基于群体感应解析特定乳酸菌共培养诱导细菌素bifidocinA合成的分子机制

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of foodborne bacteria. Previous studies showed that the synthesis of bifidocin A is induced by coculture with specific Lactobacillus plantarum and it is closely related to quorum sensing (QS). In this program, the coculture system of bacteriocin-inducer strain and bacteriocin-producer strain was established based on the construction of QS mutants of intraspecies and interspecies with key gene deletion. To make clear the cascading relationship of the bacteriocin inducer, QS signal molecules, bacteriocin producer and bacteriocin, the ecological relationship of bacteriocin inducer and producer, the regulation effect effects of the QS of intraspecies and interspecies, as well as the mode of action of inducer strain on QS signal moleculars are determined by investigating the growth of bacteriocin inducer and producer, the secretion of QS signal moleculars and the synthesis of bifidocin A. To explore the existence reason for the specificity of bacteriocin inducer, the functional genes related to the specificity are searched and verified by comparing the genomics of bacteriocin inducer and non-bacteriocin inducer and expressing differential gene in non-bacteriocin inducer. To explain the QS regulation behavior of bacteriocin induced production by coculture with specific lactic acid bacteria, the response pathways of the bacteriocin-producer to QS signal molecules are predicted by combination analysis of transcriptome and proteomics. The research results are of great scientific significance to comprehensively reveal the synthesis and regulation mechanism of bacteriocins in the in the mixed fermentation system from the molecular level and to achieve the maximum production of bacteriocin.

动物双歧杆菌BB04可代谢合成新型广谱细菌素bifidocin A，前期研究发现特定植物乳杆菌共培养可诱导该细菌素高效合成且与群体感应（QS）紧密相关。本项目拟在构建种内/种间QS关键基因缺失突变株基础上，建立细菌素诱导菌与产生菌共培养体系，考察基因敲除前后菌株生长、QS信号分子分泌及细菌素合成变化规律，分析诱导菌与产生菌生态学关系、种内/种间QS调控效应以及诱导菌对QS信号分子作用方式，明确诱导菌、QS信号分子、产生菌及细菌素间级联作用关系；通过比较特定诱导菌与其同种非诱导菌基因组差异，找寻并验证特异性相关功能基因，探明诱导菌存在特异性诱导作用的原因，进而结合转录组学与蛋白组学分析预测产生菌对QS信号分子的响应调控通路，阐释特定乳酸菌共培养诱导细菌素高效合成的群体感应调控机制。研究结果对于从分子水平全面解析混合发酵体系下乳酸菌细菌素生物合成机制以及最大限度提高细菌素产量具有重要科学意义。

分离自长寿百岁老人粪便的动物双歧杆菌（Bifidobacterium animalis）BB04可产生新型广谱细菌素双歧杆菌素A（bifidocin A），当其与植物乳杆菌（Lactiplantibacillus plantarum）1.557共培养时可诱导bifidocin A高效合成。为揭示其背后的诱导机制，构建种内群体感应（quorum sensing，QS）信号分子缺失突变株B. animalis BB04-∆bifIP和添加中间QS信号分子抑制剂D-核糖，分别阻断种内QS信号分子Bif-IP和种间QS信号分子AI-2，探究诱导菌、QS信号分子、细菌素产生菌及细菌素之间的级联作用关系，发现信号分子Bif-IP和AI-2均参与诱导菌促进细菌素产量增加的过程，且两种信号分子的作用相互独立；考察诱导菌产生诱导作用的来源，发现胞间直接接触并不是发挥诱导作用的必要条件，但诱导物需持续存在于发酵体系中；基于转录学和蛋白组学筛选具有诱导作用的关键代谢物质，发现半乳糖、甘露糖、精氨酸、半胱氨酸、谷氨酸和谷氨酰胺具有诱导bifidocin A合成的作用；并通过转录组与蛋白组初步探究共培养诱导细菌素高效合成的响应机制，发现B. animalis BB04的多种代谢途径在共培养中受到显著影响，包括：(I)PTS系统在甘露糖、半乳糖醇、纤维二糖转运中上调，PTS系统在果糖转运中下调；(II)调节参与糖酵解和其他碳水化合物途径的几种酶的表达；(III)上调涉及谷氨酸代谢的关键酶，下调涉及天冬氨酸、精氨酸和半胱氨酸代谢的多种酶；(IV)调节涉及QS和ABC运输系统的几种酶的表达；(V)上调涉及嘧啶代谢的几种酶，下调涉及嘌呤代谢的几种酶。以上研究结果可为全面揭示特定乳酸菌诱导细菌素产量增加的诱导机制，为细菌素bifidocin A的工业化生产提供重要理论依据。