Si Tong, Luo Yunzi, Bao Zehua, Zhao Huimin
†Department of Chemical and Biomolecular Engineering, ‡Department of Biochemistry, §Departments of Chemistry and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
ACS Synth Biol. 2015 Mar 20;4(3):283-91. doi: 10.1021/sb500074a. Epub 2014 May 6.
A fundamental challenge in basic and applied biology is to reprogram cells with improved or novel traits on a genomic scale. However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells. Here, we report RNA interference (RNAi)-assisted genome evolution (RAGE) as a generally applicable method for genome-scale engineering in the yeast Saccharomyces cerevisiae. Through iterative cycles of creating a library of RNAi induced reduction-of-function mutants coupled with high throughput screening or selection, RAGE can continuously improve target trait(s) by accumulating multiplex beneficial genetic modifications in an evolving yeast genome. To validate the RNAi library constructed with yeast genomic DNA and convergent-promoter expression cassette, we demonstrated RNAi screening in Saccharomyces cerevisiae for the first time by identifying two known and three novel suppressors of a telomerase-deficient mutation yku70Δ. We then showed the application of RAGE for improved acetic acid tolerance, a key trait for microbial production of chemicals and fuels. Three rounds of iterative RNAi screening led to the identification of three gene knockdown targets that acted synergistically to confer an engineered yeast strain with substantially improved acetic acid tolerance. RAGE should greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution.
基础生物学和应用生物学中的一个根本挑战是在基因组规模上对细胞进行重编程，使其具有改良的或新的性状。然而，目前在基因组规模上对细胞进行重编程的能力仅限于细菌细胞。在此，我们报告了RNA干扰（RNAi）辅助的基因组进化（RAGE），这是一种在酿酒酵母中普遍适用的基因组规模工程方法。通过创建RNAi诱导的功能降低突变体文库并结合高通量筛选或选择的迭代循环，RAGE可以通过在不断进化的酵母基因组中积累多重有益的基因修饰来持续改善目标性状。为了验证用酵母基因组DNA和趋同启动子表达盒构建的RNAi文库，我们首次在酿酒酵母中通过鉴定端粒酶缺陷突变yku70Δ的两个已知抑制子和三个新抑制子来证明RNAi筛选。然后，我们展示了RAGE在提高乙酸耐受性方面的应用，乙酸耐受性是微生物生产化学品和燃料的一个关键性状。三轮迭代RNAi筛选导致鉴定出三个基因敲低靶点，它们协同作用赋予工程酵母菌株显著提高的乙酸耐受性。RAGE应能极大地加速具有所需性状的生物体的设计和进化，并为基因组结构、功能和进化提供新的见解。
