Construction of DNA-Shuffled and Incrementally Truncated Libraries by a Mutagenic and Unidirectional Reassembly Method: Changing from a Substrate Specificity of Phospholipase to That of Lipase

A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.

本研究开发了一种可生成DNA洗牌与随机截短蛋白质文库的诱变单向重组装（mutagenic and unidirectional reassembly, MURA）技术。该技术采用脱氧核糖核酸酶I（DNase I）随机切割模板基因，再通过PCR借助单向引物对小片段进行重组装。MURA产物先后经T4 DNA聚合酶（T4 DNA polymerase）以及识别位点位于MURA引物区域的限制性内切酶处理后，利用该方法制备的沙雷氏菌属（Serratia sp.）磷脂酶A₁（phospholipase A₁）的N端截短与DNA洗牌文库，其截短片段大小呈现近乎随机的变异，同时伴随DNA洗牌相关的点突变。通过甘油三酯乳化平板开展高通量筛选（high-throughput screening）后，获得了若干具备完全脂肪酶活性的突变体（NPL变体）。对NPL变体的序列分析与脂肪酶活性测定结果显示，当N端截短区域起始于氨基酸61至71位，并伴随氨基酸取代时，该突变体的底物特异性从磷脂酶转变为脂肪酶。综上，本研究认为，结合渐进式截短与DNA洗牌的MURA方法，可为定向进化（directed evolution）实验拓展可搜索的序列空间提供助力。