title: An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.
abstract: Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium.
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Brixius-Anderko
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Hannemann
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.|Lonza AG, Visp, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Ringle
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.
authors.forename: Yogan
authors.initials: Y
authors.lastname: Khatri
authors.affiliation: Department of Biochemistry, Saarland University, Saarbrücken, Germany.
authors.forename: Rita
authors.initials: R
authors.lastname: Bernhardt
mesh_terms: D000818:Animals; D002417:Cattle; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D007211:Indoles; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014368:Tryptophanase / Q000172:deficiency*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D029968:Escherichia coli Proteins; D007211:Indoles; D011994:Recombinant Proteins; C030374:indole; D003577:Cytochrome P-450 Enzyme System; D014368:Tryptophanase
keywords: E. coli; cytochrome P450; gene disruption; indole; screening