Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis

Sandra K Hartmann; Yvonne Stockdreher; Georg Wandrey; Hamed Hosseinpour Tehrani; Thiemo Zambanini; Andreas J Meyer; Jochen Büchs; Lars M Blank; Markus Schwarzländer; Nick Wierckx

doi:10.1016/j.bbabio.2018.05.012

Online in vivo monitoring of cytosolic NAD redox dynamics in Ustilago maydis

Biochim Biophys Acta Bioenerg. 2018 Oct;1859(10):1015-1024. doi: 10.1016/j.bbabio.2018.05.012. Epub 2018 May 22.

Affiliations

¹ iAMB - Institute of Applied Microbiology (iAMB), ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany.
² Institute of Crop Science and Resource Conservation (INRES), Rheinische Friedrich-Wilhelms-Universität Bonn, Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany.
³ AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, D-52074 Aachen, Germany.
⁴ iAMB - Institute of Applied Microbiology (iAMB), ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
⁵ AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, D-52074 Aachen, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany.
⁶ Institute of Crop Science and Resource Conservation (INRES), Rheinische Friedrich-Wilhelms-Universität Bonn, Friedrich-Ebert-Allee 144, D-53113 Bonn, Germany; Institute of Plant Biology and Biotechnology (IBBP), Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany; BioSC, c/o Forschungszentrum Jülich, D-52425 Jülich, Germany. Electronic address: markus.schwarzlander@uni-muenster.de.
⁷ iAMB - Institute of Applied Microbiology (iAMB), ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany. Electronic address: nick.wierckx@rwth-aachen.de.

PMID: 29800548
DOI: 10.1016/j.bbabio.2018.05.012

Abstract

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.

Keywords: In vivo sensing; Metabolic redox balance; NADH oxidase; NADH/NAD(+); Peredox-mCherry; Ustilago.