Optimization of a Yeast RNA Interference System for Controlling Gene Expression and Enabling Rapid Metabolic Engineering
Optimization of a Yeast RNA Interference System for Controlling Gene Expression and Enabling Rapid Metabolic Engineering
复制标题
DOI：
10.1021/sb4001432
复制
发表时间：
2014-05-01
影响因子：
4.7
通讯作者：
Alper, Hal S.
中科院分区：
文献类型：
--
作者：
Crook, Nathan C.;Schmitz, Alexander C.;Alper, Hal S.
内源基因表达的减少是代谢工程的基本操作，然而目前用于基因敲减的方法（即，基因组编辑）仍然费力且缓慢，特别是在酵母中。相比之下，RNA干扰允许通过简单的质粒转化步骤进行简单且可调的基因敲除，使代谢工程师能够在花费大量成本进行基因组编辑之前在多个菌株中快速原型化敲除策略。尽管RNAi天然存在于无数的真核生物中，但它最近才在酿酒酵母中作为异源途径实施，因此尚未作为代谢工程工具进行优化。在这项研究中，我们阐明了一套设计原则，在酵母中构建发夹RNA表达盒，并实施RNA干扰，以快速确定路线，改善在这种生物体中的衣康酸生产。这里开发的方法能够快速原型化敲除策略，从而加速并降低酵母中设计-构建-测试周期的成本。
Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.