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

In shochu-making, a small amount of fermenting moromi is added to a koji/water mixture instead of yeast culture to initiate fermentation. This is a characteristic process called Sashi-moto. It is known that shochu yeast is replaced by wild yeast upon repetition of Sashi-moto. The shochu yeast strains Kagoshima No. 2 (K2), Kagoshima No. 4 (C4), and Kagoshima No. 5 (H5), but not Kagoshima No. 6 (A6), were replaced by wild yeast (strain No. S5-g). K2 and C4 were easily replaced compared to H5, and the specific growth rates of K2 and C4 were lower than that of S5-g under higher osmotic pressure. Although the specific growth rate of H5 was higher than that of S5-g, its yeast population at the stationary phase was smaller than S5-g. On the other hand, both the specific growth rate and yeast population of A6 were higher than those of S5-g. The specific growth rate of yeast would be affected by osmotic tolerance and specific characters of the yeast strain.

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

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.

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

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

Aspergillus luchuensis is used for the production of awamori and shochu, which are traditional Japanese distilled alcoholic beverages. Here, we determined the chromosome-level genome sequence of A. luchuensis RIB2601.

RNA-binding protein Nrd1 plays a role in RNA polymerase II transcription termination. In this study, we showed that the orthologous NrdA is important in global mRNA expression and secondary metabolism inAspergillusspecies. We constructed annrdAconditional expression strain using the Tet-On system inAspergillus luchuenesismut.kawachii. Downregulation ofnrdAcaused a severe growth defect, indicating that NrdA is essential for the proliferation ofA. kawachii. Parallel RNA-sequencing and RNA immunoprecipitation-sequencing analysis identified potential NrdA-interacting transcripts, corresponding to 32% of the predicted protein coding genes ofA. kawachii. Subsequent gene ontology analysis suggested that overexpression of NrdA affects the production of secondary metabolites. To clarify this, we constructed NrdA-overexpressing strains ofAspergillus nidulans,Aspergillus fumigatus, andAspergillus oryzae. Overexpression of NrdA reduced the production of sterigmatocystin and penicillin inA. nidulans, as well as that of helvolic acid and pyripyropene A inA. fumigatus. Moreover, it increased the production of kojic acid and reduced production of penicillin inA. oryzae. These effects were accompanied by almost consistent transcriptional changes in the relevant genes. Collectively, these results suggest that NrdA is the essential RNA-binding protein, which plays a significant role in global gene expression and secondary metabolism inAspergillusspecies.IMPORTANCENrd1, a component of the Nrd1–Nab3–Sen1 complex, is an essential RNA-binding protein involved in transcriptional termination in yeast. However, its role in filamentous fungi has not been studied. In this study, we characterized an orthologous NrdA in theAspergillusspecies, identified potential NrdA-interacting mRNA, and investigated the effect of overexpression of NrdA on mRNA expression inAspergillus luchuensismut.kawachii. The results indicated that NrdA controls global gene expression involved in versatile metabolic pathways, including the secondary metabolic process. We demonstrated that NrdA overexpression significantly affected the production of secondary metabolites inAspergillus nidulans,Aspergillus oryzae, andAspergillus fumigatus. Our findings are of importance to the fungal research community because the secondary metabolism is an industrially and clinically important aspect for theAspergillusspecies.

Heat shock protein 70 (HSP70) is induced by various stresses. Since HSP70 has a protein refolding activity and an anti-inflammatory activity, the HSP70 induction will help cells from harmful acute stresses. Feeding a diet containing concentrated brewed rice vinegar Kurozu (CK) diet for 5 wk resulted in an increase of HSP70 in the brains of mice. In the present study, we evaluated whether oral feeding of 25 μL CK induces HSP70 mRNA in brain and other tissues. HSP70 mRNA was significantly increased in the esophagus, small intestine, liver, and brown adipose tissue within 1 h after the oral administration of CK. A weaker induction of HSP70 mRNA was demonstrated in the stomach, large intestine, and brain. HSP70 mRNA induction returned to basal levels within 3 h after feeding. We doubted that the induction of HSP70 mRNA was caused by manual restraint of the mice during CK administration. Manual restraint of the mice did not influence HSP70 mRNA expression in intestine 1 h after these treatments. Our results suggest that transient HSP70 mRNA induction by oral feeding of CK was not caused by retention stress. There are some compounds in CK that increase HSP70 mRNA in various tissues.

