Elucidation of the biosynthesis of the hydrazine containing compound negamycin

• Applicant: Privatdozentin Dr. Efthimia Stegmann, Ph.D.
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; General Genetics and Functional Genome Biology ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 530006809

Negamycin is a specialized metabolite isolated from culture supernatants of Streptomyces purpeofuscus, now renamed Kitasatospora purpeofusca. It has antibacterial activity against Gram-negative and Gram-positive bacteria, including bacteria on the ESKAPE list (Klebsiella pneumoniae, Enterobacter sp. and Pseudomonas aeruginosa), against which new antibiotics are urgently required. Interestingly, it is more potent against Gram-negative than against Gram-positive bacteria. In addition, negamycin exhibits stop-codon readthrough activity. Such activity is valuable in the development of therapies for genetic diseases caused by mutations yielding in-frame stop codons (premature termination codon, PTC), leading to the production of truncated and non-functional proteins. This type of mutations is common, for example in some forms of the Duchenne muscular dystrophy (DMD), cystic fibrosis (CF), hemophilia A and some cancers. In these cases, the production of full-length and functional proteins could be restored to some extend by inducing the readthrough of PTCs, e.g. with negamycin. Although negamycin and its analogues show promising activities for the development of both antibiotic and PTC-readthrough inducing drugs, their characterization are hindered by the low productivity of the actinobacteria producer strains and the costly and complex chemical synthesis16. An alternative to chemical synthesis could be the establishment of negamycin production in native or in heterologous producer strains in quantities sufficient for purification and use in various assays to analyse their effectiveness. In order to achieve a stable production of negamycin and its analogues, it is necessary to identify the negamycin biosynthetic gene cluster (BGC) and to elucidate the stepwise biosynthesis. This knowledge makes it possible to use metabolic engineering approaches to optimise the yield in a targeted manner and to carry out specific structural changes. So far, nothing is known about the negamycin biosynthetic pathway. Negamycin is a hydrazide compound, a small pseudopeptide with an unusual structure containing an N-N-bond. Over the past five years, progress has been made in understanding the biosynthesis of N-N bond-containing metabolites17. However, only a limited number of biosynthetic pathways has been deciphered, and often only partially. A further moiety that is present in the structure of negamycin is β-lysine. In the literature, 2,3-lysine aminomutase has been described to catalyse the conversion of lysine to β-lysine. Our studies of negamycin offer an excellent opportunity for better characterization of the enzymes and mechanisms involved in the synthesis of N-N bond-containing metabolites, and for elucidating the individual steps in the biosynthesis of negamycin, thus establishing a new biosynthetic mechanism.

• DFG Programme: Research Grants
• International Connection: France
• Co-Investigator: Professorin Dr. Heike Brötz-Oesterhelt
• Cooperation Partner: Dr. Sylvie Lautru