酵母海藻糖酶缺失突变株的构建及其耐性

Construction and stress tolerance of trehalase mutant in Saccharomyces cerevisiae

摘要:

【目的】构建酵母海藻糖酶缺失突变株，并进行耐性分析，进一步研究海藻糖与酵母耐性之间的关系，为商业生产打下一定的基础。【方法】利用同源重组的方法，敲除了编码酸性海藻糖酶的ATH1基因和中性海藻糖酶的NTH1基因，构建了酸性海藻糖酶缺失突变株（Δath1）、中性海藻糖酶缺失突变株（Δnth1）和双缺失突变株（Δath1Δnth1），并进行了耐性分析。【结果】结合PCR和Southern blot的结果，验证了突变株构建的正确。所有突变株的海藻糖积累量和细胞密度均高于亲本，冷冻、高温、高糖和酒精耐性提高了。【结论】说明海藻糖含量与酵母耐性有一定的相关性。突变株耐性的改善，表明它们在酿造和烘焙产业中具有潜在的商业价值。

Abstract:

[Objective] Accumulation of trehalose is critical in improving the stress tolerance of Saccharomyces cerevisiae. Two enzymes are capable of hydrolyzing trehalose: a neutral trehalase (NTH1) and an acidic trehalase (ATH1). We constructed trehalase disruption mutants to provide a basis for future commercial application. [Methods] To retain the accumulation of trehalose in yeast cell, we constructed diploid homozygous neutral trehalase mutants (△nth1), acid trehalase mutants (△ath1) and double mutants (Δath1Δnth1) by using gene disruption. We tested mutants’trehalose content and their tolerance to freezing, heat, high-sugar and ethanol concentrations. [Results] These trehalase disruption mutants were further confirmed by PCR amplification and southern blot. All mutant strains accumulated higher levels of cellular treha-lose and grew to a higher cell density than the isogenic parent strain. In addition, the levels of trehalose in these mutants correlated with increased tolerance to freezing, heat, high-sugar and ethanol concentration. [Conclusion] The improved tolerance of trehalase mutants may make them useful in commercial applications, including baking and brewing. protein.