Background Red rice koji (RRK), prepared by growing Monascus species on steamed rice, has been reported to lower blood glucose levels in diabetic animal models. However, the action mechanism is not yet completely understood. Objective The objective of this study was to examine the mechanism underlying the hypoglycemic action of RRK extract in two diabetic animal models: the insulin-deficiency mice, where the insulin deficiency was induced by streptozotocin (STZ), and insulin-resistance mice, where the insulin resistance was induced by a high-fat diet (HFD). Design Low (12.5 mg/kg body weight [BW]) and high (50.0 mg/kg BW) doses of RRK extract were orally administered to the mice for 10 successive days (0.25 mL/day/mouse). The protein expression levels of glucose transporter type 4 (GLUT4) in the skeletal muscle and glucose transporter type 2 (GLUT2) in the liver were measured. Blood glucose (BG) levels of STZ-treated mice in insulin tolerance test (ITT) and BG and insulin levels of HFD-fed mice in intraperitoneal glucose tolerance test (IPGTT) were investigated. Results In the STZ-treated mice, oral administration of RRK extract lowered BG levels and food intake but increased plasma 1,5-anhydroglucitol level. Moreover, the RRK extract lowered the BG levels of STZ-treated mice as measured by ITT. In the HFD-fed mice, we confirmed that the orally administered RRK extract lowered the BG and the homeostasis model assessment index for insulin resistance. Furthermore, the RRK extract lowered the BG and insulin levels of HFD-fed mice in IPGTT. Regarding the protein levels of GLUT, the orally administered RRK extract increased the GLUT4 level in the skeletal muscle; however, the RRK extract did not alter the GLUT2 level in the liver of either the STZ-treated or the HFD-fed mice. Discussion Our study demonstrates that RRK extract can improve impaired glucose tolerance in mouse models of diabetes by enhancing GLUT4 expression in skeletal muscle. Conclusion These results suggest that RRK extract could potentially be a functional food for the treatment of diabetes mellitus.

Additional moisture in preparing red koji, Monascus-fermented rice, is a characteristic production process. To determine how additional moisture affects red koji preparation as per quality, we compared the growth of Monascus purpureus, enzyme and pigment production, and related gene expressions using our findings. We considered two kinds of red koji: one prepared with additional moisture at the middle part of the preparation and the other prepared without additional moisture. Our results showed that additional moisture did not promote the growth of M. purpureus, but it was significantly increased the pigment (red and yellow) and tended to increase the α-amylase level and saccharification power. Although adding a high amount of moisture (approximately 60% moisture content) promoted pigment production, it slightly repressed enzyme production. In contrast, adding approximately 50% moisture content promoted enzyme production. These findings showed that the additional moisture can affect the quality of red koji on the purpose. The expression of 10 pigment biosynthetic gene clusters and two glycohydrolase genes in red koji after adding moisture was analyzed through real-time qPCR. Eight genes were upregulated within 1 hr after adding water, with mppR2 being the first upregulated gene within 30 min. The expression of genes as per pigment production quickly responded to additional moisture during solid-state fermentation. Moreover, acetyl-CoA, which is a starting substrate for pigment content in red koji was increased within 3 hr after adding water. This study first described the relationship between additional moisture and expression of pigment biosynthetic genes by Monascus spp. during red koji preparation.

We developed an approach to genome editing of the white

Citrate exporter CexA plays a key role in the production of citric acid in fungi; however, its role in intracellular metabolism has remained unclear. In this study, we comparably characterized homologous cexA genes in the white koji fungus Aspergillus luchuensis mut. kawachii and the yellow koji fungus Aspergillus oryzae, which exhibit high and low abilities, respectively, to produce citric acid. Disruption of cexA caused a significant decline of both extracellular and intracellular citric acid accumulation in Aspergillus kawachii, while overexpression of the A. kawachii cexA gene (AkcexA) into A. oryzae significantly enhanced both extracellular and intracellular citric acid accumulation in A. oryzae to a level comparable to that of A. kawachii. In addition, overexpression of two intrinsic cexA homologs (AocexA and AocexB) in A. oryzae also enhanced its extracellular and intracellular citric acid accumulation. Comprehensive analysis of intracellular metabolites from an AkcexA-overexpressing strain of A. oryzae compared with its control strain identified metabolic changes associated with intracellular citric acid accumulation via the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle. Our results indicate that citric acid export enhances not only extracellular citric acid accumulation but also intracellular metabolic fluxes to generate citric acid.

