Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias, IPLA-CSIC, Villaviciosa, Spain.
Instituto de Investigación Sanitaria del Principado de Asturias, ISPA, Oviedo, Spain.
Methods Mol Biol. 2021;2278:157-182. doi: 10.1007/978-1-0716-1274-3_14.
Bifidobacteria represent an important group of (mostly) commensal microorganisms, which have enjoyed increasing scientific and industrial attention due to their purported health-promoting attributes. For the latter reason, several species have been granted "generally recognized as safe" (GRAS) and "qualified presumption of safety" (QPS) status by the Food and Drugs Administration (FDA) and European Food Safety Authority (EFSA) organizations. Increasing scientific evidence supports their potential as oral delivery vectors to produce bioactive and therapeutic molecules at intestinal level. In order to achieve an efficient utilization of bifidobacterial strains as health-promoting (food) ingredients, it is necessary to provide evidence on the molecular mechanisms behind their purported beneficial and probiotic traits, and precise mechanisms of interaction with their human (or other mammalian) host. In this context, developing appropriate molecular tools to generate and investigate recombinant strains is necessary. While bifidobacteria have long remained recalcitrant to genetic manipulation, a wide array of Bifidobacterium-specific replicating vectors and genetic modification procedures have been described in literature. The current chapter intends to provide an updated overview on the vectors used to genetically modify and manipulate bifidobacteria, including their general characteristics, reviewing examples of their use to successfully generate recombinant bifidobacterial strains for specific purposes, and providing a general workflow and cautions to design and conduct heterologous expression in bifidobacteria. Knowledge gaps and fields of research that may help to widen the molecular toolbox to improve the functional and technological potential of bifidobacteria are also discussed.
双歧杆菌是一类重要的（主要是）共生微生物，由于其被认为具有促进健康的特性，因此受到越来越多的科学和工业关注。出于后一个原因，一些双歧杆菌物种已被美国食品和药物管理局 (FDA) 和欧洲食品安全局 (EFSA) 组织授予“通常被认为是安全的”(GRAS) 和“有资格获得安全推定”(QPS) 地位。越来越多的科学证据支持它们作为口服递送载体的潜力，可在肠道水平产生生物活性和治疗性分子。为了有效地利用双歧杆菌菌株作为促进健康的（食品）成分，有必要提供其据称有益和益生菌特性背后的分子机制以及与人类（或其他哺乳动物）宿主相互作用的确切机制的证据。在这种情况下，有必要开发适当的分子工具来产生和研究重组菌株。虽然双歧杆菌长期以来一直难以进行遗传操作，但文献中已经描述了广泛的双歧杆菌特异性复制载体和遗传修饰程序。本章旨在提供关于用于遗传修饰和操作双歧杆菌的载体的最新概述，包括它们的一般特征，回顾了将其用于成功生成特定用途的重组双歧杆菌菌株的示例，并提供了一般工作流程和注意事项，以设计和进行双歧杆菌中的异源表达。还讨论了知识空白和研究领域，这些可能有助于拓宽分子工具包，以提高双歧杆菌的功能和技术潜力。
