尿素吸收型工业黄酒酵母单倍体工程菌的构建

Construction of industrial Chinese rice wine yeast haploid strain with high urea importing level

氨基甲酸乙酯是黄酒生产中的副产物,具有致癌性。尿素是氨基甲酸乙酯(EC)的主要前体物质,为降低黄酒中EC的含量,通过提高尿素转运蛋白基因DUR3的表达水平,构建了尿素吸收型工业黄酒酵母单倍体工程菌。采用融合PCR技术,将DUR3基因置于强启动子PGK1p后,构建过表达组件"URA3-PGK1p-DUR3-PGK1tURA3",转化工业黄酒酵母单倍体Na(MATa),获得单倍体工程菌Na-uD。通过实验室黄酒发酵,工程菌所酿黄酒发酵液中尿素含量降低了55.0%,EC含量降低了10.9%,其他理化指标无明显差异。因此过表达DUR3基因的工程菌Na-uD具有"吸收尿素"的能力,能够降低发酵液中尿素的含量,且无外源抗性基因的引入。

Ethyl carbamate （EC） is a carcinogen by-product in Chinese rice wine brewing. Urea is known as the main precursor of EC in Chinese rice wine. The industrial Chinese rice wine yeast engineering strain Na-uD with highly expressed DUR3 responsible for urea import was constructed, to eliminate EC in Chinese rice wine. The DUR3 gene expression cassette ＂ URA3-PGKlp-DUR3-PGKlt-URA3＂ with strong promoter PGKlp was constructed using fusion PCR, and then electro-transformed into the haploid yeast strain Na （MATa）. After lab-scale brewing of Chinese rice wine with Na-uD, the content of urea and EC showed a significant reduction to 55.0% and 10.9% , respectively. There was no difference in physical and chemical indexes between the engineered strain and parental strain N85. The study indicated that overexpression of DUR3 without foreign genes introduction was an effective way to reduce the urea content and further the EC concentration in the fermented alcohol beverages.