Department of Chemical & Biochemical Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854, United States.
Department of Genetics and Waksman Institute, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, United States.
ACS Synth Biol. 2021 Apr 16;10(4):682-689. doi: 10.1021/acssynbio.0c00449. Epub 2021 Mar 22.
Detection of azide-tagged biomolecules (, azido sugars) inside living cells using "click" chemistry has been revolutionary to the field of chemical biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called regulon, and complementary engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes. The engineered azide operon allowed highly tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01-5 mM) and rapidly induced GFP expression by over 600-fold compared to the uninduced control. Next, we showcase the superior performance of this engineered -operon over the classical -operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitating activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.
使用“点击”化学技术在活细胞内检测叠氮标记的生物分子（如叠氮糖），这一技术革新了化学生物学领域。然而，我们目前仍然缺乏合适的合成生物学工具来自动且快速地检测叠氮离子。在这里，我们开发了一种名为 regulon 的工程化合成启动子系统，以及互补的工程化菌株，用于选择性检测叠氮离子并自动诱导报告基因的下游表达。该工程化的叠氮操纵子允许在三个数量级的诱导剂叠氮离子浓度（0.01-5 mM）范围内对报告基因绿色荧光蛋白（GFP）进行高度可调的表达，与未诱导的对照相比，GFP 的表达被诱导超过 600 倍。接下来，我们展示了该工程化的 -operon 在重组蛋白生产方面相对于经典的 -operon 的卓越性能。最后，我们强调了这个合成生物学工具包如何通过使用叠氮糖作为供体底物来促进糖基工程化应用，从而为突变碳水化合物活性酶（CAZymes），即糖基合成酶的活性筛选提供便利。
