GENETIC MANIPULATION OF ACTINOMYCETES FOR THE IMPROVEMENT OF ANTIBIOTIC PRODUCTION

Antibiotics have always been considered one of the most relevant discoveries of the twentieth century, revolutionizing healthcare since their introduction. With the discovery of penicillin in 1928, the so-called "golden age of antibiotics" started. Since then, most antimicrobial classes have been isolated from microorganisms, mainly from soil Actinomycetes, known as a source of secondary metabolites. Actinomycetes are Gram-positive bacteria with a G-C-rich genome characterized by a complex life cycle. Streptomyces bacteria show an unusual fungal-like growth mode, have impressive metabolic capabilities, are ubiquity distributed in the environment, and can be readily cultured. The study of the model organism Streptomyces coelicolor has increased the knowledge of the biosynthesis and regulation of specialized metabolites and bacterial multicellular development. However, industrially relevant bacteria belong to the "rare" actinomycetes, which are less easy to cultivate than Streptomyces spp.. Antibiotic biosynthesis is subject to complex regulation processes. Typically, genes responsible for the biosynthesis of a specific antibiotic are arranged in biosynthetic gene clusters (BGCs). Genes within the cluster encode enzymes responsible for catalyzing the formation of antibiotics, regulation, immunity, and export. For the pharmaceutical industry the development of tools and techniques to optimize the yields of a specific metabolite is highly appreciated to reduce production costs. This PhD project, funded by Programma Operativo Nazionale Ricerca e Innovazione 2014-2020, aimed to investigate the molecular mechanisms involved in regulating biosynthetic pathways in Actinomycetes, contributing to enhancing the knowledge of the biosynthesis and regulation of specialized metabolites. Studies were conducted on Streptomyces coelicolor A3(2) M145 model organism and the industrially relevant rare Actinomycete Nonomuraea gerenzanensis. Specifically, the role of a novel molecular mechanism, DNA cytosine methylation (m5C), was investigated in S. coelicolor by multiple approaches, including technologies for methylome mapping. In addition, in N. gerenzanensis, the role of a pleiotropic regulator belonging to the IclR-like regulators family was investigated. Characterization of an over-iclR strain has led to identifying a negative role of the IclR factor (IclRn), which could inhibit acting directly or indirectly on pathway-specific dbv cluster regulators. Additionally, it seems to control the leuCD operon. Finally, this thesis shows the results of the morpho-physiological and bacterial genome sequencing analysis of a spontaneous mutant strain of N. gerenzanensis resistant to apramycin, which lost the parental characteristic purple/orange pigmentation and showed a decrease in the resistance to the glycopeptide A40926.