title: Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.
abstract: A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany. t.eggert@fz-juelich.de
authors.forename: Thorsten
authors.initials: T
authors.lastname: Eggert
authors.forename: Susanne Aileen
authors.initials: SA
authors.lastname: Funke
authors.forename: Nalam M
authors.initials: NM
authors.lastname: Rao
authors.forename: Priyamvada
authors.initials: P
authors.lastname: Acharya
authors.forename: Holger
authors.initials: H
authors.lastname: Krumm
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D011995:Recombination, Genetic; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism
chemical_list: D004259:DNA-Directed DNA Polymerase; D008049:Lipase