枯草芽孢杆菌嘌呤合成途径的修饰对腺苷积累的影响

Modification in de novo purine pathway for adenosine accumulation by Bacillus subtilis

摘要:

摘要:【目的】研究嘌呤操纵子中purF、purM、purN、purH和purD基因的共同过表达对枯草芽孢杆菌发酵生产腺苷的影响。【方法】利用温敏质粒pKS1，以单交换的形式增加了purF 基因在基因组上的拷贝数，同时将强启动子P43插入嘌呤操纵子中，使嘌呤合成途径中purF 基因及其下游purM、purN、purH和purD基因的表达水平得到加强，通过实时定量PCR(Realtime Quantitative PCR，RT-qPCR)测定相关基因(purF、purM、purN、purH和purD)的转录水平;通过酶活性检测分析关键酶基因扩增对PRPP转酰胺酶活性的影响;通过发酵实验考察出发菌株与工程菌株的生长、耗糖和腺苷积累情况。【结果】实时定量PCＲ 结果表明，purF、purM、purN、purH和purD基因的表达水平均有不同程度的提高，嘌呤合成途径中关键酶PRPP转酰胺酶的活性是出发菌株的2.4倍。摇瓶发酵实验发现工程菌腺苷产量较出发菌提高17.5%，糖苷转化率增加26.1%。5 L罐发酵实验表明，虽然工程菌的菌体生长受到一定的影响，但在相同发酵周期内腺苷产量比出发菌提高了9.7%。【结论】嘌呤操纵子中purF、purM、purN、purH和purD基因转录水平的增强能够提高腺 苷的产量，为通过代谢工程技术改造腺苷生产菌提供了理论依据和研究思路。

Abstract:

Abstract:［Objective］To study the effects of co-overexpression of purF，purM，purN，purH and purD genes on adenosine production in Bacillus subtilis． ［Methods］First，an extra purF gene under control of the P43 promoter was integrated into the B．subtilis chromosome at the native purF locus by single crossover，resulting in simultaneous expression of purF，purM，purN，purH and purD．Then the transcriptional levels of the five genes in the engineering strain were tested by Realtime Quantitative PCR．The activity of PRPP amidotransferase was also detected．Finally，cell growth，glucose consumption and adenosine production in engineering strain along with original strain were examined.［Results］The transcriptional analysis showed that purF and its downstream genes purM，purN，purH and purD were simultaneously upregulated at different level．The PRPP amidotransferase activity of engineering strain was about 2.4-fold that of original strain．Shake flask fermentation showed the improvement in adenosine yield and conversion ratio from glucose to adenosine (17.5% and 26.1%，respectively)．Fed-batch fermentation by the engineering strain was conducted．Compared with the original strain，adenosine accumulation of engineering strain increased within the same fermentation time．However，the cell growth of engineering strain was retarded． ［Conclusion］The co-overexpression of purF and its downstream genes purM，purN，purH and purD could enhance the adenosine yield in the culture broth． This paper could facilitate future research by providing theoretical evidence and method of metabolic engineering．