title: A generic, whole-cell-based screening method for Baeyer-Villiger monooxygenases.
abstract: Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell-based screening method was validated by screening site-saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.
authors.affiliation: Molecular Enzymology Group, University of Groningen, Groningen, The Netherlands.
authors.forename: Hanna M
authors.initials: HM
authors.lastname: Dudek
authors.forename: Petra
authors.initials: P
authors.lastname: Popken
authors.forename: Edwin
authors.initials: E
authors.lastname: van Bloois
authors.forename: Wouter A
authors.initials: WA
authors.lastname: Duetz
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D055162:Biocatalysis; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D003067:Coenzymes; D010710:Phosphates; D009249:NADP; D006899:Mixed Function Oxygenases; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C429539:NAD phosphite oxidoreductase
keywords: Baeyer-Villiger monooxygenase; Tat export; screening method; sulfoxidation; uncoupling