Cell-Surface Displayed Expression of Trehalose Synthase from Pseudomonas putida ATCC 47054 in Pichia Pastoris Using Pir1p as an Anchor Protein

Scientific Achievement

In this paper, the recombinant P.pastoris GS115/Pir1p-EGFP-TreS was successfully constructed to prove the fusion protein displayed on the P.pastoris GS115 cell surface using Pir1p as anchor protein by Western blot localizaton, fluorescence microscopy observation and flow cytometry analysis. The recombinant P.pastoris GS115/Pir1p-TreS can successfully display TreS and used to produce trehalose. The expression levels of TreS obtained in the 5 L fermenters indicate that the P_GAP system is a suitable alternative to the P_AOX1 system for large-scale production of recombinant TreS in P. pastoris GS115 by a high cell density culture, and the enzyme activity reached above 1100U/g. It can be concluded that constitutive TreS expression in a continuous culture of P.pastoris may be easy to scale up and has good prospects for decreasing production costs for large-scale industrial fermentations.

Significant and Impact

This paper revealed that the TreS can display on the P.pastoris cell surface and still had a higher catalytic activity after recycled three times, which was suitable for industrial application, especially the preparation of pharmaceutical grade trehalose products.

Research Details

•The enhanced green fluorescence protein gene (egfp) was used as the reporter protein to fusion the Pir1p gene and treS gene to construct the recombinant plasmids containing treS-egfg-Pir1p fusion gene, and electrotransformed into P.pastoris GS115 to analyze the surface display characteristics of fusion gene by Western blot, fluorescence microscopy and flow cytometry.

•In order to obtain surface active cells with high enzyme activity, the enzymatic properties of TreS displayed on the cell surface was analyzed, and the fermentation process of recombinant P.patoris GS115 containing pPICZaA-Pir1p-treS and pGAPZaA-Pir1p-treS was studied respectively.

•The cell-surface displayed TreS was used to product trehalose using high maltose syrup as substrate at pH 8.0 and 15◦C. The surface display TreS cells can be recycled for three times and the weight conversion rate of trehalose was more than 60%.