Multiplex Iterative Plasnnid Engineering for Combinatorial Optimization of Metabolic Pathways and Diversification of Protein Coding Sequences
Multiplex Iterative Plasnnid Engineering for Combinatorial Optimization of Metabolic Pathways and Diversification of Protein Coding Sequences
复制标题
用于代谢途径组合优化和蛋白质编码序列多样化的多重迭代质粒工程
DOI：
10.1021/sb400051t
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发表时间：
2013-11-01
影响因子：
4.7
通讯作者：
Zhao, Xueming
中科院分区：
文献类型：
--
作者：
Li, Yifan;Gu, Qun;Zhao, Xueming
构建复杂的生物系统通常需要组合优化以实现预期的功能。在此，我们介绍多重迭代质粒工程（MIPE），这是一种对质粒序列进行组合多样化的高效且定制化的方法。MIPE利用单链DNA介导的A. Red重组技术引入突变，使其能够同时靶向多个位点，并在一次反应中生成多达10⁷个序列的文库。我们还描述了“限制性内切酶消化介导的共选择（RD CoS）”，它使MIPE能够以大大简化的共选择程序产生更高的重组效率。为了展示这种方法，我们将MIPE应用于微调5 - 基因核黄素生物合成途径中的基因表达水平，并在不到一周的时间内成功分离出一个产量提高2.67倍的克隆。我们进一步通过同时靶向分布在750bp序列上的23个密码子，展示了MIPE对蛋白质编码序列进行高度多重多样化的能力。我们预计这种方法将有利于合成生物学和代谢工程中多种生物系统的优化。
Engineering complex biological systems typically requires combinatorial optimization to achieve the desired functionality. Here, we present Multiplex Iterative Plasmid Engineering (MIPE), which is a highly efficient and customized method for combinatorial diversification of plasmid sequences. MIPE exploits ssDNA mediated A. Red recombineering for the introduction of mutations, allowing it to target several sites simultaneously and generate libraries of up to 107 sequences in one reaction. We also describe "restriction digestion mediated coselection (RD CoS)", which enables MIPE to produce enhanced recombineering efficiencies with greatly simplified coselection procedures. To demonstrate this approach, we applied MIPE to fine-tune gene expression level in the 5-gene riboflavin biosynthetic pathway and successfully isolated a clone with 2.67-fold improved production in less than a week. We further demonstrated the ability of MIPE for highly multiplexed diversification of protein coding sequence by simultaneously targeting 23 codons scattered along the 750 bp sequence. We anticipate this method to benefit the optimization of diverse biological systems in synthetic biology and metabolic engineering.