Jha Ramesh K, Kern Theresa L, Kim Youngchang, Tesar Christine, Jedrzejczak Robert, Joachimiak Andrzej, Strauss Charlie E M
Bioscience Division, PO Box 1663, Los Alamos National Laboratory, Los Alamos NM 87545, USA
Bioscience Division, PO Box 1663, Los Alamos National Laboratory, Los Alamos NM 87545, USA.
Nucleic Acids Res. 2016 Sep 30;44(17):8490-500. doi: 10.1093/nar/gkw687. Epub 2016 Aug 17.
A whole-cell biosensor utilizing a transcription factor (TF) is an effective tool for sensitive and selective detection of specialty chemicals or anthropogenic molecules, but requires access to an expanded repertoire of TFs. Using homology modeling and ligand docking for binding pocket identification, assisted by conservative mutations in the pocket, we engineered a novel specificity in an Acinetobacter TF, PobR, to 'sense' a chemical p-nitrophenol (pNP) and measured the response via a fluorescent protein reporter expressed from a PobR promoter. Out of 10(7) variants of PobR, four were active when dosed with pNP, with two mutants showing a specificity switch from the native effector 4-hydroxybenzoate (4HB). One of the mutants, pNPmut1 was then used to create a smart microbial cell responding to pNP production from hydrolysis of an insecticide, paraoxon, in a coupled assay involving phosphotriesterase (PTE) enzyme expressed from a separate promoter. We show the fluorescence of the cells correlated with the catalytic efficiency of the PTE variant expressed in each cell. High selectivity between similar molecules (4HB versus pNP), high sensitivity for pNP detection (∼2 μM) and agreement of apo- and holo-structures of PobR scaffold with predetermined computational models are other significant results presented in this work.
利用转录因子（TF）的全细胞生物传感器是灵敏且选择性检测特定化学品或人为分子的有效工具，但需要获取更多种类的转录因子。我们通过同源建模和配体对接来识别结合口袋，并在口袋中进行保守突变辅助，对不动杆菌转录因子PobR进行工程改造，使其具有识别化学物质对硝基苯酚（pNP）的新特异性，并通过由PobR启动子表达的荧光蛋白报告基因来测量响应。在PobR的10⁷个变体中，有4个在加入pNP时有活性，其中两个突变体表现出从天然效应物4 - 羟基苯甲酸（4HB）的特异性转换。然后，其中一个突变体pNPmut1被用于构建一种智能微生物细胞，在涉及从单独启动子表达的磷酸三酯酶（PTE）的偶联测定中，对杀虫剂对氧磷水解产生的pNP做出响应。我们发现细胞的荧光与每个细胞中表达的PTE变体的催化效率相关。相似分子（4HB与pNP）之间的高选择性、对pNP检测的高灵敏度（约2 μM）以及PobR支架的脱辅基和全蛋白结构与预定计算模型的一致性是这项工作呈现的其他重要结果。
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