Masafumi Tokuoka

Genetic analysis of conidiation regulatory pathways in koji-mold Aspergillus oryzae.

Conidia of koji-mold Aspergillus oryzae are often used as starters in the fermented food industry. However, little is known about conidiation regulation in A. oryzae. To improve the productivity of conidia in A. oryzae, it is necessary to understand conidiation regulation in the strain. Therefore, we analyzed the conidiation regulatory system in A. oryzae using 10 kinds of conidiation regulatory gene disruptants. The phenotypes of AorfluG, AorflbA, AorflbB, AorflbC, AorflbD, AorflbE, AorbrlA, AorabaA, AorwetA, and AorfadA mutants are almost identical to those of the corresponding mutants in Aspergillus nidulans. The results indicated that the functions of conidiation regulatory genes are almost conserved between A. oryzae and A. nidulans. However, the severely reduced conidiation phenotype of the AorfluG disruptant in A. oryzae differs from the phenotype of the corresponding mutant in Aspergillus fumigatus in air-exposed culture conditions. These results suggest that A. oryzae, A. nidulans, and A. fumigatus have a G-protein signaling pathway and brlA orthologs in common, and only A. fumigatus has particular brlA activation pathways that are independent of the fluG ortholog. Furthermore, the analyses of AorflbA disruptant and AorfadA dominant-active mutants implicated that AorFadA-mediated G-protein signaling suppresses vegetative growth of A. oryzae.

Functional expression of the Aspergillus flavus PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid.

alpha-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavuscpaA under alpha-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS-NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module. This CpaA expression system provides us an ideal platform for PKS-NRPS functional analysis, such as adenylation domain selectivity and product releasing mechanism.

Genetic safeguard against mycotoxin cyclopiazonic acid production in Aspergillus oryzae.

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of the genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well‐known producer of aflatoxin. In contrast, the cyclopiazonic acid (CPA) biosynthetic gene (cpa) cluster in A. oryzae contains genes that have been lost in A. flavus. Through targeted gene inactivation, isolation of the corresponding metabolite, and evaluation of biological activity of the metabolite, we demonstrated that an A. oryzae‐specific gene—cpaH—mediates the conversion of CPA into the less toxic 2‐oxocyclopiazonic acid, a new analogue of CPA. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

Comprehensive analysis of dipeptides in alcoholic beverages by tag-based separation and determination using liquid chromatography/electrospray ionization tandem mass spectrometry and quadrupole-time-of-flight mass spectrometry.

Fermented foods and beverages contain several different types of dipeptides, which are believed to be important components for taste. To date, however, a method for the comprehensive analysis of dipeptides in these products has not yet been established. In this study, comprehensive analysis of dipeptides in alcoholic beverages was performed by a high-resolution separation method based on the structural characteristics of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized dipeptides as well as dipeptide quantification and structural estimation using ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and UHPLC-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS), respectively. Dipeptide content was found to differ considerably among Japanese sake, beer, and wine; UHPLC-MS/MS analysis revealed that many types of dipeptides are present in sake. Dipeptide quantification analysis identified 32 types of dipeptides within the concentration range of 1.1-97.2 μM in sake. The analysis was validated by dipeptide recovery of 64.0-107.2% (2.5 μM of standard) with a relative standard deviation of ≤33.2% from an actual alcoholic sample. Furthermore, UHPLC-Q-TOFMS analysis suggested the existence of more than 35 types of dipeptides in sake. Thus, by the combined analysis methods, we discovered that more than 60 dipeptides are present in sake. This research is the first report of dipeptide profiling of fermented alcoholic beverages by comprehensive analysis.

Simple metabolite extraction method for metabolic profiling of the solid-state fermentation of Aspergillus oryzae

Solid-state culture of microorganisms is an important style of culture both in the traditional food industry and in the modern fermentation industry. We propose here a simple method for metabolite extraction from the solid-state fermentation of a filamentous fungus, Aspergillus oryzae, which is known as rice-koji. To evaluate the efficiency of metabolite extraction, liquid chromatography-mass spectrometry (LC/MS) was used for simultaneous detection of a wide range of metabolites including amino acids, organic acids and vitamins, which are of interest in rice-koji making. Among eleven different metabolite extraction methods tested, we found that extraction using acetonitrile/water (50:50), coupled with boiling at 70 °C for 5 min, was advantageous in terms of both extraction yield and metabolism quenching. We consider that our method provides a technical basis for metabolome analysis of a solid-state fermentation.

The transfer of stable 133Cs from rice to Japanese sake

Possible contamination by radioactive cesium (Cs), released by the Fukushima Daiichi Nuclear Plant Accident in Japan on March 2011, has been a matter of concern with respect to Japanese sake made from rice grains cultivated in affected fields. In this study, the behavior of stable (133)Cs, which is a useful analogue for predicting the behavior of radioactive Cs, was investigated in the production of sake using rice grains harvested in Japan in 2010. The concentration of stable (133)Cs in the polished rice grains decreased gradually with decreasing milling ratios until a ratio of 70% was reached, and below that point, it did not change significantly. The (133)Cs concentration in the 70% polished rice was approximately 20% of that found in brown rice. The sake was brewed on a small scale using 70% polished rice, and the transfer of (133)Cs from rice to sake was examined. Approximately 30-40% of (133)Cs in the 70% polished rice was removed during the washing and the steeping of the rice grains, and approximately 40% of the (133)Cs in the 70% polished rice was transferred to the sake. If the radioactive Cs species behaves similarly, these results suggest that brown rice containing 100 Bq/kg radioactivity of Cs would generate 70% polished rice grains containing 20 Bq/kg and that the sake brewed from these grains would contain 3-5 Bq/kg.

Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis

Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.