Isolation of halophilic lactic acid bacteria possessing aspartate decarboxylase and application to fish sauce fermentation starter.

The aims of this study were to isolate halophilic lactic acid bacteria possessing aspartate decarboxylase and elucidate the property of the isolates as starter cultures for fish sauce fermentation. Seventy-four strains were isolated from fermented fish foods on aspartate indicator broth containing bromocresol purple, and all isolates were identified as Tetragenococcus halophilus and confirmed to possess the aspartate decarboxylase gene (aspD) by PCR amplification. The isolates were classified into 14 groups based on their aspD-encoding plasmid construction. Strains selected from each group and a control strain incapable of aspartate decarboxylation were inoculated into fish sauce mash as starter cultures. Isolated strains possessing aspD converted aspartate into alanine almost completely in the fish sauce mash. In addition, the strains prevented the accumulation of biogenic amines, as did the control strain, whereas various amines were accumulated in fish sauce mash without starter cultures. Sensory evaluation tests indicated that converting the sour amino acid aspartate into the sweet amino acid alanine made the fish sauce taste milder. In conclusion, the use of T. halophilus possessing aspartate decarboxylase as a fish sauce fermentation starter causes the conversion of aspartate to alanine, accompanied by taste alteration, and prevents biogenic amine accumulation in fish sauce products.

Can Interspecies Hybrid Zygosaccharomyces rouxii Produce an Allohaploid Gamete?

In soy sauce manufacturing, Candida versatilis plays a role in the production of volatile flavor compounds, such as volatile phenols, but limited accessible information on its genome has prevented further investigation regarding aroma production and breeding. Although the draft genome sequence data of two strains of C. versatilis have recently been reported, these strains are not similar to each other. Here, we reassess the draft genome sequence data for strain t-1, which was originally reported to be C. versatilis, and conclude that strain t-1 is most probably not C. versatilis but a gamete of hybrid Zygosaccharomyces rouxii Phylogenetic analysis of the D1/D2 region of the 26S ribosomal DNA (rDNA) sequence indicated that strain t-1 is more similar to the genus Zygosaccharomyces than to C. versatilis Moreover, we found that the genome of strain t-1 is composed of haploid genome content and divided into two regions that show approximately 100% identity with the T or P subgenome derived from the natural hybrid Zygosaccharomyces rouxii, such as NBRC110957 and NBRC1876. We also found a chromosome crossing-over signature in the scaffolds of strain t-1. These results suggest that strain t-1 is a gamete of the hybrid Z. rouxii, generated by either meiosis or chromosome loss following reciprocal translocation between the T and P subgenomes. Although it is unclear why strain t-1 was misidentified as C. versatilis, the genome of strain t-1 has broad implications for considering the evolutionary fate of an allodiploid.IMPORTANCE In yeast, crossing between different species sometimes leads to interspecies hybrids. The hybrid generally cannot produce viable spores because dissimilarity of parental genomes prevents normal chromosome segregation during meiotic division, leading to a dead end. Thus, only a few natural cases of homoploid hybrid speciation, which requires mating between 1n gametes of hybrids, have been described. However, a recent study provided strong evidence that homoploid hybrid speciation is initiated in natural populations of the budding yeast, suggesting the potential presence of viable 1n gametes of hybrids. The significance of our study is finding that the strain t-1, which had been misidentified as Candida versatilis, is a viable 1n gamete derived from hybrid Zygosaccharomyces rouxii.

Mechanism for Restoration of Fertility in Hybrid Zygosaccharomyces rouxii Generated by Interspecies Hybridization.

The mechanism of whole-genome duplication (WGD) in yeast has been intensively studied because it has a large impact on yeast evolution. WGD has shaped the genomic architecture of modern Saccharomyces cerevisiae; however, the mechanism for restoring fertility after interspecies hybridization, which would be involved in the process of WGD, has not been thoroughly elucidated. In this study, we obtained a draft genome sequence of the salt-tolerant yeast Zygosaccharomyces rouxii NBRC110957 and revealed that it is a hybrid lineage of Z. rouxii (allodiploid) with two subgenomes equivalent to NBRC1876. Because this allodiploid yeast can mate with other allodiploid strains and form spores, it can be a good model of restoring fertility after interspecies hybridization. We observed that NBRC110957 and NBRC1876 contain six mating-type-like (MTL) loci. There are no large deletions or deleterious mutations in MTL loci, except for several-base-pair deletions in the X region in certain MTL loci. We also assigned only one mating-type (MAT) locus that exclusively determines mating types from six MTL loci. These results suggest that it is possible to recover mating competence regardless of whether cells lose one MAT locus through random gene loss by mitotically dividing after interspecies hybridization. Moreover, we propose that perturbation of gene expression and substantial breakdown of MAT heterozygosity caused by chromosomal rearrangement at MTL loci play roles in restoring the mating competence of allodiploids. This scenario can provide a mechanism for restoring fertility after interspecies hybridization that is compatible with random gene loss models and suggests genomic plasticity during WGD in yeast.IMPORTANCE A whole-genome duplication occurred in an ancestor of the baker's yeast Saccharomyces cerevisiae The origins of this complex and multifaceted process, which requires intra- or interspecies hybridization followed by dysfunction of one mating-type (MAT) locus to regain mating competence, has not been thoroughly elucidated. In this study, we provide a mechanism for regaining fertility in an interspecies hybrid, Zygosaccharomyces rouxii The draft genome sequence analysis and mating test showed that the Z. rouxii strain used in this study is an intact interspecies hybrid, suggesting that it is possible to recover fertility regardless of whether cells lose one MAT locus.

Identification and characterization of an enzyme involved in the biosynthesis of the 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in yeast.

4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is considered a key flavor compound in soy sauce. The compound has a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. Here, we report the identification and characterization of an enzyme involved in the biosynthesis of HEMF in yeast. We fractionated yeast cell-free extract from Saccharomyces cerevisiae using column chromatography and partially purified a fraction with HEMF-forming activity. Peptide mass fingerprinting analysis showed that the partially purified fraction contains aldehyde reductase encoded by YNL134C. This reductase shares low sequence identity with enone oxidoreductase, which is responsible for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and HEMF in plants. YNL134C was expressed heterologously in Escherichia coli, and the purified protein catalyzed the formation of HEMF from the mixture of Maillard reaction products, acetaldehydes, and NADPH. Multicopy expression in S. cerevisiae resulted in increased HEMF productivity, and gene knockout of YNL134C in S. cerevisiae resulted in decreased HEMF productivity. These data suggest that the translation product of YNL134C is the HEMF-producing enzyme in yeast. Detailed analyses of an intermediate in the enzymatic reaction mixture revealed that HEMF is synthesized from (2E)-2-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone, which formed via Knoevenagel condensation between the acetaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone derived from the Maillard reaction based on ribose and glycine, by YNL134Cp in an NADPH dependent manner. Overall, this study shed light on the molecular basis for the improvement of soy sauce flavor and the biotechnological production of HEMF.