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.

Genome characterization of two novel deep-sea sediment fungi, Penicillium pacificagyrus sp. nov. and Penicillium pacificasedimenti sp. nov., from South Pacific Gyre subseafloor sediments, highlights survivability.

BACKGROUND: Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium, Aspergillus, Cladosporium, Fusarium, and Schizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients. RESULTS: Here, we present high-quality genomes of two proposed novel Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium. We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P. pacificasedimenti and 10.68% for P. pacificagyrus. A total of 12,271 genes were predicted in the P. pacificasedimenti genome and 12,568 genes in P. pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon. CONCLUSIONS: Our results provide the first constructed genomes of novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.

Efficient gene targeting in Aspergillus chevalieri used to produce katsuobushi.

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.

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.

Pseudomonas furukawaii sp. nov., a polychlorinated biphenyl-degrading bacterium isolated from biphenyl-contaminated soil in Japan.

Strain KF707T was isolated from a biphenyl-contaminated site in Kitakyushu, Japan. Analysis of 16S rRNA gene sequences, retrieved from the whole-genome sequence, revealed that the isolate was closely related to members of the genus Pseudomonas, sharing the highest sequence similarities with Pseudomonas balearica strain SP1402T (DSM 6083) (97.8 %). The DNA G+C chromosome and plasmid content of strain KF707T were 65.5 and 60.5 mol%. The major cellular fatty acids were iso-C15 :  0 and C16 : 1ω7c/C16 : 1ω6c. Polyphasic analysis indicated that strain KF707T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas furukawaii sp. nov. is proposed. The type strain is KF707T (=DSM 10086T=NBRC 110670T).

Multi-gene phylogenetic analysis reveals that shochu-fermenting Saccharomyces cerevisiae strains form a distinct sub-clade of the Japanese sake cluster.

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.

Transcriptomic analysis of temperature responses of Aspergillus kawachii during barley koji production.

The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40 °C and is then lowered to 30 °C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30 °C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40 °C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.

GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus.

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)-containing polysaccharides and glycoconjugates, including O-glycans, N-glycans, fungal-type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse-genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of ß-Galf-specific antibody EB-A2 to O-glycosylated WscA protein and galactomannoproteins. The results of an in-vitro Galf antigen synthase assay revealed that GfsA has ß1,5- or ß1,6-galactofuranosyltransferase activity for O-glycans in glycoproteins, uses UDP-d-Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal ß-galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O-glycans in the Golgi.

The putative stress sensor protein MtlA is required for conidia formation, cell wall stress tolerance, and cell wall integrity in Aspergillus nidulans.

The Mid2-like protein MtlA is a putative sensor of the cell wall integrity (CWI) signaling pathway in Aspergillus nidulans. An MtlA-EGFP fusion protein was localized at the cell surface and septa. The mtlA disruptant (∆mtlA) showed radial colony growth similar to the wild-type (wt) strain, but showed reduced conidia formation. The ∆mtlA mutant showed growth deficiency in the presence of inhibitors of cell wall synthesis. Moreover, mtlA disruption resulted in a reduction in the glucan and chitin content in the cell wall. These results suggest that MtlA plays a significant role in asexual sporulation, cell wall stress tolerance, and the maintenance of CWI in A. nidulans, but transcriptional upregulation of α-1,3-glucan synthase gene agsB induced by micafungin was observed in the ∆mtlA strain as well as the wt strain. Thus, MtlA is not essential for activation of the downstream CWI signaling pathway components identified in previous studies of Saccharomyces cerevisiae.

THI3 contributes to isoamyl alcohol biosynthesis through thiamine diphosphate homeostasis.

Isoamyl alcohol is a precursor of isoamyl acetate, an aromatic compound that imparts the ginjo aroma to sake. The isoamyl alcohol biosynthesis pathway in yeasts involves the genes PDC1, PDC5, PDC6, ARO10, and THI3 encoding enzymes that decarboxylate α-ketoisocaproic acid to isovaleraldehyde. Among these genes, THI3 is the main gene involved in isoamyl alcohol biosynthesis. Decreased production of isoamyl alcohol has been reported in yeast strains with disrupted THI3 (Δthi3). However, it has also been reported that high THI3 expression did not enhance decarboxylase activity. Therefore, the involvement of THI3 in isoamyl alcohol biosynthesis remains unclear. In this study, we investigated the role of THI3 in isoamyl alcohol biosynthesis. While reproducing previous reports of reduced isoamyl alcohol production by the Δthi3 strain, we observed that the decrease in isoamyl alcohol production occurred only at low yeast nitrogen base concentrations in the medium. Upon investigating individual yeast nitrogen base components, we found that the isoamyl alcohol production by the Δthi3 strain reduced when thiamine concentrations in the medium were low. Under low-thiamine conditions, both thiamine and thiamine diphosphate (TPP) levels decreased in Δthi3 cells. We also found that the decarboxylase activity of cell-free extracts of the Δthi3 strain cultured in a low-thiamine medium was lower than that of the wild-type strain, but was restored to the level of the wild-type strain when TPP was added. These results indicate that the loss of THI3 lowers the supply of TPP, a cofactor for decarboxylases, resulting in decreased isoamyl alcohol production.

