Hibbert Edward G, Senussi Tarik, Costelloe Sean J, Lei Wenling, Smith Mark E B, Ward John M, Hailes Helen C, Dalby Paul A
Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, UK.
J Biotechnol. 2007 Sep 30;131(4):425-32. doi: 10.1016/j.jbiotec.2007.07.949. Epub 2007 Aug 3.
We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity.
我们通过饱和诱变进行活性位点靶向定向进化，以提高大肠杆菌转酮醇酶对非磷酸化底物的活性。每组残基的选择基于与底物的结构接近程度或系统发育变异。针对羟基丙酮酸（HPA）和乙醇醛（GA）之间形成L-赤藓酮糖的反应对每个文库进行筛选，并确定与每个残基处天然序列熵相关的改良突变体的位置。在生物催化相关条件下，发现许多来自系统发育定义文库的突变体的比活性比野生型高，最高可达3倍，有趣的是，其取代残基与自然界中发现的不同。与天然底物中的磷酸基团相互作用的保守残基也产生了在非磷酸化底物上比活性提高近5倍的突变体。这些结果表明，系统发育变异的活性位点残基可用于调节对天然或结构同源底物的活性，并且不再与修饰的靶底物相互作用的保守残基是应用饱和诱变提高活性的有用位点。
