The rise of multidrug resistant bacteria and the gap in antibiotics development pose a serious threat to human health. Secondary metabolites (also termed specialized metabolites) have always been the main source of therapeutic antibiotics as well as molecules serving in various other pharmaceutical domains. They are compounds produced by bacteria, fungi or plants that are not essential for growth of the producing organism, but provide an extra advantage in difficult living conditions. As such, specialized metabolites can show antibiotic properties to combat natural enemies. As traditional screening of specialized metabolites for antibiotic properties does not yield sufficient new compounds, researchers are exploring new possibilities. Engineering of gene clusters to adapt existing metabolites has been popular, however researchers still hit the wall of nature’s ingenuity as the expected analogue is not produced or only in low amounts. In this dissertation, antibiotic research is continued in two ways. Firstly, the antibiotic activity of Pseudomonas fluorescens SWRI196 is investigated to elucidate the structure of the active compound and its biosynthetic pathway. Secondly, fundamental knowledge about gene cluster engineering is gathered, on the one hand by point mutagenesis of active sites and on the other hand by an interaction analysis, both in the kalimantacin biosynthesis cluster in P. fluorescens BCCM_ID9359. P. fluorescens SWRI196 was previously shown to exhibit antibacterial activity mainly against Gram-negative strains, and plasposon mutagenesis revealed a putative type II PKS cluster involved in the biosynthesis. In silico analysis showed the presence of a very similar cluster in two other P. fluorescens strains, a Xenorhabdus and a Pectobacterium strain. Transcription analysis showed that the cluster is transcribed as a single operon. Targeted gene deletions of eleven of the twelve individual genes in the cluster showed loss of bioactivity, indicating that these genes are essential for antibiotic production and/or activity. Only deletion of gene8 resulted in a reduced halo size, showing that the gene product is not essential for bioactivity. Gene deletion of a luxI homolog and consequent complementation by N-acyl homoserine lactones proved that transcription of the cluster is activated at least by N-decanoyl-homoserine lactone and N-dodecanoyl-homoserine lactone. Production parameters for structural elucidation were optimized and an extraction protocol was devised. Structural elucidation is still ongoing. The previously elucidated kalimantacin biosynthetic pathway was the target of point mutagenesis. Both inactivation of the ketosynthase domain KS4 and activation of the ketosynthase domain KS7b resulted in an enrichment of an unknown compound related to wildtype kalimantacin, however, none of the fermentation cultures showed the theoretically expected end product. Multiple acyl carrier proteins present in module 5 and module 9 of the assembly line appear to be redundant since inactivation of each of them individually did not result in significant loss of production. At last, the glycine-incorporating A domain of module 2 was mutated to incorporate other amino acids. An efficient cloning method was developed to introduce a genus-adapted specificity-conferring code. Predicted compounds could not be detected using LC-MS, confirming the need for extra fundamental knowledge of biosynthetic enzymes in such clusters. In the last part, a large-scale yeast two-hybrid interaction analysis was performed on all enzymes in the biosynthesis cluster. An initial interaction map showing 27 interacting partners was constructed and 17 of these interactions were confirmed in an independent yeast two-hybrid screen. This interaction map provides some first insight into the assembly of the biosynthetic pathway and can now be further investigated using other interaction techniques. To combat multidrug resistant bacteria the search for new antibacterials should be continued, as should the fundamental research on the assembly of the biosynthetic pathways. Filling the gap of knowledge on 3D structures will help targeted mutagenesis for the construction of hybrid biosynthetic clusters and might open a completely new platform for antibiotics development.