2012 Research results

1. Adenosine kinase-deficient mutant of Saccharomyces cerevisiae accumulates S-adenosylmethionine because of an enhanced methionine biosynthesis pathway

Author: Muneyoshi Kanai, Mitsunori Masuda, Yasumichi Takaoka, Hiroko Ikeda, Kazuo Masaki, Tsutomu Fujii and Haruyuki Iefuji
Abstract: To isolate an S-adenosylmethionine (SAM)-accumulating yeast strain and to develop a more efficient method of producing SAM, we screened methionine-resistant strains using the yeast deletion library of budding yeast and isolated 123 strains. The SAM content in 81 of the 123 strains was higher than that in the parental strain BY4742. We identified ADO1 encoding adenosine kinase as one of the factors participating in high SAM accumulation. The X_△ado1 strain that was constructed from the X2180-1A strain (MAT a, ATCC 26786) could accumulate approximately 30-fold (18 mg/g dry cell weight) more SAM than the X2180-1A strain in yeast extract peptone dextrose medium. Furthermore, we attempted to identify the molecular basis underlying the differences in SAM accumulation betweenX_△ado1 and X2180-1A strains. DNA microarray analysis revealed that the genes involved in the methionine biosynthesis pathway, phosphate metabolism, and hexose transport were mainly overexpressed in theX_△ado1strain compared with the X2180-1A strain. We also determined the levels of various metabolites involved in the methionine biosynthesis pathway and found increased content of SAM, tetrahydrofolate (THF), inorganic phosphate, polyphosphoric acid, and S-adenosylhomocysteine in theX_△ado1 strain. In contrast, the content of 5-methyl-THF, homocysteine, glutathione, and adenosine was decreased. These results indicated that the △ado1 strain could accumulate SAM because of preferential activation of the methionine biosynthesis pathway.
Source: Applied Microbiology and Biotechnology, 97, 1183-1190 (2013)

2. Quantitative Analysis of Total Purine Content Using the HPLC-UV Method in Beer, Low-Malt Beer, and Third-Category Beer

Author

T. Hashimoto, T. Ishihara, Y. Kakudo, K. Kaneko, T. Kenjo, Y. Masumura, M. Nakahara, W. Nakamura, S. Oshima, M. Takahashi, T. Yamamoto

Abstract

Relative repeatability standard deviation (RSDr) and repeat-ability limit (r95) for determination of total purine content using the HPLC-UV method ranged from 2.2 to 8.5% and from 2.9 to 20.6 mg/L, respectively, and were judged acceptable.
Relative reproducibility standard deviation (RSDR) and repro-ducibility limit (R95) for determination of total purine content using the HPLC-UV method ranged from 15.6 to 31.4 % and from 9.1 to 131.2 mg/L, respectively, and were judged unacceptable.

Source

Journal of American Society of Brewing Chemists, 70, 328-329 (2012)

3. The transfer of stable ¹³³Cs from rice to Japanese sake

Author: Okuda, M., Joyo, M., Tokuoka, M, Hashiguchi, T., Goto-Yamamoto, N., Yamaoka, H. and Shimoi, H.
Abstract: Possible contamination by radioactive cesium (Cs), released by the Fukushima Daiichi Nuclear Plant Accident in Japan on March 2011, has been a matter of concern with respect to Japanese sake made from rice grains cultivated in affected fields. In this study, the behavior of stable ¹³³Cs, which is a useful analogue for predicting the behavior of radioactive Cs, was investigated in the production of sake using rice grains harvested in Japan in 2010. The concentration of stable ¹³³Cs in the polished rice grains decreased gradually with decreasing milling ratios until a ratio of 70% was reached, and below that point, it did not change significantly. The ¹³³Cs concentration in the 70% polished rice was approximately 20% of that found in brown rice. The sake was brewed on a small scale using 70% polished rice, and the transfer of 133Cs from rice to sake was examined. Approximately 30-40% of ¹³³Cs in the 70% polished rice was removed during the washing and the steeping of the rice grains, and approximately 40% of the ¹³³Cs in the 70% polished rice was transferred to the sake. If the radioactive Cs species behaves similarly, these results suggest that brown rice containing 100 Bq/kg radioactivity of Cs would generate 70% polished rice grains containing 20 Bq/kg and that the sake brewed from these grains would contain 3-5 Bq/kg.
Source: Journal of Bioscience and Bioengineering, 114, 600-605 (2012)

