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ALTERNATIVE MARINE NATURAL PRODUCT SOURCES AND RESOURCE SUSTAINABILITY
Introduction
The prospecting has paid off. After hundreds of specimens from dozens of collections have been processed,
after thousands of natural products have been screened for bioactivity in the lab, one compound seems like
a real winner.
Maybe it's a potential anti-cancer drug or a future weapon in the war on HIV/AIDS. Maybe it's an
antibiotic with an apparent novel mode of action or a molecule that disrupts the cell-to-cell
communication in disease-causing bacteria. Maybe it's the next big pain medication, anti-inflamatory
agent, or enzymatic laundry detergent additive.
Whatever it is, one thing is for sure; it's good stuff and now you need more of it. Lots more.
This is the typical scenario once a marine biotechnology lab identifies a particularly promising natural
product out of the many candidates they have painstakingly extracted, isolated, and screened for activity.
In order to fully assess the pharmaceutical or industrial effectiveness of the novel compound, large
quantities are usually required.
"How Can I Get More Of This Stuff?"
Once a potential MBT-based natural product discovery has been evaluated for bioactivity and the decision
is made to proceed to the next stage in the drug development process, a critically important question
arises that must be answered: what is to be the source of continued supplies of the compound under
investigation?
Marine natural products chemists usually begin the screening process by homogenizing (think Cuisinarts and sponge daiquiris)
specimens and adding organic solvents like hexane or methanol to obtain crude liquid extracts. When and
if a specific natural product isolated from one of the homogenate extracts shows promising bioactivity,
that compound is invariably present only in exceedingly small amounts.
A first choice for obtaining more of the newly discovered target compound might be to process more of the
source material if any exists from the initial collection. If this is not possible the logical next
choice would be to return to the collection site and harvest more of the target species from the wild.
The first approach (processing material on hand) rarely yields the natural product in the amounts
required. A look at the situation regarding the prospective anti-cancer
drug bryostatin
1 serves to illustrate the point. Approximately 14 tons of the
animal source of this compound,
the bryozoan Bugula neritina, was originally required to produce less than one ounce of purified bryostatin.
As for the second approach (collecting more source material), while this solution has a reasonable
probability of success, it also has several important drawbacks. First, mounting a return collecting
expedition can be quite expensive, especially if the destination is remote and/or the required logistical
support (e.g., research vessels, manned submersibles, etc.) is substantial. The target species also may
be comparatively rare, and a return trip to a site several years after the initial collection may not turn
up specimens in the numbers required to continue chemical investigation. For example, 15 years passed
between the initial collection of the deep-water sponge Forcepia (source of the promising bioactive
lasonolides), and subsequent
relocation and follow-up collection. In this instance, the scientists were able to
relocate the target sponge species, but not all return visits to past collection sites are
so fruitful.
To further complicate matters, even if suitable populations of the target species are located, there is no
guarantee that collected specimens will yield the compound of interest that was isolated from the original
collection. This may be because the target compound is produced only seasonally, or only in response to
grazing or certain environmental conditions. It is also possible that the bioactive natural product was
not produced by the target species at all, but rather by associated microbial organisms. In the case of
sponges, for example, as much as half the dry weight of an individual may be attributed to the unseen
bacterial community residing within the pores and tissues of the sponge. If a bacterial species or strain
responsible for producing a target product is not present in the microbial assemblage of a target sponge
species on recollection, extracts derived from that specimen will not contain the target compound.
Obtainable and Sustainable?
The most critical concern regarding the continued collection of target marine organisms as natural
products source material is resource sustainability and conservation. A key issue in pursuing drug development based on a
natural product is ensuring an adequate supply of the compound while also protecting the source organism
and its habitat from overexploitation.
As a result of the U.N. Convention on Biological Diversity,
legislators, biomedical researchers, and environmental resource
managers have begun to explore various issues pertinent to preserving marine biodiversity. Central issues
include sustainable use of living marine resources, environmental and legal protection of a region's
unique biological and genetic resources, and equitable sharing of technologies and revenues resulting from
the commercial development of natural resources. Several of these aspects are examined in the "Partners in Development" subsection of this website.
Various strategies are available to alleviate the potential negative impact of continued collection of
source organisms from the wild. There are advantages and limitations to each of these, and not all
methods are applicable to all natural products. Decisions regarding the best available options are based
on a number of factors, including the complexity of the target natural product and ecology of the product source. Some of these
strategies are well established and others are still being developed.
