Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting
Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting
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DOI：
10.1093/protein/gzw032
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发表时间：
2016-09-01
影响因子：
2.4
通讯作者：
Hilvert, Donald
中科院分区：
文献类型：
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作者：
Obexer, Richard;Pott, Moritz;Hilvert, Donald
具有非自然功能的从头生物催化剂可通过计算机酶设计获得。初始设计得到的催化活性通常很低，但通过定向进化可以显著优化。然而，接近天然酶水平的速度加速只能通过多轮乏味和耗时的实验室进化才能实现。在这项工作中，我们证明了使用荧光激活液滴分类(FADS)的微流控筛选非常适合于高效优化设计的低启动活性的酶，基本上是直接从计算机出来的。我们选择了设计的逆缩醛缩酶RA95.0，它以前是通过传统的微量平板筛选而来的，并使用基于微流控的分析方法对其进行了重新优化。我们的结果表明，FADS对低至k(CAT)/K-m=0.5M-1 S(-1)的酶活性的检测是足够灵敏的。该系统的超高吞吐量使筛选大型突变文库成为可能，在该文库中，最多5个残基的簇被同时随机化。因此，可以直接识别有益突变的组合，导致催化活性在一轮进化中大幅跃升高达80倍。通过并行探索几个进化轨迹，我们确定了表现出相对增强的效率但相反的对映选择性的替代活性位点排列。
De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as k(cat)/K-m = 0.5 M-1 s(-1). The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity.