Biosynthesis of the Fluorinated Natural Product Nucleocidin in Streptomyces calvus Is Dependent on the bldA‐Specified Leu‐tRNA^UUA Molecule

Abstract

Nucleocidin is one of the very few natural products known to contain fluorine. Mysteriously, the nucleocidin producer Streptomyces calvus ATCC 13382 has not been observed to synthesize the compound since its discovery in 1956. Here, we report that complementation of S. calvus ATCC 13382 with a functional bldA-encoded Leu-tRNA(UUA) molecule restores the production of nucleocidin. Nucleocidin was detected in culture extracts by (19) F NMR spectroscopy, HPLC-ESI-MS, and HPLC-continuum source molecular absorption spectroscopy for fluorine-specific detection. The molecule was purified from a large-scale culture and definitively characterized by NMR spectroscopy and high-resolution MS. The nucleocidin biosynthetic gene cluster was identified by the presence of genes encoding the 5'-O-sulfamate moiety and confirmed by gene disruption. Two of the genes within the nucleocidin biosynthetic gene cluster contain TTA codons, thus explaining the dependence on bldA and resolving a 60-year-old mystery.

Biosynthesis of 2′-Chloropentostatin and 2′-Amino-2′-Deoxyadenosine Highlights a Single Gene Cluster Responsible for Two Independent Pathways in Actinomadura sp. Strain ATCC 39365

“…Notably, the advent of rapid and affordable DNA sequencing will certainly accelerate the traditional process for the discovery of new drugs related to PTN-related antibiotic pairs. Moreover, the enzymatic reactions of the halogenation reaction in the biosynthesis of 2=-Cl PTN, ascamycin, and nucleocidin have remained obscure for a half century (22,23), and our identifying a probable cation/H ϩ antiporter, AdaE, that plays a potential role in the chlorination in 2=-Cl PTN will not only open a door for further illumination of the entirely unknown mechanism for halogenation chemistry, but it may also be used as an enzyme probe for the discovery of more halogenated natural products.…”

Expanding beyond canonical metabolism: Interfacing alternative elements, synthetic biology, and metabolic engineering

“… Element Highlighted Compounds Organism(s) Engineered vs. Natural Titer References Fluorine Fluorosalinosporamide* Salinispora tropica Engineered 1.5 mg/L Eustaquio and Moore [167] Nucleocidin Streptomyces calvus Natural N/A Zhu et al. [42] Fluorinated rapamycin analogues* Streptomyces hygroscopicus Engineered, precursor directed 5-28 mg/L Goss et al. [44] Fluorinated tetraketide lactones* E. coli Engineered, precursor directed N/A Walker et al.…”

Radical Dehydroxymethylative Fluorination of Carbohydrates and Divergent Transformations of the Resulting Reverse Glycosyl Fluorides

“…Structurally unique reverse glycosides,aclass of saccharides with furanos-4-yl or pyranos-5-yl skeletons and N-, O-, F-, S-, or P-substituents,a re frequently found in bioactive natural products,b iological probes,a nd drug candidates. Representative examples of such substances are depicted in Figure 1, including the first fluorinated natural nucleoside nucleocidin displaying antibacterial, trypanocidal, and antiviral activity, [1] pyranos-5-yl fluorides acting as useful probes of carbohydrate-processing enzymes to investigate the mode of action of epimerases and dehydrases, [2] cytotoxic dragoci-nA, [3] and sotagliflozin, [4] which has emerged as ap otential dual inhibitor of sodium glucose co-transporter proteins 1and 2(SGLT1 and 2) for the treatment of diabetes. [5] Theu ncommon structures and characteristic biological activities of reverse glycosides have inspired the development of numerous methods for their preparation.…”