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

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