Classical strain improvement of beta-lactam producing organisms by random mutagenesis has been a powerful tool during the last century. Current insights in the biochemistry and genetics of beta-lactam production, in particular in the filamentous fungus Penicillium chrysogenum, however, make a more directed and rational approach of metabolic pathway engineering possible. Besides the need for efficient genetic methods, a thorough understanding is needed of the metabolic fluxes in primary, intermediary and secondary metabolism. Controlling metabolic fluxes can be achieved by adjusting enzyme activities and metabolite levels in such a way that the main flow is directed towards the desired product. In addition, compartmentalization of specific parts of the beta-lactam biosynthesis pathways provides a way to control this pathway by clustering enzymes with their substrates inside specific membrane bound structures sequestered from the cytosol. This compartmentalization also requires specific membrane transport steps of which the details are currently uncovered.