|Dr Ron Swanson, Associate Director of Genomics at Diversa, collecting a sample from a geothermal vent in Yellowstone National Park, Wyoming, USA. Photo courtesy of Diversa Corp.|
Stein is a Principal Scientist in charge of microbial diversity at Diversa Corporation in San Diego, California. Diversa provides its industrial customers with enzymes, and the weirder the source, the better. Enzymes are the cell's version of a chemist, capable of converting one chemical to another with astonishing efficiency and zero toxicity. They are used in the production of everything from cheese, to perfume, to stone-washed jeans Table 1. Enzymes from the single-celled bacteria that live in extreme environments should, says Stein, be able to withstand both the harsh conditions in industrial plants and the wear and tear of everday use by consumers.
The trick to this approach is direct isolation of DNA from the bacterial sample. Scientists then search for genes in the DNA that hold the code for useful enzymes. Going straight for the DNA allows you to skip the intermediate and tricky step of growing the bacteria in the laboratory.
The method seems to be yielding results. "This
is reaching out beyond organisms that are well studied,"
says Norman Pace, of the University of California at Berkeley.
"I think that is wonderful."
|Table 1: Which enzymes are used to make your favorite product?|
|Mannase||Dispersal of the galactomannans used to increase the flow of oil in a drilling operation (the other alternative is bleach). Thermophilic mannase can withstand the high temperatures at deep strata.|
|Endoglucanases and phosphatases||Increase the absorption of animal feed by breaking down glucan coatings on grain. Thermophilic enzymes survive the high-temperature extrusion process that creates the feed pellets.|
|Cellulases and xylanases||Bleaching of wood products. The enzymes loosen the structure of wood pulp. Paper manufactures can then use far less bleach, as the lignin can more easily escape from the plant matter.|
|Endoglucanases, lipases and proteases||Washing detergents. Thermophilic enzymes are useful for hospitals that wash at high temperatures for sterilization purposes; psychrophilic enzymes can be used in room-temperature washing detergents.|
|Proteases||Break down proteins in cheese to enhance flavor|
|Esterases||Create new chemicals for use in fragrances.|
|Cellulases||Soften the fibres in jeans to create stone-washed effect.|
Perhaps, then, Diversa need go no further than a San Diego garden. But Diversa scientists have faith in their search for extremophiles, the bacteria that live in harsh environments. Although 104 is a lot, 104 of pretty much the same thing may not be so useful. Isolated populations of bacteria in extreme environments probably have enzymes that can direct novel chemical reactions. And enzymes from thermophilic (heat-loving) bacteria have several potential advantages.
The first is obvious: A number of chemical processes require enzymes to act, or retain their activity, at high temperatures (see the first two entries in Table 1).
But the main stumbling block for those wishing
to use enzymes in industry has been lack of stability, not lack
of heat-resistance. Enzymes are non-toxic, biodegradable, and
great at doing only what they are told, but they go off. Diversa
recognised that thermophilic enzymes are stable enzymes. Even
if they are to be used at mild temperatures, they last far longer
than regular enzymes. They also resist the destructive effects
of organic chemicals used in other steps of the industrial process.
The centers of the vents are devoid of life, but as the temperature decreases in the surrounding chimney, core samples turn up a wealth of thermophiles. Other samples are collected using either a slurp gun, which filters water from around the vent, or collection plates seeded with chemicals that attract bacteria because the microbes see them as lunch.
The bacteria can live at temperatures of up to 113 ºC, and in conditions that are extremely salty, acidic, or alkaline.
Deep-sea marine animals, or rather the bacteria living on and in their bodies, are also a good source of biodiversity. One intriguing recent find is a worm that keeps its tail in the hot vent and its head in the cool surrounding water. Researchers are interested in the worm as a source of enzymes that can work over a broad range of temperatures.
With their similar temperatures and chemical makeup, geothermal pools are the terrestrial equivalents of deep-sea vents, at least for harvesting bacteria. Deep-sea vents and geothermal pools also share an island-like quality, isolated biologically by their unique chemistry. For example, one pool that Diversa collects from in Costa Rica, at ~95ºC and pH 0, stands in stark contrast to the surrounding rainforest. Diversa signed a bioprospecting agreement in August giving them access to Yellowstone National Park, which has over 60% of the world's geysers, hot springs and boiling mud pots.
Whale skeletons, another source of microbes, are also like isolated islands, so isolated that they are tricky to find. The US Navy helped out scientists by locating one when it was looking for a wayward Triton missile, but in other cases researchers have resorted to towing dead, beached whales out to sea and dumping them.
Isolation is not the only attraction of whale
bones. "They ooze complex lipids [greasy molecules that
line the outside of cells] over periods of up to 20 years,"
says Stein. Bacterial mats that grow on the bones are a rich source
of the lipases and esterases needed to break down the lipids,
and these two classes of enzymes are especially important to industry.
The organisms and their enzymes are adapted to the deep-sea cold;
such psychrophilic enzymes can be useful for commercial laundry
detergents. More psychrophilic enzymes are turning up in earth
and lichen samples from Icelandic and Antarctic glaciers.
Each E. coli in the library can now
make a single enzyme, which is encoded by a fragment of DNA from
a single bacterium in the isolated sample. Scientists then test
whether this enzyme does the chemical conversion that they want
it to do. Testing for the final property of the enzyme "gives
us the opportunity to select without any prior knowledge of what
the genes should look like," says Short.
Scientists have big plans for this market. "We're trying to replace enzymes already in use, and also open the door to using enzymes in other processes where they haven't been used before," says Dan Robertson, Director of Enzymology at Diversa. That approach is needed, says Pace, because the biotechnology industry has, in the past, relied on "stuff that has fallen into our pockets. There has been relatively little seeking out."
Diversa is not alone, however. Thermogen (Chicago, Illinois) has found
a number of enzymes in the species of heat-loving bacteria that
can be grown in the laboratory, and are now looking for other
bacteria in the wild. Altus Biologics, Inc. (Cambridge, Massachusetts)
has taken a very different approach to the stability problem.
They add a chemical to purified, crystallized enzymes so that
many enzyme molecules are linked together to form a stable enzyme
pellet. The cross-linked crystals are stable to heat and organic
chemicals, and have a long shelf-life.
In an area that Diversa has entered
recently the competition is fiercer still. Along with ChromaXome
(San Diego, California) and TerraGen Diversity, Inc. (Vancouver,
British Columbia), Diversa is looking to discover new drugs made
by bacteria. The companies will test natural products, the chemicals
that bacteria make either to do a job in the cell, or to kill
other bacteria (the antibiotics). "We think that the vast
majority of natural product pharmaceuticals have yet to be discovered,"
The search is based on the fact
that many related bacterial genes are clumped together. By isolating
huge fragments of DNA, Stein thinks he has a good chance of picking
up all the genes he needs to make an antibiotic in one fell swoop.
All the current antibiotics are natural products or minor variants
of natural products. With so many bacteria undiscovered, it seems
all but certain that there are more drugs just waiting to be found,
buried in anything from thermal vents to garden soil. And finding
them might be easier than trial-and-error testing of chemicals
made by humans. "Evolution has been doing a lot of tinkering
over the years," says Pace, "and you may as well take
advantage of it."