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Cloning and biochemical characterization of the hectochlorin biosynthetic gene cluster from the marine cyanobacterium Lyngbya majuscula

Posted on:2007-12-12Degree:Ph.DType:Thesis
University:Oregon State UniversityCandidate:Ramaswamy, Aishwarya VFull Text:PDF
GTID:2440390005460069Subject:Biology
Abstract/Summary:
Cyanobacteria are rich in biologically active secondary metabolites, many of which have potential application as anticancer or antimicrobial drugs or as useful probes in cell biology studies. A Jamaican isolate of the marine cyanobacterium, Lyngbya majuscula was the source of a novel antifungal and cytotoxic secondary metabolite, hectochlorin. The structure of hectochlorm suggested that it was derived from a hybid PKS/NRPS system. Unique features of hectochlorm such as the presence of a gem dichloro functionality and two 2,3-dihydroxy isovaleric acid prompted efforts to clone and characterize the gene cluster involved in hectochlorm biosynthesis.; Initial attempts to isolate the hectochlorin biosynthetic gene cluster led to the identification of a mixed PKS/NRPS gene cluster, LMcry1 , whose genetic architecture did not substantiate its involvement in the biosynthesis of hectochlorin. This gene cluster was designated as a cryptic gene cluster because a corresponding metabolite remains as yet unidentified. The expression of this gene cluster was successfully demonstrated using RT-PCR and these results form the basis for characterizing the metabolite using a novel interdisciplinary approach.; A 38 kb region putatively involved in the biosynthesis of hectochlorin has also been isolated and characterized. The hct gene cluster consists of eight open reading frames (ORFs) and appears to be colinear with regard to hectochlorin biosynthesis. An unusual feature of this gene cluster includes the presence of a ketoreductase domain in an NRPS module and appears to be the first report of such an occurrence in a cyanobacterial secondary metabolite gene cluster. Other tailoring enzymes present in the gene cluster are two cytochrome P450 monooxygenases and a putative halogenase. The juxtaposition of two ORF's with identical modular organization suggests that this gene cluster may have resulted from a gene duplication event. Furthermore, biochemical characterization of two adenylation domains from this cluster strengthens its involvement in the biosynthesis of hectochlorin.
Keywords/Search Tags:Cluster, Hectochlorin, Biosynthesis, Metabolite
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