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Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis
Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo...
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Published in: | Biochimica et biophysica acta. Bioenergetics 2018-10, Vol.1859 (10), p.1015-1024 |
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Main Authors: | , , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD+ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD+ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.
•Expression of the fluorescent NADH/NAD+ biosensor Peredox-mCherry in U. maydis•Established a non-invasive online monitoring method for NADH/NAD+ ratios•Uncovered metabolic redox dynamics under biotechnologically relevant conditions•Systemically altered metabolic redox dynamics by expression of an NADH oxidase |
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ISSN: | 0005-2728 1879-2650 |
DOI: | 10.1016/j.bbabio.2018.05.012 |