title: Use of a genetically encoded hydrogen peroxide sensor for whole cell screening of enzyme activity.
abstract: We report the use of HyPer, a genetically encoded, fluorescent sensor that reacts with hydrogen peroxide (H2O2), in a novel screen to engineer enzymes for enhanced production of H2O2. We co-expressed HyPer with cytochrome P450 BM3 variants and, using HyPer's ratiometric signal, found variants that produce greater amounts of H2O2 than the wild-type enzyme through the leakage reaction. The screen avoids lysis procedures and the addition of reagents to assay intracellular contents. Less laborious screening procedures will be useful in engineering more powerful H2O2 generators as tools in quantitative redox biology, and increasing the utility of enzymes that produce H2O2 as a by-product alongside a valuable compound.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Lim
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA sikes@mit.edu.
authors.forename: Hadley D
authors.initials: HD
authors.lastname: Sikes
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D001709:Biotechnology / Q000379:methods; D002021:Buffers; D002469:Cell Separation; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: D002021:Buffers; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D010100:Oxygen
keywords: cytochrome P450; directed evolution; genetically encoded sensor; hydrogen peroxide; reactive oxygen species