Kokuto-shochu is a traditional Japanese spirit prepared from kokuto, a non-centrifugal brown cane sugar. When manufacturing kokuto, lime is added to the sugarcane juice to accelerate the crystallization of sucrose. Although the liming process differs depending on the manufacturer, the effects of liming on the quality of kokuto-shochu are unclear. Therefore, we investigated the flavor characteristics and volatiles present in kokuto-shochu prepared from kokuto with different liming degrees. Kokuto-shochu prepared from kokuto without liming had a pronounced kokuto-like flavor with a rich taste and contained higher contents of nerolidol, nonanal, acetoin, β-damascenone, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone compared to that prepared from kokuto with liming. On the other hand, kokuto-shochu prepared from kokuto with excess liming had a comparative grassy flavor. It contained higher esters, 4-vinylguaiacol, and pyrazines compared to other shochu. The levels nerolidol, isoamyl acetate, nonanal, and acetoin were affected by the mash pH during fermentation, and thus, liming would affect the formation of such volatiles via changing the mash pH. In contrast, pyrazines, 4-vinylguaiacol, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone were not affected by mash pH, and their levels in the kokuto-shochu were consistent with those in kokuto raw materials. These results suggested that the liming process affects the levels of volatiles in kokuto-shochu by changing the mash pH and volatile levels in kokuto raw materials.

The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologous laeA genes of the white koji fungus Aspergillus luchuensis mut. kawachii (A. kawachii) to determine their role in citric acid hyperproduction. The ΔlaeA strain exhibited a significant reduction in citric acid production. Cap-analysis gene expression (CAGE) revealed that laeA is required for the gene expression of a putative citrate exporter encoding cexA, which is critical for citric acid production. Deficient citric acid production by the ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of the cexA-overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression of cexA via methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation of cexA in A. kawachii. IMPORTANCEA. kawachii has been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced by A. kawachii plays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologous laeA genes; using CAGE, complementation test, and ChIP-qPCR, it was found that laeA is required for citric acid production through the regulation of cexA in A. kawachii. The epigenetic regulation of citric acid production elucidated in this study will be useful for controlling the fermentation processes of shochu.

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

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome-level phylogeny for the strain-level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub-species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake-shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 John Wiley & Sons, Ltd.

The putative methyltransferase LaeA is a global regulator of metabolic and development processes in filamentous fungi. We characterized the homologouslaeAgenes of the white koji fungusAspergillus luchuensismut.kawachii(A. kawachii) to determine their role in the hyperproduction of citric acid. The ΔlaeAstrain exhibited a significant reduction in citric acid productivity. Cap-analysis gene expression (CAGE) revealed thatlaeAis required for the gene expression of a putative citrate exporter encodingcexA, which is critical for citric acid production. The deficit in the productivity of citric acid by the ΔlaeAstrain was rescued by the overexpression ofcexAto a level comparable with that of thecexA-overexpressing ΔcexAstrain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates the expression ofcexAvia methylation levels of the histones H3K4 and H3K9. These results indicate that LaeA is involved in citric acid production through epigenetic regulation ofcexAinA. kawachii.IMPORTANCEA. kawachiihas been traditionally used for production of the distilled spirit shochu in Japan. Citric acid produced byA. kawachiiplays an important role in preventing microbial contamination during the shochu fermentation process. This study characterized homologouslaeAgenes; using CAGE, complementation test, and ChIP-qPCR, it was found thatlaeAis required for citric acid production though regulation ofcexAinA. kawachii. The epigenetic regulation of citrate production elucidated in this study will be useful for controlling the fermentation processes of shochu.

Rice-flavor baijiu is a traditional Chinese liquor that is manufactured using a solid-state saccharification process. In the present study, we investigated the role of the process in making of rice-flavor baijiu using chemical and biological quantitative analysis approaches. More than 70% of starch in rice decomposed to glucose after saccharification. In addition, the number of fungal cells, saccharification activity, and lactic acid concentration increased. Rhizopus oryzae was identified as the major fungus proliferating under saccharification based on denaturing gradient gel electrophoresis analysis targeting the internal spacer transcribed region. Lactic acid bacteria were not detected by 16S rRNA gene-based next-generation sequencing analysis during saccharification. Conversely, R. oryzae, isolated from xiaoqu, exhibited a capacity to produce lactic acid. The results imply that the solid-state saccharification is essential not only for saccharification but also for the culture of R. oryzae, which promote saccharification activity and lactic acid production. We also investigated the most appropriate temperature for solid-state saccharification and 35°C was the optimum temperature for R. oryzae cultivation, enzyme production, and saccharification. The results could facilitate the efficient and stable manufacture of rice-flavor baijiu.

