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

U. Schwaneberg, C. Otey, P. C. Cirino, E. Farinas, F. H. Arnold

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the ω-1, ω-2, and ω-3 positions. A continuous spectrophotometric assay for P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to ω-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 DH5α 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 DH5α 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.

Original languageEnglish (US)
Pages (from-to)111-117
Number of pages7
JournalJournal of Biomolecular Screening
Volume6
Issue number2
DOIs
StatePublished - 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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