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Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD⁺ with Genetically Encoded Fluorescent Biosensors.

ABSTRACT: In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD⁺ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD⁺ binding, the fractional intensities (α _i τ _i ) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD⁺ levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.

SUBMITTER: Chang M

PROVIDER: S-EPMC5482812 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD+ with Genetically Encoded Fluorescent Biosensors.

Chang Mengfang M Li Lei L Hu Hanyang H Hu Qingxun Q Wang Aoxue A Cao Xiaodan X Yu Xiantong X Zhang Sanjun S Zhao Yuzheng Y Chen Jinquan J Yang Yi Y Xu Jianhua J

Scientific reports 20170623 1

In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD+ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD+ binding, the fractional intensities (α i τ i ) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD+ levels with a larger dyn ...[more]

PMID: 28646144

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Project description:BackgroundCofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism.ResultsThrough the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD+/NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols.ConclusionsThe ratio of NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

Dataset Information

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD⁺ with Genetically Encoded Fluorescent Biosensors.

Publications

Using Fractional Intensities of Time-resolved Fluorescence to Sensitively Quantify NADH/NAD<sup>+</sup> with Genetically Encoded Fluorescent Biosensors.

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