SUMMARY The coordination of multiple signaling pathways across different compartments of the endomembrane system is essential for cellular adaptation to the environment; however, the underlying mechanisms remain poorly understood. Previous studies have described the existence of sterol-enriched liquid-ordered (Lo) domains in the vacuole membrane of yeast, which act as platforms for signal initiation. Here, we demonstrate that vacuolar Lo domains serve as signaling platforms for the evolutionarily conserved Target of Rapamycin Complex 1 (TORC1) kinase complex. Notably, we show that TORC2, which is functionally distinct from TORC1 and localized to the plasma membrane, regulates TORC1 activity through sphingolipid metabolism and subsequent vacuolar Lo domain formation. Collectively, our findings reveal a lipid domain-mediated network that integrates two signaling pathways originating from spatially different cellular sites. HIGHLIGHTS Decreased sphingolipid levels cause characteristic phenotypes that reflect low TORC1 activity. Sphingolipid-enriched vacuolar Lo domains are required for the EGO complex-mediated activation of TORC1. Sphingolipids integrate TORC2 and TORC1 through vacuolar Lo domain formation. Vacuole fusion and acidification control TORC1 activity via sphingolipid metabolism.
This study analyzed the aroma of Pilsner-type beer by gas chromatography-olfactometry (GCO), quantified the concentrations of 76 identified odorants, and reconstituted the aroma in an ethanol/water matrix. Only nine odorants exceeded their sensory thresholds, and a 25-odorant model selected by high odor activity values failed to adequately reproduce malty/estery notes, overall aroma intensity, and similarity to the reference beer. Reconstitution with all 76 odorants demonstrated that numerous trace components, including sub-threshold odorants, act synergistically to shape the backbone of beer aroma. We further examined food-beverage compatibility at a component level using real samples. Sensory tests showed that a fish-derived metallic note emerged when beer was paired with dried squid, whereas it was suppressed with sake. The causative compound was identified as (Z)-1,5-octadien-3-one. A highly sensitive method (<1 ng L) combining SBSE extraction, PFBOA derivatization, and NCI-GC/MS/MS was established; the formation of (Z)-1,5-octadien-3-one in sake was one-seventh in beer, providing the first component-level evidence for sake's favorable food pairing.
Sake, a traditional Japanese alcoholic beverage, is produced from rice and water throughout (and increasingly outside of) Japan. Hence, methods of identifying the geographic origin of sake are urgently required. Herein, we characterized a series of sakes and the corresponding raw materials (brewing water and rice) in terms of their oxygen stable isotopic compositions (δ18O values) and contents of 22 elements, aiming to identify brewing water–specific elements suitable for determining the geographic origin of sake. Based on the results of small-scale brewing tests and correlation analyses, eight brewing water–specific elements (Li, Na, Mg, Si, Ca, V, Sr, and Ba) were identified. The contents of these elements in sake and its δ18O values showed significant positive correlations with the corresponding parameters of brewing water but not rice. Preliminary principal component analysis, multinomial logistic regression analysis, partial least squares discriminant analysis, and sparse partial least squares discriminant analysis were conducted using the contents of these eight elements in sake. For three sake production areas in Japan, the scores of discrimination analyses obtained for the eight water-specific elements were separated, and model accuracies were high. The results suggested that these contents may be used to discriminate between sake production areas and improve the accuracy of existing methods for determining the geographic origin of Japanese sake.
Sake yeast Kyokai no. 11 (K11) is an ethanol-tolerant mutant of Kyokai no. 7 (K7) and produces a higher ethanol concentration in the sake mash than K7. A previous study revealed that stress-induced genes under the control of STRE elements were upregulated in K11. To elucidate the causal mutation responsible for ethanol tolerance, we compared the genome sequences of the ethanol-tolerant mutants (K11 and K7AT2, a newly isolated ethanol-tolerant mutant of K7) with that of their parental strain, K7. We identified a shared loss of heterozygosity region in the left arm of chromosome X in both mutants. We focused on CYR1 in this region, as it encodes adenylate cyclase, which negatively regulates expression of STRE-regulated genes through the upregulation of protein kinase A. Nucleotide 2066 of CYR1 was changed from G/A (amino acids Arg/His) in K7 to A/A (amino acids His/His) in K11 and K7AT2. When the plasmid containing CYR12066G was introduced into K11 or K7AT2, the stress tolerance of the transformants decreased to the level of K7, whereas the introduction of CYR12066A had a minimal effect. Consistently, disruption of the CYR12066G allele in K7 increased stress tolerance, whereas disruption of CYR12066A decreased stress tolerance. Furthermore, when CYR1 in a laboratory haploid strain was disrupted and either the CYR12042A or CYR12042G allele of S288C (corresponding to K7CYR12066) was introduced into the disruptant, the transformants with CYR12042A showed higher stress tolerance than those with CYR12042G. We concluded that the CYR1G2066A mutation was responsible for ethanol tolerance in K11.
The thermostable proteases present in soy sauce can degrade proteins in processed foods, such as boiled eggs and fish cakes, leading to undesirable textural changes in the product. In this study, we identified DeuA, a deuterolysin-like metalloprotease from Aspergillus oryzae, as the major contributor to thermostable protease activity during soy sauce fermentation. Using CRISPR/Cas9-based genome co-editing, we generated deuA-knockout mutants and mutants of the related gene deuB, which encodes a deuterolysin-like metalloprotease, and evaluated their enzymatic activity levels under solid-state culture conditions mimicking soy sauce koji fermentation. The ΔpyrGΔdeuA strain exhibited a marked reduction in thermostable protease activity, with residual activity barely detectable in both the koji extracts and the final soy sauce, whereas knockout of deuB had no significant effect. These results indicate that DeuA is the predominant contributor to thermostable protease activity in soy sauce. The knockout of deuA did not affect other key brewing parameters such as the nitrogen or sugar contents, indicating the potential of this gene as a target for strain improvement. Our findings establish DeuA as an essential thermostable protease in soy sauce and provide a foundation for the development of brewing strains with improved industrial applicability that will not affect the textural stability of processed foods.
