An efficient method for mutant library creation inPichia pastorisuseful in directed evolution

Autor:	Manfred T. Reetz, Luciana G. de Oliveira, Layla Fernández, Pankaj Soni, Ning Jiao, Yosephine Gumulya
Rok vydání:	2010
Předmět:	Genetics Expression vector biology Chemistry Mutant Mutagenesis (molecular biology technique) biology.organism_classification medicine.disease_cause Directed evolution Biochemistry Catalysis Pichia pastoris Plasmid medicine Saturated mutagenesis Escherichia coli Biotechnology
Zdroj:	Biocatalysis and Biotransformation. 28:122-129
ISSN:	1029-2446 1024-2422
DOI:	10.3109/10242420903505834
Popis:	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.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=doi_________::bbc9f74be30dfdd2c012776b9169f3a6 https://doi.org/10.3109/10242420903505834 Zobrazit plný text záznamu Plný text ve formátu PDF Plný text ve formátu HTML
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