Optimization Of Fermentation Condition For Ribavirin And Construction Of Ribavirin Producing Strains

Ribavirin is an analog of purine nucleoside which plays important roles in the treatment of various kinds of DNA and RNA viruses. Where 1,2,4-Triazole-3-carboxamide (TCA) are added to the culture of nucleoside producing strains of purine nucleoside, and ribavirin is synthesized from endogenous nucleoside catalyzed by the natural purine nucleoside phosphorylase (PNPase, encoded by pupG) of the strain. This fermentative method is named fermentation synthesis of ribavirin.The study firstly optimizate of fermentation condition for production ribavirin by our lab stock strain. Bacillus amyloliquefaciens TA208-LM is the mutative strain of auxotroph of adenine. The adenine concentration in the fermentation broth is critical as such:too little adenine affects the growth of bacteria, while too much restrains the synthesis of key enzymes. Firstly we optimized the type of adenine donor and its appropriate content. The results indicated that Springer 2506 was the best adenine donor, and 15 g/L was optimal for ribavirin accumulation. However, at the end of fermentation the adenine concentration released from this dose of Springer 2506 was too high. Therefore, the initial quantity of Springer 2506 was reduced and extra adenine was added, the results indicated that the optimal initial adenine concentration was 47.95 mg/L and accordingly the adenine in the fermentation liquid. maintained 40-50 mg/L, which produced the highest PRPP amidotransferase activity and the ribavirin titer. This data indicated that controlling the initial addition of yeast extract and further feeding appropriate quantity of yeast extract was likely a feasible way to control adenine concentration in the fermentation. We studied Springer 2506 fed-batch fermentation in 7.5 L fermentation tank. The results indicated that when 7.5 g/L Springer 2506 was used as substrate and another 7.5 g/L fed, the ribavirin production increased by 11.29% compared to that when 15 g/L adding was added at a time.Plasmid pBSA43-pupG, that Bacillus amyloliquefaciens TA208-LM include, enhanced purine nucleoside phosphorylase catalyzing guanosine to ribavirin. It leaded to the growth slow and effect the yield of ribavirin. Thus, we used small plasmid or integrated method to reduce the growth of burden. We improved PNPase expression levels in host to increase the yield of ribavirin.In the work, pupG was overexpressed in B. amyloliquefaciens TA208, resulting in the successful construction of intracellular expression TA2081 and extracellular expression TA2082. By shake flask fermentation, extracellular expression TA2082 is more suitable for producting ribavirin than intracellular TA2081. In anther work, pupG was integrated in B. amyloliquefaciens TA208, resulting in the successful construction of B. amyloliquefaciens TA2083. By shake flask fermentation, the results showed that TA2083 than the original bacterium TA208 ribavirin yield increased by 58.3%, but there are still a large number of remaining precursor guanosine content, show that PNPase enzyme activity is not enough. Finally, in the 7.5 L fed-batch fermentations, the molar yield of ribavirin from guanosine of B. amyloliquefaciens TA2082 and TA2083 was 98.63% and 40.95%, respectively, whereas that the yield of TA208 was only 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 further constructing ribavirin producers through metabolic engineering.