High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch
High-throughput enzyme evolution in Saccharomyces cerevisiae using a synthetic RNA switch
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DOI：
10.1016/j.ymben.2012.04.004
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发表时间：
2012-07-01
影响因子：
8.4
通讯作者：
Smolke, Christina D.
中科院分区：
文献类型：
--
作者：
Michener, Joshua K.;Smolke, Christina D.
代谢工程可以利用可再生资源生产各种散装和精细化学品。这些方法常常需要多种异源酶的高活性水平。定向进化技术已被用来提高多种酶的活性，但当酶用于全细胞时可能很难应用。为了解决这一限制，我们开发了通用体内生物传感器，使用工程化的 RNA 开关将活细胞中的代谢物浓度和 GFP 表达水平联系起来。使用这样的传感器，我们通过荧光激活细胞分选（FACS）在克隆培养物或单细胞中基于荧光以高通量定量筛选大型酶库。通过反复筛选咖啡因去甲基酶文库，我们发现了有益的突变，最终使体内酶活性提高了 33 倍，产物选择性提高了 22 倍。由于适体选择策略使 RNA 开关能够轻松识别新的小分子，因此这些基于 RNA 的筛选技术适用于广泛的酶和代谢途径。 (C) 2012 Elsevier Inc. 保留所有权利。
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. (C) 2012 Elsevier Inc. All rights reserved.