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

ABSTRACT: 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.

SUBMITTER: Futagami T

PROVIDER: S-EPMC4309687 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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

Futagami Taiki T Mori Kazuki K Wada Shotaro S Ida Hiroko H Kajiwara Yasuhiro Y Takashita Hideharu H Tashiro Kosuke K Yamada Osamu O Omori Toshiro T Kuhara Satoru S Goto Masatoshi M

Applied and environmental microbiology 20150201 4

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 ...[more]

PMID: 25501485

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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.

Project description:BackgroundAlpha-amylases hydrolyze 1,4 α-glycosidic bonds of starch and produce malto-oligosaccharides. It is an important enzyme generally applied in textile, food and brewing industries. Enhancement in thermal stability and productivity of enzymes are the two most sought after properties for industrial use. The Aspergillus oryzae (Koji) has Generally Recognized as Safe (GRAS) status and safe for use in food industry. Hence, Koji strain's development for the screening of potent mutants, hyper producer of thermostable α-amylases, with desired attributes is the need of the time.ResultsA process has been developed to improve super Koji (A. oryzae cmc1) strain through γ-rays treatment. The doses i.e. 0.60, 0.80, 1.00, 1.20 & 1.40 KGy gave more than 3.0 log kill. Initially, 52 Koji mutants resistant to 1% (w/v) Triton X-100 were selected. 2nd screening was based on α-amylases hyper production and 23 mutants were sorted out by measuring clearing zones index (CI). Afterwards nine potent mutants, resistant to 2-deoxy D-glucose, were screened based on CI. These were further analyzed for thermal stability and productivity of α-amylase under submerged conditions. The mutants' M-80(10), M-100(6) & M-120(5) gave about four fold increases in α-amylases productivity. The half life of M-100(6) α-amylase at 55 °C was 52 min and was highest among the mutants. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that mutants did not produce aflatoxins. Field Emission Scanning Electron Microscopy (FESEM) of Koji mycelia depicted that exposure to gamma rays increased rigidity of the mycelium. The potent Koji mutant M-100(6) was grown on soluble starch in 10L fermenter and produced 40.0 IU ml-1 of α-amylases with specific activity of 2461 IU mg-1 protein. Growth kinetic parameters were: μ = Specific growth rate= 0.069 h-1, td = Biomass doubling time= 10.0 h, Yp/x = Product yield coefficient with respect to cell mass = 482 U g-1; qp= Specific rate of product formation= 33.29 U g-1 h-1.ConclusionIt was concluded that the developed five step screening process has great potential to generate potent mutants for the hyper production of thermostable enzymes through γ-rays mediated physical mutagenesis. The developed thermostable α-amylases of super Koji mutantM-100(6) has immense potential for application in saccharification process for maltose syrup production. Moreover, the developed five step strain's development process may be used for the simultaneous improvement in productivity and thermal stability of other microbial enzymes.

Dataset Information

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

Publications

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

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