.49

SHIRAISHI Yohei, TAKEURA Mio, OKUTSU Kayu, YOSHIZAKI Yumiko, FUTAGAMI Taiki, TAMAKI Hisanori, WAGU Yutaka, TAKAMINE Kazunori . Effect of the koji variety of Aspergillus species on the flavor formation of imo-shochu（Part 1）Differences in enzyme activity of koji and imo-shochu moromi . JOURNAL OF THE BREWING SOCIETY OF JAPAN116 ( 1 ) 39 - 48 2021

Language：Japanese Publisher：Brewing Society of Japan

To clarify the effects of the koji variety on the flavor of imo-shochu, we prepared imo-shochu with yellow, white, or black koji and investigated koji enzyme activities and chemical composition in moromi-mash. Yellow koji showed a higher α-amylase and acid carboxypeptidase activity, but lower koji acidity, β-glucosidase, cellulase, acid protease and lipase activities compared with white or black-koji. Acid-resistant α-amylase activity, which is involved in liquification of the moromi-mash under acidic conditions, was 10 times higher in black or white-koji than in yellow koji. Although the enzyme activities of white koji were similar to that of black koji, white koji had higher β-glucosidase and cellulase activities than black koji. Imo-shochu moromi prepared with white koji or black koji contained higher citric acid compared with yellow koji, and the pH of moromi was consistent with citric acid concentration. In addition, the viscosity of moromi was different among the koji types; the mash prepared with yellow koji was stickier than mash prepared with white or black-koji. This was likely caused by higher acid protease and cellulase activities of black or white-koji. Although amino acid compositions in moromi prepared with white and black koji showed similar tendencies, that with yellow koji had a different amino acid balance. Similarly, the organic acid composition in moromi differed among koji types.

DOI： 10.6013/jbrewsocjapan.116.39

Iwasaki F. . Effects of liming on the flavor of kokuto-shochu, a spirit made from non-centrifugal sugar . Journal of Bioscience and Bioengineering130 ( 4 ) 360 - 366 2020.10

Zitai Wang, Kayu Okutsu, Taiki Futagami, Yumiko Yoshizaki, Hisanori Tamaki, Takuro Maruyama, Kazufumi Toume, Katsuko Komatsu, Fumio Hashimoto, Kazunori Takamine . Microbial Community Structure and Chemical Constituents in Shinkiku, a Fermented Crude Drug Used in Kampo Medici . Frontier in Nutrition7 ( 7 ) 1 - 11 2020.7Microbial Community Structure and Chemical Constituents in Shinkiku, a Fermented Crude Drug Used in Kampo MediciReviewed

Xuan Yin, Yumiko Yoshizaki, Shugo Kurazono, Mina Sugimachi, Haruka Takeuchi, Xing-Lin Han, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Kazunori Takamine . The characterization of flavor compounds of rice-flavor baijiu, a traditional Chinese distilled liquor, compared with Japanese distilled liquor, awamori and kome-shochu . Food Science and Technology Research26 ( 3 ) 411 - 422 2020.7The characterization of flavor compounds of rice-flavor baijiu, a traditional Chinese distilled liquor, compared with Japanese distilled liquor, awamori and kome-shochuReviewed

Miyamoto A. . Sirtuin SirD is involved in α-amylase activity and citric acid production in Aspergillus luchuensis mut. kawachii during a solid-state fermentation process . Journal of Bioscience and Bioengineering129 ( 4 ) 454 - 466 2020.4

Miyamoto Aoi, Kadooka Chihiro, Mori Kazuki, Tagawa Yuki, Okutsu Kayu, Yoshizaki Yumiko, Takamine Kazunori, Goto Masatoshi, Tamaki Hisanori, Futagami Taiki . サーチュインSirDはAspergillus luchuensis mut. kawachiiの固相発酵過程におけるα-アミラーゼ活性とクエン酸産生に関与する(Sirtuin SirD is involved in α-amylase activity and citric acid production in Aspergillus luchuensis mut. kawachii during a solid-state fermentation process) . Journal of Bioscience and Bioengineering129 ( 4 ) 454 - 466 2020.4サーチュインSirDはAspergillus luchuensis mut. kawachiiの固相発酵過程におけるα-アミラーゼ活性とクエン酸産生に関与する(Sirtuin SirD is involved in α-amylase activity and citric acid production in Aspergillus luchuensis mut. kawachii during a solid-state fermentation process)

