Effect of Different Expression Approaches of Purine Nucleoside Phosphorylase on Ribavirin Accumulation
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Abstract:
Purine nucleoside phosphorylase (PNPase, encoded by pupG) is the key enzyme in the synthesis of ribavirin. The effects of pupG overexpression and integrative expression was investigated, on ribavirin production in Bacillus amyloliquefaciens TA208. The overexpression and integrative expression procedures resulted in the successful construction of B. amyloliquefaciens TA2081 and TA2082 strains. Real-time polymerase chain reaction results showed that the transcriptional levels of pupG of TA2081 and TA2082 increased significantly, but the increment of TA2081 was more obvious. PNPase activity of TA2081 and TA2082 was approximately 2- and 1.30-fold compared to that of the original strain TA208, respectively. Finally, in the 7.5 L fed-batch fermentations, the molar yields of ribavirin from guanosine, by B. amyloliquefaciens TA2081 and TA2082 were 98.63% and 40.95%, respectively, whereas that of TA208 was 20.41%. In summary, the improvement of pupG expression via either plasmid-borne or chromosomal integration approach increased ribavirin production. The results of this study would be useful for development of more efficient ribavirin producers via metabolic engineering.