Six broadly stated natural product alternative source strategies are listed below. You may select from this list to
examine each strategy in more detail.
 Controlled Harvest
Thousands of chemicals with pharmaceutical potential have been identified from marine organisms. With the enormous
potential for discovery and development of these chemicals into drugs comes the obligation to develop methods by which
these products can be supplied in a way that will not damage the ecosystem or deplete the resource.
The supply of most marine-derived chemicals is a major limiting factor to further pharmaceutical development. Usually,
the target chemical occurs in very small amounts within the source organism.
Controlled harvest is decidedly positioned at the low-tech end of the marine biotechnology spectrum. But, that doesn't mean
there is no place for this old world strategy for MBT resource acquisition, provided the demands for the
resource are relatively modest. When commercial demand outpaces sustainable harvest capacity, however, alternatives to wild
harvest must be sought.
In general, wild collection is not seen as a long-term supply option not only because of the impact on marine habitats,
but also because wild collection is generally too costly.
Since at least the Middle Ages, coastal inhabitants have harvested various seaweeds (see the "Unlikely Partners"
section of this website for more information) from natural stocks for medicinal purposes as well as for food and other uses. Seaweeds are an
excellent example of a sustainable natural marine resource. They are fast growing and require no human inputs for good yield.
Kelps and other 'rooted' intertidal seaweeds (a misnomer, as seaweeds have no true roots) can be easily harvested on rocky
shorelines, and many other species can be collected off the beaches from the wrack line. Indeterminate growth,
typical of most seaweeds, is a key trait that lends itself to sustainable harvest.
The promising anti-tumor agent Bryostatin 1 is a natural product that needed to be initially procured
through controlled wild harvest in order to proceed with product characterization and evaluation. NCI commissioned
naturalist writer and founder of the Gulf Specimen Company
Jack Rudloe in 1968 to collect many tons of the source
organism, the intertidal/subtidal bryozoan Bugula neritina to extract enough bryostatin 1 to meet research needs.
Large-scale chromatographic separation techniques eventually yielded about two-thirds of an ounce of purified bryostatin
1 from a starting amount of nearly 14 tons of harvested B. neritina (This story is detailed further elsewhere on this site). Aquaculture and laboratory synthesis were wisely pursued
as it was soon realized that wild harvest did not represent an ecologically or economically viable option for providing
the larger amounts of natural product pending clinical trials would require.
The pseudopterosins
represent a modern MBT-derived product that is still obtained from the sea through
controlled harvest. They are a diverse class of diterpenes isolated from the Caribbean sea whip Pseudopterogorgia
elisabethae. They have been shown to exhibit potent anti-inflammatory and analgesic (painkilling) activity, and are
currently used as ingrediants in a commercial skin cream.
Limited harvest of P. elisabethae in the Bahamas and elsewhere in the Caribbean has thus far been able to provide a
sustainable source of natural product for this use. But, these compounds are likely to soon come into pharmaceutical
use as well. When that happens, commercial demand will outstrip levels that can be sustainably supplied through
controlled harvest. Aquaculture, bacterial cell culture, chemical synthesis, and even, genetic engineering are being pursued as possible
future sources for these and other MBT-derived natural products.
Aquaculture of the Source Organism
Once the demand for a marine derived natural product grows, sustainable harvest of source organisms from the ocean may not provide enough of
the target compounds. Without the development of feasible alternatives,
countless promising marine natural products would remain undeveloped. Aquaculture is one way of supplying research and industry
with adequate supplies of important marine species.
The Caribbean intertidal tunicate (sea squirt) Ecteinascidia turbinata is one biomedically important species whose potential as an
aquaculture candidate is currently being explored. It produces the potent antitumor compound ecteinascidin-743
currently in Phase II clinical trials. The Spanish marine pharmaceutical company PharmaMar
S.A. is advancing techniques for farming E. turbinata. The company is also exploring laboratory chemical synthesis of Ecteinascidin-743 as
it co-develops this drug under the trade name Yondelis® with Ortho Pharmaceuticals, Inc. In the
wild, E. turbinata grows in grape-like bunches on mangrove roots and other intertidal hard surfaces. Preliminary experiments have
shown that this species can be successfully grown in aquaculture systems as well.
