噬淀粉乳杆菌阿魏酸酯酶的非典型分泌机制研究

Lactic acid bacteria usually secret a small amount of extracellular proteins, and protein-secreting tools that rely on classical secretory pathways are inefficient, which limits its utilization as cell factories for production of bioactive molecules such as antigens and pharmaceutical proteins. Therefore, it is necessary to explore new secretion mechanisms and construct novel genetic tools to increase the overproduction of heterogeneous proteins in lactic acid bacteria. In this project, high level of extracellular feruloyl esterase activity was detected in Lactobacillus amylovorus CGMCC11056. Surprisingly, no signal peptide was predicted in this feruloyl esterase, suggesting that it was transported out of the L. amylovorus cells through a new secretory pathway. Firstly, site-directed mutation and protein truncation will be applied to detect the subcellular localization of the feruloyl esterase. Based on the results to be obtained, the functional region of the feruloyl esterase which plays a secretory role will be determined. Secondly, the functional region will be fused with different reporter proteins to confirm its transport function, and the secretion efficiency will be measured. Thirdly, combining sequence mutation and structure analysis, the molecular basis of the functional region in the transport process will be explored. Fourthly, the interactive proteins during the secretion of feruloyl esterase will be analyzed by means of immunoprecipitation, mass spectrometry, gene knockout and complementation. Finally, the secretion mechanism of feruloyl esterase in L. amylovorus will be revealed, providing a theoretical basis and genetic tool for the construction of a new protein secretion system in lactic acid bacteria.

乳酸菌分泌的胞外蛋白种类少，依赖典型分泌途径开发的蛋白分泌工具效率低，从而限制了乳酸菌作为细胞工厂生产抗原、医药蛋白等生物分子的应用。因此，挖掘新的分泌机制和寻找新的遗传元件对乳酸菌功能开发具有重要意义。我们前期研究发现，噬淀粉乳杆菌CGMCC11056所产阿魏酸酯酶N端不具有典型的信号肽序列，但能被高效地分泌至胞外，预示其可能存在新的分泌机制。本项目拟采用定点突变、蛋白截短等方法，检测阿魏酸酯酶突变体的亚细胞定位，确定其引导分泌的功能区域；通过与不同报告蛋白的融合表达，测定该功能区域对蛋白大小选择性及分泌效率，证实其引导蛋白分泌的功能；结合序列随机突变和拓扑结构预测，揭示该功能区域对阿魏酸酯酶转运的分子基础；利用免疫共沉淀、质谱分析、基因敲除和回补表达等技术，探究参与阿魏酸酯酶分泌的相关辅助因子。最终揭示噬淀粉乳杆菌中阿魏酸酯酶的分泌机制，并为构建乳酸菌中高效分泌系统提供遗传操作元件。

我们前期研究发现，噬淀粉乳杆菌CGMCC11056所产阿魏酸酯酶N端不具有典型的信号肽序列，但能被高效地分泌至胞外。并且将噬淀粉乳杆菌阿魏酸酯酶在大肠杆菌中异源表达的时候，其也可被大肠杆菌所分泌。这预示着阿魏酸酯酶可能存在新的分泌机制，本项目基于这一现象开展了深入研究。首先通过定点突变构建阿魏酸酯酶活性缺失突变体，以及阿魏酸酯酶N端截短的方法，证实了阿魏酸酯酶N端20个氨基酸序列起到了引导分泌的作用，而不是由于其酯酶活性对细胞膜的降解。进一步利用与报告蛋白木聚糖酶融合表达的方法，对N端引导序列逐步缩短，表明最低8个氨基酸残基的序列仍可以引导分泌，但其效率有所下降。随后对N端20个氨基酸的引导序列进行了随机突变，构建突变库来分析序列对引导分泌活性的影响，发现以5个氨基酸为单位进行随机突变时获得的大部分突变体都不被分泌，而通过易错PCR的方式获得了一些高分泌活性突变体（突变氨基酸数目1-3不等），这表明N端引导肽的序列或结构不能变化太大，否则会导致失去引导分泌的功能。最后利用免疫共沉淀结合质谱分析，对阿魏酸酯酶转运分泌过程中的辅助因子进行了探究，共鉴定出11个与阿魏酸酯酶有相互作用且与分泌相关的蛋白，将这些蛋白和阿魏酸酯酶进行了共表达，发现都可以不同程度的提高阿魏酸酯酶的分泌水平，其中以SecB、SurA、DanK和TolC的作用最为明显。通过以上研究，我们揭示了噬淀粉乳杆菌阿魏酸酯酶非典型分泌现象的分子机制。在研究过程中，通过不同手段获得了高分泌阿魏酸酯酶的菌株，为阿魏酸酯酶的工业生产奠定了基础。同时，以N端引导肽作为遗传操作元件，构建了高效分泌系统，实现了木聚糖酶等酶制剂的高水平分泌表达。