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 10 000 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.

中文翻译：

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用于细菌的监管性NADH / NAD +氧化还原生物传感器。

NADH和NAD +辅助因子驱动数百个生化反应，它们的比率是细胞状态的关键代谢指标。传统的测定NADH / NAD +比例的方法费力，容易出错，不适合高通量筛选。我们报告了基于氧化还原反应细菌转录因子Rex克服了这些局限性的NADH / NAD +的遗传编码比例式生物传感器。我们通过调节Rex的亲和力和操作位点设计了Rex调控的大肠杆菌启动子，具有改善的生物传感器特性。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研究了在有氧运动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，九个突变体中的五个中的NADH / NAD +信号增加了3倍以上，包括NADH脱氢酶双突变体，具有6倍的升高。我们还发现，在几种常见的碳源中，与在葡萄糖上生长的大肠杆菌相比，在乙酸盐上生长的大肠杆菌显示出更高的NADH / NAD +。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指导的合并筛选富集野生型细胞中万分之一的高NADH突变体。因此，我们的Rex生物传感器报告器可实现简便，无创，高通量的氧化还原测量，以了解和设计氧化还原代谢。我们能够通过生物传感器引导的合并筛选富集野生型细胞中万分之一的高水平NADH突变体。因此，我们的Rex生物传感器报告器可实现简便，无创，高通量的氧化还原测量，以了解和设计氧化还原代谢。我们能够通过生物传感器引导的合并筛选富集野生型细胞中万分之一的高水平NADH突变体。因此，我们的Rex生物传感器报告器可实现简便，无创，高通量的氧化还原测量，以了解和设计氧化还原代谢。