Microbes make the switch: tailored bacteria need caffeine product to survive.

With some creative genetic engineering, chemists have designed bacteria that rely on a breakdown product of caffeine for their survival. The advance could eventually lead to decaffeinated coffee plants, the researchers suggest.

"The idea is to convince these microorganisms to do the chemistry that we want them to do," says Justin Gallivan, a chemist at Emory University in Atlanta. He and Shawn Desai, also of Emory, provided bacteria with a molecular switch that senses the presence of theophylline--the caffeine by-product. In response, the switch activates a gene that renders the microbes resistant to an antibiotic.

The genetic control that Gallivan designed is called a riboswitch, a segment of RNA that changes conformation when bound to certain small molecules and then turns genes on or off (SN: 4/10/04, p. 232). Riboswitches exist naturally in cells, where they regulate gene activity in response to changes in concentrations of vitamins or amino acids. Researchers have recently begun creating synthetic riboswitches that could be used as sensors or for gene therapy.

The Emory researchers incorporated the theophylline-sensitive switch into their Escherichia coli cells and grew the modified cells in the presence of an antibiotic. Without theophylline, the cells died. Even cells provided with caffeine failed to survive. However, when the researchers supplied the E. coli with the caffeine by-product, the riboswitches turned on the gene for antibiotic resistance. These cells then proliferated, the researchers report in the Oct. 20 Journal of the American Chemical Society.

"This is an important contribution. The researchers use their riboswitch in a unique way so that the survival of the cell is dependent on the function of that RNA switch," says Yale University chemist Ronald Breaker, who coined the term riboswitch.

A next step toward decaffeinated plants, says Gallivan, is to engineer the cells to produce theophylline themselves. To do that, the researchers plan to provide them with a gene for an enzyme that breaks down caffeine. When fed caffeine, the cells would produce theophylline and therefore ensure their survival in the presence of the antibiotic.

Coffee plants already produce an enzyme that naturally breaks down caffeine into theophylline. However, the process is very slow, and the gene that encodes the enzyme remains unknown. To search for that gene, the Emory group plans to insert various coffee-plant genes into bacterial hosts. Only those microbes that get the sought-after gene and make its caffeine-destroying enzyme will produce theophylline and live through antibiotic exposure.

Once the scientists find this gene, they plan to use a process called directed evolution to boost the enzyme's efficiency. They'll create millions of mutated versions of the gene and insert them into another set of bacterial hosts. As the bacteria grow, those that break down caffeine the fastest will outperform the others. The gene that codes for the fastest enzyme ultimately could be inserted into a coffee plant, yielding virtually caffeinefree coffee beans.

Gallivan says that the same technique could be used to ferret out genes associated with the synthesis or destruction of other economically important, naturally occurring compounds.