Sigma因子驱动的嗜酸乳杆菌冻干适应性进化紧密耦合转录本识别及其基本机制

Lactic acid bacteria play critical roles in health, which has been verified by human microbiology. However, they often endure low-temperature and drought stresses during the process of germplasm conservation and production. Naturally, it is important to understand the genetic mechanism of their freeze-drying adaptive evolution in the food microbiology. Possessing some unique merits deriving from its colonization in the stomach, Lactobacillus acidophilus (L. acidophilus) has recently received considerable attention. We have isolated a strain of L. acidophilus named Y1405, which presents remarkable adhesion performance. However, it is very sensitive to freeze-drying stress (the viable/total ratio <37% even after optimization of cryoprotectant; as control, an isolated L. acidophilus from commercial yogurt presented the ratio >90% under the same treatment), a necessary process for preparing starters. To surmount the obstacle, here we will focus on the identification and the mechanism of the key transcripts coupling the freeze-drying phenotype evolved by Sigma factor RpoD mutation in L. acidophilus Y1405. First, we will depict the dynamic response of freeze-drying adaptive evolution to RpoD random mutagenesis in Y1405. After screening the evolved recombinants, we will profile the transcriptome using differential RNA-seq technique. Based on the known biological principles, all related transcripts will be scanned and crossed to identify the key transcripts (not appear, new transcripts or abundance variation) coupling the freeze-drying phenotype. Finally, the mechanism of the targeted transcripts will be revealed by reverse biological strategies, such as overexpression and disruption in Y1405. When the whole project is completed, we will not only reveal the freeze-drying phenotypic biomarkers and novel pathways or targets for safe genetic modification of L. acidophilus, but also speed up the development of yogurt starters.

制备冷藏乳酸菌剂过程经常会对菌体造成低温干燥双重生理胁迫，对其进行冻干适应进化的遗传基础解析是食品微生物科学重要研究课题。前期分离出一株黏附、耐酸等益生性状优异的嗜酸乳杆菌Y1405，但其抗冻干能力差。本课题拟通过Sigma因子驱动Y1405冻干适应进化，并由转录组分析识别与进化紧密耦合转录本，对其作用机制进行研究。首先，研究Y1405冻干适应性进化对Sigma因子随机突变的响应规律；之后，由dRNA-seq技术进行对照培养及冻干刺激处理的进化前/后菌株的转录组质量表征，经比较转录组学及交叉比较，扫出冻干适应紧密耦合的转录本；最后，结合生物信息学及转录本扰动等湿实验阐明其作用机制。项目成功实施将捕获嗜酸乳杆菌抗冻干这一重要工艺适应性状紧密连锁的转录本，并阐明其基本机制，进而为抗冻干性状为目标的菌株高通量筛选提供生物标记，也为基因修饰手段进行高效安全育种提供路径或操作靶点，加快菌剂研发。

乳酸发酵菌剂的制备过程经常会对菌体造成低温及脱水干燥双重生理胁迫，对其进行冻干适应进化的遗传基础解析是食品微生物科学重要研究课题。我们前期分离出一株黏附、耐酸等益生性状优异的嗜酸乳杆菌Y1405，但其抗冻干能力差。本课题首先研究了四种冻干保护剂对Y1405存活率的影响，并在此基础上由全局转录因子Sigma的驱动，重塑了Y1405的转录谱，进而，在分子层面上对嗜酸乳杆菌的抗冻干生理机制做了初步解析。研究发现，一种天然甜味蛋白MNEI在较低剂量下可以显著提高菌体的抗冻干性能，可以作为抗冻干保护剂；在全局转录因子rpoD的突变鲁棒性的研究中，由随机突变文库筛选方法鉴定出两个rpoD突变株YL16-722及YL16-913发生了冻干适应进化，并通过PCR-测序确认了突变位点为；通过基因的水平转移研究发现， rpoD具有较强鲁棒性，在乳杆菌属内，甚至个别其它属内的rpoD可维持Y1045的正常生长；获得了Y1045 ΔrpoD ::LkrpoD突变株，显示了较强的抗冻干特性。通过dRNA-seq技术，获得了上述三个处理（MNEI、YL16-913及Y1045 ΔrpoD ::LkrpoD）的转录组数据。识别出与进化紧密耦合转录本，其中，具有显著差异转录本1633个，密切耦合关键转录本237个，其中15个属于无蛋白产物sRNA，这些差异转录本主要涉及了多糖代谢、糖及多肽转运体、氨基酸合成、脂多糖及肽聚糖的生物合成等代谢途径。进一步的遗传操作表明，MNEI可能通过与转运体作用，显著提升细胞的抗冻干特性；多糖及S层蛋白合成、谷氨酸-GABA反向转运体上调表达也显著增强细胞抗冻干性能。通过本课题研究，嗜酸乳杆菌抗冻干这一重要工艺适应性状基本机制由以往的生理生化水平，进入到了分子水平，为抗冻干性状为目标的菌株高通量筛选提供生物标记，也为基因修饰手段进行高效安全育种提供路径或操作靶点，将加快相关菌剂研发。