Molecular Ecology Laboratory, Department of Botany, Jamia Hamdard (Hamdard University), New Delhi 110062, India.
Molecular Ecology Laboratory, Department of Botany, Jamia Hamdard (Hamdard University), New Delhi 110062, India.
Biosens Bioelectron. 2014 Sep 15;59:358-64. doi: 10.1016/j.bios.2014.03.066. Epub 2014 Apr 12.
Metabolic engineering of microorganisms for production of biological molecules represent a key goal for industrial biotechnology. The metabolic engineering requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Genetically-encoded fluorescence resonance energy transfer (FRET) sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. In the present paper, we report the development of genetically-encoded FRET-based nanosensor for methionine as metabolic engineering of microbial strains for the production of l-methionine is of major interest in industrial biotechnology. In this nanosensor, methionine binding protein (MetN) from Escherichia coli (E. coli) K12 was taken and used as the reporter element of the sensor. The MetN was sandwiched between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Specificity, affinity, pH stability and metal effects was analyzed for the in vitro characterization of this nanosensor, named as FLIPM. The FLIPM is very specific to methionine and found to be stable with the pH within the physiological range. The calculated affinity (Kd) of FLIPM was 203 µM. This nanosensor successfully monitored the intracellular level of methionine in bacterial as well as yeast cell. The data suggest that these nanosensors may be a versatile tool for studying the in vivo dynamics of methionine level non-invasively in living cells.
微生物代谢工程生产生物分子是工业生物技术的一个关键目标。代谢工程需要详细了解体内代谢物和代谢中间产物的浓度和通量。基于遗传编码的荧光共振能量转移（FRET）传感器是一种用于测量活细胞中代谢物水平和相应速率变化的有前途的技术。在本文中，我们报告了基于遗传编码的 FRET 的甲硫氨酸纳米传感器的开发，因为微生物菌株的甲硫氨酸代谢工程在工业生物技术中具有重要意义。在这个纳米传感器中，我们使用了来自大肠杆菌（E. coli）K12 的甲硫氨酸结合蛋白（MetN）作为传感器的报告元件。MetN 夹在青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）之间。对这种名为 FLIPM 的纳米传感器进行了体外特异性、亲和力、pH 稳定性和金属效应分析。FLIPM 对甲硫氨酸具有特异性，并且在生理范围内的 pH 值下稳定。FLIPM 的计算亲和力（Kd）为 203 µM。这个纳米传感器成功地监测了细菌和酵母细胞内的甲硫氨酸水平。这些数据表明，这些纳米传感器可能是研究活细胞中甲硫氨酸水平的体内动力学的一种通用工具。
