High-throughput screening of enzyme libraries: Thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments
High-throughput screening of enzyme libraries: Thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments
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DOI：
10.1016/j.chembiol.2005.09.012
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发表时间：
2005-12-01
影响因子：
--
通讯作者：
Tawfik, DS
中科院分区：
文献类型：
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作者：
Aharoni, A;Amitai, G;Tawfik, DS
每个表达不同文库变体的单个细菌细胞被划分在油包水（w/o）乳液的水滴中，从而维持质粒携带的基因、编码的酶变体和该酶可能产生的荧光产物之间的联系。转化为双水包油包水 (w/o/w) 乳液后，可以通过 FACS 对这些区室进行分类，并分离活细菌细胞及其酶编码基因。我们通过筛选 > 10(7) 个血清对氧磷酶 (PON1) 突变体来证明新酶变体的定向进化，从而使硫代内酯酶活性提高 100 倍。单细胞的体外区室化 (IVC)，每个细胞携带 > 10(4) 个酶分子，体积 < 10 ferntoliter (fl)，尽管底物快速自发水解、PON1 的初始硫代内酯酶活性非常低，并且使用可扩散荧光产品，但仍然能够进行检测和选择。
Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzymecoding genes. We demonstrate the directed evolution of new enzyme variants by screening > 10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying > 10(4) enzyme molecules, in a volume of < 10 ferntoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.