Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli.
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and longchain fatty acids at the omega-1, omega-2, and omega-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was reported recently. However, this pNCA assay procedure contained steps that limited its application in high throughput screening, including expression of P450 BM-3 variant F87A in 4-ml cultures, centrifugation, resuspension of the cell pellet, and cell lysis. We have shown that permeabilization of the outer membrane of Escherichia coli DH5alpha with polymyxin B sulfate, EDTA, polyethylenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNCA. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.
Schwaneberg
U
U
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
Otey
C
C
Cirino
P C
PC
Farinas
E
E
Arnold
F H
FH
D001331:Automation; D001410:Bacillus megaterium / Q000201:enzymology*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D006900:Hydroxylation; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D008027:Light; D008826:Microbial Sensitivity Tests / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000096:biosynthesis*; D009249:NADP / Q000378:metabolism; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays
D009249:NADP; D006899:Mixed Function Oxygenases; D007521:Isocitrate Dehydrogenase