Several groups are working to advance aquaculture techniques for the bryozoan Bugula neritina, source of the anti-cancer and immunostimulant compound
Bryostatin 1. Researchers have also experienced some success in early attempts
at culturing several biomedically important sponges like the New Zealand sponge Mycale hentscheli (source of the tubulin interactive agent
peloruside A) and also the Australian octocoral Eleutherobia sp.
(source of the tubulin interactive compound eleutherobin).
The sessile, suspension-feeding habits of marine animals like those above can be exploited through in situ (in the sea) culture
strategies. Grown attached to suspended ropes or in mesh bags such organisms can filter phytoplankton and bacteria from the water
column, saving aquaculturists the trouble and cost of providing food for farmed stocks.
Marine organisms can also be farmed away from the ocean in closed culture systems. Although this is likely to be prohibitively
expensive for the commercial-scale rearing of sponges and other filter feeders, it is ideal for other species of interest to the
marine biotech fields. Large numbers of the sea hare Aplysia californica, for example, are grown
under closed culture at the NIH/University of Miami National Resource for
Aplysia Facility for use in biomedical research programs worldwide.
Cultural Considerations
Initial stocks of organisms intended for aquaculture are procured through prudent harvest from the seas. Shallow species are most
often collected using scuba, whereas collection of deepwater organisms may require the use of ROVs, or research submersibles and
their ocean-going support vessels. Species like sponges, seaweeds, corals, and some ascidians can regenerate lost biomass, so
wild-harvested specimens can be cut into smaller units for propagation through aquaculture. Cultured stocks can be harvested in a
similar manner, removing only a portion of the biomass at harvest time and allowing the remainder adequate time to to regrow.
Success in rearing biomedically important organisms through aquaculture requires a thorough understanding of the ecology of the
farmed species. For closed culture operations, the nutritional and environmental (e.g., temperature, light, etc.) requirements
must be established. For both in situ and closed-system aquaculture programs, the specific environmental conditions experienced by the
farmed stocks will affect not only growth but also production of any target natural products.
Just as controlled wild harvest can be an acceptable means of obtaining source material until demand outstrips the sustainable
supply, so too can aquaculture act as a stopgap solution to meeting research and commercial demand for biomedical marine resources
until other alternative supply pipelines are established. For example, a number of startup aquaculture operations in California
have begun growing Bugula neritina to ensure a supply of Bryostatin 1. NCI has spent more than $1 million funding research into culturing this
species in both land-based and sea-based systems. Although in situ culture of this sessile suspension feeder has proven relatively straightforward,
farmed stocks of B. neritina still represent a less than ideal source for the natural product (recall that approximately 14 tons of the organism is
needed to produce less than an ounce of bryostatin 1). Current research is therefore focused on elucidating a manageable laboratory
chemical synthesis strategy. At present, however, more than 60 steps are required for chemical synthesis of bryostatin 1.
References
Newman DJ, and GM Cragg. 2004. Advanced Preclinical and Clinical trials of natural Products and Related Compounds from marine
Sources. Current Medicinal Chemistry 11:1693-1713.
Cell Culture
Marine biotechnologists are often really only interested in collecting or culturing certain marine species as a means of
obtaining a steady supply of specific target natural products. This is considerably different than, say, the situation with
fishermen or fish farmers, in which the entire animal is the commercial product. This being the case, some marine biotech labs
have begun to look at ways of "cutting out the middle man" in the quest for sustainable supplies of marine-derived natural
products. Modern techniques of genetic engineering (see subsection below) represent perhaps the most far-reaching examples of streamlining the supply
chain for structurally complex natural products. Another promising research avenue with a similar goal is laboratory-based cell
culture.
Particularly for simple marine macroorganisms, cell culture production methods may soon become a commercial option for producing
bioactive compounds with the potential to treat human diseases. Sponges, with no tissues or organs and a level of structural
organization only slightly above that of an uncoordinated pile of cells, are most definitely the poster children for simple
marine animals. Fortunately, sponges are also amazingly prolific natural chemists; no other marine taxon has yielded the
abundance or diversity of promising novel metabolites as have the sponges.
The basic cell culture strategy for sponges is straightforward. A living sample of a sponge that produces a natural product of interest is
dissociated into individual cells that are placed in laboratory flasks. Liquid nutrients and other growth factors are added and
the individual sponge cells are stimulated to grow and divide in culture.
