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Type: Artigo de periódico
Title: An efficient method for mutant library creation in Pichia pastoris useful in directed evolution
Author: Fernandez, L
Jiao, N
Soni, P
Gumulya, Y
De Oliveira, LG
Reetz, MT
Abstract: The yeast Pichia pastoris is being increasingly used as a host for expressing enzymes on a large scale, but application in directed evolution requiring efficient expression of libraries of mutants is hampered due to the time-consuming multistep procedure which includes an intermediate bacterial host (Escherichia coli). Here we introduce a fast and highly simplified method to produce gene libraries in P. pastoris expression vectors. For the purpose of illustration, Galactomyces geotrichum lipase 1 (GGL1) was used as the catalyst in the enantioselective hydrolytic kinetic resolution of 2-methyldecanoic acid p-nitrophenyl ester, the gene mutagenesis method being saturation mutagenesis. The phosphorylated linear plasmid which is integrated in the yeast genome was obtained by combination of partially overlapped fragments using overlap-extension PCR. An intermediate bacterial host is not necessary, neither are restriction enzymes. This method is also applicable when using error-prone PCR for library creation in directed evolution.
Subject: Directed evolution
expression systems
Pichia pastoris
saturation mutagenesis
Country: Inglaterra
Editor: Taylor & Francis Ltd
Rights: fechado
Identifier DOI: 10.3109/10242420903505834
Date Issue: 2010
Appears in Collections:Artigos e Materiais de Revistas Científicas - Unicamp

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