Combinatorial library strategy for strong overexpression of the lipase from Geobacillus thermocatenulatus on the cell surface of yeast Pichia pastoris

• Enzymes can be displayed on yeast cell surface by cell surface display technique.
• Combinatorial library strategy for the lipase overexpression in yeast was developed.
• GS115/D90 exhibited 5-fold higher lipase activity was obtained by the strategy.
• GS115/D90 comprised ENO1 promoter, GAS1 secretion signal, and GAS1 anchoring protein.
• Overexpression on yeast cell surface could be achieved easily by the strategy.

The yeast cell surface display technique allows for the expression of a target protein on the yeast cell surface and has many applications such as the immobilization of enzymes and the development of biosensors. To increase the expression of the BTL2, a lipase from Geobacillus thermocatenulatus, on the cell surface of yeast Pichia pastoris, we developed a combinatorial library strategy for selecting appropriate expression cassette comprising sequences encoding a promoter, secretion signal, mature BTL2, anchoring protein, and terminator. The transformant GS115/D90, which comprised P. pastoris ENO1 promoter sequence, Hansenula polymorpha GAS1 secretion signal sequence, and Saccharomyces cerevisiae GAS1 anchoring protein gene, exhibited 5-fold higher lipase activity compared to the control strain harboring a conventional expression cassette. Using the developed strategy, an appropriate expression cassette for the strong overexpression of target proteins on the cell surface of yeast could be rapidly and easily obtained.

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