Development of molecular tools for bifidobacterium species

作为“生活品质和管理生物资源”的益生菌和益生元产品以牛奶饮料、酸奶和胶囊的形式在市场上销售。摄入益生菌对人类和动物宿主的健康有益，但目前对细菌如何发挥这些作用的了解还不是很好。虽然对乳球菌和乳杆菌等较为常见的乳酸菌有遗传学和分子工具，但对与之密切相关的双歧杆菌属（Bifidobacterium genus）在系统发育和生理（bifidus shunt）上不同的认识还很缺乏。在分子工具和机制被阐明后，益生菌的治疗应用可能会更合理。我目前的研究重点是围绕这样一个假设，即人们可以研究基础科学知识，并通过基因操纵其某些蛋白质的表达来提高益生菌菌株的性能。随着双歧杆菌遗传工具的出现，人们可以做更多的工作来了解它们在食物中生存的重要生理和代谢活动，以及它们在胃肠道系统中的有益作用。短期目标是：(1)建立食品级克隆和表达系统；(2)构建新的过表达系统，用于生产异源蛋白如胆汁盐水解酶（BSH），以验证BSH在母鸡和小鼠模型中的降胆固醇作用。3)通过克隆BSH阴性突变体的相关基因，证明可以提高益生菌菌株的性能。作为一种耐空气和耐酸的工业菌株，乳酸双歧杆菌被明确地确定为益生菌性状，目前用于益生菌酸奶中，这提出了一个挑战，因为很少有基因表达工作已经完成。开发一种创新的双歧杆菌食品级载体和表达系统的概念，以促进我们对代谢活性，基因功能和表达的理解，这将有助于研究人员和工业界评估益生菌的效果。

Probiotic and prebiotic products as "quality of life and managing biological resources" are on the market in the forms of milk based drinks, yogurts and capsules. Intake of probiotic bacteria is associated with beneficial health benefits to the human and animal hosts, but current understanding of how the bacteria actually illicit these effects is not very good. Although the genetics and molecular tools of more common dairy lactic bacteria like Lactococcus and Lactobacillus exist, knowledge is lacking behind with closely related Bifidobacterium genus, which is phylogenetically and physiologically (bifidus shunt) distinct. After the molecular tools and mechanisms are elucidated, a more rational approach to the therapeutic use of probiotics could be possible. The current focus of my research program is centered on the hypothesis that one can study fundamental scientific knowledge and improve the performance of probiotic strain by genetic manipulation of its expression of certain proteins. As genetic tools become available for bifidobacteria, more work can be done on understanding their physiology and metabolic activities important for survival in food as well as for understanding their beneficial effects in the gastrointestinal system. The short term objectives are to (1) develop food grade cloning and expression systems, (2) construct new over-expression systems for production of a heterologous protein like bile salt hydrolase (BSH) to validate BSH hypothesis for cholesterol lowering effect of Bifidoabcterium lactis strains (BSH mutant, native and over-expressed) in hens and mice models, and 3) demonstrate the hypothesis that one can improve the performance of probiotic strains by cloning the genes of interest in the respective BSH negative mutants. An air and acid tolerant industrial strain, Bifidobacterium lactis clearly identified as probiotic traits that currently used in probiotic yogurts and this presents a challenge as very little genetic expression work has been done. The concept of developing an innovative Bifidobacterium food-grade vector and expression system to advance our understanding of the metabolic activity, gene function, and expression is a novelty that will be useful for researcher and industries to evaluate the probiotic effects.