Secretion and Expression of Enzymes in the Nicotinamide Mononucleotide Synthesis Pathway and the Optimization of the Catalytic System in Escherichia coli
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Abstract:
Nicotinamide mononucleotide (NMN) is a precursor of NAD+ with important medical value. Escherichia coli BL21(DE3) expresses and secretes the enzymes nicotinamide phosphoribosyltransferase (Sfnampt), phosphoribosyl pyrophosphate kinase (Tkprs), and ribokinase (Erbks), which catalyze the synthesis of NMN through the signal peptide PelB. The enzymatic characteristics of three enzymes were analyzed, and the results showed that the optimum reaction temperatures of Sfnampt, Tkprs, and Erbks were 45 ℃, 50 ℃, and 37 ℃ respectively, and the optimum pH values were 7.5, 8.5, and 5.5 respectively. In terms of thermal stability, Sfnampt was found to have good thermal stability at 35 ℃ and poor stability at 40~55 ℃. The thermal stability of Tkprs was optimized at 40~60 ℃, whereas that of Erbks was best at 25~40 ℃ and poor at 45 ℃. The results of the enzyme kinetic analysis showed that the Vmax values of Sfnampt, Tkprs, and Erbks were 0.65, 2.37, and 12.58 μmol/(L•min), respectively, and Km values were 2.87, 25.48, and 74.13 μmol/L, respectively. Compared with the characterized Prs and Rbks, Tkprs and Erbks had better substrate affinity. The three enzymes were used for the catalytic synthesis of NMN, and the optimum catalytic conditions were 37 ℃ and pH 8.0. The substrate ATP and enzyme Sfnampt were the key factors in the reaction system. After optimizing the ratio of substrate and enzyme, 5.5 μmol/L of NMN could be obtained after 7 h of catalysis by the three enzymes. In this study, the enzymes for the pathway of NMN synthesis were determined to be successfully expressed and secreted in E. coli, which indicates a new direction for the synthesis of NMN.
