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UNLIKELY PARTNERS
INTRODUCTION
Marine biotechnology has often drawn its inspiration from some humble and unlikely sources. This is readily apparent when we stop to consider
how often it is that the most promising MBT discoveries come from 'lowly' microbes or 'primitive' invertebrates?
But, if we consider the matter further, we realize that these organisms are in reality the most likely living systems to harbor such
secrets. After all, they are the living representatives of evolutionary lines that have been quietly experimenting with various chemical
and physiological survival tricks for hundreds of millions of years. It is not surprising, therefore, that they have been able to
accumulate some rather spectacular adaptations into their otherwise unassuming forms.
The proceeding material is aimed at providing a glimpse into some of the MBT innovations that have resulted from work focusing on some of
the 'simplest' groups of organisms, including marine microbes, seaweeds, sponges, molluscs, and cnidarians (jellyfish, anemones, etc.). The MBT
efforts briefly touched on here are intended to be explored along with the more detailed descriptions of research activities and discoveries
presented on this website, in the hope that the breadth and diversity of the field of marine biotechnology might be better appreciated.
Select from the five broad biological classifications presented below to see some of the exciting marine biotech discoveries that each group has yielded.
 Marine Microbes
The sheer genetic diversity and the treasure trove of potentially useful natural products found in the microbiota of the world's oceans
are topics covered in some detail elsewhere on the site. But, a couple of other important MBT innovations involving marine prokaryotes
should also be noted.
From Whale Bones to Whiter Whites: MBT in the Wash?
The detergent industry has been searching for enzymes that can break down fats and oils under low temperature conditions. Such enzymes
could be added to laundry detergents, leading to increased efficiency and energy savings because they could dissolve greasy stains
without the need for hot water.
Marine scientists may have accidentally stumbled onto a potential source for such enzymes in the bacteria living within the bones of whale
carcasses on the sea floor.
When a large whale dies and sinks to the abyssal sea floor it represents a veritable bonanza to the food-limited deep-sea communities that
exist there. University of Hawaii researcher and professor of oceanography Craig Smith has been studying such 'whalefalls' since
the 1980s, and has long speculated that they may act as stepping stones for animals normally found near hydrothermal vents. The communities
found at various vents within an ocean basin are similar, but the vents themselves are often separated by hundreds of kilometers. Whales
can die and sink anywhere, and their carcasses produce and output sulfide as they decay over time. Sulfide-rich whale carcasses, Smith
suggests, may be similar enough to sulfide-rich vents to allow larval vent animals to colonize and use them to get their own offspring to
the next whale carcass or vent system when they reproduce as adults. Such a dispersal mechanism would have important evolutionary
implications, as whales and their ancestors have been living, dying, sinking, and rotting for at least the last 30 million years.
The accidental discovery in all this that has the attention of industry giants like Diversa Corporation and Procter & Gamble lies in the
bacteria inhabiting the sulfide-rich whale bones. The bacteria produce enzymes that can rapidly metabolize fats and proteins even in the
cold deep sea. Smith's group has a collaborative agreement with Diversa. The San Diego-based biotechnology firm has identified more
than 30 such enzymes in bacteria taken from bone and blubber samples Smith's lab has provided.
For companies like Procter & Gamble, the principal goal is putting some of these novel marine bacteria-derived enzymes to work in
money-saving coldwater detergents. But, industrial scientists also see potential applications for making cheese and developing new
food additives.
Smith points out that the bacteria enzyme-detergent aspect of his research is "a classic example of how spinoffs can come from basic
research and directions you can't anticipate."
Venting Over PCR
Polymerase chain reaction (PCR) is a laboratory tool of central importance to the field of molecular biology. It permits the rapid
synthesis of large numbers of copies of a small starting segment (as little as a single molecule) of DNA. After using heat to separate
the two strands of the starting DNA, a specialized enzyme called a DNA polymerase is used to synthesize new DNA strands complementary to
the sample strands. Repeating the process yields a geometric increase in DNA concentration until the desired quantity is obtained.
