Directed molecular evolution paves the way to decaffeinated coffee plants
Directed molecular evolution involves repeatedly screening related DNA sequences that have been spliced into yeast or bacteria to identify the sequence that codes for the best-performing protein. Scientists have a number of methods for creating the related DNA sequences, such as DNA shuffling and incremental truncation, but far fewer for subsequent screening.
The most effective current screening method for the directed evolution of enzymes involves linking the production of the desired enzyme to the survival of the bacteria. But this is restricted to screening enzymes that either provide protection against a specific toxin or that are involved in producing a compound that the bacteria cannot live without - known as auxotrophs. To extend this type of screening to a much wider range of enzymes, chemists from Emory University, Georgia, have developed a method to link a normally innocuous compound to the survival of the bacterium Escherichia coli, thereby turning it into a ’designer auxotroph’.
The researchers turned to recently discovered structures called riboswitches. These naturally occurring sections of RNA molecules bind directly to a target molecule, acting to either promote or suppress protein production. The chemists created a synthetic riboswitch by taking an artificial strand of RNA known as an aptamer, which is also able to bind to proteins and small molecules, and splicing it into a gene called lacZ that codes for the protein ?-galactosidase. They then inserted this modified lacZ gene into E. coli to see how the synthetic riboswitch would affect ?-galactosidase production.
The specific aptamer used by the chemists binds tightly to the molecule theophylline - a natural breakdown product of caffeine. They discovered that the transgenic E. coli would only produce ?-galactosidase in the presence of theophylline. So they attached the same aptamer to a gene coding for the enzyme chloramphenicol acetyl transferase (cat), which confers resistance to the antibiotic chloramphenicol. They found that E. coli containing this modified cat gene could only survive exposure to chloramphenicol if it had access to a source of theophylline.
The chemists are now using this designer auxotroph to screen enzymes produced by directed molecular evolution for their ability to break down caffeine, which could lead to the development of naturally decaffeinated coffee plants. The chemists suggest that a whole range of designer auxotrophs can easily be created by using aptamers that bind to other compounds.
S K Desai and J P Gallivan, J. Am. Chem. Soc., 2004, 126, 13247