Assembly of designed oligonucleotides as an efficient method for gene recombination: a new tool in directed evolution.
A new and practical method for gene recombination with formation of libraries of mutant genes is presented. The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information. High recombination frequency with formation of full-length products is achieved by controlled overlapping of the designed oligomers. This process (ADO) minimizes self-hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes. ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB). In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso-diacetate.
Zha
Dongxing
D
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim/Ruhr, Germany.
Eipper
Andreas
A
Reetz
Manfred T
MT
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015723:Gene Library; D006868:Hydrolysis; D008049:Lipase / Q000096:biosynthesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000138:chemical synthesis*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013237:Stereoisomerism
D009841:Oligonucleotides; D008049:Lipase