嘌呤核苷磷酸化酶（PNPase，由pupG基因编码）是合成利巴韦林的关键酶，本研究考察该酶的质粒过表达和基因组整合表达对鸟苷生产菌解淀粉芽孢杆菌（Bacillus amyloliquefaciens）TA208发酵生产利巴韦林的影响。以B. amyloliquefaciens TA208为出发菌株，分别利用基因过表达和无标记整合技术，成功构建了B. amyloliquefaciens TA2081和TA2082。通过实时定量PCR测定这3株菌pupG基因的转录水平，结果表明与B. amyloliquefaciens TA208相比，TA2081和TA2082的转录水平均有提高，但TA2081提高的更为显著；通过测定PNPase活性发现，B. amyloliquefaciens TA2081和TA2082的PNPase活力分别为出发菌株TA208的2倍和1.30倍。7.5 L分批补料发酵实验表明，与出发菌B. amyloliquefaciens TA208相比，工程菌TA2081鸟苷到利巴韦林的摩尔转化率从20.41%提高到98.63%，TA2082的转化率从20.41%提高到40.95%。综上所述，增加pupG的表达量能提高利巴韦林的产量，本研究为利巴韦林生产菌的代谢工程改造提供了理论依据和技术指导。

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.

国家自然科学基金资助项目（31100054）；天津市教委项目（20140603）