| Yeast, as an important raw material of beer brewing, is a key factor of influencing the quality of beer. On the one hand, the metabolic products of yeast contribute to pleasant tastes of beer; on the other hand, as the result of yeast autolysis, the hydrolysis products could generate unacceptable tastes in beer. Therefore, whether the beer brewing is successful depends on the yeast. To regulate the yeast autolysis performance fundamentally, it is necessary to understand the internal mechanism and detailed regulators about yeast autolysis. Besides, clarify the relationship between cell components and autolysis, which would be an efficient way to solve this problem. In this study, we overexpressed and knocked out FKS1 gene, which regulates the synthesis of β-1,3-glucan. The changes of yeast anti-autolytic and anti-stress abilities were observed. Based on these results, the relationship between β-1,3-glucan and yeast autolysis performance was established. The main contents and results are as follows:(1) Saccharomyces cerevisiae W303 was the wild-type strain. We knocked out and overexpressed FKS1 gene respectively. The knocked-out strain was constructed based on homologous recombination principle. The FKS1 gene was replaced by Kan MX and this recombinant strain was named after FKS1-QT. Meanwhile, the overexpressed strain was constructed based on 18 S r DNA sequence. The recombinant linear fragment, up-PGK1-FKS1-Kan MX-down, was transformed into W303 and the transformant was designated as FKS1-KT.(2) Analyze the yeast autolysis performance. Firstly, the overexpressed strain enhanced the accumulation of β-1,3-glucan in cell wall by 62 % compared with the wild-type. Secondly, the thickness of cell wall for the overexpressed strain was twice as much as that for wild-type strain. It showed that more accumulation of β-1,3-glucan in cell wall increased the thickness of cell wall. Finally, the results of simulation of autolysis revealed that the knocked-out strain showed serious autolysis. By contrast, the overexpressed strain was conferred the best anti-autolytic ability. These results proved that more accumulation of β-1,3-glucan can increase the anti-autolytic ability of yeast.(3) Analyze the yeast stress resistance. The overexpressed strain increased anti-stress ability at 8%(v/v) alcohol, 50 ng·m L-1 fungus inhibitor, 0.4 mol·L-1 Na Cl or starvation stress, which confirmed the potential of strains with more concentration of β-1,3-glucan in cell wall to tackle environment stresses and autolysis in high gravity brewing.(4) FKS2 or FKS3 knocked-out strain was constructed. The analysis of quantitative real-time PCR(q RT-PCR) showed that FKS1 mainly controlled the synthesis of β-1,3-glucan among the FKS family genes. FKS2 could make up the deletion of FKS1. Furthermore, the expression level of FKS1 was down-regulated because of the deletion of FKS3, which is probably the result of RPPO gene was suppressed. However, this result deserved further investigation. |
