Genetic architecture of berry aroma compounds in a QTL (quantitative trait loci) mapping population of interspecific hybrid grapes (Vitis labruscana × Vitis vinifera).

BACKGROUND: Although grapes 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 Vitis labruscana 'Campbell Early' (CE) × Vitis 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.

Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei.

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.

Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae.

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.

Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei.

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.

The potential aroma and flavor compounds in Vitis sp. cv. Koshu and V. vinifera L. cv. Chardonnay under different environmental conditions.

BACKGROUND: 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.

Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter.

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.

Fungus-specific sirtuin HstD coordinates secondary metabolism and development through control of LaeA.

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 ΔhstD 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.

Yeast diversity during the spontaneous fermentation of wine with only the microbiota on grapes cultivated in Japan.

Making wine via spontaneous fermentation without sulfur dioxide and commercial yeast (spontaneous winemaking) is increasing in recent year, but there is scant research regarding microbial communities present in Japan during spontaneous winemaking using culture-independent molecular methods. We analyzed fungal communities and populations during laboratory-scale spontaneous winemaking using sterilized labware to avoid winery-resident microbes. In the spontaneous fermentation of four grape varieties (Pinot Noir, Riesling, Koshu, and Koshusanjaku) grown in the same Japanese vineyard, our analysis of yeast and other fungal species by next-generation sequencing based on the ITS1 region demonstrated that Saccharomyces cerevisiae was eventually dominant in seven of 12 fermentation batches (three replications for each grape variety), whereas non-Saccharomyces species (e.g., Schizosaccharomyces japonicus, Lachancea dasiensis, and Hanseniaspora valbyensis) became dominant in four batches at the end of fermentation. In another batch, lactic acid bacteria (LAB) became dominant and the fermentation remained incomplete. Diverse microbes were involved in the spontaneous fermentation (particularly in Koshusanjaku), indicating that residual sugar remained and lactic and acetic acid largely increased. Compared to the control wine made with SO2 and commercial yeast, the concentration of lactic acid was 47-fold higher in the must dominated by L. dasiensis, and the concentrations of acetic acid and lactic acid were 10-fold and 20-fold higher in the must dominated by LAB, respectively. Even when indigenous S. cerevisiae became dominant, the finished wines obtained high sensory-analysis scores for complexity but low scores for varietal typicality, indicating the risk of fermentation with unselected wild yeast on the grapes grown in Japan.

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

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 (12)C(5+) ion beams with an LET of 121keV/µm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/µm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/µm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) 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 400Gy. 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 (12)C(5+) 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 >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) 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.

Modeling the sake brewing characteristics of rice from brown rice metabolites.

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 using <1 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 analyzing <1 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.

Genome sequencing and analysis of Aspergillus oryzae.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.

1H-NMR metabolomics-based classification of Japanese sake and comparative metabolome analysis by gas chromatography-mass spectrometry.

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.

Variation in the mineral composition of wine produced using different winemaking techniques.

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.

Aspergillus oryzae atfA controls conidial germination and stress tolerance.

We compared atfA and atfB, the genes encoding the respective ATF/CREB-type transcription factors in Aspergillus oryzae. The germination ratio of DeltaatfA conidia was low without any stress, unlike that of DeltaatfB conidia. The DeltaatfA conidia were more sensitive to oxidative stress than the DeltaatfB conidia, which are also sensitive to oxidative stress. We compared the gene expressions of these strains by using a DNA microarray, GeneChip. Almost all the genes regulated by atfB were also regulated by atfA, but atfA also regulated many genes that were not regulated by atfB, including some genes putatively involved in oxidative stress resistance. The level of glutamate, the major amino acid in A. oryzae conidia, was significantly low only in the DeltaatfA conidia, and the glycerol accumulation during germination was not observed only in the DeltaatfA strain. We therefore concluded that atfA is involved in germination via carbon and nitrogen source metabolism.

Author Correction: BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

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

Investigation of relationship between sake-making parameters and sake metabolites using a newly developed sake metabolome analysis method.

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 (P < 0.005) 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.

Aspergillus oryzae atfB encodes a transcription factor required for stress tolerance in conidia.

Using an Aspergillus oryzae EST database, we identified a gene encoding a transcription factor (atfB), which is a member of the ATF/CREB family. Expression of atfB was barely detectable during vegetative growth, but was readily detected during conidiation in solid-state culture. Microarray analyses showed that expression of many other genes, including catalase (catA), were downregulated in an atfB-disruptant. The expression of most of these genes was upregulated in the wild-type strain during the conidiation phase in solid-state culture, and the expression pattern was similar to that of atfB itself. In the absence of stress, e.g. heat-shock or hydrogen peroxide, the conidial germination ratios for the DeltaatfB strain and the wild-type strain were similar, but the stress tolerance of conidia carrying the DeltaatfB deletion was less than that of the wild-type conidia. CRE-like DNA motifs, which are bound by ATF/CREB proteins, were found in the promoters of most of the downregulated genes in the DeltaatfB strain. Thus, atfB appears to encode a transcription factor required for stress tolerance in conidia.

Construction of sake yeast with low production of dimethyl trisulfide precursor by a self-cloning method.

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.

Discrimination of wine from grape cultivated in Japan, imported wine, and others by multi-elemental analysis.

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.

BiFC-based visualisation system reveals cell fusion morphology and heterokaryon incompatibility in the filamentous fungus Aspergillus oryzae.

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.