Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (Delta cpxA, Delta gcvR, Delta glnL, and an unknown-gene-deleted one (Delta uk)) were able to increase the CYP154A1 activity by approximately 1.4-1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (Delta cpxA, Delta gcvR, and Delta glnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Delta uk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of Delta cpxA, Delta gcvR, and Delta glnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.