白酒发酵环境对乳杆菌细菌素群体感应合成调控行为的影响及其机制

Bacteriocin producing Lactobacillus curvatus RX-6 is naturally existed in fermented grains of Hengshui Laobaigan liquor. Previous laboratory studies showed that the synthesis of curvacin RX-6 is regulated by quorum sensing system and is an environmental conditions-sensitive process. In this program, the influence mechanism of liquor fermented grains fermentation environment on quorum sensing synthesis and regulation behavior of curvacin RX-6 is studied. The main fermentation-related environmental stress conditions affecting bacteriocin synthesis are investigated by analyzing the variation of curvacin RX-6 synthesis and activity in different fermentation stages of Laobaigan liquor fermented grains fermentation process. The coding genes of autoinducer signal peptide and two-component signal transduction protein of quorum sensing system is determined by gene targeting and chromosome walking technology. Then the effect of main stress conditions on secretion expression, molecular strucuture and induced activity of the signal peptide are studied based on no-bacteriocin producing cultures model construction, combined with real time fluorescent quantitative PCR technique (RT-qPCR), high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) and fourier transform infrared spectrometer (FTIR). To explain the effect of the main stress conditions on the interaction between the signal peptide and two-component, the two-component are inactivated and its effect on the inducer activity of signal peptide is analyzed. The research results are of great scientific significance to comprehensively reveal the synthesis and regulation mechanism of bacteriocins produced by lactic acid bacteria in the actual fermentation environment from the molecular level.

产细菌素弯曲乳杆菌 RX-6自然存在于衡水老白干酒醅中，实验室单独培养试验发现该菌代谢产细菌素表现强烈群体密度感应和环境条件依赖效应。本项目拟基于老白干酒醅发酵过程，分析乳杆菌细菌素curvacin RX-6分泌表达及活性变化规律，确定影响其合成的主要环境胁迫条件参数；通过基因定位和染色体步移技术，明确细菌素合成相关群体感应系统信号肽及双组份信号转导蛋白编码基因；基于不产细菌素培养物模型构建，并结合实时荧光定量PCR、高效液相色谱-电喷雾离子化串联质谱及傅里叶红外光谱技术，研究主要胁迫条件对信号肽分泌表达、分子结构及其诱导活性影响。通过添加组氨酸蛋白激酶抑制剂，分析双组份信号转导蛋白失活前后信号肽诱导效应强度变化，阐释胁迫条件对信号肽与双组份相互作用的影响机制。研究结果对于从分子水平全面揭示乳酸菌细菌素在实际发酵环境中的合成调控行为具有重要科学意义。

产细菌素弯曲乳杆菌 RX-6自然存在于衡水老白干酒醅中，前期研究发现该菌代谢产细菌素表现强烈群体密度感应和环境条件依赖效应。本项目基于老白干酒醅发酵过程中可能存在的多种胁迫环境条件（寡营养胁迫、渗透压胁迫、酸碱胁迫、乙醇胁迫及高低温胁迫），分析乳杆菌细菌素curvacin RX-6分泌变化规律，确定寡营养胁迫条件为1/2氮源、渗透压胁迫为2%NaCl、酸胁迫为pH 5.0时可明显刺激细菌素高效合成，且群体密度感应在其中可能发挥重要调控作用；通过构建菌株RX-6低产或不产细菌素培养模型体系，考察发酵上清浓缩液的自诱导活性，提纯并获得信号肽分子信息，确认该细菌素合成受6042.68 Da寡肽介导的群体感应调控；结合菌株全基因组测序及功能分析，确定细菌素合成及群体感应相关基因均位于染色体上，其中ORF2901（141 bp）编码信号肽基因cunIP，ORF2983（242 bp）编码组氨酸蛋白激酶基因phoP，ORF2982（467 bp）编码感应调节蛋白基因phoR；利用实时荧光定量PCR技术分析主要胁迫条件对细菌素合成及群体感应相关基因表达的影响，采用电喷雾四级杆飞行时间质谱及圆二色谱技术分析其对信号肽分子量及二级结构的影响，推测胁迫条件主要是通过刺激信号肽的合成及组氨酸蛋白激酶基因高效表达进而引发细菌素高效合成，而对信号肽的分子结构不产生影响；通过添加组氨酸蛋白激酶抑制剂，检测主要胁迫条件下信号肽基因表达及诱导效应变化，推测胁迫条件对信号肽与双组份相互作用存在影响，但是其对群体感应调控通路的影响机制有待进一步研究。以上研究结果可为全面揭示乳酸菌细菌素在实际发酵环境中的合成调控行为以及最大限度发挥产细菌素乳酸菌在发酵生产中的应用价值提供重要理论参考。

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