乳酸乳球菌有氧呼吸代谢中ATP合成机制

Lactococcus lactis is one of the mostly widely used strains in fermented dairy products, which can give the product a good texture and flavor, so it has a very high industrial and economic value. During the process of fermented dairy products manufacture, oxygen does a great harm to cells of Lactococcus lactis, it can cause the death of cells, and which directly affect the fermentation process and generation of flavor substance. According to this problem, in this study the ATP anabolic mechanism of Lactococcus lactis cultivating under respiration condition will be investigated based on one acquired Lactococcus lactis strain which can undergo respiration. First of all, the similarity of genes coding respiratory chain of Lactococcus lactis strains which can or can not undergo respiration will be compared by DNA sequencing, and then they will be cultivated under fermentation or respiration condition, subsequently the differential expression of genes related to respiratory chain and ATP anabolic pathway will be investigated by transcriptomics, and the metabolites in the fermentation broths will also by analyzed by metabolomics. And in this way the required condition for Lactococcus lactis to undergo respiration and the ATP anabolic mechanism of Lactococcus lactis under respiration condition will be illustrated, incidentally the regulation of aerobic respiration metabolism of Lactococcus lactis will be achieved, so that the damage of oxygen to lactic acid bacteria cells can be diminished, which resulting in spectacular improvements in growth rate, biomass and survival rate of Lactococcus lactis strain. Consequently, this work provides a theoretical basis and foundation for the prospect of industrialization application of Lactococcus lactis aerobic respiration metabolic regulation.

乳酸乳球菌是发酵乳制品中的重要菌株，它能赋予产品良好的质地和风味，具有极高的工业和经济价值。在发酵乳制品生产中，氧气会导致乳酸乳球菌细胞损伤甚至死亡，直接影响发酵过程和风味物质的产生。针对这一问题，本研究利用前期获得的能进行有氧呼吸代谢的乳酸乳球菌菌株，研究其有氧呼吸代谢中ATP的合成机制。首先利用DNA测序技术比较能和不能进行有氧呼吸乳酸乳球菌呼吸链相关基因的相似性，随后将它们分别在发酵和有氧呼吸条件下进行培养，然后采用转录组学技术比较呼吸链和ATP合成通路相关基因的差异表达，并利用代谢组学方法分析发酵液中代谢产物的变化，确定乳酸乳球菌能够进行有氧呼吸代谢的条件和ATP的合成机制，以期实现对乳酸乳球菌有氧呼吸代谢的调控，消除氧气对乳酸菌造成的损伤，提高乳酸乳球菌的发酵速率、生物量和存活率，为乳酸乳球菌有氧呼吸代谢调控的工业化应用前景提供理论依据和前期基础。

乳酸乳球菌是发酵乳制品中的重要菌株，它能赋予产品良好的质地和风味，具有极高的工业和经济价值。在发酵乳制品生产中，氧气会导致乳酸乳球菌细胞损伤甚至死亡，直接影响发酵过程和风味物质的产生。针对这一问题，本研究利用前期获得的能进行有氧呼吸代谢的乳酸乳球菌菌株，研究了其有氧呼吸代谢中ATP的合成机制。首先通过DNA测序、细胞色素氧化酶活性检测、转录组测序及荧光定量PCR验证确定乳酸乳球菌不能进行有氧呼吸代谢的原因并不是由于有氧呼吸链编码基因的缺陷造成的，而是由于编码血红素摄取相关基因的表达量是否上调，乳酸乳球菌有氧呼吸链相关编码基因是组成型表达，与其是否正在进行有氧呼吸代谢无关。随后利用转录组测序和生物信息学分析对KLDS4.0325在发酵和有氧呼吸代谢条件下的基因表达差异情况进行研究，并结合代谢组学关联分析研究其有氧呼吸代谢条件下糖代谢的主要途径，发现菌株进行有氧呼吸代谢时通过增加丙酮酸代谢通路和丁酸代谢通路中gap、pdh、eutD、ackA、alsS和budA基因的表达量，降低ldh基因的表达量来改变葡萄糖的代谢途径，使主要的代谢终产物由乳酸变成乳酸、乙酸和乙偶姻，从而获得更高的生物量和极强的长期存活率。随后进行的有氧呼吸代谢调控研究表明，通过优化乳酸菌的有氧呼吸代谢培养条件可以有效解决氧气对菌体细胞造成的损伤，大大提高了发酵剂用乳酸乳球菌的生长速率、存活率以及活菌数，从而为乳酸菌有氧呼吸代谢调控的工业化应用提供理论依据和技术支持，对促进我国发酵乳制品工业的发展具有重要意义。