Biosynthesis of Fluorinated Natural Product Nucleocidin
Fluorinated Natural Product
Nucleocidin is a fluorinated adenosine analogue produced by Streptomyces calvus. It is one of only six known fluorinated natural products. The 4’-fluorine substituent on nucleocidin requires a unique two-step fluorination mechanism: C-H bond cleavage followed by stereospecific glycosylation of the fluoride ion. Although the gene cluster encoding for nucleocidin has been predicted, the fluorinase gene remains unknown. The biosynthetic gene cluster of nucleocidin has been successfully cloned into a phage P1 artificial chromosome library of the S. calvus genomic DNA, yielding six clones containing the predicted gene cluster. In this thesis, the plasmids containing the nucleocidin gene cluster and large flanking genomic sequences have been introduced into Streptomyces albus and Streptomyces lividans 1326 for the purpose of heterologous expression of nucleocidin. Identification of which clones possess the ability to produce nucleocidin coupled with genomic insert sequencing data has allowed the location of the fluorinase gene to be narrowed down to a ~35 kbp region of the Streptomyces calvus genome. On a second front, an amidinotransferase enzyme encoded by the nucN gene within the predicted nucleocidin gene cluster has been successfully purified, and an initial assessment of its function within the biosynthetic pathway has been conducted.