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Aspergillus luchuensismut.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 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 counterexchange transport activity; CtpA transported citrate using countersubstrates, especiallycis-aconitate and malate, whereas YhmA transported citrate using a wider variety of countersubstrates, including citrate, 2-oxoglutarate, malate,cis-aconitate, and succinate. Disruption ofctpAandyhmAcaused deficient hyphal growth and conidium formation with reduced mycelial weight-normalized citrate production. Because we could not obtain a ΔctpAΔyhmAstrain, we constructed an S-taggedctpA(ctpA-S) conditional expression strain in the ΔyhmAbackground using the Tet-On promoter system. Knockdown ofctpA-Sin ΔyhmAresulted in a severe growth defect on minimal medium with significantly reduced acetyl coenzyme A (acetyl-CoA) and lysine levels, indicating that double disruption ofctpAandyhmAleads 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 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, 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.

The volatile compounds in three types of koji (yellow, white, and black) and steamed rice were investigated using gas chromatography-mass spectrometry. The objective of this study was to characterize each koji type based on its profile of volatile compounds. From the steamed rice and rice koji samples examined, a total of 29 volatile compounds were identified. Butanal, hexanal, dimethyl trisulfide and cyclohexyl isothiocyanate were detected only in steamed rice. The volatile profile of yellow koji (sake koji) was distinct from those of white and black kojis (shochu koji). On the basis of relative peak areas, alcohols and aldehydes were identified as being more abundant in yellow koji. White and black kojis had similar volatile profiles, and had a greater abundance of furans than yellow koji. We assume that the different suites of volatile compounds identified in shochu and sake kojis impart the respective characteristic flavours to shochu and sake.

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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 expression of a putative citrate exporter-encoding cexA gene, which is critical for citric acid production. Deficient citric acid production by a ΔlaeA strain was rescued by the overexpression of cexA to a level comparable with that of a 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. IMPORTANCE A. 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 tests, 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.

Rice koji is considered a readily accessible functional food that may have health-promoting effects. We investigated whether white, yellow, and red koji have the anti-obesity effect in C57BL/6J mice fed a high-fat diet (HFD), which is a model for obesity. Mice were fed HFD containing 10% (w/w) of rice koji powder or steamed rice for 4 weeks. Weight gain, epididymal white adipose tissue, and total adipose tissue weight were significantly lower in all rice koji groups than in the HFD-rice group after 4 weeks. Feed efficiency was significantly reduced in the yellow koji group. Blood glucose levels were significantly lower in the white and red koji groups with HOMA-R and leptin levels being reduced in the white koji group. White and red koji increased glucose uptake and GLUT4 protein expression in L6 myotube cells. These results showed that all rice koji have the anti-obesity or anti-diabetes effects although the mechanisms may differ depending on the type of rice koji consumed.