Department of Chemistry, University of Konstanz, Universitätsstraße 10, D-78457, Konstanz, Germany.
Sci Rep. 2019 Jan 24;9(1):590. doi: 10.1038/s41598-018-37233-y.
DNA polymerases the key enzymes for several biotechnological applications. Obviously, nature has not evolved these enzymes to be compatible with applications in biotechnology. Thus, engineering of a natural scaffold of DNA polymerases may lead to enzymes improved for several applications. Here, we investigated a two-step approach for the design and construction of a combinatorial library of mutants of KlenTaq DNA polymerase. First, we selected amino acid sites for saturation mutagenesis that interact with the primer/template strands or are evolutionarily conserved. From this library, we identified mutations that little interfere with DNA polymerase activity. Next, these functionally active mutants were combined randomly to construct a second library with enriched sequence diversity. We reasoned that the combination of mutants that have minuscule effect on enzyme activity and thermostability, will result in entities that have an increased mutation load but still retain activity. Besides activity and thermostability, we screened the library for entities with two distinct properties. Indeed, we identified two different KlenTaq DNA polymerase variants that either exhibit increased mismatch extension discrimination or increased reverse transcription PCR activity, respectively.
DNA 聚合酶是几种生物技术应用的关键酶。显然，自然界并没有进化出这些酶来适应生物技术中的应用。因此，对 DNA 聚合酶天然支架进行工程改造可能会产生适用于多种应用的酶。在这里，我们研究了一种两步法设计和构建 KlenTaq DNA 聚合酶突变体组合文库的方法。首先，我们选择了与引物/模板链相互作用或进化保守的氨基酸位点进行饱和诱变。从这个文库中，我们鉴定出了对 DNA 聚合酶活性影响很小的突变。接下来，我们随机组合这些功能活性的突变体，构建一个具有丰富序列多样性的第二个文库。我们推断，组合对酶活性和热稳定性影响很小的突变体，将产生具有增加突变负荷但仍保留活性的实体。除了活性和热稳定性之外，我们还筛选了文库中的两种具有独特性质的实体。事实上，我们鉴定了两种不同的 KlenTaq DNA 聚合酶变体，它们分别表现出增加的错配延伸区分或增加的反转录 PCR 活性。
