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Sake, soy sauce, miso (Japanese bean paste), and beer are made from grains. The characteristics of the grain significantly affect the quality of the final product. Many studies have been performed to evaluate the sake-brewing characteristics of rice. However, current rice analysis methods are time and labor intensive and require large samples. We developed a novel method for predicting the brewing characteristics of sake rice using1 g of sample. Brown rice extracts were analyzed by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry, and mass chromatogram data were used as explanatory variables. The objective variables were the physical and chemical properties of the rice, the enzymatic activity of the rice-koji, the fermentation properties of the sake mash, the standard analytical values of the sake, and the flavor component concentrations in the sake. Prediction models were developed using the orthogonal projections to latent structures method. The prediction performances of the models were verified, and 32 out of the 54 objective variables were used in well-performing models. In conclusion, we developed a method for predicting the rice properties and brewing characteristics from results acquired by analyzing1 g of brown rice. The method is a powerful tool for breeding new sake rice cultivars for good brewing characteristics in early generations and will improve our understanding of fluctuations in the brewing characteristics of sake rice before each sake brewing season starts.

Six categories of Japanese sake have been established by the National Tax Agency of Japan. In this system, the rice polishing ratio and the addition of alcohol are the main criteria for classification. The most common nuclear magnetic resonance (NMR) spectrometry method is 1H-NMR, and has higher throughput than gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) analysis due to its short measurement time, easy sample preparation, and high reproducibility. However, owing to the production of dominant ethanol signals, metabolome analyses have not been used for classifying Japanese sake using 1H-NMR. In this study, a technique to selectively suppress ethanol signals was used to classify Japanese sake by 1H-NMR, and a model was constructed to predict the rice polishing ratio. The results were compared to those obtained by GC–MS. The suppression of ethanol signals enabled the detection of trace components by 1H-NMR. In a principal component analysis (PCA) score plot of 1H-NMR spectra with ethanol signal suppression, PC1 was associated with both the addition of alcohol and the rice polishing ratio. Additionally, the separation of samples observed was similar when PCA score plots of 1H-NMR and GC–MS data were compared. Similarly, to predict the rice polishing ratio using partial least squares regression analysis, a model was constructed using 1H-NMR data, and showed nearly similar values for precision and predictive performance with the model constructed using GC–MS data. These results suggest that metabolomic analyses of Japanese sake based on 1H-NMR spectral patterns may be useful for classification.

Background Although grapes (Vitis labruscana × Vitis vinifera) accumulate diverse groups of volatile compounds, their genetic regulation in different cultivars remains unelucidated. Therefore, this study investigated the volatile composition in the berries of an interspecific hybrid population from a V. labruscana “Campbell Early (CE)” × V. vinifera “Muscat of Alexandria (MA)” cross to understand the relationship among volatile compounds and their genetic regulation. Then, a quantitative trait locus (QTL) analysis of its volatile compounds was conducted. Results While MA contained higher concentrations of monoterpenes and norisoprenoids, CE contained higher concentrations of C6 compounds, lactones and shikimic acid derivatives, including volatiles characteristic to American hybrids, i.e., methyl anthranilate, o-aminoacetophenone and mesifurane. Furthermore, a cluster analysis of volatile profiles in the hybrid population discovered ten coordinately modulated free and bound volatile clusters. QTL analysis identified a major QTL on linkage group (LG) 5 in the MA map for 14 monoterpene concentrations, consistent with a previously reported locus. Additionally, several QTLs detected in the CE map affected the concentrations of specific monoterpenes, such as linalool, citronellol and 1,8-cineol, modifying the monoterpene composition in the berries. As for the concentrations of five norisoprenoids, a major common QTL on LG2 was discovered first in this study. Several QTLs with minor effects were also discovered in various volatile groups, such as lactones, alcohols and shikimic acid derivatives. Conclusions An overview of the profiles of aroma compounds and their underlying QTLs in a population of interspecific hybrid grapes in which muscat flavor compounds and many other aroma compounds were mixed variously were elucidated. Coordinate modulation of the volatile clusters in the hybrid population suggested an independent mechanism for controlling the volatiles of each group. Accordingly, specific QTLs with significant effects were observed for terpenoids, norisoprenoids and some volatiles highly contained in CE berries.

