乳杆菌抗噬菌体菌株的培育及其潜在进化机制的研究

Good quality of lactic acid bacteria starter cultures and/or non-starter cultures is considered as the foundation of good fermented food products. However, bacteriophage infection of lactic acid bacteria is considered an important problem worldwide in the food fermentation industry. To overall explain the evolutionary molecular mechanisms of spontaneous phage-resistant mutants of lactobacilli, this project try to use two novel Lactobacillus virulent phages as researching objects, and screen spontaneous phage-resistant mutants. We will combine whole-genome re-sequencing technique, SMRT three generation sequencing technology and phenotypic tests to simultaneously trace the changes of phenotypes and genotypes. Through this project, we hope to fully elucidate the phage-resistant mechanisms of lactobacilli. Meanwhile, it will provide data and theory support on improving the phage-resistant properties of lactobacilli, and the stability of related fermented food products, as well as cultivating engineering strains with phage-resistant properties.

优良乳酸菌发酵剂或非发酵剂的质量是优质发酵食品生产的前提，而噬菌体污染却对食品发酵工业构成了严重的威胁。本项目拟以两株乳杆菌烈性噬菌体为研究对象，筛选自发突变的乳杆菌抗噬菌体菌株，通过基因组重测序技术和SMRT三代测序技术同时观察表型变化，以期全面解释自发突变乳杆菌菌株耐受噬菌体的分子机制。通过该项目的研究，有望全面解析乳杆菌耐受烈性噬菌体的相关机理。与此同时，这一项目的实施和完成可为提高乳杆菌噬菌体抗性及相关产品的稳定性、培育抗噬菌体工程菌株奠定坚实的理论基础，具有重要的实际指导意义。

本项目以分离自乳杆菌异常发酵液的烈性噬菌体及其宿主菌株为研究对象，采用次级感染法筛选获得自发突变抗噬菌体菌株23株。以野生株为对照，检测了其噬菌体抗性、遗传稳定性、溶原性等，确定所选育菌株为稳定可遗传的非溶原性菌株。筛选5株抗噬菌体能力强可稳定遗传的突变株，评价其耐酸耐胆盐特性、发酵性能（发酵时间、感官特性、贮藏稳定性）、营养需求等。同时，利用基因组重测序技术、RT-PCR技术，结合乳杆菌噬菌体在侵染宿主不同时期的转录组学数据及抗噬菌体菌株的相关表型数据，对其抗噬菌体机理进行了解析。结果显示，与野生株相比，抗噬菌体突变株的耐酸耐胆盐特性、发酵特性、营养需求等均无明显变化，表明其可代替野生株应用于实际生产的可能性。此外，噬菌体对抗性菌株的吸附位点并未发生改变，可正常吸附，但抗性菌株通过改变GTP焦磷酸激酶基因阻止了噬菌体基因复制及转录翻译过程，影响了噬菌体DNA及蛋白质外壳的合成，使其表型发生改变。另外，部分突变株由于ABC转运蛋白基因发生突变，影响了乳杆菌噬菌体的基因注入过程，进而使菌株产生噬菌体抗性。该研究可为提高乳杆菌发酵剂菌株噬菌体抗性及相关产品稳定性，培育抗性工程菌株提供一定的理论基础和数据支持。