Evolution of enzyme catalysts caged in biomimetic gel-shell beads
Evolution of enzyme catalysts caged in biomimetic gel-shell beads
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DOI：
10.1038/nchem.1996
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发表时间：
2014-09-01
期刊：
影响因子：
21.8
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
--
作者：
Fischlechner, Martin;Schaerli, Yolanda;Hollfelder, Florian
自然进化依赖于生物实体通过多轮多样化和选择的改进。在实验室中，定向进化已经成为开发新的和改进的生物分子的强大工具，但它受到筛选足够大的组合文库的巨大工作量和成本的限制。在这里，我们描述了生产的凝胶壳珠（GSB）的帮助下的微流体装置。这些水凝胶珠被一个包封酶、其编码DNA和荧光反应产物的壳包围。在这些人造隔室中的活性克隆可以通过荧光激活分选以>10（7）GSB/小时的速率容易地鉴定。我们使用这个系统进行定向进化的磷酸三酯酶（一种生物修复催化剂）笼在GSB和隔离20倍更快的突变体在不到一个小时。因此，我们建立了一个实际上要求不高的超高通量筛选方法，结果在功能性杂交复合材料赋予可进化的蛋白质成分。
Natural evolution relies on the improvement of biological entities by rounds of diversification and selection. In the laboratory, directed evolution has emerged as a powerful tool for the development of new and improved biomolecules, but it is limited by the enormous workload and cost of screening sufficiently large combinatorial libraries. Here we describe the production of gel-shell beads (GSBs) with the help of a microfluidic device. These hydrogel beads are surrounded with a polyelectrolyte shell that encloses an enzyme, its encoding DNA and the fluorescent reaction product. Active clones in these man-made compartments can be identified readily by fluorescence-activated sorting at rates >10(7) GSBs per hour. We use this system to perform the directed evolution of a phosphotriesterase (a bioremediation catalyst) caged in GSBs and isolate a 20-fold faster mutant in less than one hour. We thus establish a practically undemanding method for ultrahigh-throughput screening that results in functional hybrid composites endowed with evolvable protein components.