An Automated Data-Driven Pipeline for Improving Heterologous Enzyme Expression
An Automated Data-Driven Pipeline for Improving Heterologous Enzyme Expression
复制标题
用于改善异源酶表达的自动化数据驱动管道
DOI：
10.1021/acssynbio.8b00486
复制
发表时间：
2019
影响因子：
4.7
通讯作者：
Whitehead, Timothy A.
中科院分区：
文献类型：
--
作者：
Wrenbeck, Emily E.;Bedewitz, Matthew A.;Klesmith, Justin R.;Noshin, Syeda;Barry, Cornelius S.;Whitehead, Timothy A.
酶是在自然和工程生物合成途径中负责化学转化的最终实体。然而，由于固有的蛋白质稳定性差，许多天然酶的功能表达能力不佳。此外，增强稳定性的突变往往以功能受损为代价。在这里，我们展示了一种自动化的蛋白质工程策略，使用深度突变扫描结合基于多过滤器的筛选和组合突变来稳定酶并保留催化功能。我们通过改善颠茄生物碱合成途径中的III型聚酮合成酶的功能表达来验证这一策略。最好的突变体总共有8个突变，活性是野生型的25倍以上。在大肠杆菌裂解物中，融化温度提高了11.5±0.6℃，催化效率只有轻微的下降。我们表明，多滤波方法对同源建模结构保持了可接受的灵敏度，最高可达4？RMS。我们的结果突出了一种自动化的蛋白质工程工具，用于提高难以表达的酶的稳定性和溶解性，这对生物技术应用具有影响。
Enzymes are the ultimate entities responsible for chemical transformations in natural and engineered biosynthetic pathways. However, many natural enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at the cost of impaired function. Here we demonstrate an automated protein engineering strategy for stabilizing enzymes while retaining catalytic function using deep mutational scanning coupled to multiple-filter based screening and combinatorial mutagenesis. We validated this strategy by improving the functional expression of a Type III polyketide synthase from theAtropa belladonnabiosynthetic pathway for tropane alkaloids. The best variant had a total of 8 mutations with over 25-fold improved activity over wild-type inE. colicell lysates, an improved melting temperature of 11.5 ± 0.6 °C, and only minimal reduction in catalytic efficiency. We show that the multiple-filter approach maintains acceptable sensitivity with homology modeling structures up to 4 Å RMS. Our results highlight an automated protein engineering tool for improving the stability and solubility of difficult to express enzymes, which has impact for biotechnological applications.