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An efficient method for mutant library creation in Pichia pastoris useful in directed evolution

Fernández, Layla, Jiao, Ning, Soni, Pankaj, Gumulya, Yosephine, Gonzaga De Oliveira, Luciana and Reetz, Manfred T. 2010. An efficient method for mutant library creation in Pichia pastoris useful in directed evolution. Biocatalysis and Biotransformation 28 (2) , pp. 122-129. 10.3109/10242420903505834

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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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Chemistry
Publisher: Taylor and Francis Group
ISSN: 1024-2422
Last Modified: 14 Nov 2022 16:05
URI: https://orca.cardiff.ac.uk/id/eprint/153698

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