Deficiency of ß-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308.

In this study, we investigated a deleterious mutation in the ß-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial ß-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose levels during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

In vitro colonic fermentation characteristics of barley-koji differ from those of barley.

Barley-koji is prepared by inoculating barley, a beneficial prebiotic source, with the fungi Aspergillus luchuensis mut. kawachii. In this study, the prebiotic effects of barley-koji on human colonic microbiota were evaluated in vitro compared with barley, using pig feces. The enzyme-resistant fraction of the following sample groups each was added to respective fermenters: cellulose, barley (Commander and ß104), and barley-koji (Commander-koji and ß104-koji). Short-chain fatty acid and ammonia-nitrogen production increased and decreased, respectively, in barley-koji and barley groups. Furthermore, the propionate concentration increased in the barley group, showing a positive correlation with the abundance of the genus Dialister. In the barley-koji group, however, acetate and n-butyrate concentrations increased during the early stages of incubation, and the relative abundance of the genus Megasphaera was higher than those of the other genera. Therefore, this study demonstrated that barley-koji might possess beneficial physiological properties for colonic fermentation, which differ from those of barley.

The white koji fungus Aspergillus luchuensis mut. kawachii.

The white koji fungus, Aspergillus luchuensis mut. kawachii, is used in the production of shochu, a traditional Japanese distilled spirit. White koji fungus plays an important role in the shochu production process by supplying amylolytic enzymes such as α-amylase and glucoamylase. These enzymes convert starch contained in primary ingredients such as rice, barley, buckwheat, and sweet potato into glucose, which is subsequently utilized by the yeast Saccharomyces cerevisiae to produce ethanol. White koji fungus also secretes large amounts of citric acid, which lowers the pH of the shochu mash, thereby preventing the growth of undesired microbes and enabling stable production of shochu in relatively warm regions of Japan. This review describes the historical background, research tools, and recent advances in studies of the mechanism of citric acid production by white koji fungus.

LaeA Controls Citric Acid Production through Regulation of the Citrate Exporter-Encoding cexA Gene in Aspergillus luchuensis mut. kawachii.

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. kawachiiIMPORTANCEA. 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.

A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii.

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.

Mitochondrial Citrate Transporters CtpA and YhmA Are Required for Extracellular Citric Acid Accumulation and Contribute to Cytosolic Acetyl Coenzyme A Generation in Aspergillus luchuensis mut. kawachii.

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. kawachiiIMPORTANCE 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.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii.

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A. luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

Chromosome-level genome sequence data and analysis of the white koji fungus, Aspergillus luchuensis mut. kawachii IFO 4308.

Aspergillus luchuensis mut. kawachii is used primarily in the production of shochu, a traditional Japanese distilled alcoholic beverage. Here, we report the chromosome-level genome sequence of A. luchuensis mut. kawachii IFO 4308 (NBRC 4308) and a comparison of the sequence with that of A. luchuensis RIB2601. The genome of strain IFO 4308 was assembled into nine contigs consisting of eight chromosomes and one mitochondrial DNA segment. The nearly complete genome of strain IFO 4308 comprises 37,287,730 bp with a GC content of 48.85% and 12,664 predicted coding sequences and 267 tRNAs. Comparison of the IFO 4308 and RIB2601 genomes revealed a highly conserved structure; however, the IFO 4308 genome is larger than that of RIB2601, which is primarily attributed to chromosome 5. The genome sequence of IFO 4308 was deposited in DDBJ/ENA/GenBank under accession numbers AP024425-AP024433.

Insights regarding sirtuin-dependent gene regulation during white koji production.

White koji, a solid-state culture of Aspergillus luchuensis mut. kawachii using grains such as rice and barley, is used as a source of amylolytic enzymes and citric acid for the production of shochu, a traditional Japanese distilled spirit. We previously characterized changes in gene expression that affect the properties of white koji during the shochu production process; however, the underlying regulatory mechanisms were not determined. We then characterized the NAD+-dependent histone deacetylase sirtuin, an epigenetic regulator of various biological phenomena, in A. l. mut. kawachii and found that sirtuin SirD is involved in expression of α-amylase activity and citric acid accumulation. In this addendum study, we measured the NAD+/NADH redox state and found that the NAD+ level and NAD+/NADH ratio decrease during koji production, indicating that sirtuin activity declines in the late stages of koji culture. By comparing these results with transcriptomic data obtained in our previous studies, we estimate that approximately 35% of the gene expression changes during white koji production are SirD dependent. This study provides clues to the mechanism of gene expression regulation in A. l. mut. kawachii during the production of white koji.

Citrate exporter enhances both extracellular and intracellular citric acid accumulation in the koji fungi Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

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.

Making Traditional Japanese Distilled Liquor, Shochu and Awamori, and the Contribution of White and Black Koji Fungi.

The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called "honkaku shochu." It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a unique temperature control method to ensure that these three important elements, which greatly affect the taste of the produced liquor, are balanced without any excess or deficiency. This review describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan and whose market is expected to expand worldwide, with special attention paid to the koji fungi cultivation step. Furthermore, we describe the history of the koji fungi used today in the production of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also report the latest research progress on this topic.

Sirtuin SirD is involved in α-amylase activity and citric acid production in Aspergillus luchuensis mut. kawachii during a solid-state fermentation process.

Sirtuins are understood to play a significant role in growth phase-dependent gene expression. In the study presented here, we examined the sirtuins in the white koji fungus, Aspergillus luchuensis mut. kawachii (Aspergillus kawachii), to examine their role in the regulation of amylolytic enzymes and citric acid production during solid-state culture (koji). Characterization of rice koji made using five sirtuin gene disruptants indicated that these genes are involved in the amylolytic activity and acidity of rice koji; the sirD disruptant in particular showed lower levels of acid-stable α-amylase activity and citric acid production per mycelial weight in koji compared to the control strain. The sirD disruptant also showed a change in mycelial pigmentation, and had higher sensitivity to cell wall biogenesis inhibitors such as calcofluor white and Congo red and reduced conidia formation. These results indicate that SirD is required for secondary metabolism, cell wall integrity, and conidial development. Cap analysis gene expression (CAGE) and quantitative RT-PCR analysis indicated that transcriptional changes were related to the characteristic phenotype of the sirD disruptant, including a reduced transcript level of the acid-stable α-amylase gene and a citric acid exporter in rice koji. These results indicate that SirD has a significant role in global transcriptional regulation, including the production of acid-stable α-amylase and citric acid, in A. kawachii during the solid-state fermentation process.