Vitamin producing microorganisms are emerging as a natural, cost-effective and sustainable alternative to chemical vitamin production. To date, they have mainly been explored in the gut and fermented food, however, recently, the host group isolated several vaginal lactobacilli capable of vitamin B2 (over)production. Lactobacilli have a long history of safe use and are highly suitable for application as probiotics or for biofortification of foods, yet they are generally still applied as 'black boxes', without full understanding of the genes and molecules that drive their beneficial action. In this project, we will perform in silico and in vitro functional screening of the host group's large biobank of more than 1000 human bacterial isolates with the innovative goal to identify and characterize vitamin producing lactobacilli from untapped non-gut body niches (vagina, upper respiratory tract), and fermented foods. Next, to better understand and enhance the vitamin producing capacity of lactobacilli, untargeted and targeted genetic modification strategies will be implemented. A specific unique and challenging focus will be on the functionalization of the novel CRISPR-Cas9 based tool 'Prime editing' in lactobacilli for targeted genetic alterations leading to vitamin overproduction. Overproduction phenotypes, naturally isolated or resulting from genetic engineering approaches, can then be used in food/feed, as supplements and in human therapeutic applications.