Integrated genomics and phenotype microarray analysis of Saccharomyces cerevisiae industrial strains for rice wine fermentation and recombinant protein production.

The industrial potential of Saccharomyces cerevisiae has extended beyond its traditional use in fermentation to various applications, including recombinant protein production. Herein, comparative genomics was performed with three industrial S. cerevisiae strains and revealed a heterozygous diploid genome for the 98-5 and KSD-YC strains (exploited for rice wine fermentation) and a haploid genome for strain Y2805 (used for recombinant protein production). Phylogenomic analysis indicated that Y2805 was closely associated with the reference strain S288C, whereas KSD-YC and 98-5 were grouped with Asian and European wine strains, respectively. Particularly, a single nucleotide polymorphism (SNP) in FDC1, involved in the biosynthesis of 4-vinylguaiacol (4-VG, a phenolic compound with a clove-like aroma), was found in KSD-YC, consistent with its lack of 4-VG production. Phenotype microarray (PM) analysis showed that KSD-YC and 98-5 displayed broader substrate utilization than S288C and Y2805. The SNPs detected by genome comparison were mapped to the genes responsible for the observed phenotypic differences. In addition, detailed information on the structural organization of Y2805 selection markers was validated by Sanger sequencing. Integrated genomics and PM analysis elucidated the evolutionary history and genetic diversity of industrial S. cerevisiae strains, providing a platform to improve fermentation processes and genetic manipulation.

A practical comparison of the next-generation sequencing platform and assemblers using yeast genome.

Assembling fragmented whole-genomic information from the sequencing data is an inevitable process for further genome-wide research. However, it is intricate to select the appropriate assembly pipeline for unknown species because of the species-specific genomic properties. Therefore, our study focused on relatively more static proclivities of sequencing platforms and assembly algorithms than the fickle genome sequences. A total of 212 draft and polished de novo assemblies were constructed under the different sequencing platforms and assembly algorithms with the repetitive yeast genome. Our comprehensive data indicated that sequencing reads from Oxford Nanopore with R7.3 flow cells generated more continuous assemblies than those derived from the PacBio Sequel, although the homopolymer-based assembly errors and chimeric contigs exist. In addition, the comparison between two second-generation sequencing platforms showed that Illumina NovaSeq 6000 provides more accurate and continuous assembly in the second-generation-sequencing-first pipeline, but MGI DNBSEQ-T7 provides a cheap and accurate read in the polishing process. Furthermore, our insight into the relationship among the computational time, read length, and coverage depth provided clues to the optimal pipelines of yeast assembly.

Genomic and functional features of yeast species in Korean traditional fermented alcoholic beverage and soybean products.

In this review, we describe the genomic and physiological features of the yeast species predominantly isolated from Nuruk, a starter for traditional Korean rice wines, and Jang, a traditional Korean fermented soy product. Nuruk and Jang have several prevalent yeast species, including Saccharomycopsis fibuligera, Hyphopichia burtonii, and Debaryomyces hansenii complex, which belong to the CUG clade showing high osmotic tolerance. Comparative genomics revealed that the interspecies hybridization within yeast species for generating heterozygous diploid genomes occurs frequently as an evolutional strategy in the fermentation environment of Nuruk and Jang. Through gene inventory analysis based on the high-quality reference genome of S. fibuligera, new genes involved in cellulose degradation and volatile aroma biosynthesis and applicable to the production of novel valuable enzymes and chemicals can be discovered. The integrated genomic and transcriptomic analysis of Hyphopichia yeasts, which exhibit strong halotolerance, provides insights into the novel mechanisms of salt and osmo-stress tolerance for survival in fermentation environments with a low-water activity and high-concentration salts. In addition, Jang yeast isolates, such as D. hansenii, show probiotic potential for the industrial application of yeast species beyond fermentation starters to diverse human health sectors.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Metabolic Features of Ganjang (a Korean Traditional Soy Sauce) Fermentation Revealed by Genome-Centered Metatranscriptomics.

