Biosynthesis

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.

Pseudopterogorgia elisabethae is a known source of structurally interesting bioactive
metabolites. A detailed search for new, related compounds was undertaken in this study
which resulted in the isolation and characterization of more than ten new diterpenes with
serrulatane and ileabethane skeletons. Some of the new compounds isolated are closely
related terpenes with significant biological activity and others are likely to be key
biosynthetic intermediates. As a component of the development of a production method
of anti-inflammatory compounds such as seco-pseudopterosin and elisabethadione, a
synthesis of a seco-pseudoperosin aglycone from elisabethatriene was developed.

The goal of this work was to investigate the biosynthetic origins of diterpene natural products (pseudopterosins, kallolides, bipinnatins, and cembrenes) from corals of the genus Pseudopterogorgia as well as the biosynthetic pathways by which they are produced. These studies have shown that the pseudopterosins from Pseudopterogorgia elisabethae are biosynthesized within the algal symbiont (or possibly a bacterium or fungus associated with the symbiont), are not inducible by manipulation of light levels, and do not change as a result of transplantation to new locations. Studies on Pseudopterogorgia bipinnata revealed that only one chemotype is capable of biosynthesizing the kallolide family of diterpenes. The biosynthetic pathway which gives rise to the kallolides has been shown to involve members of another family of diterpenes, the bipinnatins, which coexist within the coral holobiont. Two diterpene cyclase products have been discovered within P. bipinnata chemotype A, cembrene and neocembrene, and it has been shown that neocembrene gives rise to the kallolides. Finally, the enzymatic conversion of bipinnatin J to kallolide A has shown for the first time that these compounds are in fact biogenetically related.