基于基因组尺度代谢网络重构对乳杆菌高密度发酵分子机制的研究

High cell density fermentation is the premise and key technology bottlenecks for the industrial application of Lactobacillus as the probiotic and starter culture. Lactobacillus plantarum, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. bulgaricus with important application value will be used as the research object in this project. Genome-sequencing, function annotation, and methylation identification of Lactobacillus with different high cell density fermentation characteristics will be completed by the application of the genomics re-sequencing and the SMRT three generation sequencing technology. The genetic basis study of different nutrient metabolism and high density fermentation of Lactobacillus will be carried out from sequences analyzing on the regulator, promoter, and the region of non base change. The lactobacillus genome scale metabolic network reconstruction will be performed by the genomic data analysis combined with high cell density fermentation phenotype data. In order to clarify the molecular mechanism of nutrition metabolism and high cell density fermentation, the Lactobacillus genomics scale metabolic network model will be established. The key regulatory gene targets in nutrient metabolism pathways of Lactobacillus will be identified. The study will be provided the theoretical support for high cell density fermentation and the genetic theoretical basis for improve Lactobacillus biomass through genetic engineering and metabolic flow regulation technology.

高密度发酵是乳杆菌作为益生菌和发酵剂工业化应用的前提和关键技术瓶颈。本项目拟以内蒙古农业大学乳酸菌菌种资源库中保藏的具有重要应用价值的植物乳杆菌、瑞士乳杆菌、德氏乳杆菌保加利亚亚种为研究对象，应用基因组重测序技术和SMRT三代测序技术完成高密度发酵差异乳杆菌全基因组测定、功能基因注释和甲基化修饰的研究。从调控子、上游启动子区以及非碱基变化角度分析菌株营养代谢和菌体增殖能力差异产生的遗传基础，结合乳杆菌高密度增殖培养表型数据，进行乳杆菌高密度发酵基因组尺度代谢网络重构，建立具有不同高密度发酵表型特征乳杆菌基因组尺度代谢网络模型；阐明乳杆菌种内营养代谢和高密度发酵的分子机制，鉴定乳杆菌营养代谢途径中的关键调控靶点，为乳杆菌高密度增殖培养提供理论支撑，为通过基因工程和代谢流调控技术提高乳杆菌生物量提供理论依据和遗传学基础。

项目针对对不同乳杆菌（保加利亚乳杆菌、瑞士乳杆菌、植物乳杆菌、嗜酸乳杆菌）菌株进行高密度发酵培养，并对其发酵培养基进行优化，期望在短时间内就可以获得高活力菌株。完成了25株保加利亚乳杆菌的发酵试验；完成了188株保加利亚乳杆菌的基因组重测序和GWAS分析；完成了9株保加利亚乳杆菌的甲基化分析；完成了1株鼠李糖乳杆菌和一株乳双歧杆菌的比较基因组测序分析。为了更好的对菌株进行工业化生产和应用，计划完成不同高密度发酵特性乳杆菌的全基因组测序，从中获得种内核心基因组和泛基因组，进行种内乳杆菌基因组尺度代谢网络重，建立乳杆菌高密度发酵基因组尺度代谢网络模型，为乳杆菌高密度增殖培养提供理论支撑，为通过基因工程和代谢调控技术提高乳杆菌生物量提供理论依据和调控靶点。本项目实施期间发表论文10篇，其中SCI论文8篇；授权发明专利3项；毕业博士研究生3名，硕士研究生2名，培养青年教师1人。圆满完成了任务书约定的研究内容和考核指标。