Jayaraman Muralidharan, Radhika V, Bamne Mikhil N, Ramos Ross, Briggs Rita, Dhanasekaran Danny N
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, Pennsylvania 19140, USA.
Biotechnol Prog. 2005 Sep-Oct;21(5):1373-9. doi: 10.1021/bp050104j.
One of the major stress factors during space and high-altitude flight is the oxidative damage caused by the release of reactive oxygen intermediates (ROIs) in human tissues. ROIs are released in response to several stress factors including radiation in space. Since ROIs contribute to several pathological conditions, there has been a great interest in developing a biosensor that can monitor the impact of ROIs on biological systems. Toward this goal, we sought to engineer a yeast stain that can monitor oxidative stress and be easily integrated into a biosensor platform. Saccharomyces cerevisiae respond to hyperoxidative stress by activating the expression of many proteins including the transcription factor, Yap1. Activated Yap1 primarily binds to the Yap-1 response elements in the promoters of genes that combat oxidative stress. Based on these observations, we genetically altered the Yap-1 pathway in the YCR094W BY4742 strain of S. cerevisiae by fusing the YREs in the promoter region of TRX2 gene to a cDNA-insert encoding green fluorescent protein (GFP). Exposure of this engineered yeast strain BioS-OS1 to varying levels of oxidative stress, as generated by different concentrations of H(2)O(2) or diamide, elicits robust expression of GFP that can be monitored by the fluorescence of GFP by as early as 1 h. BioS-OS1 can detect a H(2)O(2) concentration from 300 microM onward. We also show that the signaling strength of the strain can be increased by engineering multiple YREs in the upstream of the cDNA-insert encoding GFP. Thus, the results presented here demonstrate that the engineered BioS-OS yeast strain can detect ROI-generating oxidative stress and validate the use of this prototypic strain for the development of a biosensor to detect and monitor oxidative stress factors during space and high altitude flights.
太空和高空飞行期间的主要应激因素之一是人体组织中活性氧中间体（ROIs）释放所导致的氧化损伤。ROIs是对包括太空辐射在内的多种应激因素作出反应而释放的。由于ROIs会引发多种病理状况，因此人们对开发一种能够监测ROIs对生物系统影响的生物传感器产生了浓厚兴趣。为实现这一目标，我们试图构建一种能够监测氧化应激且易于整合到生物传感器平台中的酵母菌株。酿酒酵母通过激活包括转录因子Yap1在内的多种蛋白质的表达来应对高氧化应激。被激活的Yap1主要与对抗氧化应激基因启动子中的Yap-1反应元件结合。基于这些观察结果，我们通过将TRX2基因启动子区域中的YREs与编码绿色荧光蛋白（GFP）的cDNA插入片段融合，对酿酒酵母YCR094W BY4742菌株中的Yap-1途径进行了基因改造。将这种工程酵母菌株BioS-OS1暴露于不同浓度的H(2)O(2)或二酰胺所产生的不同水平的氧化应激中，最早在暴露1小时后就能通过GFP荧光监测到GFP的强烈表达。BioS-OS1能够检测到从300微摩尔起的H(2)O(2)浓度。我们还表明，通过在编码GFP的cDNA插入片段上游构建多个YREs，可以增强该菌株的信号强度。因此，此处呈现的结果表明，工程化的BioS-OS酵母菌株能够检测产生ROIs的氧化应激，并验证了该原型菌株可用于开发生物传感器，以检测和监测太空和高空飞行期间的氧化应激因素。
