Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.
Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae.
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DOI：
10.1093/nar/gkv466
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发表时间：
2015-07-27
影响因子：
14.9
通讯作者：
Boeke JD
中科院分区：
文献类型：
--
作者：
Mitchell LA;Chuang J;Agmon N;Khunsriraksakul C;Phillips NA;Cai Y;Truong DM;Veerakumar A;Wang Y;Mayorga M;Blomquist P;Sadda P;Trueheart J;Boeke JD
我们已经开发了一种用于在酿酒酵母中表达的遗传途径的方法，我们的途径组装方法称为Vegas（多功能遗传组装系统），探索了S. cerevisiae的天然能力，以执行同源性重组并有效地与终端同源性结合维加斯的工作流程，相邻途径基因与组装载体之间的终端同源性由“拉斯维加斯适配器”编码（va）序列，在酵母基因组上是序列上的正交序列，维加斯在酿酒酵母中的途径组装中，为每个基因分配了一对合适的VAS，并使用先前描述的技术来组装）。 Vegas的四，五和六基因途径可以生成酿酒酵母的细胞合成的β-胡萝卜素和紫cer蛋白，我们证明了YGG耦合到拉斯维加斯进行组合的能力。
We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by ‘VEGAS adapter’ (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.