The β-lactam antibiotics continue providing health to the world population by virtue of industrial production and discoveries of new molecules with useful activities. Sales of these remarkable compounds, including penicillins, cephalosporins, cefoxitin, monobactams, clavulanic acid, and carbapenems, have reached over $20 billion dollars per year. Strain improvement of the penicillin-producing strains of Penicillium chrysogenum has been truly remarkable, with present strains producing about 100,000 times more penicillin than the original Penicillium notatum of Sir Alexander Fleming. The traditional strain improvement programs based on random mutation and screening in combination with recombinant DNA techniques allowed an impressive β-lactam yield enhancement at industrial scale. A remarkable amount of information has been gathered on the biosynthetic enzymes involved, the pathways of biosynthesis of β-lactams as well as their regulation, and the genomics and proteomics of the producing organisms. The rational metabolic engineering of β-lactam-producing microorganisms has resulted in productivity increments and the design of biosynthetic pathways giving rise to new antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms (GMOs). Modern aspects of the processes are discussed in the present review including genetics, molecular biology, metabolic engineering, genomics, and proteomics.