Molecular genetic and enzymological techniques have been employed to study
secondary metabolite biosynthesis. These investigations have focused on two projects: the
cloning and heterologous expression of biosynthetic gene clusters from unculturable marine
organisms and the characterization of individual enzymes involved in the biosynthesis of the
antifungal agent blasticidin S.
The marine environment is proving to be a valuable source of biologically active
compounds, but problems associated with sustainable harvest, laboratory culture, and
organic synthesis make obtaining sufficient quantities of compounds for drug development
both difficult and expensive. A method has been developed for the isolation of biosynthetic
gene clusters from complex marine microbe/invertebrate associations. Using this method a
mixed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster
has been cloned from the marine sponge Jaspis splendens. The cloned gene cluster was
found to code for a PKS with three extension modules and an NRPS with three extension
modules. In addition, several open reading frames (ORFs) were identified that may be
involved in the biosynthesis of the PKS starter molecule. Partial characterization of
catalytic domains from the NRPS was also completed.
The second project centers on the characterization of enzymes involved in blasticidin
S (BS) biosynthesis. Two ORFs were identified in the BS gene cluster encoding gene
products predicted to be involved in the early steps of BS biosynthesis. The blsG gene
product has sequence similarity to lysine 2,3-aminomutase and is believed to be involved in
the formation of the β-arginine moiety of BS. A series of heterologous expression studies
were undertaken to determine the function of B1sG.
The product of blsM exhibits sequence homology with several
nucleosidetransferases. blsM was cloned from the BS gene cluster, heterologously
expressed in E. coli, and shown to catalyze the formation of cytosine using cytidine 5'-
monophosphate as the preferred substrate. Point mutations were introduced in blsM to
generate three B1sM mutant enzymes: S92D, E98A, and E98D. All three mutants lost
cytidine 5'-monophosphate hydrolysis activity. Surprisingly, the B1sM S92D mutant
exhibits cytidine deaminase activity when incubated with cytidine or deoxycytidine,
resulting in the formation of uridine and deoxyuridine, respectively. / Graduation date: 2005
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/29916 |
| Date | 11 June 2004 |
| Creators | Grochowski, Laura L. |
| Contributors | Zabriskie, T. Mark |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
Page generated in 0.0019 seconds