Investigation of the Fluorination Step in the Biosynthesis of Nucleocidin

Abstract

Natural products (NPs) are secondary metabolite compounds produced by living organisms like bacteria and fungi. NPs and their derivatives commonly have biological activity and contribute to the development of pharmaceuticals, particularly antibiotics. NPs are encoded by biosynthetic gene clusters, which are sets of genes that when collectively expressed, lead to the production of these compounds. Nucleocidin is an NP derived from adenosine and is produced by Streptomyces species. Its 4’-fluoro and 5’-O-sulfamoyl groups make nucleocidin of particular interest, as it is one of the few natural products known to contain fluorine. Additionally, the incorporation of fluorine at the 4’ position of the adenosine ring requires novel fluorine chemistry, since previously characterized fluorinases have only been known to substitute fluorine at the 5’ position. Previously, three candidate fluorinase genes from the nucleocidin biosynthetic gene cluster, ORF2, 3, and -3, were identified using a genome mining approach and cloned for gene expression in E. coli. In-vitro assays were performed with the purified fluorinase enzymes, but the results were inconclusive regarding enzymatic characterization and association with the fluorination of nucleocidin. This project builds on previous experimentation to prove the fluorination activity of these enzymes through an in-vivo system. The three candidate fluorinase genes, along with a glucosyltransferase essential to the fluorination process, were cloned into a duet-vector system and expressed in a medium supplemented with fluorine. The extracted compounds were analyzed using UPLC-MS, but the results were inconclusive. Further steps will include screening media conditions for nucleocidin production in a heterologous system, and optimizing the in-vivo expression of the potential fluorinase genes. Ultimately, further characterization will be necessary to reveal the specific role each of these candidate fluorinase enzymes plays in the biosynthesis of nucleocidin. Future exploration into the fluorination of nucleocidin offers novel insights into increasing the production efficiency of nucleocidin and its derivatives for pharmaceutical development, along with providing potential avenues for bio-catalytically introducing fluorine into new pharmaceuticals.