Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Appl Microbiol Biotechnol. 2010 Jun;87(2):647-55. doi: 10.1007/s00253-010-2505-7. Epub 2010 Mar 12.
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.
对大肠杆菌 BW25113（Keio 文库）的单基因敲除文库进行了系统筛选，以选择能够增强 CYP154A1 催化的 7-乙氧基香豆素去乙基化的突变体。经过 96 孔板高通量筛选和试管试验，四个突变体（Delta cpxA、Delta gcvR、Delta glnL 和一个未知基因缺失突变体（Delta uk））能够将 CYP154A1 活性提高约 1.4-1.7 倍，与对照菌株相比。当在大肠杆菌 BW25113 中分别敲除 cpxA、gcvR、glnL 和 uk 基因构建新的突变体时，其中三个（Delta cpxA、Delta gcvR 和 Delta glnL）表现出高的 CYP154A1 活性。然而，uk 突变体未能增强 CYP154A1 活性，表明 Keio 文库中 Delta uk 突变体的高 CYP154A1 活性是由于染色体的自发突变。Delta cpxA、Delta gcvR 和 Delta glnL 的框内缺失突变体也表现出高的酶活性，并且这些突变的互补可以降低 CYP154A1 活性。这些结果表明，酶活性的增强不是由于对其相邻基因的极性影响。据我们所知，这是首次报道通过全基因组缺失筛选来提高重组全细胞生物催化剂的活性。
