Enhancement of recombinant enzyme activity in cpxA-deficient mutant of Escherichia coli.
We have previously performed a systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) to investigate the effect of cellular components on the activity of whole-cell catalysts (Zhou et al. Appl. Microbiol. Biotechnol. in press (2010)). In the present paper, the cpxA-deficient mutant of E.coli BW25113 was found to be able to enhance the activity of cytochrome P450s, including CYP154A1 of Streptomyces coelicolor, compactin 6β-hydroxylase of Streptomyces flavus A-177, vitamin D3 hydroxylase of Pseudonocardia autotrophica, and a mutant enzyme derived from P450 BM3 of Bacillus megaterium. The cytochrome P450 activities in the cpxA-deficient mutant were 1.8-7.9 times greater than those detected with the parental strain BW25113 (DE3). The cpxA-deficient mutant also exhibited β-galactosidase activity approximately 1.5-fold greater than did the parental strain, showing that the enhancing effect was not unique to the activity of cytochrome P450s. Promoter dependency analysis revealed that the enhanced activity of the enzymes was detectable only when the genes encoding the enzymes were expressed under the control of lactose-inducible promoters. The result of real-time PCR showed that the enhancement of the enzyme activities was attributed to increased transcriptional levels of the genes coding for the enzymes.
Zhou
Ying
Y
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Minami
Takeshi
T
Honda
Kohsuke
K
Omasa
Takeshi
T
Ohtake
Hisao
H
D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D001616:beta-Galactosidase / Q000378:metabolism
D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D011494:Protein Kinases; C490475:CpxA protein, E coli; D001616:beta-Galactosidase