Rice grain structural characteristics of sake rice cultivar Hakutsurunishiki for daiginjo-shu brewing.

Hakutsurunishiki is a sake rice cultivar bred using Yamadabo (seed parent) and Wataribune 2 (pollen parent), equivalent to a Yamadanishiki sibling. This study evaluated the structural characteristics of the Hakutsurunishiki rice grain that contribute to the brewing characteristics of daiginjo-shu, via a comparison with Yamadanishiki. Hakutsurunishiki brown rice was a little heavy and had a large white core. Observing a cross-section of white rice after soaking revealed that the rice grain structure of Hakutsurunishiki was different from that of Yamadanishiki. Hakutsurunishiki white rice showed fewer voids than Yamadanishiki, promoting a slower water absorption rate. Glucose distribution in rice koji obtained by mass spectrometry imaging showed that Hakutsurunishiki rice koji, like Yamadanishiki, is tsuki-haze type, suggesting that its grain structure is suitable for making rice koji for daiginjo-shu. With these observations, we were able to clarify the structural characteristics of Hakutsurunishiki rice grain.

Artificial turn-on riboswitch to control target gene expression using a wild-type riboswitch splicing mechanism.

The thiamine pyrophospate (TPP)-dependent thiA riboswitch in Aspergillus oryzae regulates alternative mRNA splicing via TPP-riboswitch binding to reduce protein production. Based on the sequences involved in alternative splicing found in Neurospora crassa, we identified unique sequences that are thought to play a role in the alternative splicing of the thiA riboswitch. Based on the mechanism of alternative splicing regulated by the thiA riboswitch, we constructed a new TPP-dependent artificial riboswitch, which unlike the wild-type riboswitch, promotes, rather than inhibits, gene expression. We found that a target gene controlled by this turn-on artificial riboswitch can be sufficiently expressed for practical use in A. oryzae. The artificial riboswitch upregulates the target genes via TPP and can be applied as a practical tool for gene regulation.

HAP-01, the first chromogenic substrate for Aspergillus oryzae acid protease.

The first chromogenic substrate for Aspergillus oryzae acid protease, 'HAP-01', was successfully developed after evaluating the substrate specificity of the enzyme. Furthermore, with HAP-01, digestion-triggered chromophore release was employed as a novel chromogenic technique.

Analyzing the relationship between the inorganic element profile of sake dilution water and dimethyl trisulfide formation using multi-element profiling.

Dimethyl trisulfide (DMTS) is the main component of hineka, an off-flavor generated in sake during storage. Genshu, or undiluted sake, is usually diluted with water during warimizu, the process of adjusting the alcohol content of sake. In this study, we evaluated how the inorganic element composition of sake dilution water affects the DMTS-producing potential of the sake (DMTS-pp, determined as the DMTS concentration in sake stored at 70°C for 1 week after dilution) using inductively coupled plasma-mass spectrometry (ICP-MS). Partial least squares (PLS) regression analysis was conducted with the ICP-MS data as the explanatory variable and DMTS-pp as the response variable, and the selection of inorganic elements for the construction of the PLS model was performed using variable importance in projection scores. The findings confirmed that some of the compounds containing the inorganic elements extracted from the PLS regression analysis contribute to DMTS-pp.

Construction of a thiamine pyrophosphate high-producing strain of Aspergillus oryzae by overexpression of three genes involved in thiamine biosynthesis.

We have found a gene (thiP) encoding thiamine pyrophosphokinase (TPK) in the Aspergillus oryzae genome. No riboswitch-like region was found in the upstream region of thiP, although it was repressed probably by thiamine pyrophosphate (TPP) as well as thiA and nmtA, which are strictly regulated by TPP-riboswitch sequence. To improve the productivity of TPP in A. oryzae, we constructed the strain in which thiA, nmtA and thiP were overexpressed simultaneously. The resulting strain accumulated intracellular TPP 4-fold higher than did the control strain.

Roles of Mg2+ in TPP-dependent riboswitch.

We quantified the effect of Mg(2+) on thiamine pyrophosphate (TPP) binding to TPP-dependent thiA riboswitch RNA. The association constant of TPP binding to the riboswitch at 20 degrees C increased from 1.2 x 10(6) to 50 x 10(6) M(-1) as the Mg(2+) concentration increased from 0 to 1 mM. Furthermore, circular dichroic spectra under various conditions showed that 1 mM Mg(2+) induced a local structural change of the riboswitch, which might be pivotal for TPP binding. These results indicate that a physiological concentration of Mg(2+) can regulate TPP binding to the thiA riboswitch.

Thiamine-regulated gene expression of Aspergillus oryzae thiA requires splicing of the intron containing a riboswitch-like domain in the 5'-UTR.

Exogenous thiamine regulates Aspergillus oryzae thiA, which is involved in thiamine synthesis. One of the two introns in its 5'-untranslated region (5'-UTR) contains motifs (regions A and B) highly conserved among fungal thiamine biosynthesis genes. Deletion of either region relieved the repression by thiamine and thiamine inhibited intron splicing, suggesting that regions A and B are required for efficient splicing. Furthermore, transcript splicing was essential for thiA gene expression. These observations suggest a novel gene expression regulatory mechanism in filamentous fungi, in which exogenous thiamine controls intron splicing to regulate gene expression. Interestingly, regions A and B constitute a part of a thiamine pyrophosphate-binding riboswitch-like domain that has been quite recently found in the 5'-UTR of thiA.

Molecular breeding of the Mureka-non-forming sake koji mold from Aspergillus oryzae by the disruption of the mreA gene.

Mureka-non-forming sake koji molds were constructed from an Aspergillus oryzae industrial strain by the disruption of the mreA gene using a host-vector system with the ptrA gene as a dominant selectable marker. All of the mreA gene disruptants obtained retained the advantages of the host strain in terms of the brewing characteristics, while their isoamyl alcohol oxidase (IAAOD) activities were significantly lower than that of the host strain. Sake brewing was successfully carried out using the koji prepared with the disruptants, followed by storage of the resultant non-pasteurized sake (nama-shu). The isovaleraldehyde (i-Val) concentration in the sake brewed the host strain increased rapidly and reached the threshold values for mureka, 1.8 ppm and 2.6 ppm after storage at 20 degrees C for 42 d and 63 d, respectively, while those of the disruptants were less than 0.5 ppm even after storage at 20 degrees C or 30 degrees C for 63 d. In the sensory evaluation of the sake stored at 20 degrees C or 30 degrees C for 63 d, all members of the panel recognized the strong mureka flavor of the sake brewed with the host strain, while they did not detect this flavor in the sake brewed with the disruptants. Thus, we concluded that the mreA gene disruptants can be used for the production of sake in which mureka is not formed.

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae.

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.