College of Biological Engineering, Tianjin Agricultural University, Tianjin, 300384, People's Republic of China.
Tianjin Engineering Research Center of Agricultural Products Processing, Tianjin, 300384, People's Republic of China.
Microb Cell Fact. 2020 Oct 19;19(1):196. doi: 10.1186/s12934-020-01454-6.
In Saccharomyces cerevisiae, alpha-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the alpha-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear.
RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members.
The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.
在酿酒酵母中，α-葡糖苷酶（麦芽糖酶）是麦芽糖代谢中的关键酶。此外，过表达α-葡糖苷酶编码基因 MAL62 已被证明可以提高酵母在贫面团中的抗冻能力。然而，其抗冻保护机制尚不清楚。
RNA 测序（RNA-seq）显示，MAL62 的过表达增加了依赖尿苷二磷酸葡萄糖（UDPG）的海藻糖合成。通过定量逆转录聚合酶链反应（qRT-PCR）和酶活性测定证实了转录物丰度的变化。当 UDPG 依赖性海藻糖合酶活性被抑制时，MAL62 的过表达无法促进细胞内海藻糖的合成。此外，在缺乏海藻糖合成的菌株中，与 MAL62 过表达相结合的前期发酵冷冻后期的细胞存活率略有降低，这可以通过细胞内甘油浓度的增加来解释。这一结果与甘油合成途径成员转录水平的升高一致。
通过 MAL62 的过表达提高抗冻性主要是通过 UDPG 依赖性途径增加海藻糖含量来实现的，甘油也发挥了重要作用。这些发现为酵母在贫面团中对冷冻的反应机制提供了新的认识，并有助于改善工业酵母菌株。
