枯草杆菌细胞密度依赖型自诱导表达系统转录调控的分子机制

It is theoretically and practically important to develop secure, efficient and precisely controllable expression system for recombinant proteins. In the previous research, a novel cell-density-dependent auto-inducible expression system was constructed in Bacillus subtilis using promoter PsrfA. Without an inducer, the target gene could be expressed along with the cell growth coming to a certain density. Several heterologous proteins had been highly produced by this expression system. However, the mechanism of the cell-density-dependent expression has not been elucidated. The biological function of the operon containing PsrfA and the sequence structure of the PsrfA are analyzed. Based on the analysis and the related reports, the potential factors that are supposed to affect the transcriptional regulation are listed below: (1) the concentration of the extracellular molecules ComX and the CSF are supposed to induce the transcription. (2) By binding with the PsrfA, the intracellular molecular ComA is assumed to affect the topological structure of the PsrfA and the package RNA polymerase to activate the transcription. (3) The transcription factors PerR and CodY are considered to regulate the transcription by influencing the combination of ComA and RNA polymerase to the PsrfA, respectively. To verify the feasibility of these hypotheses, experiments will be carried out to elucidate the relationship between the transcription regulation and each factor, and the mechanism of the cell-density-dependent expression is expected to be elucidated. The results of this study might provide theoretical and applied references for the construction of mature auto-inducible expression system in B. subtilis.

建立安全高效、精细可控的重组表达系统具有重要的理论和应用价值。申请人前期利用启动子PsrfA建立了一种基于细胞密度的枯草杆菌自诱导表达系统，不需要添加诱导剂，细胞生长到一定密度时可启动目的基因的表达，已实现多种异源蛋白的高水平表达，但其依赖细胞密度的转录机理及调控机制尚不清楚。通过分析PsrfA所在的操纵子的生理功能及PsrfA序列结构，结合已有的研究报道，提出转录调控可能与以下因素有关：①一定浓度的胞外分子ComX和CSF诱导PsrfA转录；②胞内分子ComA结合PsrfA后通过影响PsrfA的拓扑结构及RNA聚合酶的组装激活PsrfA转录；③转录因子PerR和CodY分别通过影响ComA和RNA聚合酶与PsrfA结合调控PsrfA转录。为证实以上假说，逐一设计实验研究上述因素与转录的关系，揭示PsrfA依赖细胞密度的转录调控机制。本研究可为构建成熟的枯草杆菌自诱导表达系统提供理论基础。

本研究以枯草芽孢杆菌来源的启动子PsrfA为研究对象，研究信号分子CSF及启动子序列中的ComA、PerR、CodY结合位点序列对PsrfA表达活性的影响，探究PsrfA在E. coli和乳酸菌中的表达适应性及表达特性。主要结果：（1）体外添加含CSF的发酵上清，可提前诱导启动子表达并提高表达活性；启动子序列中的ComA、PerR、CodY结合位点可影响启动子表达活性。（2）对ComA、ComA二聚体及其结合DNA进行同源建模，分子动力学模拟显示，ComA激活后能够快速结合至靶标DNA上，结合DNA后ComA更加稳定；结合拓补结构和力场参数， ComA中D55磷酸化是ComA上DNA结合区域稳定的重要因素。（3）PsrfA可用于构建E. coli表达系统：PsrfA可在E. coli JM109和E. coli BL21中表达绿色荧光蛋白GFP和β-半乳糖苷酶LacZ。将PsrfA中的-10区突变为保守序列后，GFP和LacZ表达量增加。（4）PsrfA可用于构建乳酸菌表达系统：PsrfA可在L. casei 5257、L. plantarum 97、L. fermentum 087和W. confusa 10中表达GFP，且L. plantarum 97中GFP表达量最高；不同碳源影响菌体的生长及PsrfA的表达活性，不同氮源促进菌体生长但降低PsrfA的活性；初始pH可影响菌体生长速度、最终生物量及启动子活性，但初始pH对PsrfA表达活性的影响较小；发酵过程中当pH低于4.5时回调pH，能够提高菌体的生物量及PsrfA的表达活性。（5）PsrfA可用于控制发酵液pH：利用PsrfA在L. plantarum 97和L. casei 5257中表达NADH氧化酶，调节发酵过程中发酵液pH。对启动子-35区、-10区单独或组合突变为保守序列、RBS位点突变为保守序列和删除抑制因子CodY识别位点序列，均能提高PsrfA表达NADH氧化酶的水平。（6）PsrfA可在乳酸菌中表达枯草芽孢杆菌来源的漆酶CotA：在L. plantarum 97-06和L. casei 5257-06中可表达CotA，培养中调节发酵液的pH，L. plantarum 97-06和L. casei 5257-06中CotA酶活分别提高了2.4倍和1.6倍。