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Engineering sensitivity and specificity of AraC-based biosensors responsive to triacetic acid lactone and orsellinic acid
bioRxiv - Synthetic Biology Pub Date : 2020-09-16 , DOI: 10.1101/2020.09.15.299107
Zhiqing Wang , Aarti Doshi , Ratul Chowdhury , Yixi Wang , Costas D. Maranas , Patrick C. Cirino

We previously described the design of triacetic acid lactone (TAL) biosensor 'AraC-TAL1', based on the AraC regulatory protein. While useful as a tool to screen for enhanced TAL biosynthesis, this variant shows elevated background (leaky) expression, poor sensitivity, and relaxed inducer specificity, including responsiveness to orsellinic acid (OA). More sensitive biosensors specific to either TAL or OA can aid in the study and engineering of polyketide synthases that produce these and similar compounds. In this work, we employed a TetA-based dual-selection to isolate new TAL-responsive AraC variants showing reduced background expression and improved TAL sensitivity. To improve TAL specificity, OA was included as a 'decoy' ligand during negative selection, resulting in isolation of a TAL biosensor that is inhibited by OA. Finally, to engineer OA-specific AraC variants, the IPRO computational framework was employed, followed by two rounds of directed evolution, resulting in a biosensor with 24-fold improved OA/TAL specificity, relative to AraC-TAL1.

中文翻译:

对三乙酸内酯和奥数酸的响应的基于AraC的生物传感器的工程敏感性和特异性

我们先前描述了基于AraC调节蛋白的三乙酸内酯(TAL)生物传感器'AraC-TAL1'的设计。尽管可用作筛选增强TAL生物合成的工具,但该变体显示出较高的背景(泄漏)表达,较差的敏感性和宽松的诱导物特异性,包括对奥山酸(OA)的响应性。TAL或OA特有的更灵敏的生物传感器可以帮助研究和工程化生成这些化合物和类似化合物的聚酮化合物合酶。在这项工作中,我们采用了基于TetA的双重选择来分离新的TAL反应性AraC变体,从而显示出降低的背景表达和更高的TAL敏感性。为了提高TAL特异性,在阴性选择过程中将OA作为“诱饵”配体包括在内,从而导致OA抑制了TAL生物传感器的分离。最后,
更新日期:2020-09-16
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