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice-koji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku and Yamadanishiki. Recently, we reported the characteristic components/metabolites in sake made from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study, to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, we performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters. Finally, we demonstrated that some of the characteristic components/metabolites of sake from KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays an important role for establishment/maintenance of the quality of the final product.

In filamentous fungi, cell-to-cell recognition is a fundamental requirement for the formation, development, and maintenance of complex hyphal networks. Basically, self/compatible individuals within the fungal species are capable of fusing together, a step important for crossbreeding, which results in the formation of viable vegetative heterokaryons. Conversely, the fusion of incompatible individuals does not result in the formation of viable hyphal networks, but it often leads to growth inhibition or cell death. Even though a number of studies have been conducted to investigate such incompatibility, the understanding of the associated molecular mechanism is still limited, and this restricts the possibility of crossbreeding incompatible individuals. Therefore, in this study, the characteristics of compatibility/incompatibility in the industrial filamentous fungus, Aspergillus oryzae, were comprehensively investigated. Protoplast fusion and co-culture assays indicated the existence of a correlation between strain phylogeny and compatibility/incompatibility features. Time-course fluorescence observations were employed to investigate the types of incompatible responses that are induced at different cellular levels upon incompatible cell fusion, which eventually lead to cell death. Propidium iodide-indicated cell death, ROS accumulation, and mitochondrial fragmentation were identified as the major responses, with mitochondrial fragmentation showing the most significant subcellular change immediately after incompatible cell fusion. Furthermore, the deletions of mitochondrial fission-related genes Aofis1 and Aodnm1 in incompatible pairing alleviated cell death, indicating that mitochondrial fission is an important mechanism by which incompatibility-triggered cell death occurs. Therefore, this study provides new insights about heterokaryon incompatibility.IMPORTANCEFor a long time, it was believed that as an asexual fungus, A. oryzae does not exhibit any sexual cycle. However, the fungus has two mating types, indicating the potential for sexual reproduction besides a known parasexual cycle. Therefore, given that viable heterokaryon formation following cell fusion is an important step required for genetic crossing, we explored the mechanism of incompatibility, which restricts the possibility of cell fusion in A. oryzae. Protoplast fusion and co-culture assays led to the identification of various vegetative compatible groups. Mitochondrial fragmentation was found to be the most significant incompatible cellular response that occurred in organelles during incompatible pairing, while the deletion of mitochondrial fission-related genes was identified as a strategy used to alleviate incompatibility-triggered cell death. Thus, this study revealed a novel mechanism by which mitochondrial fission regulates incompatible responses.

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake-fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.

We developed a sake metabolome analysis method using liquid chromatography-quadrupole/time-of-flight mass spectrometry to investigate the metabolome of various types of sakes and other alcohol beverages. Our method identified 198 compounds by comparison with standard metabolites. Using this method, we investigated the relationship between several sake-making parameters and sake metabolites by conducting combination experiments of these parameters using small-scale fermentation. The results indicated that all parameters significantly affected sake metabolites (P0.005) and most peaks were affected by multiple sake-making parameters. Interestingly, the effect of the rice cultivar on sake metabolites was higher for koji rice than for kake-rice. This result suggests that the rice cultivar used has a greater effect on the characteristics of Aspergillus oryzae compared to sake yeast and affects sake metabolites. In this study, we also evaluated the combined effect of several parameters. We demonstrated the different effects of each parameter on several amino acids. The results showed a new aspect of the science of sake making. For example, the amount of α-ethylglucoside, which can affect the taste of sake, was negatively correlated with α-glucosidase activity in koji (r = -0.84). In this study, various unidentified peaks were observed; detectable peaks can be increased by analyzing additional standard reagents. Investigating these unidentified peaks and accumulating datasets for sake-making parameters will give us insight into how to improve sake taste and quality.

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice

Koshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is the most popular indigenous cultivar for wine production in Japan. However, little is known about the potential aroma compounds it contains and how environmental factors affect these. In this study, we obtained comprehensive profiles of the volatile (both glycosidically bound and free) and phenolic compounds that occur in koshu berries, and compared these with similar profiles for V. vinifera cv. chardonnay. We then compared the response of these two cultivars to bunch shading and the ripening-related phytohormone abscisic acid (ABA).; Results: Koshu berries contained significantly higher concentrations of phenolic compounds, such as hydroxycinnamic acid derivatives, and some volatile phenols, such as 4-vinyl guaiacol and eugenol, than chardonnay berries, which are thought to contribute to the characteristics of koshu wine. In addition, koshu berries had a distinctly different terpenoid composition from chardonnay berries. Shading reduced the concentration of norisoprenoid in both cultivars, as well as several phenolic compounds, particularly their volatile derivatives in koshu berries. The exogenous application of ABA induced ripening and increased the concentrations of lipid derivatives, such as hexanol, octanol, 1-nonanol, and 1-octen-3-ol. Multivariate and discriminant analyses showed that the potential aroma and flavor compounds in the berries could be discriminated clearly based on cultivar and environmental cues, such as light exposure.; Conclusion: The unique secondary metabolite profiles of koshu and their different responses to environmental factors could be valuable for developing various types of koshu wines and new cultivars with improved quality and cultural characteristics. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Differences in mineral concentrations were examined among three types of wine in the Japanese market place: Japan wine, imported wine, and domestically produced wine mainly from foreign ingredients (DWF), where Japan wine has been recently defined by the National Tax Agency as domestically produced wine from grapes cultivated in Japan. The main objective of this study was to examine the possibility of controlling the authenticity of Japan wine. The concentrations of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba, and Pb) in 214 wine samples were determined by inductively coupled-plasma mass spectrometry (ICP-MS) and ICP-atomic emission spectrometry (ICP-AES). In general, Japan wine had a higher concentration of potassium and lower concentrations of eight elements (Li, B, Na, Si, S, Co, Sr, and Pb) as compared with the other two groups of wine. Linear discriminant analysis (LDA) models based on concentrations of the 18 minerals facilitated the identification of three wine groups: Japan wine, imported wine, and DWF with a 91.1% classification score and 87.9% prediction score. In addition, an LDA model for discrimination of wine from four domestic geographic origins (Yamanashi, Nagano, Hokkaido, and Yamagata Prefectures) using 18 elements gave a classification score of 93.1% and a prediction score of 76.4%. In summary, we have shown that an LDA model based on mineral concentrations is useful for distinguishing Japan wine from other wine groups, and can contribute to classification of the four main domestic wine-producing regions of Japan.

Aspergillus oryzae is an industrially important filamentous fungus used for Japanese traditional food fermentation and heterologous protein production. Although cell fusion is important for heterokaryon formation and sexual/parasexual reproduction required for cross breeding, knowledge on cell fusion and heterokaryon incompatibility in A. oryzae is limited because of low cell fusion frequency. Therefore, we aimed to develop a BiFC system to specifically visualise fused cells and facilitate the analysis of cell fusion in A. oryzae. The cell fusion ability and morphology of 15 A. oryzae strains were investigated using heterodimerising proteins LZA and LZB fused with split green fluorescence protein. Morphological investigation of fused cells revealed that cell fusion occurred mainly as conidial anastomosis during the early growth stage. Self-fusion abilities were detected in most industrial A. oryzae strains, but only a few strain pairs showed non-self fusion. Protoplast fusion assay demonstrated that almost all the pairs capable of non-self fusion were capable of heterokaryon formation and vice versa, thus providing the first evidence of heterokaryon incompatibility in A. oryzae. The BiFC system developed in this study provides an effective method in studying morphology of fused cells and heterokaryon incompatibility in the filamentous fungal species with low cell fusion efficiency.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

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.

