解淀粉芽胞杆菌关键酶基因过表达对鸟苷积累的影响

Effects of overexpression of key enzyme genes on guanosine accumulation in Bacillus amyloliquefaciens

摘要:

摘要:【目的】研究鸟苷生物合成途径中的3 个关键酶编码基因(prs，purF，guaB)过表达对解淀粉芽胞杆菌(Bacillus amyloliquefaciens)发酵生产鸟苷的影响。【方法】利用穿梭表达载体PBE43，构建含有prs、purF和guaB 基因的单独表达载体和prs、purF 基因的串联表达载体，将它们分别转入鸟苷生产菌B.amyloliquefaciens TA208 后，通过实时定量PCR 测定各工程菌株内相关基因的转录水平;通过酶活检测分析关键酶基因扩增对肌苷酸脱氢酶活性的影响;通过摇瓶发酵实验考察工程菌株与对照菌株的生长、耗糖和鸟苷积累情况。【结果】转录分析结果表明prs、purF和guaB 基因过表达的同时都伴随着自身转录水平的显著上调。与此同时，prs 和purF 基因单独表达均轻微下调了嘌呤操纵子的转录水平，但是guaB 基因的过表达并不影响嘌呤操纵子和prs 基因的转录。酶活分析结果表明prs和purF 基因扩增并不影响肌苷酸脱氢酶的活性，guaB 基因的扩增使其活性提高了126%。摇瓶发酵实验发现prs和purF 基因的单独过表达均未促进宿主菌合成鸟苷，而含guaB 基因过表达载体的工程菌鸟苷产量较出发菌株提高20.7%。将prs和purF基因串联表达后，鸟苷产量提高14.4%，糖苷转化率增加6.8%。【结论】过表达guaB 基因能够大幅提高鸟苷产量，而prs 和purF 基因只有实现协同表达才能对宿主菌积累鸟苷产生积极影响，为通过代谢工程技术提高鸟苷产量奠定了研究基础。

Abstract:

Abstract:［Objective］ To study the effects of overexpression of key enzyme genes (prs，purF and guaB) on guanosine production in Bacillus amyloliquefaciens TA208.［Methods］ The prs，purF，guaB and prs-purF genes were inserted into constructed expression plasmid PBE43． All these constructed plasmids were electroporated into B. amyloliquefaciens TA208． The transcriptional level of various genes in the resulting strains was tested by real-time quantitative PCR． The activity of inosine 5'-monophosphate dehydrogenase in the resulting strains was detected． Finally，cell growth，glucose consumption and guanosine production of 4 engineering strains along with control strain were examined． ［Results］ The transcriptional analysis showed that overexpression of prs，purF and guaB gene accompanied by their own transcription level up-regulated． Overexpression of prs or purF genes alone slightly down-regulated the transcriptional level of purine operon，but overexpression of guaB gene independently did not disturb the transcription of prs gene and purine operon． Enzyme activity analysis showed that overexpression of prs or purF gene did not change the activity of inosine 5'- monophosphate dehydrogenase and its activity increased by 126% through overexpression of guaB gene． Finally，by fermentation flask test， we found that overexpression of prs and purF gene alone could not promote guanosine accumulation． However，overexpression of guaB gene resulted in an increase in the production of guanosine，which was 20. 7% higher than the control strain． The guanosine concentration and the conversion ratio from glucose to guanosine in the host strain containing co-expression plasmid were 14. 4% and 6. 8% higher than the control strain． ［Conclusion］Overexpression of guaB gene could enhance the guanosine yield in the culture broth． However，for prs and purF gene，only co-expression of them could lead to a significant improvement of guanosine production in B．amyloliquefaciens．It should provide a valuable insight into the construction of industrially important strains for guanosine production by metabolic engineering．