食品级枯草杆菌Sec途径高效分泌脂肪氧合酶的分子机制

It is theoretically and practically important to construct secure and efficient expression system for recombinant biological products. In our previous researches, a novel food-grade Bacillus subtilis system was constructed based on auxotrophic for lysine. In the meantime, it was the first time that a lipoxygenase gene was expressed and secreted successfully in the safe system by Sec pathway. However, the secretion efficiency was very low. Up to now, the molecular mechanism for high-level secretion efficiency has not been elucidated. It is supposed that the secretion efficiency may be related to the processing efficiency of the secretion precursor. On the other hand, it is still unknown about the factors which can affect the processing efficiency of the lipoxygenase secretion precursors by Sec way. Based on the previous studies, the potential factors that affect the processing efficiency are listed below: (1) specific recognition of the signal peptidase to the substrate and the expression level of the signal peptidase; (2) expression level of the molecular chaperone; (3) the structure domain of the secretory component protein. To verify the feasibility of these hypotheses, experiments will be carried out to elucidate the relationship between the secretion efficiency and each potential factor. The aim of this project is to solve the problem of the low secretion efficiency of food-grade B. subtilis expression system. The molecular mechanism of high-level secretion by Sec pathway will be revealed through studying the factors that affect the secretory precursor processing efficiency. Our results are expected to conquer the limitations for efficient secretion of heterologous proteins by Sec pathway, and simultaneously provide a theoretical knowledge and technical support for the constrction of food-grade Bacillus subtilis system for high-level expression and secretion of recombinant lipoxygenase.

建立安全、高效的重组生物制品表达系统具有重要的理论和应用价值。本课题组前期建立了一种基于赖氨酸营养缺陷的食品级枯草杆菌表达系统，通过Sec途径首次实现了一种脂肪氧合酶的分泌表达，但分泌效率过低。而目前，Sec途径高效分泌的分子机制还未阐明，推测分泌效率可能与分泌前体的加工效率有关。然而哪些因素影响Sec途径脂肪氧合酶分泌前体的加工效率，国内外尚无报道。在前人有关该途径研究基础上，提出加工效率可能与下列因素有关：（1）信号肽酶与"底物"的特异性识别及信号肽酶表达量；（2）分子伴侣表达量；（3）分泌途径装置蛋白结构域。为证实假说，设计实验逐一研究上述因素与分泌效率的关系。本项目针对食品级枯草杆菌分泌效率低的问题，通过研究影响前体加工效率的因素，揭示Sec途径高效分泌的分子机制。研究结果有可能突破该途径高效分泌外源蛋白的制约因素，为构建高效分泌表达脂肪氧合酶的食品级工程菌提供理论基础和技术支撑。

国内外有关枯草杆菌分泌表达外源酶基因的研究较多，而有关该表达系统高效分泌表达的分子机制研究研究较少。本研究以来源于鱼腥藻PCC7120的脂肪氧合酶基因（Ana-LOX）为靶基因，从宿主的食品级改造、表达载体的构建等方面研究影响分泌表达外源重组酶蛋白的影响因素，同时，研究了重组工程菌的产酶发酵条件，及重组酶在面制品中的应用效果。从枯草杆菌168着手，针对其蛋白酶分泌能力强的缺陷问题，利用同源重组的方法构建获得了一株赖氨酸营养缺陷型的菌株BS-PS，建立了一套无抗生素抗性标记的筛选突变菌株的方法，获得了aprE和nprE两个蛋白酶基因失活的菌株LD02，该菌株作为外源基因表达的宿主，达到食品级要求。研究发现分子伴侣、信号肽的组成和结构影响重组酶的分泌效率，构建的pHPSBR-ana-LOX工程菌胞外活性最高达到46 U/mL,分泌效率也最高为35﹪。利用自主建立的枯草杆菌无标记基因修饰技术，首次将T7噬菌体转录调控系统应用与枯草杆菌表达系统，成功实现了4种不同来源的酶基因在T7系统下得到有效转录，其中3个酶基因获得了活性表达，研究结果为挖掘新型启动子系统拓展了方向。采用Plackett-Burman设计法，建立了枯草芽孢杆菌产脂肪氧合酶的最佳工艺，酶活达到167.32U/ml。将重组Ana-LOX应用于面条、面包，结果表明重组酶对于面制品具有较好的改良效果。