Abstract:

Using Escherichia coli as the host of RihA（pyrimidine-specific ribonucleoside hydrolase）expression, uridine was efficiently transformed into uracil by biological methods. The plasmid pET22b-RihA was constructed and recombinantly expressed in E. coli BL21（DE3）, and uridine transformation was investigated. Two strategies were adopted：replacing pET22b-RihA signal peptide or/and co-expressing with molecular chaperone plasmid, for increasing the efficacy of E. coli transforming uridine. Strain F, which was co-expressed by PhoA signal peptide derived from alkaline phosphatase and molecular chaperone GroES-GroEL, presented the optimal transformation efficiency for substrate uridine. When substrate concentration was 65 g/L, uridine was almost completely transformed by strain F with 98.9% uridine conversion rate for 15 h, while conversion rate of uridine by original strain A was only 80.2%. The concentration of uridine transformed by strain F was further optimized. When adding one time volume of uridine into the fermentation broth, uridine was completely transformed in 53 h. The yield of uracil was 73.45 g/L and the uracil productive rate was 98.16%. The highest extracellular RihA activity was found in strain C co-expressed by PelB signal peptide and molecular chaperone GroES-GroEL, in which RihA activity was 10.0 times of that in original strain A. The highest total soluble RihA enzyme activity was found in strain G, which was co-expressed by PhoA signal peptide and molecular chaperone DnaK-DnaJ-GrpE, and RihA enzyme activity in strain G was 4.45 times of that in the original strain A. By optimizing the signal peptide and molecular chaperone, the conversion efficiency of uridine was improved, and an efficient method was established for the production of uracil, a drug intermediate, by whole cell catalysis of E. coli. Compared with chemical synthesis method, it avoids complicated production process steps and is environmentally friendly and green.

Key words: pyrimidine-specific ribonucleoside hydrolase RihA, uracil, biotransformation, signal peptide, molecular chaperone