Early research has demonstrated that cell cultures of some bioactive sponges prepared in this manner continue to produce the
target natural product. For example, cell cultures of the Caribbean reef sponge Axinella corrugata, source of the anti-tumor natural product
stevensine, have been successfully established and maintained using this approach.
ell Farming 101
A key advantage of a cell culture production strategy over traditional whole-organism aquaculture is that life support is greatly
simplified. Feeding and shaking or swirling to aerate cells in a flask is easier and far less expensive than establishing and
maintaining an aquaculture system to rear whole sponges. Harvesting stocks and extracting the target natural product is also
simpler.
Establishing cell cultures of various sponges that produce bioactive chemicals is also an important step towards better
understanding the cellular and molecular processes that control production of those compounds. As the bases of these processes
are discovered, it is anticipated that cell culturists will learn how best to manipulate cell cultures to optimize growth, cell
division, and production of target natural products.
Some work has also been done in an attempt to culture cells of ascidians (tunicates) like Ecteinascidia turbinata, source of the
potent anti-tumor agent ecteinascidin-743. Success has been limited thus
far, however, and at present it appears that aquaculture and possibly laboratory chemical synthesis are more promising supply avenues for these products.
There have been instances where sponge cell culture has been successful but the cultures were found incapable of producing the
target natural product. In such cases, there is the possibility that the laboratory culture environment is in need of
modification, or perhaps some needed chemical precursors must be added to the nutrient medium before biosynthesis of the target
product can occur. Another, possibly more likely explanation is that the target compound was originally produced not by the
sponge, but by associated bacteria or other microflora. If this can be demonstrated to be the case, isolation and laboratory
culture of the microbe responsible for producing the target compound may be possible (see next subsection).
Related Weblinks
Production of the Ecteinascidins, Biomedically Important Marine Natural Products, Through Cell Culture of Ecteinascidia turbinata. Marine Biotechnology Research in Florida Sea Grant, 1996-2003: An Outreach and Communication Foundation:
http://www.flseagrant.org/program_areas/biotechnology/biotech_24_rsch_prjs.htm#mb1
Marine Invertebrate Cell Culture for In Vitro Production of Compounds with Therapeutic Potential. Marine Biotechnology Research in Florida Sea Grant, 1996-2003: An Outreach and Communication Foundation:
http://www.flseagrant.org/program_areas/biotechnology/biotech_24_rsch_prjs.htm#mb5
Microbial Culture (Fermentation)
The ongoing MBT efforts of a number of research groups has revealed that some marine natural products originally isolated
from sponges, bryozoans, and other macrofauna may actually be produced by bacteria or other microorganisms that live in
association with those animal hosts. Chief among the bioactive secondary metabolites with recently demonstrated or
suspected microbial origins are several promising drug candidates, including
discodermolide,
bryostatin 1,
aplidine,
the dolastatins,
and ET-743.
As more research is done, the true microbial sources of a large number of other bioactive marine metabolites will, no doubt,
be revealed.
This finding is not entirely surprising, given the diverse microbial communities often found in and on marine animals. For
example, more than half of the dry weight of some sponges is attributable to the microflora that resides within the animal.
The marine microbial community in general is astonishingly untapped as a reservoir of novel bioactive compounds. Of all the
known bacteria that produce secondary metabolites, only about 10 percent of these come from the ocean; the remainder
have terrestrial origins. This gap is largely due to a lack of widespread research into the chemical ecology of marine
microbes. As more marine microbes are cultured and screened, more and more novel bioactivity is revealed.
If natural product-producing microorganisms can be isolated and successfully cultured, researchers can then refine culture
techniques to allow production scale-up to supply sufficient quantities for drug development purposes.
There's the rub. Culturing slow-growing marine microbes in the laboratory presents its own special challenges. Elucidation
of appropriate nutrient regimes and other environmental culture parameters has proven especially difficult for many taxa.
As a consequence, the vast majority of marine microbes remain unculturable using current techniques. This so-called "problem
of the uncultureds" has severely hampered efforts to identify and describe both free-living and macroorganism-associated
(e.g., symbiotic) marine microbial communities. It has likewise made it difficult to adequately screen these communities
for novel natural products they may produce.
"Whos Really Making This Stuff?"
Bryostatin 1 is a good example of a compound originally isolated from a marine
macroorganism (the bryozoan Bugula neritina)
that is most likely produced not by the organism itself but by a symbiotic microbial associate. An initial suggestion that
this may be the case is provided in the finding that although B. neritina is ubiquitous, only a few small and geographically
disparate populations have been shown to produce bryostatins. Another clue is found in the structural nature of the
compounds; the bryostatins are structurally and sequentially closely allied with a diverse class of natural products called
polyketides that are thus far known to originate only from microbial sources. Often structurally unrelated, the polyketides are linked
together as a class because they share common biosynthetic features including the sequence of reactions by which they are formed and the involvement of
complex polyketide synthase (PKS) multi-enzymes in their production.