While PCR techniques depend of the application of heat to split apart the DNA strands, heat generally causes big problems for enzymes
like DNA polymerases. Enzymes are proteins, and proteins tend to lose their precise 3-D form (denature) when they are heated. Form
and function are intimately associated in proteins; if a protein's shape is altered it stops working the way it is supposed to.
This problem was resolved with the discovery of a thermostable DNA polymerase enzyme produced by the thermophilic (heat loving) hot
spring bacterium Thermus aquaticus. The newly discovered enzyme was named Taq DNA polymerase.
Taq is still the workhorse DNA polymerase for most PCR applications. But, thermostability is only one requisite for suitability. The
other is a requirement that the copy fidelity rates for the DNA polymerase selected be very high (i.e., almost no mistakes are made
during DNA copying). Molecular analysis has revealed that the difference between high fidelity DNA polymerases and less accurate
versions like Taq is due to an innate 'proofreading' ability that the hi-fi enzymes posses but others do not. The presence of this
so-called exonuclease (proofreading and correcting) ability allows high fidelity DNA polymerases to spot and fix any incorrect
nucleotides that are added during DNA chain synthesis.
The search for new DNA polymerases that could take the heat and carry out a search-and-destroy on its own copying errors took researchers
back to the hot springs and to other extremophile habitats. Included among such habitats are the seafloor hydrothermal vents. And, in
fact, highly thermostable DNA polymerases with built-in copy correction have been found in multiple species of vent microbes.
For example, the archaebacterium ( Archaea) Thermococcus
litoralis is a vent community microbe that can survive at near-100° water temperatures. It produces a proofreading DNA polymerase with a copy fidelity
of up to 15 times that of Taq. The sequenced gene coding for this enzyme in T. litoralis has been inserted into cultured E. coli so that
the novel polymerase could be produced in the lab. It is now commercially available for use in high fidelity PCR applications, under the
trade names VentR® from New England BioLabs and Tli DNA polymerase® from Promega Corporation. Similar high fidelity DNA polymerases have
been derived from archaebacteria of genus Pyrococcus, including one from P. abyssi found at vents 3,000 m deep off the Fiji Islands and
under commercial production by Qbiogene Molecular Biology.
Related Weblinks
Dead whales support deep-sea colonies (Honolulu Star-Bulletin, February 15, 2004)
http://starbulletin.com/2004/02/15/news/story10.html
Genetic fingerprints yield insights into health of diverse ecosystems (Lawrence Livermore National Laboratory/DOE JGI News Release, April 21, 2005)
http://www.llnl.gov/pao/news/news_releases/2005/NR-05-04-04.html
Mussel family's missing link may have been found (Honolulu Star-Bulletin, February 22, 2000)
http://starbulletin.com/2000/02/22/news/story4.html
Seaweeds
Taken in its broadest context, marine biotechnology is a field involving several diverse technologies focused on transforming
biological resources from the ocean into new foods, drugs, chemicals, and other products benefiting humans or the environment.
In this context, perhaps no marine resource has been so successfully exploited from such an early point in history as the seaweeds.
In addition to being a dietary staple in Japan, China,
and parts of Europe well before the advent of recorded history, seaweeds have been found to be remarkably useful in a variety of ways.
Medicinal Uses
At least as far back as the Middle Ages, there were European coastal communities utilizing certain seaweeds as digestive aids, as
helminthicides (dewormers), and to treat other common ailments. This indicates that man has turned to the ocean as a potential
source of powerful medicines for far longer than we might imagine.
In the 18th and 19th centuries a crude extract made from an intertidal kelp called bladderwrack ( Fucus vesiculosus) was the main
source of dietary iodine for many European coastal communities. From the 1860s to the present this species has also been a part of
dietary regimens aimed at increasing metabolism and countering obesity. Dried and processed forms of this alga are now taken as
antirheumatic and anti-inflammatory agents as well.
