Crameri A, Raillard S A, Bermudez E, Stemmer W P
Maxygen Inc., Santa Clara, California 95051, USA.
Nature. 1998 Jan 15;391(6664):288-91. doi: 10.1038/34663.
DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination, combining useful mutations from individual genes. Libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. Template switching causes crossovers in areas of sequence homology. Our previous studies used single genes and random point mutations as the source of diversity. An alternative source of diversity is naturally occurring homologous genes, which provide 'functional diversity'. To evaluate whether natural diversity could accelerate the evolution process, we compared the efficiency of obtaining moxalactamase activity from four cephalosporinase genes evolved separately with that from a mixed pool of the four genes. A single cycle of shuffling yielded eightfold improvements from the four separately evolved genes, versus a 270- to 540-fold improvement from the four genes shuffled together, a 50-fold increase per cycle of shuffling. The best clone contained eight segments from three of the four genes as well as 33 amino-acid point mutations. Molecular breeding by shuffling can efficiently mix sequences from different species, unlike traditional breeding techniques. The power of family shuffling may arise from sparse sampling of a larger portion of sequence space.
DNA改组是一种用于定向进化的强大方法，它通过重组产生多样性，将来自单个基因的有用突变组合在一起。嵌合基因文库可以通过对一组相关基因进行随机片段化，然后在自引物聚合酶反应中重新组装片段来生成。模板切换会导致序列同源区域发生交叉。我们之前的研究使用单个基因和随机点突变作为多样性来源。另一种多样性来源是天然存在的同源基因，它们提供“功能多样性”。为了评估天然多样性是否能加速进化过程，我们比较了分别从四个头孢菌素酶基因进化获得莫拉内酰胺酶活性的效率与从这四个基因的混合库进化获得该活性的效率。经过一轮改组，分别进化的四个基因产生了八倍的改进，而四个基因一起改组则产生了270至540倍的改进，即每轮改组增加50倍。最佳克隆包含来自四个基因中三个基因的八个片段以及33个氨基酸点突变。与传统育种技术不同，通过改组进行分子育种可以有效地混合来自不同物种的序列。家族改组的强大作用可能源于对更大一部分序列空间的稀疏采样。
