Open AccessCost-Effective Whole-Cell Assay for Laboratory Evolution of Hydroxylases in Escherichia coli
Author(s) -
Ulrich Schwaneberg,
Christopher R. Otey,
Patrick Cirino,
Edgardo T. Farinas,
Frances H. Arnold
Publication year - 2001
Publication title -
slas discovery
Language(s) - English
Resource type - Journals
eISSN - 2472-5560
pISSN - 2472-5552
DOI - 10.1177/108705710100600207
Subject(s) - lysis , escherichia coli , bacillus megaterium , biochemistry , shigella dysenteriae , chemistry , chromatography , biology , bacteria , gene , genetics
Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the to-1, w-2, and c-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to co-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 DH5a 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 DH5a reduced the cofactor expense more than 10-fold. By avoiding cell lysis, re-suspension, and centrifugation, the high throughput protocol allows screening of thousands of samples per day.