Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution.
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.
Eggert
Thorsten
T
Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, 52426 Jülich, Germany. t.eggert@fz-juelich.de
Funke
Susanne Aileen
SA
Rao
Nalam M
NM
Acharya
Priyamvada
P
Krumm
Holger
H
Reetz
Manfred T
MT
Jaeger
Karl-Erich
KE
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
D004259:DNA-Directed DNA Polymerase; D008049:Lipase