半胱氨酸单细胞生物传感器的构建及应用  

Construction and application of a single-cell biosensor for detection of cysteine

作　　者：刘川 刘清岱[3] 徐宁[2] 周威[2] 刘君[2] LIU Chuan;LIU Qing-dai;XU Ning;ZHOU We;LIU Jun(College of Biotechnology, Tianjin University of Science ＆ Technology, Tianjin 300457;College of Food Engineering and Biotechnology, Tianjin University of Science ＆ Technology, Tianjin 300457;Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308)

机构地区：[1]天津科技大学生物工程学院,天津300222 [2]中国科学院天津工业生物技术研究所,天津300308 [3]天津科技大学食品工程与生物技术学院,天津300222

出　　处：《中国食品添加剂》2018年第5期147-154,共8页China Food Additives

基　　金：天津自然科学基金(17JCQNJC09600);天津自然科学基金(17JCYBJC24000)

摘　　要：半胱氨酸是一种食品添加剂,被应用于食品的增色、增香以及抗氧化。单细胞生物传感器可以用于氨基酸(如苯丙氨酸,缬氨酸等)的快速检测,但有关半胱氨酸的单细胞生物传感器还没有被报道。本研究基于转录调控因子Ccd R,构建了荧光信号与半胱氨酸浓度相偶联的单细胞生物传感器,并对其线性范围进行了检测。在0 mmol/L至32 mmol/L的半胱氨酸浓度下,荧光信号强度与半胱氨酸浓度之间具有良好的线性关系。利用构建的半胱氨酸单细胞传感器,结合等离子诱变和流式细胞分选方法,从约5万株突变菌中筛选到两株半胱氨酸高产菌,其产量分别是野生型菌株的2.1倍和3.2倍。实验结果表明构建的单细胞生物传感器可以应用于半胱氨酸的快速检测及高产菌株的筛选。Cysteine was widely used as a food additive to increase the color and flavor, and also used for antioxidation in the food industry. The single-cell biosensor can be used for rapid detection and quantification of many amino acids, including phenylalanine and valine. However, the single-cell biosensor of cysteine has not been explored so far. In this study, a new cysteine single-cell biosensor coupled with cysteine concentration and fluorescence was constructed based on the transcriptional factor CcdR, and its liner range was examined. The fluorescence intensity and cysteine concentration were highly correlated at a cysteine concentration of 0 mmol/L to 32 mmol/L. According to this single- cell biosensor of cysteine, two high cysteine-producing mutants were isolated through the combination of random mutagenesis and high-throughput screening methods. The yields of these two mutants were 2.1 and 3.2 times higher than the wild-type strain, respectively. In conclusion, these results indicate that this single cell biosensor might be used in cysteine determination and high-producing strain selection.

关 键 词：半胱氨酸 单细胞生物传感器 CcdR 荧光细胞分选 

分 类 号：O657.3[理学—分析化学] TS202.3[理学—化学]