title: In vitro flow cytometry-based screening platform for cellulase engineering.
abstract: Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 10(7) events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg).
authors.affiliation: RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.
authors.forename: Georgette
authors.initials: G
authors.lastname: Körfer
authors.affiliation: RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Pitzler
authors.affiliation: RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.
authors.forename: Ljubica
authors.initials: L
authors.lastname: Vojcic
authors.affiliation: RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.
authors.forename: Ronny
authors.initials: R
authors.lastname: Martinez
authors.affiliation: RWTH Aachen University, Worringerweg 3, D-52074 Aachen, Germany.|DWI an der RWTH Aachen e.V, Forckenbeckstraße 50, 52056 Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D002474:Cell-Free System; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D046974:Clostridium cellulovorans / Q000201:enzymology / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays
chemical_list: D002480:Cellulase