Detection Of Intracellular NADH Of Lactobacillus Paracasei Based On A Genetically Encoded Fluorescent Sensor

In this work, a method for detecting the intracellular NADH level of Lactobacillus paracasei was set up based on a novel genetically encoded fluorescent sensor Frex, which was constructed through fusing the bacterial Rex protein, specifically sensitive to NADH, with yellow fluorescent protein (cpYFP). Crystallographic studies showed that the yellow fluorescent group of this sensor would be exposed and its intensity could be real time determined by a fluorescence microplate reader, as long as it combined to NADH. With the aim of application this probe in L. paracasei for bioprocess analysis, two plasmids, pMG36e-cpYFP and pMG36e-Frex were constructed, and correspondently two recombinant strains, L. paracasei (pMG36e-cpYFP) and L. paracasei (pMG36e-Frex) were successfully obtained by electroporation. Based on the comparison of metabolic parameters, it was found that in5L bioreactor which could maintain the pH level stable at6.0, the recombinant strains had similar physiological characteristics to their parent strain during the fermentation process. Furthermore, it was found that the intracellular pH of L. paracasei during the fermentation process was approximately6.4, which satisfied the application range (pH5to8) of this sensor completely. Finally, as osmotic stress would be commonly encountered in lactic acid fermentation, it was selected as an environmental stress to verify the application of this probe in lactic acid fermentation process.In conclusion, the novel method constructed in this study could detect intracellular NADH level with the advantages of high accuracy, superior specificity, possibly in real-time and dynamically. This work started the avenue of this sensor in the bacterial kingdom, and it would provide a powerful tool for microbial cofactor engineering.