Publisher：(公社)日本生物工学会

白コウジカビAspergillus luchuensis mut. kawachiiの固相培養における、デンプン分解酵素とクエン酸産生の調節に対するサーチュインの役割を解析した。5種のサーチュイン遺伝子破壊株の性質解析により、これらの遺伝子がデンプン分解活性と酸性化に関与し、特にsirD破壊株では酸安定なα-アミラーゼ活性とクエン酸産生が低下した。sirD破壊株は菌糸の色が変化し、細胞壁合成阻害剤に高い感受性を示し、分生子形成が低下した。キャップ分析遺伝子発現と定量的RT-PCRを行ったところ、sirD破壊株において酸安定性α-アミラーゼ遺伝子とクエン酸エクスポーターの転写体レベルが低下していた。SirDはA.kawachiiの固相発酵において、酸安定性α-アミラーゼとクエン酸の産生を含む全体の転写調節に重要な役割を果たしていると考えられた。

Kadooka C. . Lae A controls citric acid production through regulation of the citrate exporter-encoding cex A Gene in Aspergillus luchuensis mut. kawachii . Applied and Environmental Microbiology86 ( 5 ) 1 - 17 2020.3

Yin X. . Manufactural impact of the solid-state saccharification process in rice-flavor baijiu production . Journal of Bioscience and Bioengineering129 ( 3 ) 315 - 321 2020.3

Yagi T. . Red rice koji extract alleviates hyperglycemia by increasing glucose uptake and glucose transporter type 4 levels in skeletal muscle in two diabetic mouse models . Food and Nutrition Research64 1 - 9 2020

Wang Z, Okutsu K, Futagami T, Yoshizaki Y, Tamaki H, Maruyama T, Toume K, Komatsu K, Hashimoto F, Takamine K . Microbial Community Structure and Chemical Constituents in Shinkiku, a Fermented Crude Drug Used in Kampo Medicine. . Frontiers in nutrition7 115 2020Microbial Community Structure and Chemical Constituents in Shinkiku, a Fermented Crude Drug Used in Kampo Medicine.

Yin Xuan, Takamine Kazunori, Yoshizaki Yumiko, Kurazono Shugo, Sugimachi Mina, Takeuchi Haruka, Han Xing-Lin, Okutsu Kayu, Futagami Taiki, Tamaki Hisanori . Characterization of Flavor Compounds in Rice-flavor baijiu, a Traditional Chinese Distilled Liquor, Compared with Japanese Distilled Liquors, awamori and kome-shochu . Food Science and Technology Research26 ( 3 ) 411 - 422 2020

Publisher：公益社団法人日本食品科学工学会

This study aimed to reveal the chemical and flavor profiles of rice-flavor baijiu by comparison with awamori and kome-shochu, traditional Japanese liquors. Rice-flavor baijiu is similar to awamori and kome-shochu with regard to ingredients and the fermentation starter. Of the 15 rice-flavor baijiu samples in this study, 3 had a light yellow to dark-brown color. Dark-brown samples had high glucose and low amino acid contents. Lactic acid was detected in all rice-flavor baijiu samples. Compared to awamori and kome-shochu, rice-flavor baijiu contained more acetic acid. Thirty-four volatile compounds in rice-flavor baijiu were identified and quantified. In all, 18 compounds in rice-flavor baijiu, 15 in awamori and 13 in kome-shochu had an odor activity value (OAV) of >1. Of these, 11 compounds showed a three-fold higher OAV in rice-flavor baijiu than in awamori and kome-shochu. Principal component analysis revealed that ethyl lactate is a key volatile compound that is distinct to rice-flavor baijiu.

DOI： 10.3136/fstr.26.411

Kadooka Chihiro, Nakamura Eri, Kubo Shingo, Okutsu Kayu, Yoshizaki Yumiko, Takamine Kazunori, Tamaki Hisanori, Futagami Taiki . Analysis of the fungal population involved in Katsuobushi production . The Journal of General and Applied Microbiology66 ( 4 ) 239 - 243 2020

Publisher：公益財団法人　応用微生物学・分子細胞生物学研究奨励会

Naturally occurring fungi have been used in the traditional production of dried bonito, Katsuobushi, in Japan. In this study, we analyzed the fungal population present during Katsuobushi production. Amplicon sequence analysis of ITS1 indicated that Aspergillus spp. are predominant throughout the production process. In addition, culture-dependent analyzes identified three species Aspergillus chevalieri, Aspergillus montevidensis, and Aspergillus sydowii, based on sequencing of benA, caM, and rpb2 genes. A. chevalieri isolates were classified into teleomorphic and anamorphic strains based on morphological analysis. A. chevarieri was the dominant species throughout the production process, whereas A. montevidensis increased and A. sydowii decreased in abundance during Katsuobushi production. Our study will enhance the understanding of fungal species involved in traditional Katsuobushi production.

