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DR. ERIC MATHUR - Diversa Corporation, San Diego, California
The Research: Using Microbes Even If You Can't Grow Them
The Diversa Corporation was born in the early '90s to capitalize
on advances made by Carl Woese and Norm Pace in the field of molecular phylogeny, the use of the structure of
molecules to gain information on an organism's evolutionary relationships. These advances were enabling cloning
of ribosomal genes from microorganisms even if they were unculturable. Common concensus for marine microbes is
that only about 1% are considered culturable by existing techniques. A group of scientists, including Diversa's
Eric Mathur, recognized that if ribosomal genes could be cloned, then so could genes that encode proteins. That
meant that the novel gene products of unculturable microbes could still be put to commercial use.
Diversa now works to identify and clone genes that produce proteins with a variety of potential industrial,
pharmaceutical, and agricultural applications. Diversa's modest goal is to tap the biodiversity of the entire
planet. As such, the company does substantial exploratory work in the ocean and on land and has amassed samples
of most of the genes from more than two million microorganisms. This is a staggering accomplishment considering
that in the last hundred years of microbiology, only about ten thousand microorganisms have been described. The
company already has a number of products on the market for industrial and aquaculture uses. Diversa also has an
active drug discovery program and a number of leads for anti-infective compounds, but none have yet made it to
human clinical trials.
 - VIDEO CLIP 1: "Diversa Corporation - Company History and Background"
 Choosing Field Locations: Siberia Can Be Hot, Too
Through its own fieldwork or that of research collaborators it funds, Diversa collects samples from a wide range of
marine locations such as Puerto Rico, Hawaii, Bermuda and Indonesia. Because the company actively seeks compounds
for so many applications, they naturally collect from a wide variety of environments. Sometimes, a particular
application may suggest a logical collection site. "When we're doing a discovery program, we'll try to go to
all environments that have the basic chemical and physical properties we're looking for," says Mathur. For
instance, one of the company's products, an enzyme marketed under the name
Luminase TM,
was recovered from a hot spring in
Kamchatka, Siberia. To reduce costs and harmful environmental impacts, the product is used in paper
pulp processing to significantly reduce the need for bleaching. Because the processing had traditionally
been done both at high temperature and high pH, the latter a condition rare in marine environments, the
company found high pH hot springs to target in their search for the right enzyme to accomplish their goals.
In other cases, broader sampling programs are required because the right area to target is not as readily
identified.
- VIDEO CLIP 2: "Worldwide Marine Research Efforts"
Evolution in a Hurry?
The group works with both cultures of living bacteria, and nucleic acids from uncultured bacteria.
"The beauty of our technique is we recover all the nucleic acids from the whole community of microorganisms
present," says Mathur. At a hydrothermal vent, for instance, a Diversa team might cut off a large chunk of
an active chimney, grind it up, and clone all the DNA for insertion into easily culturable microbes and
subsequent production of the encoded proteins. These proteins then serve as starting material for the
company's many assays. Diversa has developed techniques for extremely high throughput in its assays,
and uses plates that allow for processing hundreds of thousands of samples in a single run.
Beyond working with samples collected directly from nature, Diversa has also developed a unique group
of what the researchers refer to as evolutionary tools to create new products for analysis, or to
customize products for a specific application using natural sample collections as starting points.
One of these tools is a technique called gene reassembly, where researchers at the company chop up
genes and reassemble them to make new genes whose products can then be analyzed. "We blend genes
like a deck of cards," says Mathur. In the case of Luminase, the Diversa team found several genes
for enzymes that each had some of the characteristics desired for the pulp processing applicationÑone
had the best actual activity, one performed in the best pH range, etc. Those genes were chopped and
blended and one of the resulting variants had all the positive characteristics of the starting genes.
Another evolutionary tool is called gene site saturation mutagenesis (GSSM). In this process, specific
amino acids in a protein are systematically switched out with other amino acids and the resulting
products tested to see if they exhibit improved characteristics while retaining desirable characteristics
of the original product. With Luminase, even once Diversa created an enzyme through gene reassembly with
all the characteristics needed for the pulp processing application, except that it was not stable at the
highest target temperatures. Using GSSM, Diversa was able to modify the enzyme to create Luminase,
a version of the enzyme that could withstand much higher temperatures. The melting point of Luminase
was raised 39¡C relative to the original product.
Why Enzymes?
Though their economic value is typically not as great, enzymes offer a number of advantages for
commercialization compared to pharmaceuticals. First and foremost, enzymes can be taken to market
for industrial or other uses much more quickly than pharmaceuticals because Federal Drug Administration
approval is not needed if the a product is not intended for human consumption or use on humans.
Whereas enzymes can go from discovery to market in three years or less, drugs typically take a decade
or more. Another simplifying factor with enzymes is that their production is easier. Enzymes are
typically coded for by a single gene, while pharmaceutical compound production typically involves
a complex biosynthetic pathway that requires a number of different genes. A final attraction of
enzymes for Diversa is that they can be manipulated using the company's evolutionary tools
described above.