Scripps Institution's Margo Haygood and her colleagues
have published compelling evidence suggesting that the actual producer of bryostatin in the
drug-producing Bugula populations is a microbial symbiont, the proteobacterium denoted as Candidatus Endobugula sertula.
These authors found an apparent PKS gene fragment that was only present in the drug-producing
populations. If the microbe responsible for bryostatin production can be isolated and effectively cultured in the lab, it
may represent a viable commercial source for this important natural product. Thus far, however the proteobacterium remains
uncultured.
Alternately, the PKS gene cluster putatively coding for bryostatin synthesis could be cloned and inserted into
garden-variety laboratory microbes that can be easily propagated in the lab, in the hope that the engineered bacteria
acquire the ability to produce bryostatin. See the companion genetic engineering subsection below for more information.
Tales From the Flask
The Russell Kerr research group at Florida Atlantic University's
Center of Excellence in Biomedical and Marine Biotechnology has been exploring
strategies for the culture of symbiotic, natural product-producing dinoflagellates ( Symbiodinium sp.) isolated from tropical host octocorals.
The group has isolated and successfully grown dinoflagellate symbionts from the sea whip Eunicia fusca ostensably responsible for the biosyntthesis of the
antiinflammatory diterpenes fuscocide B and fuscol. They have also cultured dinoflagellates symbiotic with the sea whip Pseudopterogorgia elisabethae.
There is a growing body of evidence indicating that this photosynthetic symbiont is actually responsible for production of the anti-inflamatory and
analgesic pseudopterosins present in the octocoral. This natural product is
already in commercial demand as an additive in a line of skin
creams, and is likely to be employed as a pharmaceutical agent in the future if sufficient supplies can be obtained. Thus far, controlled harvest remains
the source of commercial supplies of pseudopterosins. As stated previously, this strategy is not expected to meet future demand and remain sustainable.
Part of this group's research is aimed at developing methods to stimulate dinoflagellate cultures to up-regulate the production of the target compounds.
Treating Symbiodinium cultures isolated from E. fusca with a series of known elicitors of terpene biosynthesis did lead to a significant increase in
secondary metabolite (fuscol) production. The group has also had some success in pursuing the cloning and overexpression of a specific enzyme that
catalyzes an important early step in the pseudopterosin biosynthetic pathway, and this may ultimately yield a viable up-regulation mechanism.
References
Newman DJ, and GM Cragg. 2004. Advanced preclinical and clinical trials of natural products and related compounds from marine
sources. Current Medicinal Chemistry 11:1693-1713.
Haygood, MG, and SK Davidson. 1997. Small-subunit rRNA genes and in situ hybridization with oligonucleotides specific for the bacterial
symbionts in the larvae of the bryozoan Bugula neritina and proposal of " Candidatus Endobugula sertula." Appl. Environ. Microbiol.
63:4612-4616.
Piel J, Hui D, Wen G, Butzke D, Platzer M, Fusetani N, and S Matsunaga. 2004. Antitumor polyketide biosynthesis by an uncultivated bacterial
symbiont of the marine sponge Theonella swinhoei. PNAS 101:16222-16227.
Laboratory Chemical Synthesis
There are three primary reasons for pursuing laboratory chemical synthesis of analogs for those marine-based natural products for which such a
strategy is feasible. First, synthetic analogs reduce or eliminate altogether the need for potentially damaging wild harvest of product sources to
achieve a sustainable supply. Second, for many products laboratory synthesis represents a more economically viable alternative to wild harvest,
once optimal synthetic schemes have been elucidated. And third, synthesis provides an opportunity to tweak molecular structures and actually
improve on the natural base compounds. This may be in the form of enhanced disease treatment efficacy, reduced non-target cell toxicity, or simply
through increased chemical purity compared to separations prepared from natural product extracts.
Synthetic Strategies
There are several different strategies employed in the production of synthetic analogs to marine-derived natural products. Some marine chemical
products are naturally more amenable to economical production via laboratory synthesis. This is usually related to either the overall complexity
of the model compound and/or the number and nature of the steps contained in the biosynthetic pathway. For example, a relatively simple compound
like the sponge-derived peloruside A may represent an ideal model for total synthesis, and may also be itself easily modifiable through
synthesis and substitution.