DOI： 10.2323/jgam.2019.09.003

Kadooka C. . A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii . Bioscience, Biotechnology and Biochemistry84 ( 10 ) 1 - 5 2020

Kojo T, Kawai M, Shiraishi Y, Kurazono S, Kadooka C, Okutsu K, Yoshizaki Y, IKenaga M, Futagami T, Takamine K and Tamaki H, . Effect of Maturation Time on Koji-like Smell and Volatile Compounds of Barley Miso (Japanese Soybean Paste) during Fermentation . Food Sci. Technol. Res.25 313 - 319 2019.9Effect of Maturation Time on Koji-like Smell and Volatile Compounds of Barley Miso (Japanese Soybean Paste) during FermentationReviewed

Kadooka C. . Mitochondrial citrate transporters CtpA and YhmA are required for extracellular citric acid accumulation and contribute to cytosolic acetyl coenzyme A generation in Aspergillus luchuensis mut. Kawachii . Applied and Environmental Microbiology85 ( 8 ) 2019.4

Takamine K. . Sweet potato dietary fiber . Sweet Potato: Chemistry, Processing and Nutrition 117 - 148 2019.1

Takamine K. . Sweet potato fermentation food (sweet potato shochu) . Sweet Potato: Chemistry, Processing and Nutrition 325 - 347 2019.1

Kimoto D. . Pex16 is involved in peroxisome and Woronin body formation in the white koji fungus, Aspergillus luchuensis mut. kawachii . Journal of Bioscience and Bioengineering127 ( 1 ) 85 - 92 2019.1

Kojo Toshihiro, Takamine Kazunori, Tamaki Hisanori, Kawai Mikihiko, Shiraishi Yohei, Kurazono Shugo, Kadooka Chihiro, Okutsu Kayu, Yoshizaki Yumiko, Ikenaga Makoto, Futagami Taiki . Effect of Maturation Time on Koji-like Smell and Volatile Compounds of Barley Miso (Japanese Soybean Paste) during Fermentation . Food Science and Technology Research25 ( 2 ) 313 - 319 2019

Publisher：公益社団法人日本食品科学工学会

Barley miso (Japanese soybean paste) is generally classified into either the light-colored type or the red type based on a short or long maturation time, respectively. In this study, we characterized barley miso with a focus on its koji-like smell. Sensory evaluation showed that koji-like smell was significantly reduced during the maturation process, especially after two months. However, comprehensive analysis of volatile compounds indicated that known constituents of koji-like smell, 1-octen-3-ol and phenylacetaldehyde, were present in similar and lower concentrations, respectively, in the light-colored barley miso compared to the red barley miso. In addition, most compounds, such as aldehydes and esters, were found in larger concentrations in the red barley miso than in the light-colored barley miso. These results suggest that maturation time has an important role in koji-like smell, and the contributions of decreases in 1-octen-3-ol and phenylacetaldehyde to koji-like smell during the maturation process remains unclear.

DOI： 10.3136/fstr.25.313

白石洋平、原口愛美、村田梨恵、久遼馬、奥津果優、吉﨑由美子、二神泰基、玉置尚徳、和久豊、髙峯和則 . 異なる麹菌を用いた本格焼酎の酒質の多様化 . 日本醸造学会誌113 ( 12 ) 757 - 767 2018.12異なる麹菌を用いた本格焼酎の酒質の多様化Reviewed

吉﨑由美子, 奥津果優, 二神泰基, 玉置尚徳, 鮫島吉廣, 髙峯和則 . プロテアーゼまたは細胞壁分解酵素製剤添加による紅麹焼酎製造法の改良 . 日本醸造協会誌113 ( 4 ) 265 - 272 2018.4プロテアーゼまたは細胞壁分解酵素製剤添加による紅麹焼酎製造法の改良Reviewed

髙峯和則、小島舞、奥津果優、二神泰基、玉置尚徳、吉崎由美子 . 芋焼酎の発酵および酒質に及ぼす二次醪ｐHの影響 . 日本醸造協会誌 2018芋焼酎の発酵および酒質に及ぼす二次醪ｐHの影響Reviewed

Ferdouse J. . Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast . PeerJ2018 ( MAY ) e4768 2018

Futagami T. . Multi-gene phylogenetic analysis reveals that shochu-fermenting Saccharomyces cerevisiae strains form a distinct sub-clade of the Japanese sake cluster . Yeast34 ( 10 ) 407 - 415 2017.10

奥津果優，吉﨑由美子，髙峯和則 . 栽培期間がサツマイモの品質及び芋焼酎の酒質に及ぼす影響 . 日本醸造協会誌112 ( 8 ) 543 - 549 2017.8栽培期間がサツマイモの品質及び芋焼酎の酒質に及ぼす影響Reviewed

