Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production

Abstract

cThe 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.

Characterization of amylolytic enzyme overproducing mutant of <i>Aspergillus luchuensis</i> obtained by ion beam mutagenesis

“…Rice koji was collected at the step of shimaishigoto after 30 h of cultivation and immediately frozen using liquid nitrogen. RNA was extracted from the frozen rice koji samples by a previously described method (Futagami et al, 2015). Briefly, the cells were disrupted using a hammer in liquid nitrogen to keep the samples frozen.…”

“…Because genome information of A. luchuensis RIB2601 is not available, the sequence reads were mapped to the coding sequences identified in the genome of A. luchuensis mut. kawachii IFO 4308, which is a white mutant of kuro-koji fungus (Futagami et al, 2015). Triplicate data were analyzed and the expression ratio was calculated as the log (base 2) ratio.…”

“…In addition, the genes were analyzed for gene ontology (GO) (Data Set S2 in supplemental file 2). GO enrichment analysis was performed using a PERL script as described previously (Ashburner et al, 2000;Futagami et al, 2015;Harris et al, 2004). The RNA-seq data were deposited in the Gene Expression Omnibus (GEO) under accession number GSE92237.…”

“…The real-time RT-PCR primers were designed using genetic information on A. luchuensis mut. kawachii IFO 4308 (Futagami et al, 2015). The following primers were used: ALamyA-RT-F and ALamyA-RT-R for acid-labile amyA, ASamyA-RT-F and ASamyA-RT-R for acid-stable amyA, glaA-RT-F and glaA-RT-R for glaA, AK10307-RT-F and AK10307-RT-R for AKAW_10307, AK660-RT-F and AK660-RT-R for AKAW_00660, and actin-RT-F and actin-RT-R for the actin gene (Table S1 in supplemental file 3).…”

Marker recycling system using the <i>sC</i> gene in the white koji mold, <i>Aspergillus luchuensis</i> mut. <i>kawachii</i>

“…The genome sequence of A. kawachii was recently reported, and data regarding A. kawachii gene expression during the shochu brewing process is also available (Futagami et al, 2015). With these genome and transcriptome data, the sC marker recycling system may be useful for molecular breeding of A. kawachii.…”