Overexpression of the DHA1 family, ChlH and ChlK, leads to enhanced dicarboxylic acids production in koji fungi, Aspergillus luchuensis mut. kawachii and Aspergillus oryzae.

The white koji fungus Aspergillus luchuensis mut. kawachii secretes substantial amounts of citric acid through the expression of the citric acid exporter CexA, a member of the DHA1 family. In this study, we aimed to characterize 11 CexA homologs (Chl proteins) encoded in the genome of A. luchuensis mut. kawachii to identify novel transporters useful for organic acid production. We constructed overexpression strains of chl genes using a cexA disruptant of the A. luchuensis mut. kawachii as the host strain, which prevented excessive secretion of citric acid into the culture supernatant. Subsequently, we evaluated the effects of overexpression of chl on producing organic acids by analyzing the culture supernatant. All overexpression strains did not exhibit significant citric acid accumulation in the culture supernatant, indicating that Chl proteins are not responsible for citric acid export. Furthermore, the ChlH overexpression strain displayed an accumulation of 2-oxoglutaric and fumaric acids in the culture supernatant, while the ChlK overexpression strain exhibited the accumulation of 2-oxoglutaric, malic and succinic acids. Notably, the ChlH and ChlK overexpression led to a substantial increase in the production of 2-oxoglutaric acid, reaching approximately 25 mM and 50 mM, respectively. Furthermore, ChlH and ChlK overexpression also significantly increased the secretory production of dicarboxylic acids, including 2-oxoglutaric acid, in the yellow koji fungus, Aspergillus oryzae. Our study demonstrates that overexpression of DHA1 family gene results in enhanced secretion of organic acids in koji fungi of the genus Aspergillus.

Homozygous gene disruption in diploid yeast through a single transformation.

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

Characterization of aroma profiles of kokuto-shochu prepared from three different cultivars of sugarcane.

Kokuto-shochu is a traditional Japanese spirit prepared from kokuto, obtained by evaporating water from sugarcane (Saccharum officinarum L.) juice. To clarify the effects of sugarcane cultivars on the sensory quality of kokuto-shochu, we investigated the flavor characteristics and composition of volatiles in kokuto-shochu prepared from kokuto using three different sugarcane cultivars, NiF8, Ni15, and RK97-14. Furthermore, experiments were conducted by using the cultivars collected between 2018 and 2020 to observe annual variations in their properties. The amino acid content of the three kokuto varieties did not differ significantly, but the amino acid content of NiF8 was two to five times higher than that of RK97-14, which was the same for all samples collected in the selected years. The browning degrees of kokuto were also higher in NiF8, and they were positively correlated to the amino acid contents of kokuto. The kokuto-like aroma of shochu made from Ni15 was stronger than that of shochu made from RK97-14. The concentration of ethyl lactate in shochu made from Ni15 was higher, however, the concentration of guaiacol was the lowest in the three cultivars' products. Shochu made from NiF8 had the highest levels of Maillard reaction products (MRPs; pyrazines and furans), ß-damascenone, and guaiacol amounts. In contrast, shochu made from RK97-14 tended to have a fruity flavor, and lower MRP levels than those made from NiF8. Thus, it was shown that sugarcane cultivars affect the sensory characteristics and volatiles in kokuto-shochu.

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.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

Genome sequence of the white koji mold Aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu.

The filamentous fungus Aspergillus kawachii has traditionally been used for brewing the Japanese distilled spirit shochu. A. kawachii characteristically hyperproduces citric acid and a variety of polysaccharide glycoside hydrolases. Here the genome sequence of A. kawachii IFO 4308 was determined and annotated. Analysis of the sequence may provide insight into the properties of this fungus that make it superior for use in shochu production, leading to the further development of A. kawachii for industrial applications.

Putative stress sensors WscA and WscB are involved in hypo-osmotic and acidic pH stress tolerance in Aspergillus nidulans.

Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in Aspergillus nidulans. Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be N- and O-glycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The wscA disruptant (ΔwscA) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the ΔwscA strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the ΔwscA strain. A similar inhibition was observed in the wscB disruptant strain, but to a lesser extent. In addition, a double wscA and wscB disruptant (ΔwscA ΔwscB) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of α-1,3-glucan synthase genes (agsA and agsB) was significantly altered in the wscA disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of wsc disruption. Moreover, the transient transcriptional upregulation of the agsB gene via MpkA signaling was observed in the ΔwscA ΔwscB strain to the same degree as in the wt strain. These results indicate that A. nidulans Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast Saccharomyces cerevisiae and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.