Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering
Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering
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工程调节子可实现自主叠氮离子生物传感、重组蛋白生产和体内糖工程
DOI：
10.1021/acssynbio.0c00449
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发表时间：
2021
影响因子：
4.7
通讯作者：
Chundawat, Shishir P.
中科院分区：
文献类型：
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作者：
Bandi, Chandra Kanth;Skalenko, Kyle S.;Agrawal, Ayushi;Sivaneri, Neelan;Thiry, Margaux;Chundawat, Shishir P.
利用“点击”化学检测活细胞内的叠氮化合物标记的生物分子(例如叠氮糖)是化学生物学领域的革命性进展。然而，目前我们仍然缺乏合适的合成生物学工具来自主、快速地检测叠氮离子。在这里，我们开发了一种名为cynRegon的工程合成启动子系统，并与大肠杆菌工程菌株互补，以选择性地检测叠氮离子并自主诱导报告基因的下游表达。工程氰氮化物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)范围内高度可调的报告绿色荧光蛋白(GFP)表达，并快速诱导GFP表达，与未诱导的对照相比，GFP表达增加了600倍以上。接下来，我们展示了这种工程环状操纵子相对于经典的丙交酯操纵子在重组蛋白生产中的优越性能。最后，我们强调这个合成生物学工具包如何通过促进以叠氮糖为供体底物的突变碳水化合物活性酶(CAZymes)的活体活性筛选来实现基于糖工程的应用。
Detection of azide-tagged biomolecules (e.g., 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 calledcynregulon, and complementaryEscherichia coliengineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes. The engineeredcynazide 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 engineeredcyn-operon over the classicallac-operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitatingin vivoactivity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.