Aspergillus luchuensis mut. kawachii (A. kawachii) produces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterized A. kawachii CtpA and YhmA, which are homologous to the yeast Saccharomyces cerevisiae mitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified from A. kawachii and reconstituted into liposomes. The proteoliposomes exhibited only counterexchange transport activity; CtpA transported citrate using countersubstrates, especially cis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate, cis-aconitate, and succinate. Disruption of ctpA and yhmA caused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpA ΔyhmA strain, we constructed an S-tagged ctpA (ctpA-S) conditional expression strain in the ΔyhmA background using the Tet-On promoter system. Knockdown of ctpA-S in ΔyhmA resulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption of ctpA and yhmA leads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of acetate or lysine, which could remedy the acetyl-CoA level. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in acetyl-CoA biogenesis in A. kawachii. IMPORTANCE Citrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters from Aspergillus luchuensis mut. kawachii. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidium formation, cytosolic acetyl-CoA synthesis, and citric acid production. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

We investigated the contribution of the complex expressions of two α-amylases, namely acid-stable α-amylase (AS-amylase) and acid-labile α-amylase (AL-amylase), from Aspergillus kawachii to the microstructure of koji and the brewing. AL-amylase was found to be the primary contributor to the decomposition of starch in the early stage of koji making. From the middle stage, both α-amylases decomposed the starch in a coordinated manner, and at the final stage, acid-stable α-amylase and glucoamylase decomposed the starch granules. Characterization of koji prepared by the single disruptions of AS-amylase or AL-amylase genes, double disruption of both α-amylase genes, and triple disruption of two α-amylase and glucoamylase genes in A. kawachii cells indicated that both α-amylases can work in a synergistic manner to decompose starches during koji making. AL-amylase was found, for the first time, to play an important role in starch decomposition in koji. The speed of alcohol fermentation and ester contents of the fermented mash were higher the mash prepared the control strain, followed by single, double, and triple disruptants. These results indicate that the microstructure of koji plays a role in promoting alcohol fermentation and flavor development.

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.

Shochu, a traditional Japanese distilled liquor, makes use of rice koji, which is koji mould grown on rice grain. Rice koji is an essential ingredient of Japanese liquors such as shochu, and it plays a role as a source of the enzyme to degrade starch. However, there has been no research on the effect of rice koji on the flavour of shochu. Therefore, in this study, the volatile compounds in shochu derived from rice koji were investigated. Two shochu samples were prepared to assess the contribution of rice koji to the flavour. One shochu sample was prepared from rice koji, yeast and water (rice koji-shochu). The other shochu sample was obtained from steamed rice and various enzymes instead of rice koji (enzyme-shochu), along with yeast and water. The volatile compounds were analysed by gas chromatography–mass spectrometry (GC-MS) and GC-MS/olfactometry with aroma extract dilution analysis. The results showed that enzyme-shochu had a higher flavour dilution value of dimethyl trisulphide and hexanal, whereas rice koji-shochu had a higher flavour dilution value of some ester compounds that imparted aromatic odours such as fruity. Some unknown peaks representing compounds that impart characteristic odours such as soda, potato, lavender, and tea-like were specifically detected in rice koji-shochu. The concentrations and calculated odour active values of 14 compounds were measured. These results showed that isovaleraldehyde, ethyl caprylate, ethyl caproate and ethyl 2-methylbutyrate played an important role in imparting the specific odour of rice koji-shochu. Copyright © 2016 The Institute of Brewing & Distilling

