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

成果快讯|2019-03-12



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 PGAP system is a suitable alternative to the PAOX1 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%.



· 通讯作者 ·

王腾飞

职称:教授

研究方向:传统微生物发酵与酿造技术研究;发酵废物的微生物体系处理与再利用研究;工业微生物表达体系的改造,优化与产业设计;工业用酶的新酶挖掘与分子改造及酶的高效表达研究

Email:wangtengfei1981@163.com

王腾飞
· 课题组名称 ·

· 联系我们 ·

联系我们

kfg@qlu.edu.cn

寻求合作

yunlingwe@163.com

· 分享 ·
×关闭