酿酒酵母SRP40基因对细胞耐受性影响的研究

Effect of SRP40 gene on cell tolerance of Saccharomyces cerevisiae

【目的】本论文研究酿酒酵母srp40^(39)突变基因对酵母细胞异丁醇耐受性的影响。【方法】首先,以酿酒酵母野生型W303-1A和突变株EMS39染色体DNA为模板克隆野生型SRP40基因和srp40^(39)突变基因;然后,将野生型SRP40基因和srp40^(39)突变基因分别连接到质粒YCplac22上,构建质粒YCplac22-SRP40和YCplac22-srp40^(39)。将质粒YCplac22-SRP40、YCplac22-srp40^(39)以及YCplac22空质粒分别转化入野生型酿酒酵母W303-1A中,分别得到W303-1A-SRP40工程菌、W303-1A-srp40^(39)工程菌和W303-1A-control工程菌。将3株工程菌分别置于含1.0%异丁醇、1.3%异丁醇、8.0%乙醇和0.5%异戊醇的CM培养基中进行发酵,测定细胞密度(OD_(600))和生长情况,并计算2–10 h的比生长速率(μ)。将3株工程菌于55°C热激4 min后做稀释实验(dilution),观察它们在平板上的生长情况。最后,对野生型SRP40基因、srp40^(39)突变基因的氨基酸序列进行生物信息学分析。【结果】在不含异丁醇(0%异丁醇)的培养基中,3株工程菌无明显差异。在含1.0%异丁醇和1.3%异丁醇的CM培养基中,发酵24 h工程菌W303-1A-srp40^(39)的细胞密度分别是工程菌W303-1A-SRP40的1.12倍和1.06倍,是工程菌W303-1A-control的1.10倍和1.10倍;工程菌W303-1A-srp40^(39)的比生长速率(μ)分别是工程菌W303-1A-SRP40的1.07倍和1.10倍,是对照菌W303-1A-control的1.10倍和1.10倍。在含8.0%乙醇和0.5%异戊醇的CM培养基中,发酵24h工程菌W303-1A-srp40^(39)的细胞密度分别是工程菌W303-1A-SRP40的1.12倍和1.01倍,是对照菌W303-1A-control的1.17倍和1.07倍;工程菌W303-1A-srp40^(39)的比生长速率(μ)分别是工程菌W303-1A-SRP40的1.37倍和1.07倍,是对照菌W303-1A-control的1.31倍和1.09倍。工程菌W303-1A-srp40^(39)在热激后生长仍优于其他2株菌。生物信息学分析发现,Srp40^(39)蛋白与硅藻的胸膜蛋白-1具有相似的结构。【结论】本研究发现酿酒酵母srp40^(39)突变基因能够提升细胞的异丁醇耐受性,此外对提升细胞的乙醇耐受性、异戊醇耐受性和耐热性也有一定的作用。同时,我们发现Srp40^(39)蛋白与硅藻的胸膜蛋白-1具有相似的结构,说明其可能具有胸膜蛋白-1的功能,推测该蛋白在细胞壁维持方面有作用。这些研究将为提高酿酒酵母对乙醇、异丁醇、异戊醇和高温等的抗逆性提供新思路。

[Objective] In this study, the effect of the mutant srp40^(39) gene on isobutanol tolerance of Saccharomyces cerevisiae was studied. [Methods] Firstly, the wild-type SRP40 gene and the mutant srp40^(39) gene were respectively cloned from the chromosome DNAs of the wild-type strain W303-1 A and the mutant EMS39 of S. cerevisiae. Then, SRP40 and srp40^(39) were respectively ligated to YCplac22 to construct the recombinant plasmids YCplac22-SRP40 and YCplac22-srp40^(39). The obtained recombinant plasmids and the YCplac22 empty plasmid were respectively transformed into the wild-type strain W303-1 A to construct the engineering strains W303-1 A-SRP40, W303-1 A-srp40^(39), and W303-1 A-control. The three engineering strains were then fermented in the complete media containing 1.0% isobutanol, 1.3% isobutanol, 8.0% ethanol, and 0.5% isoamyl alcohol, respectively. The cell density(OD_(600)) was measured, and the specific growth rate in 2–10 h was calculated. The three engineering strains were heated at 55 °C for 4 min and then diluted for observation of cell growth on the plate. Finally, the amino acid sequences of SRP40 and srp40^(39) were analyzed by bioinformatics tools.[Results] In the medium without isobutanol(0% isobutanol), the three strains showed no significant difference. After fermentation for 24 h in the complete media containing 1.0% isobutanol and 1.3%isobutanol, W303-1 A-srp40^(39) showed the cell density 1.12 and 1.06 times that of W303-1 A-SRP40, and 1.10 and 1.10 times that of W303-1 A-control, respectively;the specific growth rate of W303-1 A-srp40^(39) was 1.07 and 1.10 times as high as that of W303-1 A-SRP40, and 1.10 and 1.10 times as high as that of W303-1 A-control, respectively. After fermentation for 24 h in the complete media containing 8.0% ethanol and 0.5% isoamyl alcohol, W303-1 A-srp40^(39) showed the cell density 1.12 and 1.01 times that of W303-1 A-SRP40, and 1.17 and 1.07 times that of W303-1 A-control, respectively;W303-1 A-srp40^(39) showed the specific growth rate 1.37 and 1.07 times as high as that of W303-1 A-SRP40, and 1.31 and 1.09 times as high as that of W303-1 A-control, respectively. Moreover,W303-1 A-srp40^(39) still grew better than the other two strains after heat shock. The bioinformatics analysis showed that Srp40^(39) protein had a similar structure to the pleuralin-1 of diatom. [Conclusion]We found that the srp40^(39) mutant gene can enhance the tolerance of S. cerevisiae to isobutanol, and plays a role in enhancing the tolerance to ethanol, isoamyl alcohol, and heat. Srp40^(39) protein has a similar structure with the pleuralin-1 of diatom, which indicates that Srp40^(39) protein may have the function of pleuralin-1 and play a role in cell wall maintenance. These findings will provide new ideas for improving the tolerance of S. cerevisiae to ethanol, isobutanol, isoamyl alcohol, and high temperature.