Disruption of YCP4 enhances freeze-thaw tolerance in Saccharomyces cerevisiae.

OBJECTIVE: This study aimed to identify genes related to freeze-thaw tolerance and elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. RESULTS: In this study, one tolerant strain exposed to freeze-thaw stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be YCP4. In addition, this phenotype related to freeze-thaw tolerance was confirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed a freeze-thaw tolerance by reducing the intracellular level of reactive oxygen species and the activation of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12. CONCLUSIONS: Disruption of YCP4 in S. cerevisiae results in increased tolerance to freeze-thaw stress.

Antarctic yeasts: analysis of their freeze-thaw tolerance and production of antifreeze proteins, fatty acids and ergosterol.

BACKGROUND: Microorganisms have evolved a number of mechanisms to thrive in cold environments, including the production of antifreeze proteins, high levels of polyunsaturated fatty acids, and ergosterol. In this work, several yeast species isolated from Antarctica were analyzed with respect to their freeze-thaw tolerance and production of the three abovementioned compounds, which may also have economic importance. RESULTS: The freeze-thaw tolerance of yeasts was widely variable among species, and a clear correlation with the production of any of the abovementioned compounds was not observed. Antifreeze proteins that were partially purified from Goffeauzyma gastrica maintained their antifreeze activities after several freeze-thaw cycles. A relatively high volumetric production of ergosterol was observed in the yeasts Vishniacozyma victoriae, G. gastrica and Leucosporidium creatinivorum, i.e., 19, 19 and 16 mg l- 1, respectively. In addition, a high percentage of linoleic acid with respect to total fatty acids was observed in V. victoriae (10%), Wickerhamomyces anomalus (12%) and G. gastrica (13%), and a high percentage of alpha linoleic acid was observed in L. creatinivorum (3.3%). CONCLUSIONS: Given these results, the abovementioned yeasts are good candidates to be evaluated for use in the production of antifreeze proteins, fatty acids, and ergosterol at the industrial scale.

Effect of Lactylated Gluten and Freeze-Thaw Cycles on Frozen Dough: From Water State and Microstructure.

The influence of lactylated gluten and Freeze-Thaw Cycles on the water state, microstructure, and quality of frozen steamed bread dough was investigated. After three freeze-thaw cycles (3F/T), the specific volume of steamed bread with sodium lactate-treated gluten increased by 18.34% compared with the blank group and 5.73% compared with the wheat gluten (WG) group. Compared with wheat gluten, the texture properties of steamed bread with lactylated gluten increased significantly. Changes in rheological properties demonstrated that the frozen dough's viscoelasticity increased significantly. The lactylated gluten could reduce water mobility and decrease the content of freezable water in frozen dough. Moreover, the free sulfhydryl (SH) content increased, revealing that the protein was depolymerized. Based on the microstructure and corresponding protein network analysis (PNA), the total area and the number of protein network connection points of the dough adding lactylated gluten were significantly higher than those of the blank group and the WG group. In conclusion, lactylated gluten enhanced the freeze-thaw tolerance of frozen dough.

Metabolomics analysis of freeze-thaw tolerance enhancement mechanism of ε-poly-l-lysine on industrial yeast.

Antimicrobial polycationic peptide ε-poly-l-lysine (ε-PL) enhanced the freeze-thaw tolerance of industrial yeast; the enhancement mechanism of ε-PL on yeast was studied. Results showed that a ε-PL coating was observed in ε-PL-treated yeast. After 4 times of freeze-thaw, the cell viability, glycerol content, and CO2 production of 0.6 mg/mL ε-PL-treated yeast were higher than those of untreated yeast, specifically, the cell viability of ε-PL-treated yeast was 87.6%, and that of untreated yeast was 68.5%. Metabolomic results showed that the enhancement mechanism of ε-PL on yeast was related to the promotion of cell membrane-related fatty acid synthesis pathways before freeze-thaw treatment, and the promotion of trehalose biosynthesis and glycerophospholipid metabolism pathways after freeze-thaw. Furthermore, ε-PL induced inhibition of the tricarboxylic acid cycle, resulting in a longer stationary phase at the beginning of the freeze-thaw and ultimately providing a higher level of freeze-thaw stress tolerance than untreated yeast.

Hsp104 contributes to freeze-thaw tolerance by maintaining proteasomal activity in a spore clone isolated from Shirakami kodama yeast.

The supply of oven-fresh bakery products to consumers has been improved by frozen dough technology; however, freeze-thaw stress decreases the activity of yeast cells. To breed better baker's yeasts for frozen dough, it is important to understand the factors affecting freeze-thaw stress tolerance in baker's yeast. We analyzed the stress response in IB1411, a spore clone from Saccharomyces cerevisiae Shirakami kodama yeast, with an exceptionally high tolerance to freeze-thaw stress. Genes encoding trehalose-6-phosphate synthase (TPS1), catalase (CTT1), and disaggregase (HSP104) were highly expressed in IB1411 cells even under conditions of non-stress. The expression of Hsp104 protein was also higher in IB1411 cells even under non-stress conditions. Deletion of HSP104 (hsp104Δ) in IB1411 cells reduced the activity of the ubiquitin-proteasome system (UPS). By monitoring the accumulation of aggregated proteins using the ΔssCPY*-GFP fusion protein under freeze-thaw stress or treatment with proteasomal inhibitor, we found that IB1411 cells resolved aggregated proteins faster than the hsp104Δ strain. Thus, Hsp104 seems to contribute to freeze-thaw tolerance by maintaining UPS activity via the disaggregation of aggregated proteins. Lastly, we found that the IB1411 cells maintained high leavening ability in frozen dough as compared with the parental strain, Shirakami kodama yeast, and thus will be useful for making bread.