文献类型：期刊文献

英文题名：Engineering Transcription Factor XylS for Sensing Phthalic Acid and Terephthalic Acid: An Application for Enzyme Evolution

作者：Li, Jiawei[1];Nina, Mario Roque Huanca[1];Zhang, Xiaoyan[1];Bai, Yunpeng[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2022

卷号：11

期号：3

起止页码：1106

外文期刊名：ACS SYNTHETIC BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000776274500009)】；

基金：This work was financially sponsored by the National Key Research and Development Program of China (2016YFA0204300), the National Natural Science Foundation of China (No. 22078096), and the Natural Science Foundation of Shanghai (18ZR1409900).

语种：英文

外文关键词：biosensor; transcription factor engineering; phthalic acid; terephthalic acid; enzyme evolution; biodegradation

摘要：Poly(ethylene terephthalate) (PET) and phthalate esters (PAEs) are used extensively as plastics and plasticizers. Enzymatic degradation of PET and PAEs has drawn great attention in recent years; however, evolution of PET- and PAE-degrading enzymes is still a big challenge, partly because of the lack of an effective screening method to detect phthalic acid (PA) and terephthalic acid (TPA), which are the main hydrolysis products of PAEs and PET. Here, by directed evolution of a promiscuous transcription factor, XylS from Pseudomonas putida, we created two novel variants, XylS-K38R-L224Q and XylS-W88C-L224Q, that are able to bind PA and TPA and activate the downstream expression of a fluorescent reporter protein. Based on these elements, whole-cell biosensors were constructed, which enabled the fluorimetric detection of as little as 10 mu M PA or TPA. A PAE hydrolase, GoEst15, was preliminarily engineered using this new biosensor, yielding a mutant GoEst15-V3 whose activity toward dibutyl phthalate (DBP) and p-nitrophenyl butyrate was enhanced 2.0- and 2.5-fold, respectively. It was shown that 96.5% DBP (5 mM) was degraded by GoEst15-V3 in 60 min, while the wild-type enzyme degraded only 55% DBP. This study provides an effective screening tool for directed evolution of PAE-/PET-degrading enzymes.