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

作者：Fernandez Layla; Jiao Ning; Soni Pankaj; Gumulya Yosephine; De Oliveira Luciana Gonzaga; Reetz Manfred T^*

来源：Biocatalysis and Biotransformation, 2010, 28(2): 122-129.

DOI：10.3109/10242420903505834

• Directed evolution
• expression systems
• Pichia pastoris
• saturation mutagenesis

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.

• 出版日期2010-3