芳香氨基酸转氨酶Aro9过表达增强酿酒酵母耐冻性能

Overexpression of aromatic amino acid aminotransferase Aro9 enhances freeze tolerance of Saccharomyces cerevisiae

摘要:

为提高酵母在冷冻环境的存活率，降低冷冻面团的品质劣变。本研究探究芳香氨基酸转氨酶（Aro9）在酵母抗冻的作用，以期提高其在冷冻胁迫下的存活率。通过构建aro9过表达菌株（LKF-1-Aro9）与aro9敲除菌株（LKF-1-ΔAro9），验证Aro9对酿酒酵母抗冻能力的影响。LKF-1在-20 ℃保存2、5、10和15 d存活率分别为60.76%、56.40%、13.76%、8.63%。LKF-1-Aro9对应为79.97%、67.03%、43.80%、29.30%。经3 d冻藏后，LKF-1，LKF-1-Aro9和LKF-1-ΔAro9到达平台期的时间分别为14 h，12 h，21 h。扫描电镜结果显示，在-20 ℃保存7 d后，LKF-1-Aro9的细胞表面结构保持更为完整。此外，在-20 ℃保存3 d后LKF-1和LKF-1-Aro9经碘化吡啶（PI）染色后荧光强度分别为4096.00，3336.67，在-20 ℃保存后LKF-1的细胞膜比LKF-1-Aro9受到更大的破坏。同时LKF-1和LKF-1-Aro9胞内脯氨酸分别为0.35和2.19 μg?mg-1。LKF-1-Aro9具有更多的脯氨酸对冷冻环境具有更好的耐受性。同时，LKF-1和LKF-1-Aro9的经结晶紫染色OD595分别为0.2424和0.2796，LKF-1-Aro9分泌更多的生物膜对抗外界环境。实验表明，aro9基因的过表达提高耐冻性作用与强化细胞膜和生物膜、促进保护性物质脯氨酸的积累密切相关。

Abstract:

To improve yeast survival under freezing conditions and reduce quality deterioration of frozen dough, this study investigated the role of aromatic amino acid aminotransferase (Aro9) in yeast cryotolerance, aiming to enhance survival under freezing stress. An aro9-overexpressing strain (LKF-1-Aro9) and an aro9 knockout strain (LKF-1-ΔAro9) were constructed to evaluate the effect of Aro9 on the freezing tolerance of Saccharomyces cerevisiae. The survival rates of LKF-1 after 2, 5, 10, and 15 days of storage at -20 ℃ were 60.76%, 56.40%, 13.76%, and 8.63%, respectively, whereas those of LKF-1-Aro9 were 79.97%, 67.03%, 43.80%, and 29.30%, respectively. After 3 days of frozen storage, the times required for LKF-1, LKF-1-Aro9, and LKF-1-ΔAro9 to reach the stationary phase were 14 h, 12 h, and 21 h, respectively. Scanning electron microscopy revealed that LKF-1-Aro9 maintained a more intact cell surface structure after 7 days of storage at -20°C. Furthermore, after 3 days of storage at -20°C, the fluorescence intensities of LKF-1 and LKF-1-Aro9 after PI staining were 4096.00 and 3336.67, respectively, indicating that the cell membrane of LKF-1 suffered more severe damage than that of LKF-1-Aro9. After storage at -20°C, the intracellular proline contents of LKF-1 and LKF-1-Aro9 were 0.35 and 2.19 μg·mg?1, respectively, suggesting that the higher proline accumulation in LKF-1-Aro9 conferred greater tolerance to freezing stress. And crystal violet staining showed OD??? values of 0.2424 and 0.2796 for LKF-1 and LKF-1-Aro9, respectively, indicating that LKF-1-Aro9 produced more biofilm to withstand adverse environmental conditions. In conclusion, overexpression of the aro9 gene effectively enhances the freezing tolerance of S. cerevisiae. This improvement is closely associated with strengthened cell membrane and biofilm formation, as well as increased accumulation of the protective compound proline.