| The emergence of antibiotic-resistant bacteria has created an urgent need for new antibiotics. Streptomyces species produce more than 80% of all currently used commercial antibiotics. The organisms make these antibiotics when their metabolism switches from primary to secondary metabolism. Surprisingly, recent genome sequences of actinomycetes and fungi revealed the existence of many ?sleeping? antibiotic biosynthesis clusters, but no clues are available how these clusters can be switched on. Thus, a novel approach to make new antibiotics available concerns the understanding of what triggers the metabolic switch and antibiotic production. It is envisioned that by manipulating this switch or the signaling compounds that trigger this switch, the novel ?sleeping? antibiotic biosynthesis clusters can be awakened. We focus our research on the role of small signaling molecules called -butyrolactones. These compounds are found in more than 60% of Streptomyces species, where they play an important role in the regulation of antibiotic production, acting as ?pheromones? in the biology of streptomycetes. Although the first -butyrolactone was identified already 27 years ago, the biosynthesis pathway for these molecules has only been investigated during the last two years, and is still only partially known. This proposal concerns ScbA, the protein that synthesizes the -butyrolactones, which may act as the key to the metabolic switch. In addition, preliminary evidence suggests that ScbA, besides its function as an enzyme, has also a role as a regulator, which is exciting and an unique feature of this protein. For the first time, the 3D structure of ScbA and several active site point mutants will be analysed to determine the exact enzymatic function of ScbA. Furthermore, the regulatory function of ScbA will be uncoupled from the enzymatic activity so that this function can be characterized in vivo. The timing of antibiotic production (metabolic switch), transcriptional, and translational analysis will be carried out on several ScbA point mutants, which will replace the wild type protein at the chromosomal location. |