It has been reported that the concentrations of minerals in wines can be used to discriminate their geographical origin. However, some winemaking techniques may also affect the mineral concentration of the final product. In this study, we examined the effects of various winemaking techniques, including (i) fining, (ii) aging with oak tips, (iii) maceration with grape skins, (iv) chaptalization and acidification, and (v) yeast nutrient addition for alcohol fermentation, on the concentration of 18 minerals (Li, B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb) in a total of 154 wine samples using grapes from different production areas. Among the various winemaking techniques, maceration with grape skin had the largest effect on mineral content, significantly changing the concentrations of 17 or 18 elements (B, Na, Mg, Si, P, S, K, Ca, Mn, Co, Ni, Ga, Rb, Sr, Mo, Ba and Pb). Fining treatment using bentonites had the second largest effect, altering the concentrations of 14 elements (Li, B, Na, Si, P, S, Ca, Co, Ga, Rb, Sr, Mo, Ba and Pb). However, in principal component analysis using all data (n = 154), the wine samples were clearly clustered according to grapes used in the experiments rather than the winemaking technique. In conclusion, some winemaking techniques significantly altered the concentration of some minerals in wine; however, the principal factor influencing wine mineral composition seems to be its geographical origin.

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae. Inter-strain expression of sequence-diverse HET domain genes severely inhibited growth in an incompatible strain pair of Aspergillus oryzae, and SNPs responsible for the growth inhibition were identified.

BACKGROUNDKoshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is the most popular indigenous cultivar for wine production in Japan. However, little is known about the potential aroma compounds it contains and how environmental factors affect these. In this study, we obtained comprehensive profiles of the volatile (both glycosidically bound and free) and phenolic compounds that occur in Koshu berries, and compared these with similar profiles for V. vinifera cv. Chardonnay. We then compared the response of these two cultivars to bunch shading and the ripening-related phytohormone abscisic acid (ABA).RESULTSKoshu berries contained significantly higher concentrations of phenolic compounds, hydroxycinnamic acid derivatives, and some volatile phenols, such as 4-vinyl guaiacol and eugenol, than Chardonnay berries, which are thought to contribute to the characteristics of Koshu wine. In addition, Koshu berries had a distinctly different terpenoid composition from Chardonnay berries. Shading reduced the concentrations of norisoprenoid in both cultivars, as well as several phenolic compounds particularly their volatile derivatives in Koshu. The exogenous application of ABA induced ripening and increased the concentrations of lipid derivatives, such ashexanol, octanol, nonanol, and 1-octen-3-ol. Linear discriminant analysis showed that the aromatic potential could be discriminated clearly based on cultivar, bunch shading, and ABA application.CONCLUSIONThe unique secondary metabolite profiles of Koshu and their different responses to environmental factors could be valuable for developing various styles of Koshu wines and new cultivars with improved quality and cultural characteristics.

Koshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is the most popular indigenous cultivar for wine production in Japan. However, little is known about the potential aroma compounds it contains and how environmental factors affect these. In this study, we obtained comprehensive profiles of the volatile (both glycosidically bound and free) and phenolic compounds that occur in koshu berries, and compared these with similar profiles for V. vinifera cv. chardonnay. We then compared the response of these two cultivars to bunch shading and the ripening-related phytohormone abscisic acid (ABA).Koshu berries contained significantly higher concentrations of phenolic compounds, such as hydroxycinnamic acid derivatives, and some volatile phenols, such as 4-vinyl guaiacol and eugenol, than chardonnay berries, which are thought to contribute to the characteristics of koshu wine. In addition, koshu berries had a distinctly different terpenoid composition from chardonnay berries. Shading reduced the concentration of norisoprenoid in both cultivars, as well as several phenolic compounds, particularly their volatile derivatives in koshu berries. The exogenous application of ABA induced ripening and increased the concentrations of lipid derivatives, such as hexanol, octanol, 1-nonanol, and 1-octen-3-ol. Multivariate and discriminant analyses showed that the potential aroma and flavor compounds in the berries could be discriminated clearly based on cultivar and environmental cues, such as light exposure.The unique secondary metabolite profiles of koshu and their different responses to environmental factors could be valuable for developing various types of koshu wines and new cultivars with improved quality and cultural characteristics. © 2018 Society of Chemical Industry.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Dimethyl trisulfide (DMTS) is the primary component responsible for "hineka", the stale aroma of Japanese sake. Deletion of the MRI1 or MDE1 gene of sake yeast, encoding 5'-methylthioribose-1-phosphate isomerase and 5'-methylthioribulose-1-phosphate dehydratase, respectively, has been reported to greatly reduce the amount of DMTS precursor (DMTS-P1) in sake and to suppress the formation of DMTS during storage. In this study, we constructed sake yeast strains lacking MRI1 gene function by a self-cloning method. Two methods were applied: in one, a stop codon was introduced in the MRI1 ORF by point mutation; in the other, the entire MRI1 ORF was deleted from the genome. In both methods, a plasmid vector containing drug-resistance and counter-selectable markers was used to introduce the mutation. We successfully obtained the strains, which did not contain the plasmid sequences, by both methods. Small-scale sake brewing tests using these SC strains (strains obtained by the self-cloning method) found that DMTS-P1 was hardly detected in sake brewed with SC strains, and DMTS production after sake storage was greatly reduced as compared with the parent strain. The components of brewed sake were almost the same between the SC and parent strains. These results suggest that SC strains can produce sake with higher flavor stability without changing the sake brewing properties.

Numerous strains of Aspergillus oryzae are industrially used for Japanese traditional fermentation and for the production of enzymes and heterologous proteins. In A. oryzae, deletion of the ku70 or ligD genes involved in non-homologous end joining (NHEJ) has allowed high gene targeting efficiency. However, this strategy has been mainly applied under the genetic background of the A. oryzae wild strain RIB40, and it would be laborious to delete the NHEJ genes in many A. oryzae industrial strains, probably due to their low gene targeting efficiency. In the present study, we generated ligD mutants from the A. oryzae industrial strains by employing the CRISPR/Cas9 system, which we previously developed as a genome editing method. Uridine/uracil auxotrophic strains were generated by deletion of the pyrG gene, which was subsequently used as a selective marker. We examined the gene targeting efficiency with the ecdR gene, of which deletion was reported to induce sclerotia formation under the genetic background of the strain RIB40. As expected, the deletion efficiencies were high, around 60~80%, in the ligD mutants of industrial strains. Intriguingly, the effects of the ecdR deletion on sclerotia formation varied depending on the strains, and we found sclerotia-like structures under the background of the industrial strains, which have never been reported to form sclerotia. The present study demonstrates that introducing ligD mutation by genome editing is an effective method allowing high gene targeting efficiency in A. oryzae industrial strains.

Sake is a Japanese traditional alcoholic beverage, which is produced by simultaneous saccharification and alcohol fermentation of polished and steamed rice by Aspergillus oryzae and Saccharomyces cerevisiae. About 300 compounds have been identified in sake, and the contribution of individual components to the sake flavor has been examined at the same time. However, only a few compounds could explain the characteristics alone and most of the attributes still remain unclear. The purpose of this study was to examine the relationship between the component profile and the attributes of sake. Gas chromatography coupled with mass spectrometry (GC/MS)-based non-targeted analysis was employed to obtain the low molecular weight component profile of Japanese sake including both nonvolatile and volatile compounds. Sake attributes and overall quality were assessed by analytical descriptive sensory test and the prediction model of the sensory score from the component profile was constructed by means of orthogonal projections to latent structures (OPLS) regression analysis. Our results showed that 12 sake attributes [ginjo-ka (aroma of premium ginjo sake), grassy/aldehydic odor, sweet aroma/caramel/burnt odor, sulfury odor, sour taste, umami, bitter taste, body, amakara (dryness), aftertaste, pungent/smoothness and appearance] and overall quality were accurately explained by component profiles. In addition, we were able to select statistically significant components according to variable importance on projection (VIP). Our methodology clarified the correlation between sake attribute and 200 low molecular components and presented the importance of each component thus, providing new insights to the flavor study of sake.

