分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

The sectionalized DNA shuffling: an effective tool for molecular directed evolution of Meiothermus ruber TreS

摘要:

【目的】红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase, M-TreS)将麦芽糖转化生成海藻糖只需一步反应, 且具有很好的热稳定性及pH耐受性, 是潜在的工业生产海藻糖的酶源。为了提高该酶的性能, 有必要对其进行定向进化。【方法】M-TreS基因(M-treS)大小为2 889 bp。该蛋白质分子本身具有很大的进化空间, 但是却不宜进行全长基因shuffling。分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法。该方法分为三步: (1) 用两对引物分别扩增目的基因的上游片段和下游片段; (2) 上下游片段各自进行shuffling; (3) 利用重叠延伸PCR连接上下游突变群, 建立完整基因的突变文库。【结果】结合易错PCR, 通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株。序列分析表明, 该突变株共有6个位点发生了氨基酸的替代, 其中一个来自易错突变, 2个来自同源重组, 3个为随机突变。【结论】分段DNA shuffling是进化大分子蛋白质的有效方法。

Abstract:

[Objective] The gene M-treS from Meiothermus ruber CBS-01 encodes a trehalose synthase of 962 amino acids, named M-TreS. To improve its catalytic activity, we constructed a method of molecular directed evolution, the sectionalized DNA shuffling. [Methods] Through two PCR steps with two pairs of partially complementary primers, the M-treS gene was parted into two sections. After the two sections shuffled respectively, a whole gene was obtained through the complementarity of the primers. This method was more feasible, with higher mutability than normal DNA shuffling. [Results] Mutants were obtained after one round of the sectionalized DNA shuffling, in combination with error-prone PCR. The best mutant enzyme contained 6 amino acid substitutions, whose catalytic activity and efficiency were 1.6-fold and 2-fold of that of the wild type, respectively. In the 6 amino acid substitutions, 5 were caused by homologous recombination, and one by error-prone PCR. [Conclusion] This study indicates that the sectionalized DNA shuffling is an effective tool for molecular directed evolution of macromolecular proteins.