Labunskyy Vyacheslav M, Suzuki Yo, Hanly Timothy J, Murao Ayako, Roth Frederick P, Gladyshev Vadim N
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, La Jolla, California 92037.
G3 (Bethesda). 2014 Apr 28;4(7):1183-91. doi: 10.1534/g3.114.010868.
Being a simple eukaryotic organism, Saccharomyces cerevisiae provides numerous advantages for expression and functional characterization of proteins from higher eukaryotes, including humans. However, studies of complex exogenous pathways using yeast as a host have been hampered by the lack of tools to engineer strains expressing a large number of genetic components. In addition to inserting multiple genes, it is often desirable to knock out or replace multiple endogenous genes that might interfere with the processes studied. Here, we describe the "insertion Green Monster" (iGM) set of expression vectors that enable precise insertion of many heterologous genes into the yeast genome in a rapid and reproducible manner and permit simultaneous replacement of selected yeast genes. As a proof of principle, we have used the iGM method to replace components of the yeast pathway for methionine sulfoxide reduction with genes encoding the human selenoprotein biosynthesis machinery and generated a single yeast strain carrying 11 exogenous components of the selenoprotein biosynthetic pathway in precisely engineered loci.
作为一种简单的真核生物，酿酒酵母为表达和功能表征包括人类在内的高等真核生物的蛋白质提供了诸多优势。然而，以酵母为宿主研究复杂的外源途径受到了限制，因为缺乏用于构建表达大量遗传元件的菌株的工具。除了插入多个基因外，通常还需要敲除或替换可能干扰所研究过程的多个内源基因。在此，我们描述了“插入式绿色怪物”（iGM）表达载体集，该载体集能够以快速且可重复的方式将许多异源基因精确插入酵母基因组，并允许同时替换选定的酵母基因。作为原理验证，我们已使用iGM方法，用编码人类硒蛋白生物合成机制的基因替换酵母中甲硫氨酸亚砜还原途径的组分，并在精确工程化的位点生成了一个携带硒蛋白生物合成途径11个外源组分的单一酵母菌株。
