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Gene expression profile of high amylolytic enzyme producing mutant strain of Aspergillus luchuensis obtained by ion beam mutagenesis

ABSTRACT: 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.

ORGANISM(S): Aspergillus luchuensis

PROVIDER: GSE92237 | GEO | 2017/03/14

SECONDARY ACCESSION(S): PRJNA356975

REPOSITORIES: GEO

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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.

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Gene expression profile of high amylolytic enzyme producing mutant strain of Aspergillus luchuensis obtained by ion beam mutagenesis

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