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

dc.creatorFernandez, L
dc.creatorJiao, N
dc.creatorSoni, P
dc.creatorGumulya, Y
dc.creatorDe Oliveira, LG
dc.creatorReetz, MT
dc.date2010
dc.dateMAR
dc.date2014-11-20T00:18:17Z
dc.date2015-11-26T18:07:59Z
dc.date2014-11-20T00:18:17Z
dc.date2015-11-26T18:07:59Z
dc.date.accessioned2018-03-29T00:50:06Z
dc.date.available2018-03-29T00:50:06Z
dc.identifierBiocatalysis And Biotransformation. Taylor & Francis Ltd, v. 28, n. 2, n. 122, n. 129, 2010.
dc.identifier1024-2422
dc.identifierWOS:000274855300004
dc.identifier10.3109/10242420903505834
dc.identifierhttp://www.repositorio.unicamp.br/jspui/handle/REPOSIP/54362
dc.identifierhttp://www.repositorio.unicamp.br/handle/REPOSIP/54362
dc.identifierhttp://repositorio.unicamp.br/jspui/handle/REPOSIP/54362
dc.identifier.urihttp://repositorioslatinoamericanos.uchile.cl/handle/2250/1293717
dc.descriptionThe 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.
dc.description28
dc.description2
dc.description122
dc.description129
dc.descriptionDeutsche Forschungsgemeinschaft [1170]
dc.descriptionFunda ao de Amparo a Pesquisa do Estado de Sao Paulo
dc.descriptionDeutsche Forschungsgemeinschaft [1170]
dc.languageen
dc.publisherTaylor & Francis Ltd
dc.publisherAbingdon
dc.publisherInglaterra
dc.relationBiocatalysis And Biotransformation
dc.relationBiocatal. Biotransform.
dc.rightsfechado
dc.rightshttp://journalauthors.tandf.co.uk/permissions/reusingOwnWork.asp
dc.sourceWeb of Science
dc.subjectDirected evolution
dc.subjectexpression systems
dc.subjectPichia pastoris
dc.subjectsaturation mutagenesis
dc.subjectBt107 Lipase-i
dc.subjectOverlap Extension
dc.subjectHeterologous Proteins
dc.subjectExpression
dc.subjectMutagenesis
dc.subjectTransformation
dc.subjectSystem
dc.subjectGene
dc.subjectRecombination
dc.subjectSelection
dc.titleAn efficient method for mutant library creation in Pichia pastoris useful in directed evolution
dc.typeArtículos de revistas


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