Cost-effective whole-cell assay for laboratory evolution of hydroxylases in Escherichia coli

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hyd roxylation of medium- and long-chain 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 rec ently. 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 pe llet, and cell lysis, We have shown that permeabilization of the outer memb rane of Escherichia coli DH5 alpha with polymyxin B sulfate, EDTA, polyethy lenimine, or sodium hexametaphosphate results in rapid conversion of 12-pNC A. A NADPH-generating system consisting of NADP(+), D/L-isocitric acid, and the D/L-isocitrate dehydrogenase of E. coli DH5 alpha reduced the cofactor expense more than 10-fold. By avoiding cell lysis, resuspension, and centr ifugation, the high throughput protocol allows screening of thousands of sa mples per day.