Fibinger Michael P C, Davids Timo, Böttcher Dominique, Bornscheuer Uwe T
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Straße 4, D-17491, Greifswald, Germany.
Davids Biotechnologie GmbH, Röntgenstraße 3, D-93055, Regensburg, Germany.
Appl Microbiol Biotechnol. 2015 Nov;99(21):8955-62. doi: 10.1007/s00253-015-6686-y. Epub 2015 May 22.
Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 10(6) cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.
基于自然选择和进化适应中的适者生存，开发了一种智能高通量系统，用于从大型突变体文库中筛选活性卤代烷脱卤酶变体。只有活性酶变体能够水解有毒的卤代烷以促进生长，而无活性的突变体则会因有毒化合物而饥饿或死亡。借助这一强大工具，可在数天内筛选庞大的酶突变体文库。筛选过程无需任何难以合成的人工底物，且这些底物与卤代烷脱卤酶的典型底物相似。基于卤代烷脱卤酶DhaA或DhlA的无活性变体成功筛选出了三个规模超过10⁶个细胞的饱和文库，以获得活性酶。与无活性变体相比，活性野生型酶的富集倍数约为340倍。此外，这种筛选方法可应用于连续定向进化实验，以富集表达适应性卤代烷脱卤酶的细胞。