In the eukaryotic cell, histone deacetylases (HDACs) play key roles in the regulation of fundamental cellular process such as development regulation, stress response, secondary metabolism and genome integrity. Here, we provide a comprehensive phenotypic analysis using HDAC disruptants in Aspergillus oryzae. Our study revealed that four HDACs, hdaA/Aohda1, hdaB/Aorpd3, hdaD/Aohos2 and hst4/AohstD were involved in stress response, cell wall synthesis and chromatin integrity in A. oryzae. Osmotic stress sensitivity of HDAC disruptants differed between plate cultures and liquid cultures, suggesting that HDACs adapt to the difference environmental conditions. Using a common A. oryzae fermentation medium, rice-koji, we also characterized HDACs related to growth and enzyme production to investigate which HDACs will be required for adaptation to environmental conditions and stress resistances. Because HDACs are widely conserved, our study has broad applications and may inform work with filamentous fungi and other eukaryote.

The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin.

The sirtuins are members of the NAD + -dependent histone deacetylase family that contribute to various cellular functions that affect aging, disease, and cancer development in metazoans. However, the physiological roles of the fungus-specific sirtuin family are still poorly understood. Here, we determined a novel function of the fungus-specific sirtuin HstD/ Aspergillus oryzae Hst4 (AoHst4), which is a homolog of Hst4 in A. oryzae yeast. The deletion of all histone deacetylases in A. oryzae demonstrated that the fungus-specific sirtuin HstD/AoHst4 is required for the coordination of fungal development and secondary metabolite production. We also show that the expression of the laeA gene, which is the most studied fungus-specific coordinator for the regulation of secondary metabolism and fungal development, was induced in a Δ hstD strain. Genetic interaction analysis of hstD / Aohst4 and laeA clearly indicated that HstD/AoHst4 works upstream of LaeA to coordinate secondary metabolism and fungal development. The hstD/Aohst4 and laeA genes are fungus specific but conserved in the vast family of filamentous fungi. Thus, we conclude that the fungus-specific sirtuin HstD/AoHst4 coordinates fungal development and secondary metabolism via the regulation of LaeA in filamentous fungi.

Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.

Grapes ( Vitis spp.) produce diverse polyphenolic compounds, which are phytochemicals that contribute to human health. In this study, the polyphenolic profiles of the red–purple berries of two wild grape species native to Japan, Vitis ficifolia and V. coignetiae , and their interspecific hybrid cultivars were investigated and compared with the profiles of V. vinifera and V. × labruscana cultivars. Proanthocyanidins (PAs) were present at lower concentrations in both skins and seeds of wild grape species and their hybrid cultivars than those in V. vinifera cultivars. They also differed in their composition, consisting mainly of epicatechin in wild grape species, but containing considerable amounts of both epigallocatechin in the skins and epicatechin gallate in the seeds of V. vinifera . In contrast, V. ficifolia varieties and their hybrid cultivars accumulated high concentrations of diverse anthocyanins, and whose compositions of anthocyanins and flavonols differed between species in their degree of modification by glucosylation, acylation, methylation and B-ring hydroxylation. Principal component analysis (PCA) indicated that the polyphenolic constituents clearly separate V. vinifera and V. × labruscana cultivars from the wild grape species as well as between wild grape species, V. coignetiae and V. ficifolia . Intermediate compositions were also observed in the hybrid cultivars between these wild grape species and V. vinifera .

