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.