Abstract
Analyses of intracellular NADPH concentrations are prerequisites for the design of microbial production strains and process optimization. mBFP was described as metagenomics derived, blue fluorescent protein showing NADPH-dependent fluorescence. Characterization of mBFP showed a high specificity for binding of NADPH (KD 0.64 mM) and no binding of NADH, the protein exclusively amplified fluorescence of NADPH. mBFP catalyzed the NADPH-dependent reduction of benzaldehyde and further aldehydes, which fits to its classification as short chain dehydrogenase. For in vivo NADPH analyses a codon-optimized gene for mBFP was introduced into Corynebacterium glutamicum WT and the phosphoglucoisomerase-deficient strain C. glutamicum Δpgi, which accumulates high levels of NADPH. For determination of intracellular NADPH concentrations by mBFP a calibration method with permeabilized cells was developed. By this means an increase of intracellular NADPH concentrations within seconds after the addition of glucose to nutrient-starved cells of both C. glutamicum WT and C. glutamicum Δpgi was observed; as expected the internal NADPH concentration was significantly higher for C. glutamicum Δpgi (0.31 mM) when compared to C. glutamicum WT (0.19 mM). Addition of paraquat to E. coli cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor.
Highlights
The redox state of cells is represented by the ratio of the internal concentrations of the pyridine nucleotides NADH and NADPH and their corresponding oxidized forms NAD+ and NADP+ (Blacker et al, 2014)
The metagenome derived blue fluorescent protein mBFP exhibits blue fluorescence upon binding of NADPH independently of the availability of oxygen (Hwang et al, 2012). mBFP was here produced with E. coli, which carries the plasmid pCN_mBFP for constitutive high expression of mBFP in E. coli strains (Spaans et al, 2015)
Plasmid encoded mBFP allowed to measure fast changes of NADPH concentrations in both C. glutamicum and E. coli strains and most importantly it allowed to analyze increases as well as decreases of NADPH concentrations in real time. It has to be denoted, that each use of mBFP requires calibration of the signals by permeabilization of the cell and incubation with different NADPH concentrations as the mBFP derived fluorescence signal depends both on the internal NADPH concentration as well as the amount of mBFP, which varies
Summary
The redox state of cells is represented by the ratio of the internal concentrations of the pyridine nucleotides NADH and NADPH and their corresponding oxidized forms NAD+ and NADP+ (Blacker et al, 2014). These ubiquitous cofactors are present in cells only in catalytic amounts, efficient recycling is required for the maintenance of viability and antioxidant protection. To increase productivity and yields of microbial production strains current metabolic engineering strategies aim at an improved NADPH availability such as the redirection of the metabolism toward the PPP or introduction of transhydrogenases (Kabus et al, 2007; Lindner et al, 2013; Wang et al, 2017). Research is limited due to the technically challenging and labor-intensive analysis of internal NADPH concentrations (Lu et al, 2018)
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