The taste and quality of soy sauce, a fermented liquid condiment popular worldwide, is greatly influenced by microbial metabolism during fermentation. To investigate the fermentative features of ganjang (a Korean traditional soy sauce), ganjang batches using meju (fermented soybean) bricks and solar salts were prepared, and organic compounds, microbial communities, metagenomes, and metatranscriptomes of ganjang were quantitively analyzed during fermentation. Polymeric compound analysis in the ganjang treated with/without microbial inhibitors revealed that indigenous enzymes of meju bricks might be primarily responsible for degrading polymeric compounds. Through metagenome binning and microbe sequencing, 17 high-quality genome sequences representing all major ganjang microbiota were obtained, and their transcriptional expressions were quantitatively analyzed by mapping metatranscriptome reads normalized by spike-in RNA sequencing to the 17 genomes, which revealed that microbial metabolism might primarily occur while meju bricks are in the ganjang solution and decrease significantly after the removal of meju bricks. Metabolic pathways for carbohydrates, proteins, and lipids of the major ganjang microbiota were reconstructed, and their metabolic genes were transcriptionally analyzed, revealing that facultative lactic acid fermentation by Tetragenococcus was the major fermentation process active in the ganjang fermentation and that aerobic respiration by facultatively aerobic bacteria such as Chromohalobacter, Halomonas, and Marinobacter was also an important metabolic process during fermentation. Although the abundances of Fungi and the corresponding transcriptional expression levels were generally much lower than those of Bacteria, our analysis suggests that yeasts such as Debaryomyces and Wickerhamomyces might be in large part responsible for producing biogenic amines and flavors. IMPORTANCE The taste and quality of soy sauce, a popular fermented liquid condiment worldwide, is greatly influenced by microbial metabolism during fermentation. Spontaneous fermentation of ganjang (a Korean traditional soy sauce) in a nonsterile environment leads to the growth of diverse bacteria and fungi during fermentation, making it difficult to understand the mechanism of ganjang fermentation. Genome-centered metatranscriptomic analysis, combined with organic compound analysis, quantitative metagenome and metatranscriptome analyses, and metabolic pathway reconstruction and expressional analysis of the major ganjang microbiota during fermentation, would provide comprehensive insights into the metabolic features of ganjang fermentation.

Short bZIP homologue of sulfur regulator Met4 from Ogataea parapolymorpha does not depend on DNA-binding cofactors for activating genes in sulfur starvation.

The acquisition of sulfur from environment and its assimilation is essential for fungal growth and activities. Here, we describe novel features of the regulatory network of sulfur metabolism in Ogataea parapolymorpha, a thermotolerant methylotrophic yeast with high resistance to harsh environmental conditions. A short bZIP protein (OpMet4p) of O. parapolymorpha, displaying the combined structural characteristics of yeast and filamentous fungal Met4 homologues, plays a key role as a master regulator of cell homeostasis during sulfur limitation, but also its function is required for the tolerance of various stresses. Domain swapping analysis, combined with deletion analysis of the regulatory domains and genes encoding OpCbf1p, OpMet28p, and OpMet32p, indicated that OpMet4p does not require the interaction with these DNA-binding cofactors to induce the expression of sulfur genes, unlike the Saccharomyces cerevisiae Met4p. ChIP analysis confirmed the notion that OpMet4p, which contains a canonical bZIP domain, can bind the target DNA in the absence of cofactors, similar to homologues in other filamentous fungi. Collectively, the identified unique features of the O. parapolymorpha regulatory network, as the first report on the sulfur regulation by a short yeast Met4 homologue, provide insights into conservation and divergence of the sulfur regulatory networks among diverse ascomycetous fungi.

Correlation between serum homocysteine concentration and severity of mitral valve disease in dogs.

OBJECTIVE To measure serum homocysteine concentrations in dogs with myxomatous mitral valve disease (MMVD) and identify any association between this variable and stage of MMVD. ANIMALS 53 client-owned dogs with MMVD and 10 healthy control Beagles. PROCEDURES Dogs with MMVD were allocated to 3 groups in accordance with the staging system for chronic valvular heart disease in dogs and cats of the American College of Veterinary Internal Medicine. Blood samples were collected from all dogs, and serum homocysteine and cardiac troponin 1 concentrations were measured by enzyme immunoassay and chemiluminescence immunoassay, respectively. Analyte values were tested for associations with each other and with stage of MMVD. RESULTS A significant correlation was identified between serum homocysteine concentration and stage of MMVD. Mean ± SD concentrations were 6.72 ± 1.65 µmol/L for control dogs, 13.37 ± 4.16 µmol/L for dogs with stage B MMVD, 18.86 ± 6.73 µmol/L for dogs with stage C disease, and 28.26 ± 4.48 µmol/L for dogs with stage D disease. In addition, serum homocysteine concentration was correlated with serum cardiac troponin 1 (r = 0.34) and creatinine (r = 0.46) concentrations, systolic blood pressure (r = 0.57), and left atrium-to-aortic root ratio (r = 0.28), all of which were positively correlated with stage of MMVD. CONCLUSIONS AND CLINICAL RELEVANCE Serum homocysteine concentrations of dogs with MMVD were significantly higher than those of control dogs, and significant correlations were identified between these values and several risk factors for heart failure. Measurement of serum homocysteine concentration may be useful in the prediction of severity of disease in dogs with MMVD.