Goldbeck Oliver, Eck Alexander W, Seibold Gerd M
Institute of Microbiology and Biotechnology, Ulm University, Ulm, Germany.
Institute for Biochemistry, University of Cologne, Cologne, Germany.
Front Microbiol. 2018 Oct 24;9:2564. doi: 10.3389/fmicb.2018.02564. eCollection 2018.
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 ( 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 NADPH analyses a codon-optimized gene for mBFP was introduced into WT and the phosphoglucoisomerase-deficient strain Δ, 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 WT and Δ was observed; as expected the internal NADPH concentration was significantly higher for Δ (0.31 mM) when compared to WT (0.19 mM). Addition of paraquat to cells carrying mBFP led as expected to an immediate decrease of intracellular NADPH concentrations, showing the versatile use of mBFP as intracellular sensor.
细胞内NADPH浓度的分析是设计微生物生产菌株和优化工艺的先决条件。mBFP被描述为源自宏基因组学的蓝色荧光蛋白，显示出依赖NADPH的荧光。mBFP的表征显示其对NADPH（解离常数为0.64 mM）的结合具有高度特异性，且不结合NADH，该蛋白仅放大NADPH的荧光。mBFP催化了NADPH依赖的苯甲醛及其他醛类的还原反应，这与其作为短链脱氢酶的分类相符。为了进行NADPH分析，将一个密码子优化的mBFP基因导入野生型（WT）和磷酸葡萄糖异构酶缺陷型菌株Δ中，该菌株会积累高水平的NADPH。为了通过mBFP测定细胞内NADPH浓度，开发了一种通透细胞的校准方法。通过这种方法，观察到在向野生型和Δ型饥饿营养细胞中添加葡萄糖后的几秒钟内，细胞内NADPH浓度增加；正如预期的那样，与野生型（0.19 mM）相比，Δ型的细胞内NADPH浓度显著更高（0.31 mM）。向携带mBFP的细胞中添加百草枯，正如预期的那样，导致细胞内NADPH浓度立即下降，这表明mBFP作为细胞内传感器具有广泛的用途。
