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New approach for producing novel antibiotics demonstrated

Stanford scientists have developed a new approach for making novel antibiotics that may aid in the fight against the growing drug resistance of many strains of bacteria.

The achievement, which is reported in the July 18 issue of the journal Science, involves the use of genetically engineered enzymes to produce new types of polyketides, a family of molecules that are found in a number of antibiotic, immunosuppressant and anti-cancer drugs.

Stanford postdoctoral fellow John R. Jacobsen performed the work with the support of Chaitan Khosla, associate professor of chemical engineering. C. Richard Hutchinson from the University of Wisconsin-Madison and David E. Cane from Brown University provided synthetic compounds that were crucial for the research.

The approach involves genetically doctoring the enzymes that cells use to produce polyketides so that they will accept and process synthetic compounds, rather than the chemical compounds that they normally use. The enzymes thus can be forced to produce unnatural polyketides, many of which exhibit anti-bacterial properties.

The enzymes, called polyketide synthases, produce these molecules in a process somewhat similar to a chemical assembly line, typically with more than 30 steps. They start with relatively simple molecules, called precursors, and combine them into molecules of increasing complexity until polyketides are formed in the final step. The molecules produced in one step become the precursors for the subsequent step.

A major obstacle the researchers had to overcome was competition from natural feedstocks, molecules that the cell produces for the enzyme's consumption. Jacobsen solved the problem by altering the enzyme to block the first step in its synthesis pathway. This kept the enzyme from producing natural precursors for the second and subsequent steps. That allowed the scientists to operate the enzyme without interference, by providing it with synthetic molecules resembling the precursors for its later steps.

The researchers performed this delicate genetic surgery on the polyketide synthase that produces erythromycin. They then injected synthetic precursors into the cells that contained the altered enzyme and produced several unnatural polyketides. These compounds exhibited an antibacterial potency comparable to the well-known antibiotic in laboratory tests, the scientists report.

"We expected to find other obstacles once we had this initial problem solved, but we haven't seen any that are serious," Khosla said. "We've been pleasantly surprised by how tolerant the entire system appears to be toward unnatural substrates. The overall process seems to work remarkably well."

The process represents the latest twist on the production of novel polyketides pioneered in Khosla's laboratory using a process called "combinatorial biosynthesis." Previously, Khosla and his students worked out the biosynthesis rules that the polyketide-making enzymes follow. With this knowledge they used genetic engineering techniques to make slight alterations in the enzymes, causing them to produce new polyketides. Khosla and his collaborators have used this method successfully to make hundreds of new polyketides.

Now the ability to produce these compounds by altering the enzyme's feedstocks promises to multiply the power of this basic approach many-fold. Researchers should be able to generate hundreds of new compounds for each one that they have previously created. This could eliminate a major drawback to the commercial adoption of combinatorial biosynthesis: the limited number of compounds that can be produced.

When drug companies begin looking for a new drug and have no place to start, they begin by screening thousands of natural products in search of a "lead" molecule that exhibits some of the desired activity. If a library containing thousands of synthetic polyketides can be generated using genetic engineering, it could become an attractive alternative source of lead molecules for new antibiotics, immunosuppressants and anti-cancer drugs.

The research was supported by the National Science Foundation, the National Institutes of Health, and the David and Lucile Packard Foundation.


By David F. Salisbury

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