Real Time Monitoring of NADPH Concentrations in Corynebacterium glutamicum and Escherichia coli via the Genetically Encoded Sensor mBFP

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 (K _D 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.

Keywords: Corynebacterium glutamicum; Escherichia coli; NADPH; biosensor; redox state; short chain dehydrogenase.

FIGURE 1

Dependence of dehydrogenase activity of purified mBFP from its substrate benzaldehyde (A) and its cofactor NADPH (B); different concentrations of benzaldehyde (0.5–12.5 mM; (A) and NADPH (0.5–180 μM; (B) were tested in presence of 200 μM NADPH and 12.5 mM benzaldehyde, respectively. Data represent mean values from three independent measurements, data were fitted according to the Michaelis–Menten equation.

FIGURE 2

Effects of purified mBFP on fluorescence of NADPH, NADH, NADP⁺, and NAD⁺ (each at 0.5 mM) at excitation with 395 nm and emission at 451 nm. Data represent mean values and SDs of three independent measurements.

FIGURE 3

Thermal shift assays for the analysis of the cofactor preference of mBFP. Melting temperatures of apo-mBFP without (–) or in the presence of (1 mM each) NADPH, NADH, NADP⁺, or NAD⁺ (A). Dependence of mBFP melting temperature on the concentration of its cofactor NADPH (B), NADPH concentrations of 0–10 mM were tested. Melting temperatures were determined by heating from 40 to 70°C in 0.5°C steps, and unfolding was monitored as described in Section “Materials and Methods.” Data represent mean values and SDs of three independent measurements, data in (B) were fitted according to the Michaelis–Menten equation.

FIGURE 4

Analyses of changes of mBFP fluorescence in starved cells of C. glutamicum WT (pEKEx2-mBFPopt) [filled circles] and C. glutamicum Δpgi (pEKEx2-mBFPopt) [open circles] upon addition of the substrate glucose (indicated by the arrow) (A). In situ calibration of mBFP derived signals by the use CTAB permeabilized cells from C. glutamicum WT (pEKEx2-mBFPopt) [filled circles] and C. glutamicum Δpgi (pEKEx2-mBFPopt) [open circles] (B) in presence of different amounts of added NADPH. Steady state levels of intracellular NADPH concentrations calculated based on in situ calibrations in C. glutamicum WT (pEKEx2-mBFPopt) and C. glutamicum Δpgi (pEKEx2-mBFPopt) before and after glucose addition (C). For panels (A,B) one representative experiment of a series of three independent experiments is shown. Data in (C) represent mean values and SDs of three independent experiments.

FIGURE 5

Analyses of changes of NADPH concentrations in cells of E. coli DH5α (pEKEx2_mBFPopt) upon addition of the substrate glucose (indicated by the arrow, 100 mM) and consecutive addition of different amounts of paraquat (8 mM paraquat—gray circles, 16 mM paraquat—white triangles, no addition of paraquat—black circles) (A). In situ calibration of mBFP derived signals by the use CTAB permeabilized cells from E. coli DH (pEKEx2_mBFPopt) in presence of different amounts of added NADPH (B). One representative experiment of a series of three independent experiments is shown.