Project description:A koji mold Aspergillus kawachii is used for making a Japanese distilled spirit, shochu. During making shochu, A. kawachii is grown in a solid-state culture comprised of steamed grains such as rice or barley, termed koji, to convert the starch to glucose and also to produce citric acid. During this process, cultivation temperature of A. kawachii is generally controlled by raising to 40M-BM-0C and then lowering to 30M-BM-0C. The cultivation at 40M-BM-0C and the following at lower temperature of 30M-BM-0C has important roles in the elevated activity of amylase and starting an accumulation of a large amount of citric acid, respectively. In this study, we investigated the effect of temperature on the gene expression of A. kawachii when barley was used as ingredient for making koji. The results of DNA microarray and gene ontology analysis showed that the expression of genes involved in the metabolic processes of glycerol, trehalose, and pentose phosphate that branch from glycolysis was down-regulated by shifting the cultivation temperature from 40M-BM-0C from 30M-BM-0C. In addition, reduction in the expression of the genes related with heat shock responses and increasing in the expression of the genes related with amino acid transport after the temperature lowering were found to be significant change. These results suggested that the cultivation at 40M-BM-0C is stressful event for the A. kawachii and the heat adaption lead to the depression of citric acid accumulation through activation of the branching pathways from glycolysis. The gene expression profile obtained in this study will help to understand the gene regulation during koji-making process to optimize A. kawachii as industrial microorganism. Gene expression of Aspergillus kawachii in barley koji was measured at 25, 26.5, and 44 hours at two different temperature control. Three independent experiments were performed at each time (25, 26.5, or 44 hours) using different barley koji for each expreriment.
Project description:A koji mold Aspergillus kawachii is used for making a Japanese distilled spirit, shochu. During making shochu, A. kawachii is grown in a solid-state culture comprised of steamed grains such as rice or barley, termed koji, to convert the starch to glucose and also to produce citric acid. During this process, cultivation temperature of A. kawachii is generally controlled by raising to 40°C and then lowering to 30°C. The cultivation at 40°C and the following at lower temperature of 30°C has important roles in the elevated activity of amylase and starting an accumulation of a large amount of citric acid, respectively. In this study, we investigated the effect of temperature on the gene expression of A. kawachii when barley was used as ingredient for making koji. The results of DNA microarray and gene ontology analysis showed that the expression of genes involved in the metabolic processes of glycerol, trehalose, and pentose phosphate that branch from glycolysis was down-regulated by shifting the cultivation temperature from 40°C from 30°C. In addition, reduction in the expression of the genes related with heat shock responses and increasing in the expression of the genes related with amino acid transport after the temperature lowering were found to be significant change. These results suggested that the cultivation at 40°C is stressful event for the A. kawachii and the heat adaption lead to the depression of citric acid accumulation through activation of the branching pathways from glycolysis. The gene expression profile obtained in this study will help to understand the gene regulation during koji-making process to optimize A. kawachii as industrial microorganism.
Project description:We report that the RNA-binding protein NrdA has a significant role on the global mRNA expression in the white koji fungus, Aspergillus luchuensis mut. kawachii. We constructed the nrdA conditional expression strain using the Tet-On system in A. kawachii. Downregulation of nrdA caused severe growth defect, indicating that NrdA is essential for proliferation of A. kawachii. Parallel RNA-seq and RNA immunoprecipitation (RIP)-seq analysis identified potential NrdA-interacted transcripts, corresponding to 32% of predicted protein coding genes of A. kawachii. Subsequent gene ontology analysis suggested that overexpression of NrdA affects a variety of metabolic activity including secondary metabolite production.
Project description:Sirtuin is considered to play a significant role in the growth phase-dependent gene expression. In this study, we characterized sirtuin in the white koji fungus, Aspergillus kawachii, to examine their role on regulation of amylolytic enzymes and citric acid productions during the solid-state culture (koji). Characterization of rice-koji made using five putative sirtuin gene disruptants indicated that they are involved in amylolytic activity and acidity of rice koji; especially the sirD disruptant showed lower levels of α-amylase activity and citric acid production per mycelial weight in the koji compared the control strain. In addition, the sirD disruptant also showed a change in mycelial pigmentation, higher sensitivity to cell wall biogenesis inhibitor such as calcofluor white and Congo red, and reduced conidia formation, indicating that SirD is required for secondary metabolism, cell wall integrity, and conidial development. The cap analysis gene expression (CAGE) indicated that the transcriptional changes related to the characteristic phenotype of the sirD disruptant (e.g., a reduced transcript level of acid-stable α-amylase gene and a citric acid exporter) in rice koji. These results indicated that SirD has a significant role on the global transcriptional regulation including productions of α-amylase and citric acid in A. kawachii during the solid-state fermentation process.
Project description:LaeA, a putative methyltransferase, is a global regulator for metabolic and development process in filamentous fungi. We characterized the laeA homologous genes in the white koji fungus, Aspergillus luchuensis mut. kawachii (A. kawachii) to examine their role in citric acid production. The ΔlaeA strain showed a significant reduction in the citric acid productivity. The cap-analysis gene expression (CAGE) revealed the laeA is required for the gene expression of a putative citrate exporter encoding cexA, which has a critical role for the citric acid production. The deficient citric acid productivity of the ΔlaeA strain was remedied by overexpression of cexA to a comparative level to that of the cexA overexpressing ΔcexA strain. In addition, chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) analysis indicated that LaeA regulates gene expression of the citrate exporter encoding cexA gene via histone H3K4 and histone H3K9 methylation levels. These results indicate that LaeA is involved in the citric acid production through epigenetic regulation of cexA in A. kawachii.
Project description:To investigate the roles of epigenetic regulator homologs, heterochromatin protein 1 (HepA) and a putative methyltransferase (LaeA), in the citric acid produciton by Aspergillus luchuensis mut. kawachii, we constructed the ΔhepA and ΔlaeA strains. We then performed gene expression profiling analysis using data obtained from RNA-seq of Aspergillus luchuensis mut. kawachii control, ΔhepA, and ΔlaeA strains.
Project description:Aspergillus luchuensis is a kuro (black) koji mold that have been used as starch degrader for awamori and shochu making industries in Japan. In this study, we investigated the effect of ion beam irradiation on the A. luchuensis RIB2601 and successfully obtained a high starch degrading mutant strain U1. Strain U1 showed reduced growth rate, whereas showed higher α-amylase, glucoamylase, and α-glucosidase activities per mycelial growth than those of wild type strain both in the agar plate and rice koji. In addition, strain U1 showed higher N-acetylglucosamine content in the cell wall and showed higher sensitivity to calcofluor white, indicating deficient cell wall integrity. Interestingly, the analysis of secreted protein showed higher expression of acid labile α-amylase (AmyA) and glucoamylase (GlaA) in strain U1 although the real-time-RT-PCR analysis indicated that the transcriptional levels of the amyA and glaA genes are not changed significantly. These results suggested that the high amylolytic activity of strain U1 is attributed to the high AmyA and GlaA expression level, but it is not through transcriptional regulation of the amyA and glaA genes. Furthermore, the RNA-seq analysis indicated that strain U1 shows transcriptional change of at least 604 genes related to oxidation-reduction, transport, and glucosamine-containing compound metabolic processes, which may be involved in the deficient cell wall integrity of strain U1.