Natural selection is daily and hourly scrutinizing, throughout the world, every variation, even the slightest; rejecting that which is bad, preserving and adding up all that is good; silently and insensibly working, whenever and wherever opportunity offers, at the improvement of each organic being in relation to its organic and inorganic conditions of life.

                                    Charles Darwin, On the Origin of Species

Charles Darwin wrote these words almost 150 years ago to describe what is now generally acknowledged as the central organizing principle of modern biology: natural selection. When we read "every variation," we naturally think of DNA and genomics, although Darwin knew nothing of the gene. At the time of the publication of On the Origin of Species, Gregor Mendel was in the midst of growing and crossing pea plants in the garden of the St. Thomas Monastery in Moravia. After counting and sorting hundreds of thousands of garden peas, Mendel reported his results suggesting the nature of the elements of heredity and was then ignored for almost 40 years, until long after his death in 1884.

The nascent ideas proposed by Darwin and Mendel have emerged as the cornerstone of our understanding of life. At a very practical level, directed molecular evolution - an extension of the discoveries of both Darwin and Mendel, which I like to think would have given them both enormous delight - is providing many highly useful enzymes and enzyme products that have never existed in nature. These custom-designed catalysts and chemicals are being used in agriculture, drug discovery, food processing, and industrial chemistry. Directed molecular evolution works at the microtiter-plate level to produce novel enzymes through the same process of variation and selection described by Darwin, but in a breathtaking timescale of days or weeks instead of the millennia required by natural selection.

Diversa Corporation, a biotechnology company located in San Diego, California, has a business plan based upon the use of directed molecular evolution. Using this approach, they have created new, highly specific, and custom-designed enzymes and even entire novel synthetic pathways for the production of small organic molecules with drug potential. Diversa scientists search for genes that encode useful enzymes from microbes that exist in extreme environments; then they use the paradigm of variation and selection to further fine-tune these naturally occurring enzymes.

Searching for useful enzymes and compounds from microbes, even from extreme environments, is not novel. However, Diversa has added a new and useful twist to the process. Microbiologists estimate that the vast majority of naturally occurring microbes are not capable of being grown in culture. "A single sample may contain 10,000 different microbes," says Jay Short, Diversa's president and chief executive officer, "but only a tiny number, maybe 0.1% of the total microorganisms in such a sample, can be isolated and grown under laboratory conditions." Short explains that it is sometimes very difficult to establish in a laboratory setting the conditions for which some microorganisms have become exquisitely adapted, a particular problem for organisms isolated from extreme environments. Also, some organisms seem to require metabolites from other organisms to grow.

The very act of isolating the microorganism removes it from the complex chemical environment it needs to survive and reproduce. To circumvent these problems Diversa scientists simply bypass attempts to isolate and grow the microbes in the laboratory. Instead, they isolate DNA directly from a raw sample, usually just a few tablespoons of soil, sediment, or muck, and then use it to build gene-expression libraries in one well-defined, easy-to-propagate bacterial host. This host organism, according to Diversa, allows the production of numerous variants of the parent gene, is amenable to high-throughput screening protocols, and is appropriate for scale-up to produce large quantities of enzymes or metabolites.

Diversa obtains its raw materials from a wide variety of sources. It has agreements in place to allow collection of samples from hot springs and geysers at Yellowstone National Park and in Iceland, the salt marshes of the New Jersey Meadowlands, and tropical rainforests in Costa Rica. Diversa literally prospects worldwide for useful microbial genes, including such exotic venues as the decaying bowels of a whale carcass sitting on the bottom of the sea, a site which recently provided one of their most interesting enzymes. Diversa estimates that its microbial gene libraries contain the DNA from over 1,000,000 microorganisms isolated from such sources, which far exceeds the approximately 10,000 microorganisms that have been described in the scientific literature.

Once the gene for a useful enzyme is discovered from a natural source, a rapid version of natural selection is used to optimize it for a particular use in a specific environment. Diversa uses gene site saturation mutagenesis and gene reassembly to produce hundreds of thousands of variants of the natural enzyme. Gene site mutagenesis allows the production of a family of different genes, each of which will produce a protein with one amino acid difference at a series of defined positions in the naturally occurring amino acid sequence. Gene reassembly allows whole portions of a specific gene, or even the genes of a complex synthetic pathway, to be rearranged. Using these techniques, scientists can rapidly produce hundreds of thousands to millions of different variations of a genome. Such an approach removes the need for a detailed understanding of the enzyme in order to optimize it for a particular purpose. Instead, it mimics the trial and error process of nature.

It is useless to generate libraries of millions of genes and then create hundreds of thousands of variants if there is no way to carefully select for the optimal product. Nature usually does this slowly over very long periods of time and many generations, but Diversa claims its proprietary technology, biopanning, can streamline this process by screening over one billion genes per day, to rapidly identify the optimal enzyme for a particular environmental condition.

Using these approaches, Diversa has evolved and brought to market its first product, an enzyme that is used in the recovery of oil fields, to be followed by a second generation enzyme that will be useful for recovery of higher-temperature oil wells. This application is projected to bring in $10 to $20 million per year to the young company. Recently, Diversa has produced enzymes useful in chemical synthesis applications that also involve exposure to high heat. In this case, the enzymes can be renatured after the heating step to regain their catalytic activity. In the future, Diversa hopes to develop additional enzymes that can be used as additives to detergents; in corn, textile, and pulp and paper processing; and for agricultural uses of crop protection and yield enhancement. In addition, Diversa hopes to discover "non-natural" natural products that will be candidates for anti-microbials, anti-fungals, and anti-virals.