During the past decade, the bacterium Klebsiella pneumoniae drew attention and growing concern because of its capacity to share with other bacterium features such as the last-resort antibiotic carbapenem resistance. It is part of the healthy gut microbiota. It has been shown that antibiotherapy and gut inflammation induce a bloom of Klebsiella pneumoniae population that can further lead to blood infection. The limited therapeutic options available to tackle this bacterium call for a better understanding of its interaction with the host and its immune system. Following the selection of genes related to antibiotic resistance and amino acid metabolism from RNA sequencing data obtained in vivo, we hereby establish a protocol to delete one or multiple genes from Klebsiella pneumoniae. We generated and characterized 4 different single knocked-out mutants that did not result in any noticeable phenotype. However, we are confident that by this approach we will uncover important pathogenetic factors of this threatening infectious agent, thus providing novel targets to tackle it in patients.