Rahayu Y. . Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentation . Food Chemistry224 398 - 406 2017.6

奥津果優, 門岡千尋, 小城章裕, 吉崎由美子, 二神泰基, 玉置尚徳, 高峯和則 . 中国及び韓国の市場品「神麹」における菌叢と含有成分の実態調査 . 生薬学雑誌71 ( 1 ) 41 - 48 2017.2中国及び韓国の市場品「神麹」における菌叢と含有成分の実態調査

Publisher：(一社)日本生薬学会

中国および韓国で入手した市場品の神麹15種をサンプルとし、菌叢、酵素活性(アミラーゼ、リパーゼ、プロテアーゼ)およびフェルラ酸含量について調査した。Denaturing gradient gel electrophoresis(DGGE)解析の結果、神麹には乳酸菌や枯草菌など複数の微生物が存在し、製品によって微生物の種類が大きく異なることが判明した。また、半数以上のサンプルでアミラーゼ活性およびリパーゼ活性を認め、全サンプルでフェルラ酸の含有を認めた。色調により暗褐色系、白色系、黄褐色系の3群に分類して主成分分析した結果、酵素活性が検出されなかったのは全て黄褐色群で、明度が低いほどフェルラ酸含量が高い傾向であった。以上より、神麹は、色調によって酵素活性およびフェルラ酸含量の予測が可能であると考えられた。

白石洋平, 安藤有加, 奥津果優, 吉﨑由美子, 二神泰基, 玉置尚徳, 和久豊, 髙峯和則 . 芋焼酎の香気形成に及ぼすアミノ酸の影響 . 日本醸造協会誌112 ( 8 ) 563 - 568 2017芋焼酎の香気形成に及ぼすアミノ酸の影響Reviewed

白石洋平, 安藤有加, 奥津果優, 吉﨑由美子, 二神泰基, 玉置尚徳, 和久豊, 髙峯和則 . 芋焼酎醪へのプロテアーゼ剤添加による揮発成分と官能評価への影響 . 日本醸造協会誌112 ( 7 ) 517 - 523 2017芋焼酎醪へのプロテアーゼ剤添加による揮発成分と官能評価への影響Reviewed

Yen Yen Sally Rahayu,· Yumiko Yoshizaki,· Keiko Yamaguchi, Kayu Okutsu, Taiki Futagami, Hisanori Tamaki, Yoshihiro Sameshima, Kazunori Takamine . Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentation . Food Chem224 398 - 406 2017Key volatile compounds in red koji-shochu, a Monascus-fermented product, and their formation steps during fermentationReviewed

奥津果優，門岡千尋，小城章裕，吉﨑由美子，二神泰基，玉置尚徳，髙峯和則 . 中国及び韓国の市場品「神麹」における菌叢と含有成分の実態調査 . 生薬学雑誌71 ( 1 ) 41 - 48 2017中国及び韓国の市場品「神麹」における菌叢と含有成分の実態調査Reviewed

Kojo Toshihiro, Iwashita Kazuhiro, Onoue Masahira, Okutsu Kayu, Yoshizaki Yumiko, Takamine Kazunori, Futagami Taiki, Mori Kazuki, Tamaki Hisanori, Kadooka Chihiro, Komohara Marisa, Onitsuka Shiori, Tanimura Miya, Muroi Yukiko, Kurazono Shugo, Shiraishi Yohei, Oda Ken . Characterization of amylolytic enzyme overproducing mutant of Aspergillus luchuensis obtained by ion beam mutagenesis . The Journal of General and Applied Microbiology63 ( 6 ) 339 - 346 2017

Publisher：公益財団法人　応用微生物学・分子細胞生物学研究奨励会

Aspergillus luchuensis is a kuro (black) koji fungus that has been used as a starch degrader for the awamori- and shochu-making industries in Japan. In this study, we investigated the effect of ion beam irradiation on A. luchuensis RIB2601 and obtained a high starch-degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas it showed higher α-amylase, glucoamylase, and α-glucosidase activities on a mycelial mass basis than the wild type (wt) strain both on agar plates and in rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and higher sensitivity to calcofluor white, suggesting a deficiency in cell wall composition. Interestingly, produced protein showed higher expression of acid-labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1, although real-time RT-PCR indicated no significant change in the transcription of the amyA or glaA gene. These results suggested that the high amylolytic activity of strain U1 is attributable to a high AmyA and GlaA production level, but the elevated production is not due to transcriptional regulation of the corresponding genes. Furthermore, RNA-seq analysis indicated that strain U1 shows transcriptional changes in at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall composition of strain U1.

DOI： 10.2323/jgam.2017.02.004