A Resource for Cloning and Expression Vectors Designed for Bifidobacteria: Overview of Available Tools and Biotechnological Applications.

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.