Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites
Genomic engineering of Escherichia coli by the phage attachment site-based integration system with mutant loxP sites
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DOI：
10.1016/j.procbio.2012.08.022
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发表时间：
2012-12-01
影响因子：
4.4
通讯作者：
Chao, Yun-Peng
中科院分区：
文献类型：
--
作者：
Chiang, Chung-Jen;Saini, Mukesh;Chao, Yun-Peng
利用质粒进行大肠杆菌的代谢工程是一个难题，通过开发大肠杆菌基因组工程工具箱解决了这个问题。杆菌该工具箱包括基于attP位点的整合载体和基于attB位点的模板载体，其配备有突变型loxP位点（即，LE* 和RE*）。前者载体促进乘客基因整合到attB位点，而后者允许创建新的attB位点。因此，插入的载体骨架侧接LE* 和RE* 位点，并且可以被Cre拯救。在此基础上，首次在大肠杆菌中构建了聚3-羟基丁酸的生物合成途径。杆菌通过对可观察到的整合子表型进行评分，结果显示基因整合效率可达100%。此外，对E. coli中获得无质粒生产菌株。结果，生产菌株能够从葡萄糖（20 g/L）稳定过量生产正丁醇（3.7 g/L）。最后，外切葡聚糖酶在E.携带celY基因的多个基因组拷贝的大肠杆菌。总体而言，它表明了我们的方法的承诺，为循环改善E。杆菌(C)2012爱思唯尔有限公司保留所有权利。
Metabolic engineering of Escherichia coli using plasmids is problematic, which is addressed by developing a toolbox for genomic engineering of E. coli. This toolbox includes the attP site-based integration vectors and the attB site-based template vectors, equipped with mutant loxP sites (i.e., LE* and RE*). The former vectors facilitate integration of passenger genes into attB sites while the latter allows creation of new attB sites. Consequently, the inserted vector backbone is flanked by LE* and RE* sites and can be rescued by Cre. Based on this approach, the biosynthetic pathway of poly(3-hydroxybutyric acid) was first built in E. coli. By scoring the observable phenotype of integrants, the result revealed that the efficiency of gene integration could reach 100%. In addition, reconstruction of the n-butanol-synthesizing pathway in E. coli resulted in a plasmid-free producer strain. As a consequence, the producer strain was able to stably overproduce n-butanol (3.7 g/L) from glucose (20 g/L). Finally, exoglucanase was overexpressed in E. coli that carried multiple genomic copies of the celY gene. Overall, it indicates a promise of our method for cycling improvement of E. coli. (C) 2012 Elsevier Ltd. All rights reserved.