Other seaweeds are now also commonly encountered as herbal medicines. Irish moss or carrageen ( Chondrus crispus), the principal
source of carrageenan (see below), has medicinal properties that include antiseptic and blood-thinning abilities, and its processed
products are also administered as expectorants, nasal lubricants, emollients, and digestive aids. Products from Laminaria and
Sargassum species are traditional Chinese cancer treatments, and dried Laminaria has also been used by obstetricians in China to
speed up cervical dilation during labor. A coralline hard alga (genus Corallina) has been employed in bone-replacement therapy, and
preliminary studies suggest that extracts taken from certain red algae may inhibit the herpes simplex virus.
Agriculture
Seaweed played a key role in some of the earliest agricultural engineering. Agriculture was extremely difficult in coastal western
Ireland and on nearby islands like the Aran Islands because there was very little topsoil covering the harsh limestone substrate.
The sparse soil that was present had a very high clay content making it ill-suited for cultivation. The resilient farmers of that
region several centuries ago took matters into their own hands and developed the science and art of "landmaking." Up the barren
rocky slopes they would carry load after load of kelp harvested off of the beach, to be combined with sand and clay and laid out on
top of the bare rock. The slowly decaying seaweed was a source of fertilizer for planted crops and the farmers
became the early practitioners of organic agriculture. Such use of seaweeds continues today in Ireland, Scotland, Brittany, and
elsewhere.
Modern seaweed-based fertilizers and agrochemicals in production include dried seaweed meal, liquid extracts, and marl (seaweed-soil
mixtures). As worldwide concern grows over the use of artificial chemical fertilizers and pesticides, sustainable organic products
as these are expected to gain an appreciable market share. In Great Britain, roadsides and other disturbed areas are revegetated
using a mixture of crude brown algae macerate or extract and grass seed that is formed into a paste and sprayed onto revegetation
sites. The algal paste binds the seed on place, stabilizes the soil, retains moisture, and acts as a fertilizer.
Various forms of seaweeds are also used in livestock feeds or as feed supplements. Historically many seaweeds were used in this
manner, particularly as winter feeds when suitable terrestrial forage material was scarce.
Industrial Applications
Seaweeds were economically important industrial resources in the historic past. Various red algae were collected from western
European seashores for use as fabric dyes several hundred years ago, and there is evidence that similar practices in the
Mediterranean date to the pre-Christian era. Irish moss historically has been used as a clarifying agent in the brewing of British
ale, and the practice is still in use by traditional and craft brewers.
The use of algal products in industry has never been greater than it is right now. Natural gelatinous colloids such as agar,
carrageenan (Both from red algae), and algin (from brown algae, Division Phaeophyta) are in modern use in a tremendous array of
products. They are natural stabilizing, emulsifying and thickening agents, found in toothpaste, ice cream, cosmetics, canned
products, animal-free gelatins, sauces and salad dressings, whipped toppings, cheeses and yogurt, and many other products we
encounter on a daily basis. Agar is used as a standard growth medium in microbial culture labs, and purified agarose gel is used as
support scaffolding in electrophoresis and chromatography applications.
Alternative Renewable Fuels
At the height of the United States energy crisis in the early 1980s, skyrocketing oil and gas prices prompted the government and
private gas companies to explore alternative energy sources. Some of the funding at that time led to the initiation of "energy from
biomass" research studies. Some of these studies examined the feasibility using bacterial fermentation of seaweed to produce energy.
The projects demonstrated that seaweed, a wonderfully renewable source of biomass, could be partially digested by bacteria in
the absence of oxygen to produce clean-burning methane "biogas." Interest in pursuing such projects on a larger scale leading to
commercial viability waned when crude prices dropped, but the concepts themselves were proven sound. Small-scale commercial
seaweed-to-energy ventures do currently exist in parts of the world (see seblinks below).
