Department of Bioengineering, 1200 E. California Blvd., MC 210-41, California Institute of Technology, Pasadena, CA 91125, USA.
Metab Eng. 2012 Jul;14(4):306-16. doi: 10.1016/j.ymben.2012.04.004. Epub 2012 Apr 25.
Metabolic engineering can produce a wide range of bulk and fine chemicals using renewable resources. These approaches frequently require high levels of activity from multiple heterologous enzymes. Directed evolution techniques have been used to improve the activity of a wide range of enzymes but can be difficult to apply when the enzyme is used in whole cells. To address this limitation, we developed generalizable in vivo biosensors using engineered RNA switches to link metabolite concentrations and GFP expression levels in living cells. Using such a sensor, we quantitatively screened large enzyme libraries in high throughput based on fluorescence, either in clonal cultures or in single cells by fluorescence activated cell sorting (FACS). By iteratively screening libraries of a caffeine demethylase, we identified beneficial mutations that ultimately increased the enzyme activity in vivo by 33 fold and the product selectivity by 22 fold. As aptamer selection strategies allow RNA switches to be readily adapted to recognize new small molecules, these RNA-based screening techniques are applicable to a broad range of enzymes and metabolic pathways.
代谢工程可以使用可再生资源生产各种大宗化学品和精细化学品。这些方法通常需要多种异源酶具有很高的活性。定向进化技术已被用于提高多种酶的活性，但当酶在完整细胞中使用时，该技术可能难以应用。为了解决这一限制，我们使用工程 RNA 开关开发了可推广的体内生物传感器，将代谢物浓度与活细胞中的 GFP 表达水平联系起来。使用这种传感器，我们可以通过荧光在高通量水平上对大型酶文库进行定量筛选，无论是在克隆培养物中还是通过荧光激活细胞分选 (FACS) 在单个细胞中进行筛选。通过对咖啡因脱甲基酶文库的反复筛选，我们鉴定出了有益的突变，最终使酶的体内活性提高了 33 倍，产物选择性提高了 22 倍。由于适体选择策略允许 RNA 开关很容易适应识别新的小分子，因此这些基于 RNA 的筛选技术适用于广泛的酶和代谢途径。