Martensitic transformation is the phase transformation accompanying orderly shear deformation without atomic diffusion. The structures made by martensitic transformation are classified as thin plate, lens or lath in steels. The mechanism by which the hierarchic microstructure in the lath martensite phase forms has heretofore not been understood. We have made clear the mechanism by considering, independently, two plastic deformations using the slip deformation model proposed by Khachaturyan, and present herein a deformation matrix for each of the six crystallographic variants in a packet of the hierarchic structure. Our results are quantitatively consistent with experimental results for the Kurdjumov–Sachs (K-S) crystal orientation relationship and habit plane. Furthermore, the important points of our study are as follows: the origin of the sub-block structure and the specific combination of the sub-block structure are clarified; the laths existing in a block can be explained; and deviations between the di...

The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.

Five bitter-tasting peptides were isolated from charcoal-untreated sake, following a Sepabeads resin separation, an initial reverse-phase chromatography (RP-HPLC), a gel permeation-chromatography, and a second RP-HPLC. The isolated peptides consisted of six to thirteen amino acid residues. The N-termini were uniformly pyroglutamate residues. Based on the rice protein database, the peptides were derived from two different N-termini of the rice glutelin acidic subunit. One of them was reported as a prolyl endopeptidase inhibitor. The thirteen amino acid peptides in charcoal-untreated ginjyo-type sakes were lower than that in charcoal-untreated jyunmai-type sakes. The thirteen amino acid peptides were not detected in the commercial ordinary-type sake analyzed. The concentration of analyzed peptides of nine to thirteen amino acid residues in charcoal-untreated sake exceeded their preliminary estimated sensory threshold values, suggesting that they contribute to the sensory quality of charcoal-untreated sake.

The separation of insoluble solids from wastewater from shochu distillation is very difficult because of their high viscosity. Thus a large amount of shochu waste has been dumped into the ocean. The purpose of this study is to establish a new separation technique for shochu waste. Filamentous fungus was inoculated to shochu waste at 1×106 cells/ml and the culture solution was incubated for several days. After that, the culture solution was filtered on a filter paper for 15 minutes using an aspirator. Accordingly the shochu waste was condensed to about 6 to 10 times. We consider that the elongated filaments of fungi should grasp and aggregate the solid particles in shochu waste, and this is expected to be the major accelerated separation mechanism in this study. Aspergillus oryzae RIB128 showed the highest separation efficiency among in the tested strains. With the treatment of shochu waste with filamentous fungi, the pH of shochu waste was changed from 3.9 to 7 by the decrease of organic acid. Organic carbon, nitrogen, and phosphorus decreased to about 70%, 60%. and 90%, respectively.

Filamentous fungi have been widely exploited for the homologous and heterologous protein production, because of the high capacity of their protein secretion machinery. However, the production of heterologous proteins is often limited while the production of homologous proteins can be very high. Various researches have reported the methods for overcoming this problem and some techniques, such as the fusion gene system, improve the production of heterologous proteins. Recently, the molecular biological study of solid-state culture attracts the attention, because the long history of biological studies has shown that the productivity of protein in the solid-state culture frequently exceeds the productivity of protein in the submerged culture. The recent researches of solid-state culture have revealed the new aspects of protein production in filamentous fungi. Solid-state specific gene expression was observed in the glaB and pepA genes of Aspergillus oryzae. A GC-box and HSE element of the glaB promoter region affected solid-state specific gene expression of this gene. Solid-state culture-specific release of enzymes from the cell wall was also observed in the production of beta-glucosidases in Aspergillus kawachii. Extracellular soluble polysaccharide (ESP) from A. kawachii was concerned with the location of beta-glucosidases. Moreover, ESP and the cell wall fraction of A. kawachii were shown to be involved in the stability of beta-glucosidases. The knowledge of the molecular biology of solid-state culture should provide new approaches for the production of both homologous and heterologous proteins in both submerged culture and solid-state culture of filamentous fungi.