On the other hand, a natural product like with 60 or more steps
required for complete synthesis may never be economically
produced in its entirety by synthetic chemists. Analogs of the more complex or unusual compounds might be produced through a semi-synthetic
strategy that starts with and builds on one or more precursor molecules. Fortunately, the precursor molecules can come from sources other than the
original marine source of the target product. For example, efficient semi-synthetic production of ecteinascidin 743 (ET-743) has been
attained by using the closely related compound safracin B as a starting point. This natural product is produced by an easily culturable
pseudomonad bacterium, allowing sustainable and cost-effective semisynthetic production of ET-743.
Successful laboratory synthesis of an impressive number of marine natural products and/or closely related analogs has been reported, and this
number is expected to continue to grow in coming years.
The dolastatin natural product family is a prime example. Although
cyanobacterial (blue-green 'algae') origin of the dolastatins
has been demonstrated, the structural simplicity of the compounds makes total synthesis a more viable option for supplying clinical trials than
microbial culture. Two synthetic dolostatin derivatives, TZT-1027 (soblidotin) and LU-1037993 (cemadotin), are currently in Phase II
human clinical trials in the US and abroad. Several more recent synthetic dolastatin-derived products also exist further back in the
evaluation and development pipeline.
IMPROVING ON NATURE'S DESIGNS?
Total or partial laboratory synthesis of marine product analogs is a powerful tool because it can yield chemical variants of the natural products
that are perhaps better than the original in terms of pharmacologic potential.
In such cases, the natural compounds still provide the source of inspiration in the form of unique molecular structures with significant
bioactivity. These products (or their synthetic equivalents) have enormous value as "scaffolds" that can be built upon using combinatorial
synthesis to try to produce molecules with qualities that make them superior to the original (natural) product. The synthetic compounds produced
in this manner may exhibit improved bioactivity, stability, or solubility. They may also prove to be less toxic in some instances than their
natural counterparts.
A good example is seen in the development of a synthetic analog to the cyanobacterium-derived natural product curacin A. Early
laboratory work suggested that the compound was a powerful tubulin interactive agent, but insolubility issues impeded effective delivery in live
animal models. However, semi-synthesis based on the original parent molecule has yielded several bioactive variants with enhanced solubility that
are currently in preclinical evaluation as drug candidates.
Such improvements are not guaranteed, however, as recent work with synthetic variants of the cone snail peptide ziconotide (Prialt®)
illustrates. Chemists working for compound licensee Neurex, Inc. (since acquired by Elan Corporation)
synthesized more than 200 ziconotide variants, but eventually decided that the original marine-derived structure (identical to their synthetic analog MVIIA)
exhibited the most desirable bioactivity.
Laboratory synthetic routes of production also allow the combinatorial formation of chimeric molecules—novel products created by joining the base
structures of two or more unrelated compounds. For example, base structures from the coral-derived sarcodictyins and eleutherobins have been synthetically combined in this manner.
References
Jin M and RE Taylor. 2005. Total Synthesis of (+)-Peloruside A. Org Lett 7:1303-1305.
Newman DJ, and GM Cragg. 2004. Advanced preclinical and clinical trials of natural products and related compounds from marine
sources. Current Medicinal Chemistry 11:1693-1713.
Rayl AJS. 1999. Oceans: Medicine Chests of the Future? The Scientist 13:1
Related Weblinks
Moving Beyond Natural Products: Organic synthetic chemistry amplifies the potential of natural products as drug leads.
Cemical and Engineering News, October 13 2003.
http://pubs.acs.org/cen/coverstory/8141/8141pharmaceuticals4.html
Genetic Engineering
Modern molecular and genetic techniques represent the next giant leap for marine natural products research. Among the many exciting
possibilities is the promise that these techniques will allow the development of sustainable and economical alternate sources of valuable
marine-based natural products.
The field itself is only thirty or so years old, commencing with the monumental work of Stanley Cohen and Herb Boyer demonstrating that DNA extracted
from one species could be successfully inserted into the genome of another, completely unrelated species. This discovery is widely seen as marking the
birth of genetic engineering.
