A Regulatory NADH/NAD plus Redox Biosensor for Bacteria
A Regulatory NADH/NAD plus Redox Biosensor for Bacteria
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DOI：
10.1021/acssynbio.8b00485
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发表时间：
2019-02-01
影响因子：
4.7
通讯作者：
Raman, Srivatsan
中科院分区：
文献类型：
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作者：
Liu, Yang;Landick, Robert;Raman, Srivatsan
NADH和NAD+辅因子驱动数百种生化反应，它们的比例是细胞状态的关键代谢标志物。测量NADH/NAD+比率的传统测定方法费力，容易出现不准确，并且不适合高通量筛选。我们报告了一种基于氧化还原反应细菌转录因子雷克斯的基因编码的比例生物传感器，克服了这些限制。我们设计了一个Rex调控的E.通过调节雷克斯和操纵位点的亲和力，使大肠杆菌启动子具有改进的生物传感器特性。由于NADH在有氧呼吸过程中被氧化，因此我们使用生物传感器-报告器来研究在有氧呼吸过程中去除呼吸链酶对NADH/NAD+比率的影响。我们发现，与野生型相比，9个突变体中的5个中的NADH/NAD+信号增加超过3倍，包括具有6倍升高的NADH脱氢酶双突变体。在几种常见的碳源中，E.与大肠杆菌相比，在乙酸盐上生长的大肠杆菌具有更高的NADH/NAD+。在葡萄糖上生长的大肠杆菌。作为高通量氧化还原筛选的概念验证，我们能够通过生物传感器引导的合并筛选在野生型细胞中富集1/10000的高NADH突变体。因此，我们的雷克斯生物传感器-报告器能够实现简便、无创、高通量的氧化还原测量，以了解和设计氧化还原代谢。
NADH and NAD+ cofactors drive hundreds of biochemical reactions, and their ratio is a key metabolic marker of cellular state. Traditional assays to measure the NADH/NAD+ ratio is laborious, prone to inaccuracies, and not suitable for high-throughput screening. We report a genetically encoded ratiometric biosensor for NADH/NAD+ based on redox-responsive bacterial transcription factor Rex that overcomes these limitations. We engineered a Rex-regulated E. coli promoter with improved biosensor characteristics by tuning the affinity of Rex and the operator site. Since NADH is oxidized during aerobic respiration, we used the biosensor-reporter to investigate the effect of removing respiratory chain enzymes on NADH/NAD+ ratio during aerobiosis. We found that the NADH/NAD+ signal increased in five of the nine mutants by over 3-fold compared to wildtype, including an NADH dehydrogenase double mutant with 6-fold elevation. We also found that among several common carbon sources, E. coli grown on acetate exhibited higher NADH/NAD+ compared to E. coli grown on glucose. As a proof-of-concept for high-throughput redox screening, we were able to enrich high NADH mutants present at 1 in 10000 among wildtype cells by biosensor-guided pooled screen. Thus, our Rex biosensor-reporter enables facile, noninvasive, high-throughput redox measurement to understand and engineer redox metabolism.