Zhang Xinjie, He Peng, Tao Yong, Yang Yi
Wei Sheng Wu Xue Bao. 2013 Nov 4;53(11):1195-204.
High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering.
We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected.
mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein.
The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.
构建由内部核糖体进入位点（IRES）介导的异源蛋白在酿酒酵母中的高效表达系统，用于酿酒酵母在代谢工程中的其他应用。
构建包含Pilv5、Padh2和Ptdh3启动子的共表达盒（启动子-mCherry-TIF4631 IRES-URA3），并将该共表达盒重组到W303-1B-A的基因组中。筛选URA3+转化子。通过比较转化子中mCherry平均荧光值的差异，检测共表达盒中三种启动子的作用。通过实时定量PCR测定基因组中目的基因的拷贝数。在无选择压力下连续传代培养转化子，分析其遗传稳定性。为验证共表达盒的应用，将mCherry的开放阅读框替换为β-半乳糖苷酶（LACZ）和木糖还原酶（XYL1）。检测β-半乳糖苷酶和木糖还原酶的酶活性及产量。
在含有Pilv5启动子的共表达盒转化子中，mCherry表达水平最高。含有Pilv5的转化子中整合到基因组的DNA片段最高拷贝数为47。工程菌株表现出良好的遗传稳定性。木糖还原酶在含有Pilv5启动子和TIF4631 IRES的共表达盒中成功表达。转化子WIX-10中的最高酶活性为0.209 U/mg粗蛋白。β-半乳糖苷酶也成功表达。酶活性最高的转化子是WIL-1，酶活性为12.58 U/mg粗蛋白。
由Pilv5启动子和TIF4631 IRES介导的系统可在酿酒酵母中高效表达异源蛋白。本研究为酿酒酵母中异源蛋白的表达提供了新策略，并为该系统在酵母代谢工程中的应用提供了充分的实验依据。