Since that early research was conducted, the technologies allowing renewable fuels to be derived from algal other biomass have
steadily matured. Currently there is considerable interest in using pyrolysis or thermal decomposition of seaweed to yield energy. Pyrolysis
uses heat and the absence of oxygen to decompose biomass leading to formation of gas, liquid, and solid
products that are all usable as fuel. The pyrolysis products can also be sold to chemical or industrial industries.
With oil and gas prices once again on the rise, a continued growing interest in clean and renewable energy, and the inevitable
depletion of the world's fossil fuels, seaweed is likely to again become an important focus of alternative energy production
strategies.
Related Weblinks
NUI, Galway, Seaweed Site
http://www.seaweed.ie/defaultwednesday.html
Seaweeds/Carageenan Industry Profile (Philippines)
http://www.dticebu.net.ph/03_a_04.html
DOE Biomass Program Home Page
http://www.eere.energy.gov/biomass
Energy from kelp on Vancouver Island, British Columbia
http://www.islandnet.com/pwacvic/koppel05.html
Sponges
Sponges have been key targets of "drugs from the sea" MBT research projects since the 1950s, when compounds isolated
from sponges off the coast of Florida led to the development of the antiviral drug Acyclovir (Zovirax®) and Cytarabine
(Cytosar®), used to treat non-Hodgkin's lymphoma. Research into some of the novel chemistry of a Caribbean sponge also
eventually led to the development of the antiretroviral agent AZT (Retrovir®), the first important drug to be rolled
out in the fight against HIV.
Since this time, several intriguing natural products isolated from
sponges have shown promise for human drug development. The 1987
discovery by the Harbor Branch Oceanographic Institution's Division of
Biomedical Research of the natural product discodermalide
from a Bahamian deep-sea sponge ( Discodermia dissoluta). The
characterization and subsequent licensing of discodermalide to Novartis
for development as an anti-cancer drug is the focus of a companion Research Spotlight
profile piece on this website. Discodermalide is currently in stage
two
human clinical trials.
Another sponge-derived metabolite that shows promise as an anti-cancer therapy is halichondrin B. This compound was
first isolated from a Japanese sponge ( Halichondria okadai), and has also been found in a deep-sea sponge from New
Zealand ( Lissodendoryx sp.). NCI is conducting preclinical trials with
Halichondrin B which has been shown to be effective against certain melanomas and leukemias. A shallow Pacific sponge
from Palau ( Stylotella aurantium) has also yielded a natural product that may soon be used to treat osteoarthritis.
Unlike many complex marine metabolites with interesting bioactive properties, this compound is structurally relatively
simple and may therefore be easily (and economically) synthesized for eventual clinical use.
Among the more exciting marine biomedical discoveries potentially on a fast track to commercial production are a number
of sponge-derived compounds that may prove effective as treatments against malaria and tuberculosis. A research group
in the laboratory of Mark T. Hamann (scientist interview profile
here) at
the University of Mississippi School of Pharmacy have found that a group of marine alkaloids called manzamines include
compounds effective in treating malaria in mouse models and TB in a laboratory setting. The compounds are doubly
intriguing because in addition to impacting the target pathogen (e.g., killing the malaria parasite), they stimulate
the immune system (in the test mice) that helps clear the parasite from the system. Also encouraging is the fact that
relatively large quantities of the products can be extracted from sponges, allowing the possibility that sponges could
be grown through aquaculture to produce the bioactive compounds in quantity.
Since manzamines have now been found in more than 20 different sponge species, Hamann believes the compounds may
actually be produced by symbiotic bacteria and not the sponges themselves. This may be the case for discodermalide and
halichondrin B as well. If this is the case, the bacteria producing the target compounds may eventually be isolated
and cultured independent of its host sponge to yield sufficient quantities of the chemicals for research, clinical
trials, and possible commercial development.
In addition to the many promising biomedical discoveries that marine sponges have yielded, this ancient animal phylum
is also a source of inspiration in other MBT fields, including biomimetic approaches to materials engineering.
