Graduate Thesis Or Dissertation
 

Biosynthetic studies of fungal diterpene antibiotics

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  • The isoprenoid pathway is one of the major biosynthetic pathways of secondary metabolites. Isoprenoids with a C₂₀ skeleton are known as diterpenoids and are derived from the common precursor, geranylgeranyl diphosphate (GGPP). This dissertation describes approaches to discover the diterpene synthase enzymes which provide the backbone structures for pleuromutilin and sordarin, two diterpene antibiotics produced by the fungi Pleurotus passeckerianus Pilat and Sordaria araneosa Cain, respectively. A greater understanding of these synthase enzymes may allow us to eventually manipulate the corresponding genes, creating new scaffolds for bioactive molecules. In this dissertation, attempts to identify the biosynthetic gene clusters, including the diterpene synthase genes, for the formation of pleuromutilin and sordarin are described. Three methods were used to locate the diterpene synthase genes. The first approach involved the use of PCR (Polymerase Chain Reaction) with degenerate primers designed from identified and putative fungal diterpene synthase genes. A variety of conditions and degenerate primers were utilized, but no desired PCR products were found. The reason that this method did not work might be the limited overall knowledge of fungal diterpene synthases; the degenerate primers might not be suitable for P. passeckerianus and S. araneosa. The second method attempted to locate the diterpene synthase genes through a related isoprenoid biosynthetic gene, the ggs genes (GGPP synthase genes). P. passeckerianus and S. araneosa ggs genes were used to try to locate the biosynthetic genes of pleuromutilin and sordarin by probing and screening a genomic DNA library and by genome walking methods. Three ggs gene fragments were identified from P. passeckerianus and two fragments were identified from S. araneosa. The BLAST searches strongly supported their ggs gene identities. About 10 kb of sequence extending from each fragment was obtained. However, no gene associated with the pleuromutilin or sordarin biosyntheses was found. This result and a recent review suggest that fungal diterpene gene clusters do not necessarily need a dedicated ggs. So although the ggs approach has worked for several fungal clusters, it does not appear to work for all fungal diterpenes. The biosynthesis genes for the formation of pleuromutilin and sordarin appear to belong to the category that is not clustered with a dedicated ggs gene. The third method utilized was Suppression Subtractive Hybridization (SSH), which is a technique used to find differentially expressed genes. This method was tried on the fungus P. passeckerianus. A subtracted library was formed from cDNA prepared from a 2nd day culture that did not produce pleuromutilin and from cDNA from a 7th day culture that produced pleuromutilin. The genes involved in the biosynthesis of pleuromutilin should be among the genes in the subtracted library. The PCR amplification results suggest that the SSH method has worked and identified a number of possible candidate genes. Several clones from the subtracted library have been sent for sequencing to find out whether or not genes associated with pleuromutilin biosynthesis are present. In conclusion, three methods have been attempted to identify the diterpene synthase genes for the formation of pleuromutilin and sordarin. Although no diterpene biosynthetic genes have been found to date, these studies will provide a solid foundation for further studies in this area.
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