The rise of multi-drug resistant bacteria is leading to a shortage of effective antibiotic treatments. One of the priority pathogens that requires urgent research and development of new antibiotics is the opportunistic Gram-negative bacterium Pseudomonas aeruginosa, which is largely responsible for nosocomial infections that commonly affect immunocompromised patients. In this study, we focus on the enzyme diaminopimelate decarboxylase (DAPDC) from P. aeruginosa as a novel antibiotic target. DAPDC catalyses the irreversible decarboxylation of meso-diaminopimelate to form L-lysine, which is critical for bacterial protein synthesis. However, studies employing transposon libraries present conflicting evidence for the essentiality of the DAPDC-encoding gene for bacterial survival. To address this issue, we performed for the first time, a complete knockout of the P. aeruginosa DAPDC-encoding gene using a two-step allelic exchange method. The knockout resulted in a lethal phenotype that could only be rescued with supplementation of high micromolar concentrations of L-lysine. Given its essentiality in this pathogen, we subsequently cloned, expressed and purified the recombinant protein. Employing circular dichroism spectroscopy and analytical ultracentrifugation experiments, we determined that the protein adopts a mixed alpha/beta fold dimeric structure in solution. The enzymatic activity of P. aeruginosa DAPDC was also examined, yielding a Michaelis-Menten constant for meso-diaminopimelate of 0.73 mM. We are currently optimising the conditions to crystallise the protein, which will be used to screen for novel inhibitors using in silico approaches. We envisage that inhibitors of DAPDC may represent a novel class of antibacterials against this clinically important pathogen.