Isolation of sake yeast strains from Ariake Sea tidal flats and evaluation of their brewing characteristics.

Screening for new sake yeasts can expand the sensory diversity of sake, due to their production of metabolites that characterize sake's aroma and taste. In this study, mud from tidal flats in the Ariake Sea was screened for Saccharomyces cerevisiae strains with ethanol productivity suitable for sake brewing, and the brewing characteristics of isolated strains were evaluated. Five strains (H1-1, H1-2, H1-3, H3-1, and H3-2) classified as S. cerevisiae were isolated. Karyotype analysis by pulsed-field gel electrophoresis showed that five isolated strains were closely related to sake yeast strains (K7, K701, K9, K901, and Y52) instead of laboratory yeast strain. Results of small-scale brewing tests including sake yeast strains K701, K901, and Y52 showed that the five isolated strains have fermentation activity comparable to sake yeast strains. Principal component analysis (PCA) revealed that the five isolated strains produce higher levels of ethyl caproate and lower levels of acidic compounds than sake yeasts. In addition, isolated strains H3-1 and H3-2 produce higher levels of isoamyl acetate and lower levels of acetic acid than other isolated strains. Consequently, five S. cerevisiae strains that have high fermentation activity and differ from common sake yeast strains in terms of brewing characteristics were successfully isolated from the Ariake Sea.

Development of Monascus purpureus monacolin K-hyperproducing mutant strains by synchrotron light irradiation and their comparative genome analysis.

Monascus purpureus have been used for making koji and other fermented foods and supplements. M. purpureus characteristically produces monacolin K (MK), a secondary metabolite that competitively inhibits cholesterol synthesis. Synchrotron light irradiation was applied to induce mutation in the strain KUPM5 to improve the MK-producing ability of M. purpureus strain KUPM5. Screening by a bioassay utilizing sensitivities to yeast Saccharomyces cerevisiae from 936 colonies allows isolating three mutant strains: SC01, SC02, and SC03. These mutant strains and the parental strain KUPM5 were subjected to make koji using rice, and their metabolites were compared. All strains SC01, SC02, and SC03 in koji showed higher production of MK than the strain KUPM5. Particularly, the SC02 strain produced MK threefold higher than KUPM5 and maintained the production capabilities of other metabolites, including red, yellow, and orange pigments, mycelial contents, and α-amylase activity comparable to those of the strain KUPM5. Comparative genome analysis among strain KUPM5 and the mutants revealed that synchrotron light irradiation introduced mutations in approximately 90% of the total genes, including SNV, MNV, and indel mutations. The frequencies of SNV substitution in the whole genome occupied 68.96% of all mutations, of which 92.38% were transversions and 7.62% were transitions. This study, therefore, proved the synchrotron light irradiation was highly efficient for the strain improvement of a filamentous fungus, M. purpureus, and provided insights into the properties of mutation in the fungus by this mutagen.

The genome of the diatom Chaetoceros tenuissimus carries an ancient integrated fragment of an extant virus.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

Breeding sake yeast and identification of mutation patterns by synchrotron light irradiation.

Sake yeast is one of the important factors that characterize the aroma and taste of sake. To obtain sake yeast strains with different metabolic capabilities from other strains, breeding of a sake yeast is an effective way. In this study, sake yeast strain Y5201 was mutagenized by synchrotron light irradiation to obtain the mutant strains showing different brewing characteristics from parental strain Y5201, and comparative genome analysis between strain Y5201 and mutant strains was performed to identify mutation points and patterns induced by synchrotron light irradiation. Screening with the drug-resistant and fermentation tests selected the nine mutants (C18, C19, C29, C50, C51, C52, C54, T25, and T49) from the mutagenized Y5201 cells. Principal component analysis results based on the analysis of the small-scale brewing test metabolites showed that the mutant strain C19 was different from other strains, which had higher productivity of ethyl caproate and isoamyl acetate than those of the Y5201. Comparative genome analysis revealed that mutants by synchrotron light irradiation had a higher diversity of single nucleotide substitutions and a higher frequency of Indel (insertion/deletion) in these DNA than ethyl methanesulfonate and UV irradiation. These results suggest that synchrotron light irradiation is an effective and unique mutagen for yeast breeding.

Induction of mutation in Monascus purpureus isolated from Thai fermented food to develop low citrinin-producing strain for application in the red koji industry.

Red koji is produced from cultivating rice with Monascus strains that contain various types of fungal secondary metabolites, such as red pigments and monacolin K. Monascus strain also produces citrinin-a mycotoxin. In this study, Monascus purpureus KUPM5 isolated from the Thai fermented food, sufu, was mutagenized to reduce its citrinin production using UV irradiation, NTG treatment, and a combination of UV and NTG. Screening of the mutants using plate bioassay based on the inhibitory effect against Bacillus subtilis enables the selection of 10 mutants. The mutant strains KS301U and KS302U showed an 80% reduction in citrinin production in red koji compared with the wild type (wt), and maintained the ability to produce red pigments similar to the wild type. Activities of enzymes, α-amylase, protease, and lipase, from red koji extract produced by the mutant strain KS302U, were higher than those of the wt, whereas those of the mutant strain KS301U were similar to those of the wt. Consequently, strains KS301U and KS302U were successfully selected as strains suitable for producing red koji and fermented food.