Development of sake yeast haploid set with diverse brewing properties using sake yeast strain Hiroshima no. 6 exhibiting sexual reproduction.

Among sake yeast strains, Kyokai no. 7 (K7) and its closely related strains (K7 group) are predominantly used because of their excellent brewing properties. In the sake industrial sector, the need for various types of yeast strains is high. Although crossbreeding is an effective method for generating genetic diversity that should result in diverse characteristics, most K7 group strains lack normal sporulation ability, including the ability to undergo meiotic chromosomal recombination, which leads to difficulties in crossbreeding. Accordingly, the improvement of sake yeast strains primarily depends on mutagenesis and suitable selection in a stepwise manner. Our recent study revealed that the long-preserved sake yeast strain Hiroshima no. 6 (H6) does not belong to the K7 group despite genetically being extremely similar. In addition, H6 exhibited normal sporulation. Thus, we isolated haploid cells from H6 and mated them with previously isolated haploid cells of K7 group strains. The crossbred diploid strains had normal sporulation ability; hence, we performed tetrad analysis. The brewing characteristics of the obtained haploid set were extremely diverse. Principal component analysis based on the volatile and organic acid components measured using small-scale sake brewing tests revealed that the haploid strains derived from each diploid strain displayed a characteristic distribution. Thus, we demonstrated the availability of genetic crossbreeding using H6 with sporulation ability to facilitate both the development of novel sake yeast strains with many desirable characteristics and analyses of the function of sake yeast.

Characteristic analysis of the fermentation and sporulation properties of the traditional sake yeast strain Hiroshima no.6.

General sake yeasts (e.g., Kyokai no.7, K7) show high fermentation ability and low sporulation frequency. Former is related to stress-response defect due to the loss-of-function of MSN4 and RIM15. Later is mainly caused by low IME1 expression, leading to difficulty in breeding and genetic analysis. Sake yeast Hiroshima no.6 (H6), which had been applied for sake fermentation, has sporulation ability. However, its detailed properties have not been unveiled. Here we present that the fermentation ability of H6 is suitable for sake brewing, and the precursor of dimethyl trisulfide in sake from H6 is low. MSN4 but not RIM15 of H6 has the same mutation as K7. Our phylogenetic analysis indicated that H6 is closely related to the K7 group. Unlike K7, H6 showed normal sporulation frequency in a partially RIM15-dependent manner, and IME1 in H6 was expressed. H6 possesses excellent properties as a partner strain for breeding by crossing.

A region corresponding to second aspartate-rich motif in tryptophan isoprenylating enzyme, ComQ, serves as a substrate-binding site.

Posttranslational isoprenylation of a tryptophan residue identified from Bacillus quorum sensing pheromone, ComX pheromone, is unique and essential for the bioactivity. A modifying enzyme, ComQ, forms ComX pheromone from the ComX precursor and isoprenyl pyrophosphate and exhibits moderate similarity to isoprenyl pyrophosphate synthases. We investigated non-conserved region in ComQ, corresponding to isopentenyl pyrophosphate binding region of the synthases, using in vitro cell-free isoprenylation. These results suggested that the only conserved aspartic acid residue in the region of ComQ is critical for enzyme activity and responsible for ComX binding. Our findings should contribute to basic understanding of the mechanism of tryptophan isoprenylation.

Phage-displayed peptide library screening for preferred human substrate peptide sequences for transglutaminase 7.

Transglutaminases are a family of enzymes that catalyze cross-linking reactions between proteins. Among the members, there is currently no information regarding the substrate preferences of transglutaminase 7 (TG7), that would clarify its physiological significance. We previously obtained several highly reactive substrate peptide sequences of transglutaminases from a random peptide library. In this study, we screened for preferred substrate sequences for TG7 from a phage-displayed 12-mer peptide library. The most preferred sequence was selected based on reactivity and isozyme specificity. We firstly exhibited the tendency for the preference of substrate sequence for TG7. Then, using the most efficient peptide, Z3S, we established an in vitro assay system to assess enzymatic activity of TG7.

Identification of a highly reactive substrate peptide for transglutaminase 6 and its use in detecting transglutaminase activity in the skin epidermis.

Mammalian transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent crosslinks between glutamine and lysine residues in proteins. These catalytic reactions play roles in several essential biological processes, including blood coagulation, skin formation, and stabilization of the extracellular matrix. Among the members of this family, factor XIII and TGs 1-5 have been characterized well, but very little is known about the novel members TG6 and TG7. Recently, however, autoantibodies against TG6 were found in a patient with gluten ataxia, a disease caused by enzymatically modified gluten-derived peptides in neuronal cells. To characterize the possible physiological functions of TG6, in this study we screened a phage-displayed random peptide library to find highly reactive glutamine donor substrate peptides. From several candidate peptides, one sequence, designated Y25, appeared to have the highest reactivity. The Y25 sequence also has apparent isozyme specificity when evaluated by incorporation of the labeled glutamine acceptor substrate as a fusion protein with glutathione-S-transferase. Also, the sequence retained high reactivity as well as the isozyme specificity in the peptide form. Analyses with the biotin-labeled and fluorescence-labeled peptides showed TG6 to be an active enzyme and react to specific substrates in the skin, which is consistent with the results of the expression pattern of its transcripts.