Exploration of Methods to Identify Biosynthetic Genes and the Origin of Biosynthesis in Marine Octocorals

The overall goal of this research was to isolate key genes involved in the
diterpene biosynthesis from Euniceafusca and Erythropodium caribaeorum using
molecular biology techniques. The initial goal was to use fuscol induced cell
cultures of Symbiodinium sp. isolated from E. fusca and to develop an approach
based on differential display of mRNA-reverse transcription-PeR. Together with
inverse PCR, these techniques ultimately provided a full-length farnesyl
diphosphate synthase sequence. Functional expression of this enzyme was
demonstrated with the addition of appropriate substrates and confirmed by
chromatography. From this data, degenerate primer based PCR was used to
isolate putative geranylgeranyl diphosphate biosynthetic genes from E.
caribaeorum. Both chemical and genetic examinations of Pseudopterogorgia
elisabethae eggs and their associated Symbiodinium sp. were employed to identify
the biosynthetic origin of their diterpenes. Terpene content and biosynthetic
capabilities of azooxanthellae eggs demonstrated the presence of pseudopterosins
but also indicated that the eggs were not capable of producing these compounds.
Likewise, no correlation could be observed for the phylogenetic relationships
inferred for the Symbiodinium sp., with that of the terpene chemistry present in P.
elisabethae. This finding leads us to speculate about an additional source of
terpene production within this coral.
Based on these and other recent findings suggesting symbiotic bacteria as
the source of secondary metabolites from marine invertebrates, bacterial
assemblages from E. caribaeorum were examined. This study revealed
considerable phylogenetic bacterial diversity within this coral and the
identification of several bacteria known to produce terpenes in other organisms.