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

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


Author Fernandez, Layla
Jiao, Ning
Soni, Pankaj
Gumulya, Yosephine
De Oliveira, Luciana Gonzaga
Reetz, Manfred T.
Title An efficient method for mutant library creation in Pichia pastoris useful in directed evolution
Formatted title
An efficient method for mutant library creation in Pichia pastoris useful in directed evolution
Journal name Biocatalysis and Biotransformation   Check publisher's open access policy
ISSN 1024-2422
1029-2446
Publication date 2010
Sub-type Article (original research)
DOI 10.3109/10242420903505834
Open Access Status Not yet assessed
Volume 28
Issue 2
Start page 122
End page 129
Total pages 8
Place of publication Abingdon, Oxfordshire, United Kingdom
Publisher Taylor & Francis
Language eng
Formatted 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.
Keyword Directed evolution
Expression systems
Pichia pastoris
Saturation mutagenesis
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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