Related Weblinks
RSC Chemistry Science Network Sponge Page
http://www.chemsoc.org/
exemplarchem/entries/2002/Gup%20Hup%20Tan%20-%20Marine%20Biodiversity/sponges.htm
Cnidarians
A couple of compounds that have been extracted from members of Phylum Cnidaria (corals, anemones, jellyfish and their allies) are
examined in a bit more detail elsewhere on this site. These include the potent
cancer cell inhibitor eleutherobin, and a chemical family
known as the pseudopterosins that display powerful
analgesic and anti-inflamatory properties. Eleutherobin is derived from a Western Australian soft coral ( Eleutherobia sp.) while the pseudopterosins were
initially extracted from a Caribbean sea whip ( Pseudopterogorgia elisabethae).
Thus far, however, the most heavily exploited MBT discovery from a cnidarian source centered not on animals inhabiting tropical reefs
but on their luminescent, floating, distant cousins the jellyfish residing within the water column.
Know That Glow
The alien appearance and bioluminescent glow of many jellyfishes makes public aquarium displays featuring these ancient animals
popular with visitors. But that eerie living light has also been a source of intrigue and inspiration for scientists studying the
animals. Curiosity about why and how these animals glow has, in turn, lead to a series of important biotechnology breakthroughs.
In 1962 Princeton biologist Osamu Shimomura, working with the jellyfish Aequoria victoria discovered the protein responsible for the
animal's bioluminescence. Shimomura's research led to the discovery of the protein GFP (for "glowing fluorescent protein"). This
jellyfish protein glows blue when calcium is present in the environment and also glows green when struck by light in the blue to
near-UV range (near-UV is commonly called "black light").
Three decades later, Woods Hole Oceanographic Institute scientist Douglas Prasher found the gene that coded for the protein.
Subsequently, Martin Chalfie of Columbia University successfully incorporated the jellyfish GFP gene into a viral plasmid vector
and then inserted that into bacteria. Confirmation that the gene had been successfully inserted came when the cultures began glowing
on exposure to near-UV light, evidence that the transgenic cells were producing the novel jellyfish protein.
GFP as a Reporter Gene
Because GFP is coded for by a single gene that can be inserted into other organisms, and is a gene product whose presence can be visually
ascertained, it has become it a powerful research tool now in use in an array of settings. The marine-derived GFP gene and its protein
product have led to the creation of headline-grabbing transgenic organisms like glowing pet fish,
mice, and rabbits. More important, however, they have become immensely important as a serious and groundbreaking
molecular research tool.
One important application of the GFP gene is as a 'reporter gene.' Incorporated into a vector along with another gene that is the one
researchers are actually interested in, the vector can be inserted and incorporation success can be quickly assessed by visually
testing for GFP. For example, groundbreaking transgenic techniques such as Honolulu transgenesis can be used to insert novel genetic material into oocytes that can
then be transplanted into a foster mother. This technique was perfected using the GFP gene because, successful incorporation
of the novel genes can be confirmed by using high-sensitivity photosensors to see if test subjects glow under black light.
Glowing Fish, Polluted Water, and Cancer-Causing Genes
Although they made headlines as novelty pets and/or 'Frankenfish' (depending on one's point of view), genetically modified glowing
fish are popular and flexible research tools. In particular, transgenic freshwater zebrafish ( Danio rerio) have been extensively
utilized since the first ones were created in a laboratory 20 years ago. Zebrafish have many natural qualities that make suitable as
research organisms. They are small, hardy vertebrates that reach sexual maturity at three months of age, they produce hundreds of
transparent, externally developing eggs bi-monthly, and offspring develop from embryo to free-swimming larvae in just five days. Such
favorable attributes place zebrafish high on the list of useful research animals alongside traditional standbys like fruit flies and
laboratory mice.
Once GFP-containing vectors are successfully inserted to zebrafish embryos, these animals are capable of passing the engineered
traits on to their offspring when they become reproductively mature. This is important because it makes mass-production of research
organisms (as well as pets for aquarists) cost effective.
