乳酸菌胞外多糖结构表征及关键基因调控机制研究

The studies on bioactive exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are hot points at present. However, there is a lack of study on the structural characterization and key gene expression regulation, which affects the relationship analysis between structure and bioactivity, and makes it difficult to increase the EPS yield by the gene regulation methods. The bioactive EPS produced by lactobacillus casei LC2W was obtained by the previous research, then the separation and purification process of EPS was determined, and finally the complete genome sequence of actobacillus casei LC2W was obtained. Based on the current study, the primary structure of bioactive EPS is further investigated using partial acid hydrolysis and methylation analysis, combined with HPAEC-PAD, GC-MS and 1D & 2D NMR spectroscopy techniques, and the monosaccharide composition, linkages of glycosidic bonds and side chains attached to the EPS backbone are determined. Advanced structural characterizations of bioactive EPS are described by reasoning the characteristic parameters of polysaccharide chains, such as symmetry and screw pitch. The morphology and gel network of bioactive EPS molecules are investigated using the atomic force microscope (AFM).The related genes which involve in the EPS synthesis and the corresponding synthesis mechanisms are determined by the bioinformatics analysis of lactobacillus casei LC2W genome and gene knockout techniques. The regulation mechanisms of related genes on EPS synthesis are elucidated by inactivating or activating the key genes using the molecular biology methods, with the purpose of high yield of EPS.

乳酸菌胞外多糖具有重要的生理活性，是目前科学研究的热点，但是胞外多糖结构和关键基因表达调控研究薄弱，影响了多糖构效关系的分析，也难以通过基因调控手段提高乳酸菌制品活性胞外多糖含量。本课题前期研究获得了产活性多糖的干酪乳杆菌LC2W，确定了对胞外多糖分离纯化工艺，完成了LC2W全基因序列测定，本课题在此研究基础之上进一步研究胞外多糖组分的一级结构，确定其单糖组成、糖苷键的连接方式等；研究胞外多糖组分的高级结构，分析多糖的分子内成键方式和分子构型，确定对称性、螺距等螺旋体参数，观察多糖分子形貌和凝胶网络结构；通过对干酪乳杆菌LC2W生物信息学分析和基因敲除实验，确定胞外多糖合成相关基因及相应合成机制；利用分子生物学手段沉默或激活关键基因，阐释胞外多糖相关基因的调控机制，以实现高产胞外多糖的目的。

干酪乳杆菌是一种乳制品中常用的乳酸菌，本项目在前期研究的基础上，以课题组筛选出的干酪乳杆菌LC2W为研究对象，通过发酵罐发酵制备LC2W产胞外多糖（LCP），采用现代仪器分析手段对LCP的结构特征、分子构象和流变特性进行系统的分析；在此基础上，通过对LC2W 生物信息学分析和基因编辑手段，研究了胞外多糖合成相关基因及相应合成机制。结果表明：LC2W产胞外多糖LCP存在三个组分，其中LCP1和LCP3是主要的级分，LCP1为含有乙酰基团的多糖链，具有降血糖和免疫调节活性，而LCP3是一种以蛋白为主的糖蛋白成分；LCP1是由T-Rhap，3-Rhap，T-Glcp，4-Glcp，3,4,6-Glcp和2,6-Galp等糖残基组成的高支化结构，其中Rhap上连接有-NAc和-OAc基团，根据NMR分析结果，推断了LCP1的重复单元结构；采用光散射、HPSEC、圆二色谱、AFM和分子模拟技术等手段证明LCP1是一种高支化的无规卷曲构象，在水溶液中易发生分子聚集而呈现球形结构，浓溶液呈现低粘度、不成胶的性质，且溶液粘度对温度敏感。抗性实验确定了10 μg/mL红霉素作为LC2W的筛选条件，对LC2W电转条件进行了优化，使得LC2W的电转化效率达到10^5（个/ugDNA）；发现了LC2W中携带天然质粒pLC2W，其对胞外多糖合成无显著影响，基于此构建了新的大肠杆菌-乳酸菌穿梭质粒－pUE-F1，对pUE-F1进行宿主范围和稳定性分析，发现其能够在多种乳酸菌中工作并保持较好的稳定性；通过pUE-F1建立了一套应用于干酪乳杆菌中的基于CRISPR-Cas9的基因编辑工具，该工具与传统方法相比，具有高效、操作方便等优点；应用该基因编辑工具，成功构建了干酪乳杆菌胞外多糖基因簇基因缺失菌株和过表达菌株，证明胞外多糖基因簇中葡萄糖基转移酶基因LC2W_2179和两个多糖合成基因（LC2W_2188和LC2W_2189）对胞外多糖的产量有影响，通过敲除或过表达这些基因，可对LC2W胞外多糖的合成进行调控。该研究成果将对我国乳酸菌代谢产物合成与调控研究产生积极的影响，为构建优质乳酸菌资源、优化乳酸菌发酵乳生产工艺、提高发酵乳功能活性奠定理论基础。