title: A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.
abstract: Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Pitzler
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Georgette
authors.initials: G
authors.lastname: Wirtz
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vojcic
authors.affiliation: DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056 Aachen, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Hiltl
authors.affiliation: DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056 Aachen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Böker
authors.affiliation: EW-Nutrition GmbH, Enzyme Technology, Nattermannalle 1, 50829 Köln, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany; DWI-Leibniz Institut für Interaktive Materialien, Forckenbeckstraße 50, 52056 Aachen, Germany. Electronic address: u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015723:Gene Library; D005949:Glucose Oxidase / Q000378:metabolism*; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D011092:Polyethylene Glycols / Q000737:chemistry; D058105:Polymerization; D012235:Rhodamines / Q000737:chemistry*; D015007:Yersinia / Q000201:enzymology
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D005949:Glucose Oxidase; D010832:6-Phytase; C029773:rhodamine B