The discovery of Cohen and Boyer made it possible to turn laboratory microbial cultures into custom-made chemical factories producing natural compounds for
biomedical or industrial use. In 1978, Boyer utilized the technique to insert the human gene coding for the production of insulin into
bacteria, making possible for the first time a sustainable, low-cost supply of this life-saving drug. Since this time, a number of
important drugs (including several antibiotics) have come to be produced in this manner.
Applications in MBT
There is now a considerable amount of work being conducted on these research fronts in the fields of marine biotechnology as well.
Recombinant techniques that allow scientists to insert genes of interest from source organisms into easy to grow strains of laboratory
bacteria may soon alleviate supply shortages of several marine microbial natural products. Heterologous expression, the production of target products by
genetically modified microbes, represents a welcome addition to the list of potential alternate sources for these compounds.
The anticancer agent bryostatin
1 is, again, a relevant example. Bryostatin 1 belongs to a chemical class known as the
polyketides. These are structurally diverse compounds produced by a variety of soil and marine microorganisms, including several
bacterial symbionts isolated from marine invertebrate hosts. Despite their diversity, the polyketides are all produced using similar
chemical precursors and modular enzymes called polyketide synthase
(PKS). Naturally occurring polyketides have already proven
important as pharmaceutical compounds; several valuable commercial antibiotics (e.g., erythromycin, the tetracyclines, and Zithromax®),
anti-cancer drugs (e.g., doxorubicin), immunosuppressents (e.g., rapamycin), and others are all polyketides.
Since identifying the probable microbial symbiont source and the gene fragment responsible for the production of the molecule, Margo Haygood and associates have directed
considerable efforts toward transferring the PKS gene fragment that codes for bryostatin 1 biosynthesis
into readily-culturable microbial strains in order to pursue sustainable production of the drug through laboratory fermentation.
Another multifinctional enzyme family that has been implicated in natural product synthesis is the non-ribosomal peptide synthetase (NRPS)
family. These systems direct the synthesis of complex nonribosomal
peptides from simple amino acid monomer precursors, accomplished in a manner similar to that seen in syntheses coordinated by modular PKS
systems. In many cases NRPS and PKS systems work in concert to produce elaborate hybrid natural products (the cyanobacterium-derive natural
molluscicide barbamide is an example). As with
PKS-assembled natural products, bioactive compounds synthesized through the direction NRPS systems may eventually be made available via
insertion of NRPS gene fragments into garden variety culturable microbes.
The bioprospecting program of the Bermuda Biological Station for Research is pursuing similar research avenues. They are developing techniques for cloning
genes from the 'metagenome' (gene complex consisting of the genes of host macrofauna and associated microbial symbionts) of sponges and other marine invertebrates and
inserting them into laboratory bacterial strains. The hope is that target natural products can be sustainably produced using such a
strategy, even if the microbial agents responsible for them themselves remain unculturable, or even unidentified. Leveraging the genetic
diversity of the marine environment at large via the emerging science of ecological genomics is at the forefront of emerging challenges for the fields of marine biotechnology.
An ambitious MBT molecular genetics initiative with great commercial potential is that of the biotechnology company Kosan Biosciences. This company uses genetic engineering techniques to create novel versions of important bioactive polyketide compounds.
Kosan uses a variety of strategies to produce polyketide products by manipulating the genes involved in their specific biosynthetic
pathways. In some cases, they look to alter the polyketide source microorganisms at the gene level. In other cases they isolate and insert
PKS gene clusters (with or without modification) into recombinant culture systems to facilitate heterologous polyketide production.
The company's lead compounds include the novel anti-cancer agent (non marine-derived) OS-862 (epothilone D), currently in Phase
II human clinical development. The
company also has developed a large number of novel discodermolide analogs that
are currently under preclinical investigation, including trials in animal
models). Because cost-effective total synthesis of discodermolide remains elusive and laboratory fermentation of the source microorganism
is not yet practical, genetically engineered sources of this and other structurally complex compounds are likely to prove valuable as
clinical development and evaluation continue.
While heterologous production of marine natural products via engineered culture systems gets around the problem of "the uncultureds," the
cutting edge techniques employed will, no doubt, continue to present their own formidable challenges.
References
Chang Z, Flatt P, Gerwick WH, Nguyen VA, Willis CL, and DH Sherman. 2002. The barbamide biosynthetic gene cluster: a novel
marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an
unusual trichloroleucyl starter unit. Gene 296:235-247.
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Related Weblinks
The Birth of Biotech (Wisconsin Technology Network)
http://wistechnology.com/article.php?id=1118
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