地衣芽孢杆菌中木糖操纵子受葡萄糖胁迫的转录调控特性

The transcriptional regulation characteristics of xylose-inducible promoter in Bacillus licheniformis

木糖操纵子是芽孢杆菌中常用的表达元件,但目前对其认识只停留在静态机理层面,关于其在发酵过程中转录调控特性的研究还鲜见报道.利用qPCR技术探究在葡萄糖胁迫下地衣芽孢杆菌木糖诱导的木糖异构酶基因在发酵过程中的转录水平,考察菌体的生长状态,并通过二硝基水杨酸(DNS)法及高效液相色谱(High performance liquid chromatography,HPLC)法测定发酵过程中糖浓度变化.结果显示:在实验所设置的地衣芽孢杆菌代谢相对稳定的条件下,地衣芽孢杆菌木糖启动子转录强度在稳定期以前均呈增加的趋势,在对数生长末期或稳定前期转录强度最高,约是7 h时的14倍,随后呈下降趋势;进一步研究发现20-180 g/L葡萄糖浓度均对其表现为抑制,且抑制程度一致,当葡萄糖含量极少或者没有而木糖存在的情况下,启动子转录强度极高.本研究表明以地衣芽孢杆菌为宿主的木糖诱导系统在菌体对数生长末期诱导效果最佳;当环境中葡萄糖含量极少或者没有而木糖存在的情况下,更有利于启动子表达;结果对利用木糖诱导型重组地衣芽孢杆菌诱导发酵有一定的启示与指导意义.

The xylose operon is a commonly used as a expression element in Bacillus spp., but our present understanding of its function remains only at the level of its static mechanism, whereas studies on its transcriptional regulation characteristics in the fermentation process have only been done rarely. Obtaining further understanding of this operon's characteristics during the fermentation process should help to provide a scientific basis for the application of xylose-inducible expression systems in the fermentation process. In this study, the dinitrosalicylic acid(DNS) method was used to control the glucose content during the fermentation process, and then quantitative reverse transcriptase polymerase chain reaction(RT-qPCR) technology and high-performance liquid chromatography(HPLC) were used to accurately analyze the residual sugar content and quantify the transcription levels of the operon gene, respectively, in samples collected during the fermentation process. As a result, the glucose concentration was controlled within a desired range so that the growth and metabolism of Bacillus licheniformis in the samples were stable. Meanwhile, a systematic method was established for detecting the expression levels of the xyloseinducible promoter gene during the fermentation process, which revealed the transcriptional regulation characteristics of the xylose-inducible promoter gene in the fermentation process. The transcription level of the xylose-inducible promoter gene increased until the stationary phase, with the highest transcription level attained at the end of the logarithmic growth or prestabilization phase, which was increased by about 14 times after 7 h, and then after this it began to decline. Further, glucose concentrations of 20-180 g/L inhibited the transcription of this promotor gene, and the degree of inhibition was consistent across this concentration range. When the glucose content was low or zero, and xylose was present, the transcription level of the promoter was extremely high. The results of this study indicated that the xylose-inducible system with Bacillus licheniformis as the host has the best induction effect at the end of the logarithmic growth phase of microbial fermentation, and when there is little or no glucose in the environment and xylose is present conditions are even more favorable for promoter expression. These findings are significant because they could be used to guide the optimization of the application of inducible fermentation by xylose-induced recombinant Bacillus licheniformis.