- VIDEO CLIP 3: "Industrial Enzyme Discovery and Drug Discovery"
Going for the Glow
Fluorescent genes are absolutely essential to a number of biotechnological applications. One reason is that the
genes that code for them can be attached to other genetic material so that if the genes are expressed, easily
detectable fluorescent proteins are also produced. The biotechnology industry is always seeking new fluorescent
proteins that might enable new applications or improve on existing ones. Because these proteins are so valuable
and also happen to be reasonably common in certain marine environments, Diversa has done extensive work in such
locations as Bermuda and Costa Rica in search of these proteins. This work involves using special filtered
lights that, when viewed through different filters, allow fluorescence in organisms to be easily spotted.
Fluorescence occurs when an animal or object absorbs light of one color and then reemits light of, or glows,
another color. Once fluorescent organisms are spotted, Diversa teams then take samples back to the laboratory
and isolate the genes responsible for the fluorescence. This process led to the discovery of proteins that
are brighter and more robust than those previously available, which Diversa has licensed to the Amgen
Corporation for commercialization.
Hot, Hot, Hot and Deep, Deep, Deep
Hydrothermal vents have proven productive locations for Diversa's search for enzymes with industrial applications.
One key area of study has been a search for enzymes that break down carbohydrates found in otherwise undigestable
agricultural waste. Breaking this material down converts it into fermentable sugars that can then be used for ethanol
production. Diversa has a product called Ultra-Thin for this use that is nearing market approval. It was created
using the gene reassembly technique on material from uncultured organisms. Conventional processing of biomass,
which occurs in nature at a pH of about 4.5, utilizes enzymes that will only work at significantly higher pH.
Processing therefore requires an expensive, environmentally unfriendly conversion of the material to a higher
pH for breakdown using caustic additives. Diversa's product works well in the 4.5 pH range, eliminating the need
for this cumbersome step and reducing costs as well as energy requirements for ethanol production. Through
related work in collaboration with Switzerland-based Syngenta, Diversa is also exploring the possibility that
the gene coding for their enzyme could be inserted in corn so that the enzyme is automatically present in crops
that will be used for ethanol production, making conversion even more efficient. Reducing the energy required
to produce ethanol has been a major goal of the U.S. Department of Energy in order to make widespread use of
ethanol more feasible. "I think this is really, really important," says Mathur, "that we start looking at
methods to really leverage biotechnology and marine biotechnology to start producing renewable fuels and
renewable energy and stop having to work with oil in the ground."
Diversa has also found enzymes that may prove useful for biomass conversion through studies of organisms that
grow on whale carcasses that fall to the deep seafloor. Other enzymes isolated from this work in development
also appear to be very good at breaking down fats, which could mean a range of potential applications from use
in laundry detergents to the production of vegetable oils.
Another hydrothermal vent-derived Diversa product, called
Pyrolase TM,
which became available in 2002, was developed to allow recovery of oil from "dry" wells through a process called
hydraulic fractionation. This technique involves pumping sand mixed with a viscous carbohydrate such as guar
to hold it together into a dry well, and then the well is pressurized. This process forces sand into cracks
in a well, allowing oil from surrounding but previously blocked areas to seep in. But once the oil is flowing
again the viscous liquid makes recovery difficult. Pyrolase breaks down this material but can be added to the
sand-guar mix because it is only activated at higher temperatures, such as those found deep in the ground. As
temperatures rise, therefore, the enzyme activates, breaks down the guar, and allows recovery of oil that flows in.
Still another hydrothermal vent product called ThermalAce TM, now licensed to Invitrogen, is a DNA polymerase
enzyme that works at higher than average temperatures for use with the ubiquitous polymerase chain reaction
process.
- VIDEO CLIP 4: "Chemical Diversity in Unlikely Places: Whale Falls and More"
Key to the Lox... Disease
Salmon rickettsial syndrome (SRS) is a disease that can result in the death of up to 40% of salmon raised in
aquaculture facilities. The condition can be treated effectively with antibiotics, but a growing number of
consumers and countries are demanding that food be free of antibiotics. So, Diversa has identified and
recently commercialized a vaccine under the name Bayovac that is derived from a protein the company discovered
and is the first such treatment for SRS. The product, which has proven highly effective, is produced through
a partnership with Bayer Animal Health. Diversa has a number of other vaccines in various stages of
development for use in the aquaculture industry with a focus on treating conditions in salmon and related
species, but with some work related to shrimp aquaculture as well.
- VIDEO CLIP 5: "More Thoughts on Enzymes and Drugs; Customizing Gene Products"
Educational Background
Mathur began with a BS in biology from the University of California, Riverside, then spent 15 years in basic
research with the Scripps Research Foundation. From there, he shifted to commercial biotech work. He began with
Strategene, based in La Jolla, Calif., then came to Diversa. Mathur's work, like Diversa's, stretches beyond the
bounds of marine biotechnology, but he was nonetheless always fascinated with the ocean and is an avid scuba diver.
He also has some interesting extracurricular research interests. For example, he sits on a number of boards
including that of the Search for Extraterrestrial Intelligence (SETI), as well as the space studies board for
the prevention of foreign contamination of Mars. He is also part of the Sloan Foundation's Working Group for
the Census of Marine Microbes.
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