Viña-Gonzalez Javier, Gonzalez-Perez David, Alcalde Miguel
Department of Biocatalysis, Institute of Catalysis, CSIC.
Department of Biocatalysis, Institute of Catalysis, CSIC;
J Vis Exp. 2016 Apr 1(110):e53761. doi: 10.3791/53761.
Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.
在为生物技术应用设计酶时，酿酒酵母中的定向进化具有许多吸引人的优势，该过程涉及突变文库的构建、克隆和表达，并与体内高频同源DNA重组相结合。在此，我们以真菌芳基醇氧化酶（AAO）为例，介绍一种在酵母中创建和筛选突变文库以提高其总活性的方案。通过随机诱变和体内DNA重组对两个蛋白质片段进行聚焦定向进化。每个片段两侧约50 bp的突出端允许AAO融合基因在线性化载体中正确重新组装，从而产生一个完整的自主复制质粒。基于Fenton反应的灵敏高通量测定法在酿酒酵母上清液中筛选富含功能性AAO变体的突变文库。这里描述的酿酒酵母文库构建的一般过程可以很容易地应用于进化许多其他真核基因，避免额外的PCR反应、体外DNA重组和连接步骤。