4. Lethal and mutagenic effects of ion beams and gamma-rays in Aspergillus oryzae

Author: Yoshiyuki Toyoshima,, Akemi Takahashi, Hisaki Tanaka, Jun Watanabe, Yoshinobu Mogi, Tatsuo Yamazaki, Ryoko Hamada, Kazuhiro Iwashita, Katsuya Satoh, Issay Narumi
Abstract: Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was ¹²C⁵⁺ ion beams with an LET of 121 keV/μm. The ¹²C⁵⁺ ion beams had a 3.6-times higher lethal effect than low-LET (0.2 keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. ¹²C⁶⁺ ion beams with an LET of 86 keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to ¹²C⁶⁺ ion beams increased with an increase in dose and reached 3.47 × 10^-3 at 700 Gy. In the dose range from 0-700 Gy, ¹²C⁵⁺ ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67 × 10^-3) at 400 Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between ¹²C⁵⁺ ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions > 2 bp was generally low, deletions > 20 bp were characteristic for ¹²C⁵⁺ ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation.
Source: Mutation Research, 740, 43-49 (2012)

5. Analysis of Sake Component Presented to Sake Contests in 2011

Author: Katsumi MATSUMARU, Atsuko ISOGAI, Akiko FUJITA, Shigetoshi SUDO, Yasuzo KIZAKI
Source: Report of the Research Institute of Brewing, 184, 1-15 (2012)

6. Analysis of Traditional Shochu Presented to the 34th Contest in 2011

Author: Hisashi FUKUDA, Osamu MIZUTANI, Muneyoshi KANAI, Masayuki TAKAHASHI, Yasuzou KIZAKI
Source: Report of the Research Institute of Brewing, 184, 16-25 (2012)

7. Concentration of Ethyl Carbamate in Stone fruit Spirits and Liqueurs Sold in Japan

Author: Tomokazu HASHIGUCHI, Hanae IZU, Katsumi MATSUMARU
Source: Report of the Research Institute of Brewing, 184, 26-28 (2012)

8. Stable Isotope Analysis of Commercially Supplied Honkaku Shochu

Author: Hanae IZU, Tomokazu HASHIGUCHI, Sachie HORII, Shigetoshi SUDO, Katsumi MATSUMARU
Abstract: In this study, we examined the carbon stable isotope ratio (δ¹³C) of alcohol and oxygen stable isotope ratio (δ¹⁸O) of water in commercially-supplied Honkaku Shochu (Mugi Shochu, Imo Shochu, Kome Shochu, Soba Shochu, Kokuto Shochu, Sakekasu Shochu, Awamori) to confirm them as a potential index for verifying the geographical and raw material origins. Small-scale Kome Shochu brewing experiment revealed that the material water affects the δ¹⁸O value of water in it at around 25% alcohol. The δ¹⁸O values of water in Honkaku Shochu are correlated with the latitude (R²=0.67) regardless of the kind of raw materials of that. The δ¹³C values of alcohol in Kokuto Shochu (-17.6 ± 0.8‰) made from brown sugar are higher than those in other Honkaku Shochu made from rice, barley, sweet potato, buckwheat, and Sake lees. Mugi Shochu made from domestic barley (-26.2 ± 0.8‰) is characterized by lower δ¹³C of alcohol than that has no indication of geographical origin of barley (-24.8 ± 1.4‰). Imo Shochu made from Imo Koji (-28.4 ± 0.2‰) is characterized by lower δ¹³C of alcohol than that from Kome Koji (-27.8 ± 0.4‰). These results suggest that the δ¹³C values of alcohol and δ18O values of water in Honkaku Shochu are partially useful for the confirmation of the geographical and raw material origins.
Source: BUNSEKI KAGAKU, 61, 643-647 (2012)