Aspergillus luchuensismut. kawachiiproduces a large amount of citric acid during the process of fermenting shochu, a traditional Japanese distilled spirit. In this study, we characterizedA. kawachiiCtpA and YhmA, which are homologous to the yeastSaccharomyces cerevisiaemitochondrial citrate transporters Ctp1 and Yhm2, respectively. CtpA and YhmA were purified fromA. kawachiiand reconstituted into liposomes. The proteoliposomes exhibited only counter-exchange transport activity; CtpA transported citrate using counter substrates especially forcis-aconitate and malate, whereas YhmA transported citrate using a wider variety of counter substrates, including citrate, 2-oxoglutarate, malate,cis-aconitate, and succinate. Disruption ofctpAandyhmAcaused deficient hyphal growth and conidia formation with reduced mycelial weight–normalized citrate production. Because we could not obtain a ΔctpAΔyhmAstrain, we constructed actpA-Sconditional expression strain in the ΔyhmAbackground using the Tet-On promoter system. Knockdown ofctpA-Sin ΔyhmAresulted in a severe growth defect on minimal medium, indicating that double disruption ofctpAandyhmAleads to synthetic lethality; however, we subsequently found that the severe growth defect was relieved by addition of lysine. Our results indicate that CtpA and YhmA are mitochondrial citrate transporters involved in citric acid production and that transport of citrate from mitochondria to the cytosol plays an important role in lysine biogenesis inA. kawachii.IMPORTANCECitrate transport is believed to play a significant role in citrate production by filamentous fungi; however, details of the process remain unclear. This study characterized two citrate transporters fromAspergillus luchuensismut. kawachii. Biochemical and gene disruption analyses showed that CtpA and YhmA are mitochondrial citrate transporters required for normal hyphal growth, conidia formation, and citric acid production. In addition, this study provided insights into the links between citrate transport and lysine biosynthesis. The characteristics of fungal citrate transporters elucidated in this study will help expand our understanding of the citrate production mechanism and facilitate the development and optimization of industrial organic acid fermentation processes.

We characterized Pex16 in Aspergillus luchuensis mut. kawachii to examine the role of peroxisomes on citric acid production during the shochu-fermentation process. Rice koji made using a Δpex16 strain exhibited no significant change in citric acid accumulation but a 1.4-fold increase in formic acid production. Microscopic observation of mRFP-SKL (a peroxisome protein marker) showed that pex16 disruption decreased the number of dot-like structures per hyphal cell to 5% of the control. Pex16-GFP exclusively co-localized with mRFP-SKL throughout the hyphae including the very close position to the septal pore. Moreover, the Δpex16 strain was hypersensitive to calcofluor white, which appeared to induce bursting of the hyphal tip and translocation of mRFP-SKL signals to the septal pore. These results indicate that Pex16 does not play a role in citric acid accumulation but is significantly involved in peroxisome and Woronin body formation in Aspergillus kawachii.

Imo-shochu is a Japanese traditional spirit made from sweet potatoes. Characteristic volatile compounds in imo-shochu are mainly derived from the sweet potato and its flavour significantly depends on the quality and cultivar of sweet potato used. Thus, the effects of the cultivation period of sweet potatoes on sensory characteristics and composition of volatile compounds of imo-shochu were investigated. Sweet potatoes (cv. Koganesengan) used in this study were harvested at 120, 150 or 180 days after planting, and each sample was used to prepare imo-shochu. The imo-shochu samples were evaluated by eight panellists in a blind study, who ranked them on the basis of various odour and taste attributes. Rank sums were calculated and data were analysed using the Friedman test. The compositions of the volatile compounds in the imo-shochu samples were analysed using gas chromatography–mass spectrometry (GC-MS). Sensory evaluations showed that a longer cultivation period of the sweet potatoes enhanced the floral aroma and characteristic taste of imo-shochu. In addition, imo-shochu prepared with the sample cultivated for 150 days was evaluated to have a sweeter taste than that prepared with the other samples. The GC-MS analysis showed that imo-shochu prepared with the sample cultivated for 180 days contained a lower concentration of monoterpene alcohols, but higher concentrations of β-damascenone, rose oxide, and fatty acid esters than the imo-shochu prepared with the sample cultivated for 120 days. These differences in the composition of the volatile compounds affected the sensory qualities of the imo-shochu. Copyright © 2016 The Institute of Brewing & Distilling

In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungusAspergillus oryzae. During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy,A. oryzae, andGrifola frondosaglycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition ofA. oryzaeandG. frondosaglycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast.

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.

Here, we report a draft genome sequence of Saccharomyces cerevisiae strain Kagoshima no. 2, which is used for brewing shōchū, a traditional distilled spirit in Japan. The genome data will facilitate an understanding of the evolutional traits and genetic background related to the characteristic features of strain Kagoshima no. 2.

Here, we report a draft genome sequence of Saccharomyces cerevisiae strain Kagoshima no. 2, which is used for brewing shōchū, a traditional distilled spirit in Japan. The genome data will facilitate an understanding of the evolutional traits and genetic background related to the characteristic features of strain Kagoshima no. 2.