植物乳杆菌ST发酵酸茶促进儿茶素生物转化的分子机制研究

Catechins are the main bioactive compounds in tea. In our previous study, pickled tea submerged fermented with Lactobacillus plantarum ST isolated from De’ang pickled tea has shown greater immunomodulatory effects than has crude tea. The contents of total catechins and the major monomeric catechins such as epicatechin and epigallocatechin were higher than those in crude tea. These results suggest that Lactobacillus plantarum ST improve catechins biotransformation in pickled tea with novel modes of action. The over-arching objective of this proposal is to investigate the molecular mechanism involved in Lactobacillus plantarum ST improving catechins biotransformation in pickled tea. To meet this challenge, objectives to be met include (a) Investigation of transcriptional profiles of Lactobacillus plantarum ST during submerged fermentation of pickled tea and screening of the candidate genes involved in catechins biotransformation and (b) Validation of the roles of the candidate genes in catechins biotransformation by construction of deletion mutants as wells as heterologous expression in a Lactobacillus plantarum strain with low bioconversion efficiency and (c) Expression of the recombinant critical enzymes bioconversing catechins in E. coli and investigation of their roles in catechins biotransforamtion. This research will elucidate the modes of Lactobacillus plantarum ST improving catechins biotransformation in pickled tea and provide insight into the mechanisms underlying the enhanced bioactivities of pickled tea fermented by Lactobacillus plantarum. This research will also emphasize the possibility of developing novel tea products and extending the tea industry.

儿茶素是茶叶主要生物活性物质。申请人前期发现与毛茶相比，德昂酸茶中自主分离的植物乳杆菌ST发酵酸茶，儿茶素单体EGC和EC含量、儿茶素总量和免疫调节功能显著增加，表明ST具有促进儿茶素生物转化的新机制。因此，本研究以ST液态发酵酸茶为研究对象，研究发酵过程中ST转录谱变化，筛选ST生物转化儿茶素关键酶候选基因；构建ST儿茶素关键酶基因缺失株，并将关键酶基因在儿茶素转化效率低的菌种中异源表达，与相应野生株对比分析儿茶素含量变化，对ST生物转化儿茶素关键酶基因进行功能验证；将儿茶素关键酶基因在大肠杆菌中重组表达、纯化和序列分析，利用重组蛋白催化主要儿茶素合成前体物质，研究发酵过程中前体物质、中间产物和儿茶素的变化规律，阐明ST发酵酸茶促进儿茶素生物转化的分子机制。研究结果对揭示植物乳杆菌发酵提高酸茶生物学功效的作用机制，丰富茶叶产品、拓展茶产业结构，具有重要的理论和实际意义。

儿茶素是茶叶主要生物活性物质。申请人前期发现与毛茶相比，德昂酸茶中自主分离的植物乳杆菌ST发酵酸茶，儿茶素单体EGC和EC含量显著增加，表明ST具有促进儿茶素生物转化的新机制。本项目以茶汤和RPMI1640-茶多酚液体培养基为发酵基质，研究了植物乳杆菌ST发酵过程中儿茶素EC和EGC含量和转录谱变化，筛选ST生物转化儿茶素关键酶基因；将关键酶基因在儿茶素转化效率低的植物乳杆菌DA9菌株中异源表达，与相应野生株对比分析儿茶素含量变化，对ST生物转化儿茶素关键酶基因进行功能验证；将儿茶素关键酶基因在大肠杆菌中重组表达、纯化和序列分析，利用重组蛋白催化主要儿茶素合成前体物质。结果显示，与未发酵组相比，植物乳杆菌ST液态发酵酸茶和RPMI1640-茶多酚培养基提高了儿茶素EC和EGC含量，差异表达基因富集在氨基酸合成和ABC转运蛋白信号通路，差异表达基因经RT-PCR验证后，筛选获得莽草酸激酶（AroL）和预苯酸脱氢酶(TyrA2)2个候选基因进行功能验证；与野生株比较，植物乳杆菌DA9- AroL过表达株发酵茶多酚促进了EC含量，重组表达后的莽草酸激酶蛋白对茶多酚无直接催化作用。上述研究结果显示，莽草酸激酶参与了植物乳杆菌ST生物转化儿茶素EC，为阐明植物乳杆菌ST生物转化儿茶素的分子机制提供了理论依据。