9. Taste-Guided Fraction and Instrumental Analysis of Hydrophobic Compounds in sake

Author: Hashizume, K., Ito, T., Shimonohashi, M., Kokita, A., Tokiwano, T., Okuda, M.
Abstract: Taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA) method. All of the high TD factor fractions showed a bitter or astringent taste in common, but their taste character were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionations, using RP-HPLC and instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of peptide of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl esters of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably in the compounds of high hydrophobicity.
Source: Bioscience, Biotechnology, and Biochemistry., 76, 1291-1295 (2012)

10. Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

Author: Kazuhiko Imamura, Yoshihito Tsuyama, Terukage Hirata, Sumihiro Shiraishi, Kazutoshi Sakamoto, Osamu Yamada, Osamu Akita, Hitoshi Shimoi
Abstract: WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.
Source: Applied Environmental Microbiology, 78, 6996-7002 (2012)

11. The influence of vanadium on the components of hineka

Author: Atsuko ISOGAI, Ryoko KANDA, Hiroshi IWATA, Shigetoshi SUDO
Abstract: We investigated the suppression effect of the activated carbons and filtering aids on the formation of dimethyl trisulfide (DMTS), one of the main components of hineka, during the storage of sake. Although the activated carbons did not have this effect, two kinds of filtering aids, celite®, greatly reduced the DMTS producing potential. We searched for the active component in the celite and found that vanadium suppressed the formation of DMTS. Meanwhile, the addition of vanadium to sake increased the formation of aldehydes during storage. As a result of sensory evaluation, vanadium suppressed the formation of the sulfury aroma but increased the caramel/sweet aroma. The application of vanadium for the suppression of all components of hineka may be difficult; however, in view of aged sake, it might be promising as it accelerated the maturation of aroma without increasing the sulfury aroma.
Source: J. Brew. Soc. Japan, 107, 443-450 (2012)

12. Light quality affects flavonoid biosynthesis in young berries of Cabernet Sauvignon grape

Author: Kazuya Koyama, Hiroko Ikeda, Puspa Raj Poudel, Nami Goto-Yamamoto
Abstract: Biosynthesis of phenolic compounds is known to be sensitive to light environments, which reflects the possible role of these compounds for photoprotection in plants. Herein, the effects of UV and visible light on biosynthesis of flavonoids was investigated, i.e., proanthocyanidins (PAs) and flavonols, in young berry skins of a red-wine grape, Vitis vinifera cv. Cabernet Sauvignon. Shading with light-proof boxes from the flowering stage until 49 days after treatment (DAT) partially decreased PA concentrations, and completely decreased flavonol concentrations in the berry skins. Shading decreased the transcript abundance of a flavonol-related genes more remarkably than those of PA-related genes. In addition, light exclusion influenced the composition of PAs, such as the decrease in the proportion of trihydroxylated subunits and the mean degree of polymerization (mDP) within PAs. However, solar UV exclusion did not affect the concentration and composition of PAs, whereas this exclusion remarkably decreased the flavonol concentration. Consistently, UV exclusion did not influence the transcript levels of PA-related genes, whereas it dramatically decreased that of flavonol-related genes. These findings indicated a different light regulation of the biosynthesis of these flavonoids in young berry skins of wine grape. Visible light primarily induces biosynthesis of PAs and affects their composition, whereas UV light specifically induces biosynthesis of flavonols. Distinct roles of members of a MYB transcription factor family for light regulation of flavonoid biosynthesis were proposed.
Source: Phytochemistry, 78, 54-64 (2012)

13. A loss-of-function mutation in the PAS kinase Rim15p is related to defective quiescence entry and high fermentation rates in Saccharomyces cerevisiae sake yeast strains

