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United States Department of Agriculture

Agricultural Research Service

Title: An Episomal Expression Vector for Screening Mutant Gene Libraries in Pichia Pastoris

Authors
item Lee, Charles
item Williams, Tina
item Wong, Dominic
item Robertson, George

Submitted to: Plasmid Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 1, 2004
Publication Date: March 1, 2004
Citation: Lee, C.C., Williams, T.G., Wong, D., Robertson, G.H. 2004. An episomal expression vector for screening mutant gene libraries in pichia pastoris. Plasmid Journal. 54(1):80-85.

Interpretive Summary: It is very important to develop improved enzymes that are used in industrial processes. For instance, multiple enzymes are used in the conversion of biomass to fuel and other useful chemicals. The current industrial protocols for biomass conversion are not cost competitive with fossil fuel-derived products such as petroleum. In order to reduce the nation's reliance upon fossil fuels, it is imperative to improve the overall efficiency of biomass conversion. Developing techniques to improve enzyme activity will increase the efficiency of the biomass conversion and other industrial processes. In this report, we describe the creation of an expression vector that can be used in the yeast Pichia pastoris. This vector allows for improved high throughput screening of large libraries of enzyme variants, some of which will have improved activity relative to the native enzyme.

Technical Abstract: Screening mutant gene libraries is a powerful technique for isolating improved enzyme variants. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, commercially-available P. pastoris expression vectors are all designed as integrating plasmids and hence are not as amenable to high throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, that includes an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the a-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.

Last Modified: 8/22/2014
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