Transgenic zebrafish possessing the GFP gene have been developed by researchers at the University of Singapore for application as
pollution indicators in natural water bodies. In
this instance, the inserted GFP gene has been associated with a promoter gene that regulates when GFP is to be synthesized.
Painstaking work is being done to identify promoters that switch on GFP production in the presence of specific environmental
contaminants like estrogen and heavy metals.
The Singapore research group has also successfully incorporated another gene, RFP ("red fluorescent protein"), into their zebrafish. This one,
taken from a marine coral, produces a protein that fluoresces red. The team believes that ultimately they will be able to produce fish with
introduced genes coding for as many as five different colors. These would be associated with specific promoters and the result would
be a fish that turned different colors in the presence of various environmental pollutants.
Another group out of Penn State's Hershey Medical Center is using similar transgenic zebrafish to try to detect cancer-causing changes
to an animal's genes detect cancer-causing changes to an animal's genes. In this
case, the GFP gene is associated with a
promoter DNA sequence that is highly susceptible to frame-shift mutations similar to those associated with several types of cancer.
The current goal of this research is to produce a line of zebrafish whose cells glow blue when they are normal, but fluoresce green if
a frameshift mutation has occurred after exposure to a known or suspected carcinogen. A key benefit to utilizing such an assay animal
is that the precise locations of the induced mutations within the test animals can be visually identified.
Of Mice and Men
Mammalian cancer research models exploiting the fluorescent cnidarian proteins and their genes have also been developed. AntiCancer Inc., a commercial company in San Diego, has successfully developed more than 100
different mouse lines that can be used to visualize tumor expression, growth, and metastasis. The original mouse line selected for
use was nude (hairless) and also immunodeficient. Naked mice were chosen so that glowing GFP/RFP contained in the animals' cells
could be detected without sacrificing them. Immunodeficiency was required because human tumor tissues and cancerous cell lines needed
to be surgically grafted into the mice without triggering a histoincompatibility response. Prior to surgical implantation the
GFP and/or RFP genes are incorporated into the human cancerous tissues.
The result is a mammalian laboratory model designed to have certain cancers whose expression and progression can be visually assessed
in real time by placing the test animals in a light box and looking for the areas of the body that fluoresce when illuminated with
black light. Of central importance in ensuring the mouse cancer models have relevance to human health, the clinical progressions of tumor growth,
vascularization, metastasis, etc., in mice are nearly identical to the way these cancers develop in humans. These
novel research animals are commercially available and they are being used in a variety of ways. Most important, perhaps, new
anti-cancer drugs can now be screened for their effectiveness in arresting specific types of cancer in living mammalian subjects.
Related Weblinks
JNCI NEWS article: Jellyfish Protein Gives New Glow to Tumor Imaging
http://jncicancerspectrum.oupjournals.org/cgi/content/full/jnci;93/13/976
CNN.com article: Scientists use jellyfish DNA to detect cancer
http://www.cnn.com/TECH/computing/9908/06/t_t/carcinogen.tester
Wired News article: Why Goldfish Might Turn Blue
http://www.wired.com/news/technology/0,1282,49185,00.html
Molluscs
Hopefully the preceding information has led to a new appreciation for some of the unlikely scientific partners that have emerged from
the sea. But, can teaming up with a shell-less snail really ever be considered a good career move?
For Nobel laureate Eric Kandel the match has proved to be a
very good one. Kandel discovered that the sea hare Aplysia could be used as a model organism to examine the physiological basis for
learning. Since the early 1960s he and co-workers have used Aplysia to study processes like sensitization and habituation, and also
the development of short and long term memory. Through this seminal research it was deduced that the creation of short term memories
depends on functional changes in existing neural synapses, while the creation of long term memories is contingent upon the production
of novel neuroproteins and a change in the number of physical synaptic connections.
Erik Kandel was (jointly) awarded the 2000 Nobel Prize in Physiology and
Medicine for his discoveries with Aplysia concerning signal transduction in the nervous system.
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