Author: Daisuke Watanabe, Yuya Araki, Yan Zhou, Naoki Maeya, Takeshi Akao, and Hitoshi Shimoi
Abstract: Sake yeast cells have defective entry into the quiescent state, allowing them to sustain high fermentation rates. To reveal the underlying mechanism, we investigated the PAS kinase Rim15p, which orchestrates initiation of the quiescence program in Saccharomyces cerevisiae. We found that Rim15p is truncated at the carboxyl terminus in modern sake yeast strains as a result of a frameshift mutation. Introduction of this mutation or deletion of the full-length RIM15 gene in a laboratory strain led to a defective stress response, decreased synthesis of the storage carbohydrates trehalose and glycogen, and impaired G1 arrest, which together closely resemble the characteristic phenotypes of sake yeast. Notably, expression of a functional RIM15 gene in a modern sake strain suppressed all of these phenotypes, demonstrating that dysfunction of Rim15p prevents sake yeast cells from entering quiescence. Moreover, loss of Rim15p or its downstream targets Igo1p and Igo2p remarkably improved the fermentation rate in a laboratory strain. This finding verified that Rim15p-mediated entry into quiescence plays pivotal roles in the inhibition of ethanol fermentation. Taken together, our results suggest that the loss-of-function mutation in the RIM15 gene may be the key genetic determinant of the increased ethanol production rates in modern sake yeast strains.
Source: Applied and Environmental Microbiology, 78, 4008-4016 (2012)

14. Lack of endoplasmic reticulum 1,2-α-mannosidase activity that trims N-glycan Man₉GlcNAc₂ to Man₈GlcNAc₂ isomer B in a manE gene disruptant of Aspergillus oryzae

Author: Takeshi Akao, Akinori Yahara, Kazutoshi Sakamoto, Osamu Yamada, Osamu Akita and Takashi Yoshida
Abstract: The gene manE, encoding a probable class I endoplasmic reticulum 1,2-α-mannosidases (ER-Man), was identified from the filamentous fungus Aspergillus oryzae due to similarity to orthologs. It removes a single mannose residue from Man₉GlcNAc₂, generating Man₈GlcNAc₂ isomer B. Disruption of manE caused drastic decreases in ER-Man activity in A. oryzae microsomes.
Source: Journal of Bioscience and Bioengineering, 113, 438-441 (2012)

15. Modified Cre-loxP Recombination in Aspergillus oryzae by Direct Introduction of Cre Recombinase for Marker Gene Rescue

Author: Osamu Mizutani, Kazuo Masaki, Katsuya Gomi and Haruyuki Iefuji
Abstract: Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.
Source: Applied and Environmental Microbiology, 78, 4126-4133 (2012)

16. Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production

Author: Yu Sasano, Daisuke Watanabe, Ken Ukibe, Tomomi Inai, Hitoshi Shimoi, Hiroshi Takagi
Abstract: Lignocellulosic biomass is a promising source for bioethanol production, because it is abundant worldwide and has few competing uses. However, the treatment of lignocelllulosic biomass with weak acid to release cellulose and hemicellulose generates many kinds of byproducts including furfural and 5-hydroxymethylfurfural, which inhibit fermentation by yeast, because they generate reactive oxygen species (ROS) in cells. In order to acquire high tolerance to oxidative stress in bioethanol yeast strains, we focused on the transcription activator Msn2 of Saccharomyces cerevisiae, which regulates numerous genes involved in antioxidative stress responses, and constructed bioethanol yeast strains that overexpress Msn2 constitutively. The Msn2-overexpressing bioethanol strains showed tolerance to oxidative stress, probably due to the high-level expression of various antioxidant enzyme genes. Unexpectedly, these strains showed ethanol sensitivity compared with the control strain, probably due to imbalance of the expression level between Msn2 and Msn4. In the presence of furfural, the engineered strains exhibited reduced intracellular ROS levels, and showed rapid growth compared with the control strain. The fermentation test in the presence of furfural revealed that the Msn2-overexpressing strains showed improvement of the initial rate of fermentation. Our results indicate that overexpression of the transcription activator Msn2 in bioethanol yeast strains confers furfural tolerance by reducing the intracellular ROS levels and enhances the initial rate of fermentation in the presence of furfural, suggesting that these strains are capable of adapting rapidly to various compounds that inhibit fermentation by inducing ROS accumulation. Our results not only promise to improve bioethanol production from lignocellulosic biomass, but also provide novel insights for molecular breeding of industrial yeast strains
Source: Journal of Bioscience and Bioengineering, 113, 451-455 (2012)