Directed Evolution of Transcriptional Factor BreR for Improved Substrate Specificity Toward Deoxycholic Acid and Ursodeoxycholic Acid

IF 3.1 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Biotechnology Journal Pub Date : 2025-06-09 DOI:10.1002/biot.70058

Jianping Xu, Jian Zhang, Xiaotong Wang, Yue Hao, Jinghai Wang, Baiyu Su, Jingyan Yin, Zhiguo Wang, Qian Wang, Qingsheng Qi

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Abstract

Bile acids are endogenous organic acids in mammals that are crucial for digestion and human health. Diverse bile acids serve distinct roles in physiological processes. Deoxycholic acid (DCA) is acknowledged as a biomarker linked to liver and gallbladder disorders, while ursodeoxycholic acid (UDCA) serves as a therapeutic agent for gallstones and various biliary conditions. In this study, we developed a high-throughput screening apparatus utilizing growth coupling and fluorescence sorting to evolve DCA biosensor BreR, and altered its major substrate-binding site to yield mutant I125P, which significantly enhanced the specificity of its DCA response. Simultaneously, we evolved the BreR mutant in response to UDCA. The introduction of 50 mM UDCA resulted in a 235.7% rise in fluorescence intensity, so offering a novel technique for the expedited detection of UDCA. This work establishes an engineering strategy for reprogramming biosensor specificity and provides a foundational platform for UDCA biosensor deployment.

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转录因子BreR定向进化提高对脱氧胆酸和熊脱氧胆酸的底物特异性

胆汁酸是哺乳动物体内的内源性有机酸，对消化和人体健康至关重要。不同的胆汁酸在生理过程中起着不同的作用。去氧胆酸（DCA）被认为是与肝脏和胆囊疾病相关的生物标志物，而熊去氧胆酸（UDCA）可作为胆结石和各种胆道疾病的治疗剂。在本研究中，我们开发了一种高通量筛选装置，利用生长偶联和荧光分选进化DCA生物传感器BreR，并改变其主要底物结合位点，产生突变体I125P，显著提高了其DCA反应的特异性。同时，我们进化出BreR突变体来应对UDCA。50 mM UDCA的引入使荧光强度提高了235.7%，为UDCA的快速检测提供了一种新的技术。这项工作建立了一个重新编程生物传感器特异性的工程策略，并为UDCA生物传感器的部署提供了一个基础平台。

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来源期刊

Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine

CiteScore

8.90

自引率

2.10%

发文量

123

审稿时长

1.5 months

期刊介绍： Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.