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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, October 2004 by Alexandra Goho .

"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: Available to subscribers at
[www.sciencenews.org]). 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.

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