Terpenes--Synthesis

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.

The marine environment is a prolific source of novel compounds for therapeutic
use due to the complex biological and chemical diversity. Throughout the past 30-40
years, over 15,000 natural products have been discovered from the oceans, many of
which display a broad range of potential clinical and commercial applications. Many
marine invertebrates are sessile organisms that lack physical protection, and which
chemical defense may be a possible explanation for these secondary metabolites. Despite
the promise marine natural products have as potent pharmaceutical agents, one of the
major factors delaying clinical use is the supply issue. These bioactive compounds are
often found in trace amounts in the host organism, which makes harvesting from the reefs
unfeasible. A general goal in our lab was to investigate the biosynthesis of secondary
metabolite terpenes to ultimately provide a production method of these potent marine
derived compounds. Eleutherobin and desmethyleleutherobin are diterpenes isolated from the
Caribbean soft coral Erythropodium caribaeorum. These extremely valuable anticancer
agents disrupt cell division by polymerizing and stabilizing microtubules, and have
demonstrated tumor tissue selectivity toward selected breast, renal, ovarian and lung
cancer cell lines. Determining the first intermediate in terpene biosynthesis is the initial
step in developing a biotechnological production method of these cytotoxic agents. We
investigated the complex chemistry of this coral using a radioactivity-guided isolation
procedure, and isolated and partially characterized a diterpene hydrocarbon from E.
caribaeorum.
The close association between marine invertebrates, zooxanthellae and numerous
bacteria gives rise to the question of the identity of the producer of secondary metabolites
in marine organisms. If the symbiont produces these therapeutic agents, cell culture
methods could be employed to supply the compounds rather than obtaining them from
coral reefs. Sesquiterpenes have been isolated from the gorgonian Plexaurella spp.,
however, no investigations concerning host/symbiont contribution of the sesquiterpenes
have been reported. We investigated the biosynthetic source of terpenes in this coral, and
experimental evidence indicates that bacteria are responsible for sesquiterpene
production. We also examined sesquiterpene variation of Plexaurella spp. from various
locations, and found sesquiterpene content to vary within and between species,
identifying Plexaurella as a chemically indistinguishable genus.

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.