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CONTACT: Stanford University News Service (650) 723-2558
COMMENT:Prof. Chaitan Khosla, Chemical Engineering (415) 723-6538; Rembert Pieper, Chemical Engineering (415) 723-8770

Complex members of drug-rich family of proteins created in test tube

STANFORD -- The antibiotics tetracycline and erythromycin, the anticancer agent daunomycin, the immunosuppressant rapamycin and the veterinary products monensin and avermectin are just a few of the dozens of drugs that come from a family of proteins produced by soil and marine organisms.

Cells create these compounds, called polyketides, in an assembly process that requires anywhere from 10 to more than 100 different enzymes. Over the past 30 years, attempts to recreate the more complex members of this pharmaceutically rich family in the test tube have proven unsuccessful. But a research team led by Chaitan Khosla, Stanford professor of chemical engineering, now report achieving this goal.

Writing in the Nov. 16 issue of the journal Nature the scientists describe the cell-free synthesis of two polyketides, including the parent molecule of the antibiotic erythromycin A.

In a related article in an upcoming issue of the Journal of the American Chemical Society, Khosla and his colleagues report that cell-free synthesis allowed them to produce a type of polyketide not known in nature, and can provide important new information about the molecular assembly process.

The researchers working with Khosla were Stanford postdoctoral fellow Rembert Pieper, and Guanglin Luo and David E. Cane from Brown University.

According to Khosla, the motivation for duplicating the polyketide manufacturing process outside the cell was to learn more about how these molecules are assembled. "It's like eating chocolate without a wrapper versus eating it with a wrapper - you get a much better feeling for what you're doing if you do it without the wrapper," he said.

But the scientists had a practical reason for their research as well. Synthesizing these molecules outside the cell makes it possible to create types of polyketides that don't exist in nature.

"You are no longer limited to the confines of cellular metabolism in terms of what molecules are taken up by the assemblies and what molecules are spat out by these assemblies," Khosla said.

Because these molecules historically have been an extremely rich source of medicinally active agents, the more novel polyketides that scientists can synthesize, the greater the odds of finding interesting new drugs, he added.

The researchers used fairly standard techniques in protein chemistry: purifying individual components and carefully putting them back together. Compared to previous researchers, the group benefited from the powerful genetic engineering tools that have been developed in recent years. As a result, it was easy for them to manipulate the enzymes, called polyketide synthases, that create these molecules. Using recombinant DNA techniques, they included the genetic material that produced all the enzymes that they needed in a single organism. That allowed them to remove all the enzymes from the cell in a single step.

Nevertheless, getting the enzymes to produce polyketides in the test tube was not a simple matter. Pieper, who did most of the laboratory work, has half a dozen notebooks full of unsuccessful attempts that ultimately lead him to the successful combination of steps and conditions that make the process work.

"It sounds like alchemy, and to some...

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